Multispecific binding protein compositions and uses thereof

Abstract

Described herein are single-chain polypeptide compositions comprising variable domains linked by a linker, folded into a conformation such that the variable domains form two effector cell antigen interaction domains (E) wherein each effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single-chain variable fragment, and at least one of the effector cell antigen interaction domains is linked to one or more target interaction domains. Methods of use of the single chain polypeptides are also described herein.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 307,584, filed on February 7, 2022, which is incorporated by reference in its entirety. Summary of the invention

[0003] Disclosed herein is a single-chain polypeptide comprising a variable domain connected by a linker, folded into a conformation such that the variable domain forms two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single-chain variable fragment, and at least one of the effector cell antigen interaction domains is connected to one or more target interaction domains. In some embodiments, the target interaction domain is bound 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 a tumor microenvironment. In some embodiments, the target interaction domain comprises an antibody, an antibody fragment, a ligand, or a cofactor. In some embodiments, the antibody or antibody fragment of the target interaction domain comprises Fab, Fab′, F(ab′)2, Fv fragment, single-chain Fv (scFv), tandem single-chain Fv ((scFv)2, dual-affinity retargeting antibody, bispecific antibody (diabody), VHH single domain antibody, TriTac, Adnectin, Anticalin, Avimer, Fynomer, Kunitz domain, knottin, Affibody or DARPin. In some embodiments, one or more target interaction domains comprise one or more target cell antigen interaction domains that bind to a target cell antigen. In some embodiments, the target cell antigen interaction domain binds to more than one target cell antigen. In some embodiments, the target cell antigen interaction domain is a bispecific or multispecific antibody or antibody fragment. In some embodiments, the target cell antigen interaction domain comprises a VHH single domain antibody (TVHH). In some embodiments, the single chain polypeptide does not contain a crystallizable fragment (Fc) structure. Domain. In some embodiments, the two variable domains (EV and EV) of the single-chain variable fragment include 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, at least one EVL of the effector cell antigen interaction domain connected to one or more target cell antigen interaction domains is connected to at least one TVHH, and the EVH of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to at least one TVHH. In some embodiments, at least one TVHH connected to EVL binds to the same target antigen as at least one TVHH connected to EVH. In some embodiments, at least one TVHH connected to EVL binds to the same target antigen as at least one TVHH connected to EVH and has the same complementary determining region (CDR) sequence. In some embodiments, at least one TVHH connected to EVL binds to the same target antigen as at least one TVHH connected to EVH and has the same complementary determining region (CDR) sequence. In some embodiments, at least one TVHH connected to EVL binds to the same target antigen as at least one TVHH connected to EVH and does not have a common complementary determining region (CDR) sequence.In some embodiments, at least one TVHH connected to EVL binds to a different target antigen than at least one TVHH connected to EVH. In some embodiments, EVL and EVH are connected to the same number of TVHHs. In some embodiments, EVL is connected to a different number of TVHHs compared to the number of TVHHs connected to EVH. In some embodiments, at least one EVL connected to the effector cell antigen interaction domain of one or more target cell antigen interaction domains is connected to one TVHH, and at least one EVH connected to the effector cell antigen interaction domain of one or more target cell antigen interaction domains is connected to one TVHH. In some embodiments, at least one EVL connected to the effector cell antigen interaction domain of one or more target cell antigen interaction domains is connected to one TVHH, and at least one EVH connected to the effector cell antigen interaction domain of one or more target cell antigen interaction domains is connected to two TVHHs. In some embodiments, at least one EVL connected to the effector cell antigen interaction domain of at least one or more target cell antigen interaction domains is connected to one TVHH, and at least one EVH connected to the effector cell antigen interaction domain of one or more target cell antigen interaction domains is connected to three TVHHs. In some embodiments, the EVH of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to one TVHH, and the EVL of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to two TVHHs. In some embodiments, the EVH of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to one TVHH, and the EVL of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to three TVHHs. In some embodiments, the EVH of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to two TVHHs, and the EVL of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to two TVHHs. In some embodiments, the EVH of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to three TVHHs, and the EVL of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to three TVHHs.In some embodiments, the EVH of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to four TVHHs, and the EVL of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to four TVHHs. In some embodiments, the EVH of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to five TVHHs, and the EVL of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to five TVHHs. In some embodiments, the EVH of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to six TVHHs, and the EVL of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to six TVHHs. In some embodiments, the EVH of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to seven TVHHs, and the EVL of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to seven TVHHs. In some embodiments, the EVH of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to eight TVHHs, and the EVL of at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to eight TVHHs. In some embodiments, at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to one target cell antigen interaction domain. In some embodiments, the C-terminus of one or more target cell antigen interaction domains is connected to at least one of the effector cell antigen interaction domains. In some embodiments, the N-terminus of one or more target cell antigen interaction domains is connected to at least one of the effector cell antigen interaction domains. In some embodiments, at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to more than one target cell antigen interaction domain. In some embodiments, all of the more than one target cell antigen interaction domains bind to the same target antigen. In some embodiments, all of the more than one target cell antigen interaction 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 interaction domains bind to the same target antigen and at least two of the more than one target cell antigen interaction domains do not have a common CDR sequence. In some embodiments, at least two of the more than one target cell antigen interaction domains bind to different target antigens. In some embodiments, at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to two target cell antigen interaction domains. In some embodiments, the C-termini of the two target cell antigen interaction domains are connected to one of the effector cell antigen interaction domains. In some embodiments, the N-termini of the two target cell antigen interaction domains are connected to one of the effector cell antigen interaction domains. In some embodiments, at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to three target cell antigen interaction domains. In some embodiments, the C-termini of the three target cell antigen interaction domains are connected to one of the effector cell antigen interaction domains. In some embodiments, the N-termini of the three target cell antigen interaction domains are connected to one of the effector cell antigen interaction domains. In some embodiments, at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected 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 domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-EV-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-TVHH-TVHH-EV-EV-EV-EV; EV-EV-EV-EV-EV-TVHH-TVHH-TVHH; TVHH-EV-EV-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;

[0004] 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. In some embodiments, the variable domains and one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH; or TVHH-EV-EV-EV-EV-TVHH. In some embodiments, the variable domains and one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH. In some embodiments, the variable domains and one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-EV-EV-EV-EV-TVHH-TVHH. In some embodiments, the variable domains and the one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which the (-) is the linker: 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 domains and the one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which the (-) is the linker: TVHH-EVL-EVH-EVL-EVH; TVHH-EVH-EVL-EVH-EVL;

[0005] 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;

[0006] TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH;TVHH-TVHH-EVL-EVH-EVL-EVH-EVL;EVL-EVH-EVL-EVH-TVHH-TVHH;EVH-EVL-EVH-EVL-TVHH-TVHH;TVHH-TVHH-TVHH-TVHH-TVHH;TVHH-TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH-EVL-EVH;EVL-EVH-EVL-EVH-TVHH-TVHH;EVH-EVL-EVH-TVHH-TVHH;TVHH-EVL-

[0007] 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-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. In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which the (-) is a linker: 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-EVH-EVL-EVH-EVL-TVHH. In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which the (-) is a linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH; or TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH. In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-EVL-EVH-EVL-EVH-TVHH; or TVHH-EVH-EVL-EVH-EVL-TVHH. In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH. In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH.In some embodiments, the variable domain and one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-EVL-EVH-EVL-EVH-TVHH. The single-chain polypeptide of claim 53, wherein the variable domain and one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-EVH-EVL-EVH-EVL-TVHH. In some embodiments, the linker connecting two effector cell antigen interaction domains (E) to each other comprises an internal linker. In some embodiments, the effector cell antigen interaction domain that is not directly connected to one or more target cell antigen interaction domains comprises a terminal linker connecting EVL and EVH. In some embodiments, each internal linker is 6-8 amino acids in length. In some embodiments, the terminal linker is 15 or 16 amino acids in length. In some embodiments, each effector cell antigen interaction 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 CD3δ, CD3γ or CD3ε. In some embodiments, the effector cell antigen is on the effector cell. In some embodiments, the effector cell comprises T cells, NK cells or macrophages. In some embodiments, the target cell antigen interaction domain (T) interacting with the target cell is bound to the target cell antigen. In some embodiments, when on cells associated with the disease state, the target cell antigen is clustered in lipid rafts, and when on cells unrelated to the disease state, the target cell antigen is not clustered in lipid rafts. In some embodiments, when on cells associated with the disease state, the target cell antigen is clustered, and when on cells unrelated to the disease state, the target cell antigen is not clustered. In some embodiments, when on cells associated with the disease state, the target cell antigen is 100nm or less, and when on cells unrelated to the disease state, the target cell antigen is not within 100nm. In some embodiments, the target cell antigen is 75 nm or less when on cells associated with a disease state, and the target cell antigen is not within 75 nm when on cells not associated with a disease state. In some embodiments, the target cell antigen is 50 nm or less when on cells associated with a disease state, and the target cell antigen is not within 50 nm when on cells not associated with a disease state. In some embodiments, the target cell antigen is 25 nm or less when on cells associated with a disease state, and the target cell antigen is not within 25 nm when on cells not associated with a disease state.In some embodiments, when on cells associated with a disease state, the target cell antigen is 10 nm or less, and when on cells not associated with the disease state, the target cell antigen is not within 10 nm. In some embodiments, when on cells associated with a disease state, the target cell antigen is 5 nm or less, and when on cells not associated with the disease state, the target cell antigen is not within 5 nm. In some embodiments, when on cells associated with a disease state, the target cell antigen is 2 nm or less, and when on cells not associated with the disease state, the target cell antigen is not within 2 nm. In some embodiments, when on cells associated with a disease state, the target cell antigen is in close proximity, and when on cells not associated with the disease state, the target cell antigen is not in close proximity. In some embodiments, when on cells associated with a disease state, the target cell antigen is a dimer, trimer, tetramer or oligomer, and when on cells not associated with the disease state, the target cell antigen is a monomer or dimer. In some embodiments, the target cell antigen is part of a cell signaling complex when on a cell associated with a disease state, and the target cell antigen is not part of the same cell signaling complex when on a cell not associated with a disease state. In some embodiments, the target cell antigen is part of a cell signaling complex when on a cell associated with a disease state, and the target cell antigen is not part of a cell signaling complex when 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.

[0008] Disclosed herein is a single-chain polypeptide comprising a variable domain connected by a linker, folded into a conformation such that the variable domain forms one or more effector cell antigen interaction domains (E), wherein the effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single-chain variable fragment, and the effector cell antigen interaction domain is directly connected to one or more target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH), wherein the single-chain polypeptide does not comprise an antibody constant domain. Disclosed herein is a single-chain polypeptide comprising a variable domain connected by a linker, folded into a conformation such that the variable domain forms two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single-chain variable fragment, and one of the effector cell antigen interaction domains is directly connected to one or more target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH), wherein the single-chain polypeptide does not comprise an antibody constant domain. In some embodiments, the two variable domains (EV and EV) of the single-chain variable fragment include the variable heavy chain (EVH) of the single-chain variable fragment and the variable light chain (EVL) of the single-chain variable fragment. In some embodiments, one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to one target cell antigen interaction domain. In some embodiments, one target cell antigen interaction domain C-terminus is connected to one of the effector cell antigen interaction domains. In some embodiments, one target cell antigen interaction domain N-terminus is connected to one of the effector cell antigen interaction domains. In some embodiments, one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to two target cell antigen interaction domains. In some embodiments, two target cell antigen interaction domains C-terminus are connected to one of the effector cell antigen interaction domains. In some embodiments, two target cell antigen interaction domains N-terminus are connected to one of the effector cell antigen interaction domains. In some embodiments, one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to three target cell antigen interaction domains. In some embodiments, three target cell antigen interaction domains C-terminus are connected 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 connected to one or more target cell antigen interaction domains is connected to four target cell antigen interaction domains. In some embodiments, the C-termini of the four target cell antigen interaction domains are connected to one of the effector cell antigen interaction domains. In some embodiments, the N-termini of the four target cell antigen interaction domains are connected to one of the effector cell antigen interaction domains. In some embodiments, the EVL of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to one TVHH, and the EVH of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to one TVHH. In some embodiments, the EVL of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to one TVHH, and the EVH of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to two TVHHs. In some embodiments, the EVL of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to one TVHH, and the EVH of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to three TVHHs. In some embodiments, the EVH of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to one TVHH, and the EVL of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to two TVHHs. In some embodiments, the EVH of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to one TVHH, and the EVL of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to three TVHHs. In some embodiments, the EVH of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to two TVHHs, and the EVL of one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to two TVHHs.In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) 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-TV-EV-EV-EV; EV-EV-EV-EV-TVHH-TVHH-TVHH; TVHH-TVHH-TVHH-TVHH-E V-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.In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) 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-TVH H; 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-E VL-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-EV H-EVL-TVHH;TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH;TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH;TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH;TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH;TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH;TVHH-TVHH-TVHH-EVL-EVH-EVL-TVHH;TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH;or TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH.In some embodiments, the linker connecting two effector cell antigen interacting domains (E) to each other comprises an internal linker.In some embodiments, the effector cell antigen interaction domain that is not directly connected to one or more target cell antigen interaction domains comprises a terminal joint connecting EVL and EVH. In some embodiments, the length of each internal joint is 6-8 amino acids. In some embodiments, the length of the terminal joint is 15 or 16 amino acids. In some embodiments, both effector cell antigen interaction domains are directly connected to the target cell antigen interaction domain. In some embodiments, both effector cell antigen interaction domains are N-terminally and C-terminally connected to the target cell antigen interaction domain. In some embodiments, both effector cell antigen interaction domains comprise a terminal joint connecting EVL and EVH. In some embodiments, the length of the terminal joint is 15 or 16 amino acids. In some embodiments, the variable domains and target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: 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-TVHH-EVL-EVH-TVHH.

[0009] In some embodiments, each effector cell antigen interaction domain is bound 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 CD3δ, CD3γ or CD3ε. In some embodiments, the effector cell antigen is on the effector cell. In some embodiments, the effector cell comprises T cells, NK cells or macrophages. In some embodiments, the target cell antigen interaction domain (T) interacting with the target cell is bound to the target cell antigen. In some embodiments, when on cells associated with a disease state, the target cell antigen is clustered in lipid rafts, and when on cells unrelated to the disease state, the target cell antigen is not clustered in lipid rafts. In some embodiments, when on cells associated with a disease state, the target cell antigen is clustered, and when on cells unrelated to the disease state, the target cell antigen is not clustered. In some embodiments, when on cells associated with a disease state, the target cell antigen is a dimer, trimer, tetramer or oligomer, and when on cells not associated with the disease state, the target cell antigen is a monomer or dimer. In some embodiments, when on cells associated with a disease state, the target cell antigen is part of a cell signaling complex, and when on cells not associated with the disease state, the target cell antigen is not part of the same cell signaling complex. In some embodiments, when on cells associated with a disease state, the target cell antigen is part of a cell signaling complex, and when on cells not associated with the disease state, the target cell antigen is not part of a cell signaling complex. In some embodiments, the target cell antigen comprises CD33, FAP, EGFR, HER2 or EpCAM. In some embodiments, cells associated with a disease state are myeloid cells, fibroblasts or cancer cells.

[0010] Disclosed herein is a pharmaceutical composition comprising: (a) the single-chain polypeptide of any one of the above embodiments; and (b) a pharmaceutically acceptable excipient.

[0011] Disclosed herein is an isolated recombinant nucleic acid encoding a single-chain polypeptide according to any one of the above embodiments.

[0012] Disclosed herein is a vector comprising the isolated nucleic acid according to the above embodiments.

[0013] Disclosed herein is a host cell comprising the isolated nucleic acid of the above embodiments or the vector according to the above embodiments.

[0014] Disclosed herein is a method of treating cancer comprising administering to a subject in need thereof a single-chain polypeptide according to any one of the above embodiments. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is a hematological cancer.

[0015] Disclosed herein are methods 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.

[0016] Disclosed herein are methods 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.

[0017] 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.

[0018] Disclosed herein are methods 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.

[0019] 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 embodiments.

[0020] 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 embodiments.

[0021] Disclosed herein is a single-chain polypeptide comprising two variable domains (EV and EV) of a single-chain variable fragment connected by a joint, folded into a conformation to form an effector cell antigen interaction domain (E), wherein the effector cell antigen interaction domain is connected to two or more target cell antigen interaction domains that interact with a target cell by one or more joints, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH), wherein the single-chain polypeptide does not include an antibody constant domain. In some embodiments, the two variable domains (EV and EV) of the single-chain variable fragment include a variable heavy chain (EVH) of a single-chain variable fragment and a variable light chain (EVL) of a single-chain variable fragment. In some embodiments, two or more target cell antigen interaction domains are C-terminally connected to an effector cell antigen interaction domain. In some embodiments, two or more target cell antigen interaction domains are N-terminally connected to an effector cell antigen interaction domain. In some embodiments, two or more target cell antigen interaction domains are N-terminally and C-terminally connected to an effector cell antigen interaction domain. In some embodiments, an effector cell antigen interaction domain is connected to two target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to three target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to four target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to five target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to six target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to two target cell antigen interaction domains and the variable domain and the two target cell antigen interaction domains are arranged according to the following N-terminal to C-terminal arrangement, in which (-) is a joint: 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.In some embodiments, the effector cell antigen interaction domain is linked to three target cell antigen interaction domains and the variable domain and the three target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: 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-EVH-EVL-TVHH-TVHH; TVHH-EVH-EVL-TVHH-TVHH; TVHH-EVL-TVHH-TVHH; or TVHH-TVHH-EVH-EVL-TVHH. In some embodiments, the effector cell antigen interaction domain is linked to four target cell antigen interaction domains and the variable domain and the four target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-TVHH-TVHH-TVL-EVH; EVH-EVL-TVHH-TVHH-TVHH-TVHH; EVL-EVH-TVHH-TVHH-TVHH-TVHH; TVHH-TVHH-TVHH-TVHH; VHH-TVHH-EVH-EVL; TVHH-TVHH-TVHH-EVL-EVH-TVHH; TVHH-EVH-EVL-TVHH-TVHH-TVHH; TVHH-EVL-EVH-TVHH-TV HH-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 interaction domain is linked to five target cell antigen interaction domains and the variable domain and the five target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the 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-E VL-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-T VHH-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.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 ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVL-EVH; EVH-EVL-TVHH-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-TVL-EVH-TVHH; TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH TVHH-TVHH-TVHH-TVHH-TVHH;TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH;TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH-TV HH;TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH-TVHH;TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH;TVHH-TVHH-TVHH-TVHH-EVL-TVHH-TVHH;TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH;or TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH.In some embodiments, the effector cell antigen interaction domain is connected to six target cell antigen interaction domains and the variable domains and the six target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH;or EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH. In some embodiments, the effector cell antigen interaction domain comprises a terminal joint connecting EVL and EVH. In some embodiments, the terminal joint is 15 or 16 amino acids in length. In some embodiments, one or more joints connecting two or more target cell antigen interaction domains comprise an internal joint. In some embodiments, each internal joint is 6-8 amino acids in length. In some embodiments, the effector cell antigen interaction domain is bound 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 CD3δ, CD3γ or CD3ε. In some embodiments, the effector cell antigen is on the effector cell. In some embodiments, the effector cell comprises a T cell or a NK cell or a macrophage. In some embodiments, the target cell antigen interaction domain (T) interacting with the target cell is bound to the target cell antigen. In some embodiments, when on cells associated with a disease state, the target cell antigen is clustered in lipid rafts, and when on cells unrelated to the disease state, the target cell antigen is not clustered in lipid rafts. In some embodiments, when on cells associated with a disease state, the target cell antigen is clustered, and when on cells unrelated to the disease state, the target cell antigen is not clustered. In some embodiments, when on cells associated with a disease state, the target cell antigen is a dimer, trimer, tetramer or oligomer, and when on cells unrelated to the disease state, the target cell antigen is a monomer or dimer. In some embodiments, when on cells associated with a disease state, the target cell antigen is part of a cell signaling complex, and when on cells not associated with the disease state, the target cell antigen is not part of the same cell signaling complex. In some embodiments, when on cells associated with a disease state, the target cell antigen is part of a cell signaling complex, and when on cells not associated with the disease state, the target cell antigen is not part of a cell signaling complex. In some embodiments, the target cell antigen comprises CD33, FAP, EGFR, HER2, or EpCAM. In some embodiments, the cells associated with the disease state are myeloid cells, fibroblasts, or cancer cells. In some embodiments, the single-chain polypeptide has an effector cell antigen interaction domain.

[0022] 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.

[0023] Disclosed herein is an isolated recombinant nucleic acid encoding a single-chain polypeptide according to any one of the above embodiments.

[0024] Disclosed herein is a vector comprising the isolated nucleic acid according to the aforementioned embodiments.

[0025] Disclosed herein is a host cell comprising the isolated nucleic acid according to any one of the preceding embodiments or the vector according to the preceding embodiments.

[0026] Disclosed herein is a method for 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. In some embodiments, it is a method for 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 is a method for 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 is a method for 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 is a method for 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 is a method for 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 for treating a viral infection, comprising administering a single-chain polypeptide according to any one of the preceding embodiments to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The novel features of the invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description of illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings, in which:

[0028] Figure 1A and Figure 1B Certain non-limiting embodiments of the single-chain polypeptides of the present disclosure are described, in which the single-chain polypeptide comprises variable domains connected by a linker, folded into a conformation such that the variable domains form two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single-chain variable fragment, and one of the effector cell antigen interaction domains is connected to two target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH). The variable domains and target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: Figure 1A EVL-EVH-EVL-EVH-TVHH-TVHH; and Figure 1B EVH-EVL-EVH-EVL-TVHH-TVHH.

[0029] Figure 2A and Figure 2BCertain non-limiting embodiments of the single-chain polypeptides of the present disclosure are described, in which the single-chain polypeptide comprises variable domains connected by a linker, folded into a conformation such that the variable domains form two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EVL and EVH) of a single-chain variable fragment, and one of the effector cell antigen interaction domains is connected to four target cell antigen interaction domains that interact with target cells, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH). The variable domains and target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: Figure 2A EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH-TVHH; and Figure 2B EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH-TVHH.

[0030] Figure 3A and Figure 3B Certain non-limiting embodiments of the single-chain polypeptides of the present disclosure are described, in which the single-chain polypeptide comprises variable domains connected by a linker, folded into a conformation such that the variable domains form two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EVL and EVH) of a single-chain variable fragment, and one of the effector cell antigen interaction domains is connected to four target cell antigen interaction domains that interact with target cells, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH). The variable domains and target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: Figure 3A TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH; and Figure 3B TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH.

[0031] Figure 4A and Figure 4BCertain non-limiting embodiments of the single-chain polypeptides of the present disclosure are described, in which the single-chain polypeptide comprises variable domains connected by a linker, folded into a conformation such that the variable domains form two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single-chain variable fragment, and one of the effector cell antigen interaction domains is connected to a target cell antigen interaction domain that interacts with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH). The variable domains and the target cell antigen interaction domain are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: Figure 4A EVL-EVH-EVL-EVH-TVHH; and Figure 4B EVH-EVL-EVH-EVL-TVHH.

[0032] Figure 5A and Figure 5B Certain non-limiting embodiments of the single-chain polypeptides of the present disclosure are described, in which the single-chain polypeptide comprises variable domains connected by a linker, folded into a conformation such that the variable domains form two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EVL and EVH) of a single-chain variable fragment, and one of the effector cell antigen interaction domains is connected to three target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH). The variable domains and target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: Figure 5A EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH; and Figure 5B EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH.

[0033] Fig. 6A and Figure 6BCertain non-limiting embodiments of the single-chain polypeptides of the present disclosure are described, in which the single-chain polypeptide comprises variable domains connected by a linker, folded into a conformation such that the variable domains form two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EVL and EVH) of a single-chain variable fragment, and one of the effector cell antigen interaction domains is connected to two target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH). The variable domains and the target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: Fig. 6A TVHH-EVL-EVH-EVL-EVH-TVHH; and Figure 6B TVHH-EVH-EVL-EVH-EVL-TVHH.

[0034] Fig. 7A and Figure 7B The T cell killing activity of exemplary single chain polypeptide constructs in a cytotoxicity assay on MCF7 cancer cells is demonstrated.

[0035] Fig. 8A and Figure 8B The T cell killing activity of exemplary single chain polypeptide constructs in a cytotoxicity assay on SKBR3 cancer cells is demonstrated.

[0036] Fig. 9 T cell activation by exemplary single-chain polypeptide constructs in MCF7 cancer cells is demonstrated.

[0037] Fig.10 T cell activation by exemplary single chain polypeptide constructs in SKBR3 cancer cells is demonstrated. DETAILED DESCRIPTION

[0038] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the features described herein will be obtained by reference to the following detailed description which sets forth illustrative examples of the principles in which the features described herein are utilized and the accompanying drawings, wherein:

[0039] Disclosed herein are single-chain polypeptides comprising variable domains connected by a linker, which fold into a conformation such that the variable domains form two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single-chain variable fragment, and at least one of the effector cell antigen interaction domains is connected to one or more target interaction domains. In some embodiments, the target interaction 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 a tumor microenvironment. In some embodiments, the target interaction domain comprises an antibody, an antibody fragment, a ligand, or a cofactor. In some embodiments, the antibody or antibody fragment of the target interaction domain comprises Fab, Fab′, F(ab′)2, Fv fragment, single-chain Fv (scFv), tandem single-chain Fv ((scFv)2, dual-affinity retargeting antibody, bispecific antibody, VHH single domain antibody, TriTac, Adnectin, Anticalin, Avimer, Fynomer, Kunitz domain, knottin, affinity body or DARPin.

[0040] In some embodiments, one or more target interaction domains include one or more target cell antigen interaction domains that bind to a target cell antigen. In some embodiments, the target cell antigen interaction domain binds to more than one target cell antigen. In some embodiments, the target cell antigen interaction domain is bispecific or multispecific. In some embodiments, the target cell antigen interaction domain includes a VHH single domain antibody (TVHH). In some embodiments, the single-chain polypeptide does not contain a crystallizable fragment (Fc) domain.

[0041] Described herein are single chain polypeptides comprising variable domains connected by a linker, which fold into a conformation such that the variable domains form two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single chain variable fragment, and one of the effector cell antigen interaction domains is directly linked to one or more target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH), wherein the single chain polypeptide does not comprise an antibody constant domain.

[0042] definition

[0043] In the following description, certain specific details are set forth to provide a comprehensive understanding of various embodiments. However, those skilled in the art will appreciate that the embodiments provided can be practiced without these details. Unless the context otherwise requires, throughout the specification and subsequent claims, the words "comprise" and its variants such as "comprises" and "comprising" should be interpreted in an open, inclusive sense, i.e., "including but not limited to". Unless the content clearly states otherwise, as used in this specification and the appended claims, the singular forms "one", "a kind of" and "the" include plural indicators. It should also be noted that, unless the content clearly states otherwise, the term "or" is generally used with its meaning including "and / or". In addition, the titles provided herein are only for convenience and do not explain the scope or meaning of the claimed embodiments.

[0044] As used herein, the term "about" refers to an amount that is around 10% or less of the stated amount.

[0045] As used herein, the terms "individual," "patient," or "subject" refer to an individual diagnosed with, suspected of having, or at risk of developing at least one disease for which the described compositions and methods are useful. 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.

[0046] 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 provided polypeptides and antibody chains and other peptides (e.g., connectors and binding peptides)) may include amino acid residues (including natural and / or non-natural amino acid residues). The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, as long as the protein maintains the desired activity, the polypeptide may contain modifications to the native or natural sequence. These modifications may be deliberate (e.g., by site-directed mutagenesis), or may be accidental (such as by mutations in the host producing the protein or errors caused by PCR amplification).

[0047] The percentage of sequence identity (%) relative to the reference polypeptide sequence is the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence after comparing the sequences and introducing gaps when necessary to achieve maximum percentage sequence identity, and not considering any conservative substitutions as a part of sequence identity. For determining the comparison of the purpose of percentage amino acid sequence identity, it can be realized in various known ways (for example, using publicly available computer software (such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software)). Suitable parameters for comparing sequences can be determined, including the algorithm required for maximum comparison on the entire full length of the compared sequences. However, for the purpose of this article, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the US Copyright Office, Washington DC, 20559, together with user documents, where it is registered with US Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and are unchanged.

[0048] Where ALIGN-2 is employed for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A relative to, with or to a given amino acid sequence B (or it may be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity relative to, with or to a given amino acid sequence B) is calculated as follows: 100 multiplied by 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 the program's alignment of A and B, and where 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, then the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A. Unless expressly stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the previous section.

[0049] In some embodiments, amino acid sequence variants of the polypeptides provided herein are contemplated. Variants are generally different from the polypeptides specifically disclosed herein in terms of one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring, or may be, for example, synthetically generated by modifying one or more of the above-mentioned polypeptide sequences of the present invention and evaluating one or more biological activities of the polypeptides described herein and / or using any of a number of known techniques. For example, improving the binding affinity and / or other biological properties of the antibody may be desirable. The amino acid sequence variants of the antibody may be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions of residues in the amino acid sequence of the antibody and / or insertions and / or substitutions of residues into residues. Any combination of deletions, insertions and substitutions may be performed to obtain the final construct, as long as the final construct has the desired characteristics, for example, antigen binding.

[0050] In some embodiments, "antigen binding domain" or "antigen interaction domain" refers to an immunoglobulin derivative with antigen binding properties, i.e., an immunoglobulin polypeptide or fragment thereof containing an antigen binding site. In some cases, the binding domain includes the variable domain of an antibody or fragment thereof. Each antigen binding domain is formed by an antibody (i.e., an immunoglobulin, a variable heavy chain domain (VH), and an antibody variable light chain domain (VL) that binds to the same epitope), and the variable heavy chain domain (VH) includes three heavy chain complementary determination regions (CDRs): CDR1, CDR2, and CDR3; and the variable light chain domain (VL) includes three light chain complementary determination regions (CDRs): CDR1, CDR2, and CDR3. In some cases, the binding domain according to some embodiments herein lacks an immunoglobulin constant domain. In some cases, the variable light chain and heavy chain domains that form the antigen binding site are covalently linked to each other (e.g., covalently linked by a peptide connector), or in other cases, the variable light chain and heavy chain domains are non-covalently linked to each other to form an antigen binding site. Binding domains and interaction domains also refer to antibody fragments or antibody derivatives, including, for example, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv, tandem single-chain Fv ((scFv)2, bispecific T cell engagers Dual-affinity retargeting antibodies (DARTs) TM ), double antibody, IgG molecules, single domain antibodies (eg, VHH), TriTacs, etc. Furthermore, in some cases, the binding domain is multivalent, i.e., has two, three or more binding sites for one or more antigens.

[0051] In certain embodiments, the antigen binding domain or antigen interaction domain includes a sequence derived from the complementary determining region of an antibody. As known in the art, the terms "complementarity determining region" and "CDR" synonymous with "hypervariable region" or "HVR" refer to discontinuous sequences of amino acids in the variable region of an antibody, which confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) has 3 CDRs, and each light chain variable region (CDR-L1, CDR-L2, CDR-L3) has 3 CDRs. As known in the art, "framework region" and "FR" refer to the non-CDR portions of the variable regions of heavy and light chains. Generally, each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4) has 4 FRs, and each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4) has 4 FRs.The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("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 January; 27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plückthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 June 8; 309(3):657-70, ("Aho" numbering scheme); and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modelling antibodies on the WEB," Protein Eng. 2000 December; 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.

[0052] The boundary of given CDR or FR can change depending on the scheme used for identification. For example, the Kabat scheme is based on structural comparison, and the Chothia scheme is based on structural information. The numbering of both Kabat and Chothia schemes is based on the most common antibody region sequence length, and insertion is accommodated by inserting letters (for example, "30a"), and disappearance occurs in some antibodies. These two schemes place certain insertions and deletions ("insertion / deletion (indel)") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures, and is similar to the Chothia numbering scheme in many respects.

[0053] In certain embodiments, the antigen binding domain or antigen interaction domain includes a sequence derived from the complementary determining region of an antibody. The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of a primary antibody (VH and VL, respectively) generally have similar structures, with each domain including four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains may be used to separate antibodies that bind to a specific antigen from antibodies that bind to an antigen, to screen the library of complementary 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)).

[0054] In certain embodiments, antigen binding domains or antigen interaction domains are "humanized". "Humanized" polypeptides or antibodies are polypeptides or antibodies 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. Humanized antibodies may optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a humanized non-human antibody, which is generally used 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 replaced by corresponding residues from a non-human antibody (e.g., an antibody derived from CDR residues), such as to restore or improve antibody specificity or affinity.

[0055] Human antibodies are among the polypeptides and antibodies provided. A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or a non-human source utilizing a human antibody library or other human antibody-encoded sequences, including a human antibody library. The term excludes humanized forms of non-human antibodies that include non-human antigen binding regions, such as non-human antigen binding regions in which all or substantially all CDRs are non-human.

[0056] The polypeptide described herein can be encoded by nucleic acid. Nucleic acid is a polynucleotide comprising two or more nucleotide bases. In certain embodiments, nucleic acid is a component of a vector, which can be used to transfer a polypeptide encoding a polynucleotide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid that has been connected to it. One type of vector is a genomic integration vector or "integrated vector", which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episome" vector, for example, a nucleic acid capable of extrachromosomal replication. The vector capable of directing the expression of genes operably connected to them is referred to herein as an "expression vector". Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, etc. In an expression vector, the vector regulatory elements (such as promoters, enhancers, polyadenylation signals) used to control transcription can be derived from mammals, microorganisms, viruses, or insect genes. The ability to replicate in a host (usually given by a replication origin) and selection genes that promote transformant recognition can be additionally incorporated. A vector derived from a virus can be used, such as a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, etc. Plasmid vectors can be linearized to integrate into chromosomal locations. The vector can include sequences that direct specific site integration into a defined position or restricted site set (e.g., AttP-AttB recombination) in the genome. In addition, the vector can include sequences derived from transposable elements.

[0057] As used herein, the terms "homology", "homology" or "homology percentage" used herein to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, revised as Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formulas are incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). The homology percentage of a sequence can be determined using the latest version of BLAST as of the date of filing of this application.

[0058] Nucleic acids encoding polypeptides described herein can be used to infect, transfect, transform or otherwise transfect cells appropriate for the nucleic acid, thereby enabling the production of polypeptides for commercial or therapeutic use. Standard cell lines and methods for producing antibodies or polypeptides from large-scale cell cultures 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 used to produce a polypeptide or antibody, and is a Chinese hamster ovary cell (CHO) cell, an NS0 mouse myeloma cell, or Cells. In certain embodiments, nucleic acids encoding antibodies are integrated into the genomic loci of cells for producing polypeptides or antibodies. In certain embodiments, methods of making antibodies are described herein, comprising culturing cells containing nucleic acids encoding antibodies under in vitro conditions sufficient to allow production and secretion of the antibody.

[0059] In certain embodiments, described herein is a master cell bank comprising: (a) a mammalian cell line comprising a nucleic acid encoding an antibody or polypeptide described herein integrated at 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 capable of withstanding freezing by liquid nitrogen or a temperature equivalent to liquid nitrogen, and is stored at a temperature equivalent to liquid nitrogen.

[0060] Also described herein is a method for making polypeptides described herein. Such methods are included in incubating cells or cell lines comprising nucleic acids encoding polypeptides in cell culture medium under conditions sufficient to allow antibody expression and secretion, and further harvesting the polypeptides from the cell culture medium. Harvesting may further comprise one or more purification steps to remove living cells, cell debris, non-target proteins or polypeptides, undesirable salts, buffers, and culture medium components. In certain embodiments, additional purification steps include centrifugation, ultracentrifugation, protein A purification, protein G purification, protein A / G purification, or protein L purification, size exclusion chromatography, hydrophobic interaction chromatography, and / or ion exchange chromatography.

[0061] Single-chain polypeptide composition

[0062] Disclosed herein are single-chain polypeptides that are folded to form a three-dimensional binding structure. The single-chain polypeptide comprises an antigen binding domain that can bind 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 is bispecific and trivalent (e.g., comprising three antigen binding domains). In certain embodiments, the single-chain polypeptide is trivalent relative to an effector cell antigen or a target cell antigen. In certain embodiments, the single-chain polypeptide is bispecific and bivalent, and in other cases is bispecific and tetravalent (e.g., comprising four antigen binding domains). In certain embodiments, the single-chain polypeptide may be tetravalent relative to an effector cell antigen or a 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 an immune cell (e.g., T cell, NK cell, or NKT cell). Some non-limiting embodiments of single-chain polypeptides are discussed in the following paragraphs.

[0063] In certain embodiments, described herein are single-chain polypeptides comprising variable domains connected by a linker, which fold into a conformation such that the variable domains form one or more effector cell antigen interaction domains (E), wherein the effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single-chain variable fragment, and the effector cell antigen interaction domain is directly linked to one or more target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH), wherein the single-chain polypeptide does not comprise an antibody constant domain.

[0064] In certain embodiments, described herein are single-chain polypeptides comprising variable domains connected by a linker, which fold into a conformation such that the variable domains form two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single chain variable fragment, and one of the effector cell antigen interaction domains is directly linked to one or more target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH), wherein the single chain polypeptide does not comprise an antibody constant domain.

[0065] In certain embodiments, 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.

[0066] Branched three-dimensional configuration of the target cell-antigen interaction domain

[0067] In some embodiments, an EVL connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to at least one TVHH, and an EVH connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to at least one TVHH. In some embodiments, at least one TVHH connected to EVL binds to the same target antigen as at least one TVHH connected to EVH. In some embodiments, at least one TVHH connected to EVL binds to the same target antigen as at least one TVHH connected to EVH and has the same complementary determining region (CDR) sequence. In some embodiments, at least one TVHH connected to EVL binds to the same target antigen as at least one TVHH connected to EVH and does not have a common complementary determining region (CDR) sequence. In some embodiments, at least one TVHH connected to EVL binds to a different target antigen than at least one TVHH connected to EVH.

[0068] In some embodiments, EVL and EVH are connected to the same number of TVHHs. In some embodiments, EVL is connected to a different number of TVHHs than the number of TVHHs connected to EVH.

[0069] In some embodiments, an EVL that is linked to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is linked to one TVHH, and an EVH that is linked to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is linked to one TVHH.

[0070] In some embodiments, the EVL connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to one TVHH, and the EVH connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to two TVHHs.

[0071] In some embodiments, the EVL connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to one TVHH, and the EVH connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to three TVHHs.

[0072] In some embodiments, the EVH connected to one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to one TVHH, and the EVL connected to one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to two TVHHs.

[0073] In some embodiments, the EVH connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to one TVHH, and the EVL connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to three TVHHs.

[0074] In some embodiments, the EVH connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to one TVHH, and the EVL connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to one TVHH. Fig. 6A and 6B Single chain polypeptides are exemplified in which an EVH linked to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is linked to one TVHH, and an EVL linked to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is linked to one TVHH.

[0075] In some embodiments, an EVH connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to two TVHHs, and an EVL connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to two TVHHs. Figure 3A and 3B Single chain polypeptides are exemplified in which an EVH linked to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is linked to two TVHHs, and an EVL linked to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is linked to two TVHHs.

[0076] In some embodiments, the EVH connected to one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to three TVHHs, and the EVL connected to one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to three TVHHs.

[0077] In some embodiments, an EVH connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to four TVHHs, and an EVL connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to four TVHHs.

[0078] In some embodiments, an EVH connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to five TVHHs, and an EVL connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to five TVHHs.

[0079] In some embodiments, an EVH connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to six TVHHs, and an EVL connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to six TVHHs.

[0080] In some embodiments, an EVH connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to seven TVHHs, and an EVL connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to seven TVHHs.

[0081] In some embodiments, an EVH connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to eight TVHHs, and an EVL connected to one of the effector cell antigen interaction domains of one or more target cell antigen interaction domains is connected to eight TVHHs.

[0082] Linear three-dimensional configuration of the target cell antigen interaction domain

[0083] In some embodiments, one of the effector cell antigen interaction domains that is linked to one or more target cell antigen interaction domains is linked to one target cell antigen interaction domain. In some embodiments, one target cell antigen interaction domain is C-terminally linked to one of the effector cell antigen interaction domains. In some embodiments, one target cell antigen interaction domain is N-terminally linked to one of the effector cell antigen interaction domains. Figure 4A and 4B Single chain polypeptides are exemplified in which one target cell antigen interacting domain is C-terminally linked to one of the effector cell antigen interacting domains.

[0084] In some embodiments, at least one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to more than one target cell antigen interaction domain. In some embodiments, all of the more than one target cell antigen interaction domains are bound to the same target antigen. In some embodiments, all of the more than one target cell antigen interaction domains are bound to the same target antigen and all have the same CDR sequence. In some embodiments, all of the more than one target cell antigen interaction domains are bound to the same target antigen and at least two of the more than one target cell antigen interaction domains do not have a common CDR sequence. In some embodiments, at least two of the more than one target cell antigen interaction domains are bound to different target antigens.

[0085] In some embodiments, one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to two target cell antigen interaction domains. In some embodiments, two target cell antigen interaction domains are C-terminally connected to one of the effector cell antigen interaction domains. Figure 1A and 1B Single chain polypeptides are exemplified in which two target cell antigen interacting domains are C-terminally linked to one of the effector cell antigen interacting domains.

[0086] In some embodiments, one of the effector cell antigen interaction domains that is linked to one or more target cell antigen interaction domains is linked to three target cell antigen interaction 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. Figure 5A and 5B Exemplified are single chain polypeptides in which three target cell antigen interacting domains are C-terminally linked to one of the effector cell antigen interacting domains.

[0087] In some embodiments, one of the effector cell antigen interaction domains connected to one or more target cell antigen interaction domains is connected to four target cell antigen interaction domains. In some embodiments, the four target cell antigen interaction domains are C-terminally connected to one of the effector cell antigen interaction domains. Figure 2A and 2B Single chain polypeptides are exemplified in which four target cell antigen interacting domains are C-terminally linked to one effector cell antigen interacting domain.

[0088] Arrangement of single-chain polypeptide from N-terminus to C-terminus

[0089] Listed below are additional configurations and specific embodiments of the arrangement of target cell antigen interaction domains (T) or effector cell antigen interaction domains (E). In certain embodiments, the single-chain polypeptide comprises one or more target cell antigen interaction domains (T) and one or more effector cell antigen interaction domains (E), wherein the effector cell antigen interaction domain and the one or more target cell antigen interaction domains are arranged according to any one or more of the following N-terminal to C-terminal arrangements, in which (-) 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; HH-TVHH-TVHH; TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV; EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH; TVHH-EV-EV-EV-EV-EV-TVHH; TVHH-EV-EV-EV-EV-TVHH-TVH H; 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.

[0090] In some embodiments, the variable domain and one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangements, 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-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.

[0091] In some embodiments, the variable domains and one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH; or TVHH-EV-EV-EV-EV-TVHH. In some embodiments, the variable domains and one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH. In some embodiments, the variable domains and one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-EV-EV-EV-EV-TVHH-TVHH.

[0092] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH.

[0093] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EV-EV-EV-EV-TVHH.

[0094] In certain embodiments, the single-chain polypeptide comprises one or more target cell antigen interaction domains (T) and one or more effector cell antigen interaction domains (E), wherein the one or more effector cell antigen interaction domains and the one or more target cell antigen interaction domains are ordered according to one or more of the following N-terminal to C-terminal arrangements, in which (-) 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; TV HH-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-E VH-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-TVH H;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;TV HH-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.

[0095] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which the (-) is a linker: 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-EVH-EVL-EVH-EVL-TVHH. In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which the (-) is a linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH; or TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH. In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-EVL-EVH-EVL-EVH-TVHH; or TVHH-EVH-EVL-EVH-EVL-TVHH. In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH. In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH. In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EVL-EVH-EVL-EVH-TVHH. In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EVH-EVL-EVH-EVL-TVHH.

[0096] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH.

[0097] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH.

[0098] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EVL-EVH-EVL-EVH-TVHH.

[0099] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EVH-EVL-EVH-EVL-TVHH.

[0100] In some embodiments, the variable domains and target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: 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-TVHH-EVL-EVH-TVHH.

[0101] In some embodiments, the variable domains and target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EVH-EVL-TVHH-TVHH-EVH-EVL-TVHH.

[0102] In some embodiments, the variable domains and target cell antigen interacting domain are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EVL-EVH-TVHH-TVHH-EVH-EVL-TVHH.

[0103] In some embodiments, the variable domains and target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EVH-EVL-TVHH-TVHH-EVL-EVH-TVHH.

[0104] In some embodiments, the variable domains and target cell antigen interacting domain are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EVL-EVH-TVHH-TVHH-EVL-EVH-TVHH.

[0105] Additional configuration

[0106] Disclosed herein are single-chain polypeptides comprising two variable domains (EV and EV) of a single-chain variable fragment connected by a linker, which fold into a conformation to form an effector cell antigen interaction domain (E), wherein the effector cell antigen interaction domain is connected by one or more linkers to two or more target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH), wherein the single-chain polypeptide does not comprise an antibody constant domain.

[0107] In some embodiments, 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.

[0108] In some embodiments, two or more target cell antigen interacting domains are C-terminally linked to an effector cell antigen interacting domain. Figure 4B Single-chain polypeptides are exemplified in which six target cell antigen interaction domains are C-terminally linked to an effector cell antigen interaction domain. In some embodiments, two or more target cell antigen interaction domains are N-terminally linked to an effector cell antigen interaction domain. In some embodiments, two or more target cell antigen interaction domains are N-terminally and C-terminally linked to an effector cell antigen interaction domain. Figure 4A Exemplified are single chain polypeptides in which six target cell antigen interacting domains are N-terminally and C-terminally linked to an effector cell antigen interacting domain.

[0109] In some embodiments, the effector cell antigen interaction domain is connected to two target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to three target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to four target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to five target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to six target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to seven target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to eight target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to nine target cell antigen interaction domains. In some embodiments, the effector cell antigen interaction domain is connected to ten target cell antigen interaction domains.

[0110] Arrangement of single-chain polypeptide from N-terminus to C-terminus

[0111] Additional configurations and specific embodiments of the arrangement of target cell antigen interaction domains (T) or effector cell antigen interaction domains (E) are listed below.

[0112] In some embodiments, the effector cell antigen interaction domain is linked to two target cell antigen interaction domains and the variable domain and the two target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: 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.

[0113] In some embodiments, the effector cell antigen interaction domain is linked to three target cell antigen interaction domains and the variable domain and the three target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: 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-EVH-EVL-TVHH-TVHH; TVHH-EVH-EVL-TVHH-TVHH; TVHH-EVL-TVHH-TVHH; or TVHH-TVHH-EVH-EVL-TVHH.

[0114] In some embodiments, the effector cell antigen interaction domain is linked to four target cell antigen interaction domains and the variable domain and the four target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-TVHH-TVHH-TVL-EVH; EVH-EVL-TVHH-TVHH-TVHH-TVHH; EVL-EVH-TVHH-TVHH-TVHH-TVHH; TVHH-TVHH-TVHH-TVHH; VHH-TVHH-EVH-EVL; TVHH-TVHH-TVHH-EVL-EVH-TVHH; TVHH-EVH-EVL-TVHH-TVHH-TVHH; TVHH-EVL-EVH-TVHH-TV HH-TVHH; TVHH-TVHH-TVHH-EVH-EVL-TVHH; TVHH-TVHH-EVL-EVH-TVHH-TVHH; or TVHH-TVHH-EVH-EVL-TVHH-TVHH.

[0115] In some embodiments, the effector cell antigen interaction domain is linked to five target cell antigen interaction domains and the variable domain and the five target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the 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-E VL-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-T VHH-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.

[0116] 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 ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVL-EVH; EVH-EVL-TVHH-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-TVHH-EVL-EVH-TVHH; TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH TVHH-TVHH-TVHH-TVHH-TVHH;TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH;TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH-TV HH;TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH-TVHH; TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH; TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH;

[0117] In some embodiments, the effector cell antigen interaction domain is linked to six target cell antigen interaction domains and the variable domain and the six target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH; or EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH.

[0118] In some embodiments, the effector cell antigen interaction domain is linked to six target cell antigen interaction domains and the variable domain and the six target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH.

[0119] 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 ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH.

[0120] Adapter sequence

[0121] In certain embodiments, the linker connecting two effector cell antigen interaction domains (E) to each other comprises an internal linker.

[0122] In certain embodiments, an effector cell antigen interacting domain that is not directly linked to one or more target cell antigen interacting domains comprises a terminal linker connecting EVL and EVH.

[0123] In some embodiments, the effector cell antigen interaction domain comprises a terminal linker connecting EVL and EVH.

[0124] In some embodiments, one or more linkers connecting two or more target cell antigen interacting domains comprise an internal linker.

[0125] 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 is about 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 5-29, 6-29, 7-29, 8-29, 9-29, 10-29, 5-28, 6-28, 7-28, 8-28, 9-28, 10-28, 5-27, 6-27, 7-27, 8-27, 9-2 7, 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, 10 -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 length of each internal linker is 6-8 amino acids.

[0126] 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 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 10-29, 11-29, 12-29, 13-29, 14-29, 15-29, 10-28, 11-28, 12-28, 13-28, 14-28, 15-28, 10-27, 11-27, 12-27, 1 3-27, 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, 12 -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 cases, the terminal joint has a length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In certain embodiments, the terminal joint has a length of 15 or 16 amino acids. In some cases, the terminal joint has a length of about 16 amino acids.

[0127] In some embodiments, the length of the internal joint is about 25 amino acids to about 45 amino acids. For example, the length of the internal joint is about 25-45, 26-44, 27-43, 28-42, 29-41, 30-40, 31-39, 32-38, 33-37 or 34-36 amino acids in length. In some embodiments, the length of the internal joint is about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids in length. In some embodiments, the length of the internal joint is 35 amino acids.

[0128] In some embodiments, the length of the terminal joint is about 25 amino acids to about 45 amino acids. For example, the length of the terminal joint is about 25-45, 26-44, 27-43, 28-42, 29-41, 30-40, 31-39, 32-38, 33-37 or 34-36 amino acids. In some embodiments, the length of the terminal joint is about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids. In some embodiments, the length of the terminal joint is 35 amino acids.

[0129] In some embodiments, the linker is sized to implement pairing of the heavy and light chain variable domains to form two effector cell antigens that interact in a single polypeptide chain, eliminating mispairing, preventing aggregation and multi-species, diffusion during purification, and thus facilitating manufacturing.

[0130] 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 thereto (e.g., a sequence having 0-2 amino acid modifications, substitutions or deletions).

[0131] Table 1. Exemplary linker sequences

[0132] describe Amino acid sequence (N to C) SEQ ID NO: Connector-1(L1) GGSGGS 1 Connector-2(L2) GGGGSGGGGSGGGGS 2 Connector-3(L3) GGSGGS 3 Connector-4(L4) GGGGSGGGG 4 Connector-5(L5) GGGGSGGGG 5 Connector-6(L6) AAAGS 6

[0133] Effector cell antigen binding domain

[0134] In some embodiments, the effector cell antigen interaction domain binds to an effector cell antigen. In some embodiments, CD3 is CD3δ, CD3γ or CD3ε. In some embodiments, the effector cell antigen is on an effector cell. In some embodiments, the effector cell comprises a T cell or a NK cell or a macrophage. In some embodiments, the effector cell antigen comprises CD3, CD16a or death receptor 5 (DR5).

[0135] CD3 binding domain

[0136] 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.

[0137] In some embodiments, EVH comprises an amino acid sequence according to Table 2 or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity). In some embodiments, EVH comprises a CDR1 sequence STYAMN (SEQ ID NO: 13) or a sequence having 0-2 amino acid modifications, substitutions or deletions. In some embodiments, EVH comprises a CDR2 sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 14) or a sequence having 0-2 amino acid modifications, substitutions or deletions. In some embodiments, EVH comprises a CDR3 sequence HGNFGNSYVSWFAY (SEQID NO: 15) or a sequence having 0-2 amino acid modifications, substitutions or deletions. In some embodiments, EVH comprises a CDR3 sequence HGNFGNSYVSYFAY (SEQ ID NO: 16) or a sequence having 0-2 amino acid modifications, substitutions or deletions.

[0138] In some embodiments, the EVL comprises an amino acid sequence according to Table 3 or a sequence substantially identical thereto (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 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 GTNCRAP (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 ALWYSNL (SEQ ID NO: 19) or a sequence having 0-2 amino acid modifications, substitutions, or deletions.

[0139] Table 2. Amino acid sequences of anti-CD3 variable heavy chain domains (the amino acid sequences of variable heavy chain CDR1, CDR2 and CDR3 are in bold and underlined)

[0140]

[0141] Table 3. Amino acid sequences of anti-CD3 variable light chain domains (the amino acid sequences of variable light chain CDR1, CDR2 and CDR3 are in bold and underlined)

[0142]

[0143] In some cases, the CDR1, CDR2, CDR3 from EVH or EVL are derived from other CD3 antibodies, such as, for example, UCHT1, muromonab-CD3 (OKT3), otelizumab (TRX4), teplizumab (MGA031), vesiculomab (Nuvion), etc.

[0144] In some aspects, described herein are anti-CD3 binding molecules comprising a humanized or fully human (ie, human-derived) anti-CD3 binding domain. In some embodiments, described herein are anti-CD3 binding molecules comprising a camelid or llama anti-CD3 binding domain.

[0145] In certain instances, a CD3 binding site has moderate or low affinity as a monomer and has higher affinity and / or specificity as a bivalent molecule due to avidity.

[0146] CD16a binding domain

[0147] 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.

[0148] In some embodiments, the EVH comprises an amino acid sequence according to SEQ ID NO:20, or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0149] In some embodiments, the EVL comprises an amino acid sequence according to SEQ ID NO:21, or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0150] In some aspects, described herein are anti-CD16a binding molecules comprising a humanized or fully human (ie, human-derived) anti-CD16a binding domain.

[0151] Table 4. Amino acid sequences of anti-CD16a VH or VL domains

[0152]

[0153] Death receptor 5 binding domain

[0154] In some embodiments, EVH comprises a variable heavy chain that binds to a single chain variable fragment of DR5, and EVL comprises a variable light chain that binds to a single chain variable fragment of DR5.

[0155] In some embodiments, the EVH comprises an amino acid sequence according to SEQ ID NO:22-26, or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0156] In some embodiments, the EVL comprises an amino acid sequence according to SEQ ID NO:27-31, or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0157] In some aspects, described herein are anti-DR5 binding molecules comprising a humanized or fully human (ie, human-derived) anti-DR5 binding domain.

[0158] Table 5. Amino acid sequences of anti-DR5 VH or VL domains

[0159]

[0160] Target cell antigen interaction domain

[0161] In some embodiments, the target cell antigen interaction domain (T) interacting with the target cell is bound to the target cell antigen. In some embodiments, when on cells associated with a disease state, the target cell antigen is clustered in lipid rafts, and when on cells unrelated to the disease state, the target cell antigen is not clustered in lipid rafts. In some embodiments, when on cells associated with a disease state, the target cell antigen is clustered, and when on cells unrelated to the disease state, the target cell antigen is not clustered. In some embodiments, when on cells associated with a disease state, the target cell antigen is 100nm or less, and when on cells unrelated to the disease state, the target cell antigen is not within 100nm. In some embodiments, when on cells associated with a disease state, the target cell antigen is 75nm or less, and when on cells unrelated to the disease state, the target cell antigen is not within 75nm. In some embodiments, when on cells associated with a disease state, the target cell antigen is 50 nm or less, and when on cells not associated with the disease state, the target cell antigen is not within 50 nm. In some embodiments, when on cells associated with a disease state, the target cell antigen is 25 nm or less, and when on cells not associated with the disease state, the target cell antigen is not within 25 nm. In some embodiments, when on cells associated with a disease state, the target cell antigen is 10 nm or less, and when on cells not associated with the disease state, the target cell antigen is not within 10 nm. In some embodiments, when on cells associated with a disease state, the target cell antigen is 5 nm or less, and when on cells not associated with the disease state, the target cell antigen is not within 5 nm. In some embodiments, when on cells associated with a disease state, the target cell antigen is 2 nm or less, and when on cells not associated with the disease state, the target cell antigen is not within 2 nm.

[0162] In some embodiments, when on cells associated with a disease state, the target cell antigen is in close proximity, and when on cells not associated with the disease state, the target cell antigen is not in close proximity. In some embodiments, when on cells associated with a disease state, the target cell antigen is a dimer, trimer, tetramer or oligomer, and when on cells not associated with the disease state, the target cell antigen is a monomer or dimer. In some embodiments, when on cells associated with a disease state, the target cell antigen is part of a cell signaling complex, and when on cells not associated with the disease state, the target cell antigen is not part of the same cell signaling complex. In some embodiments, when on cells associated with a disease state, the target cell antigen is part of a cell signaling complex, and when on cells not associated with the disease state, the target cell antigen is not part of a cell signaling complex. In some embodiments, 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.

[0163] CD33 binding domain

[0164] 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 according to SEQ ID NOs: 32-43, SEQ ID NOs: 72-74, or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0165] Table 6. Amino acid sequences of anti-CD33 VHH single domain antibodies

[0166]

[0167] HER2 binding domain

[0168] In some embodiments, the TVHH comprises a VHH single domain antibody that binds to HER2. In some embodiments, the TVHH comprises an amino acid sequence according to SEQ ID NO: 44-46, or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0169] Table 7. Amino acid sequences of anti-HER2 VHH single domain antibodies

[0170]

[0171] EGFR binding domain

[0172] In some embodiments, the TVHH comprises a VHH single domain antibody that binds to EGFR.In some embodiments, the TVHH comprises an amino acid sequence according to SEQ ID NO: 47-49, or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0173] Table 8. Amino acid sequences of anti-EGFR VHH single domain antibodies

[0174]

[0175] FAP binding domain

[0176] In some embodiments, the TVHH comprises a VHH single domain antibody that binds to FAP. In some embodiments, the TVHH comprises an amino acid sequence according to SEQ ID NO: 51, SEQ ID NO: 65-67, or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0177] Table 9. Amino acid sequences of anti-FAP VHH single domain antibodies

[0178]

[0179] EpCAM binding domain

[0180] In some embodiments, the TVHH comprises a VHH single domain antibody that binds to EpCAM.In some embodiments, the TVHH comprises an amino acid sequence according to SEQ ID NO: 68-71, or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0181] Table 10. Amino acid sequences of anti-EpCAM VHH single domain antibodies

[0182]

[0183] Exemplary Single Chain Polypeptide (SCP) Compositions

[0184] SCP 1

[0185] In some embodiments, the single-chain polypeptide comprises two target cell antigen interaction domains. In some embodiments, the variable domain and the two target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: EVH-EVL-EVH-EVL-TVHH-TVHH. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment bound to CD3, and EVL comprises a variable light chain of a single-chain variable fragment bound to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody bound to CD33. In some embodiments, the single-chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 52. A summary of the amino acid sequences of the domains of SCP 1 is provided in Table 11.

[0186] Table 11. Summary of SCP 1 sequences

[0187]

[0188] SCP 2

[0189] In some embodiments, the single-chain polypeptide comprises two target cell antigen interaction domains. In some embodiments, the variable domain and the two target cell antigen interaction domains are arranged according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: EVL-EVH-EVL-EVH-TVHH-TVHH. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment bound to CD3, and EVL comprises a variable light chain of a single-chain variable fragment bound to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody bound to CD33. In some embodiments, the single-chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 53. A summary of the amino acid sequences of the domains of SCP 2 is provided in Table 12.

[0190] Table 12. Summary of SCP 2 sequences

[0191]

[0192] SCP 3

[0193] In some embodiments, the single-chain polypeptide comprises two target cell antigen interaction domains. In some embodiments, the variable domain and the two target cell antigen interaction domains are arranged according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: EVH-EVL-EVH-EVL-TVHH-TVHH. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment bound to CD3, and EVL comprises a variable light chain of a single-chain variable fragment bound to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody bound to CD33. In some embodiments, the single-chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 54. A summary of the amino acid sequences of the domains of SCP 3 is provided in Table 13.

[0194] Table 13. Summary of SCP 3 sequences

[0195]

[0196]

[0197] SCP 4

[0198] In some embodiments, the single-chain polypeptide comprises two target cell antigen interaction domains. In some embodiments, the variable domain and the two target cell antigen interaction domains are arranged according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: EVL-EVH-EVL-EVH-TVHH-TVHH. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment bound to CD3, and EVL comprises a variable light chain of a single-chain variable fragment bound to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody bound to CD33. In some embodiments, the single-chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 55. A summary of the amino acid sequences of the domains of SCP 4 is provided in Table 14.

[0199] Table 14. Summary of SCP 4 sequences

[0200]

[0201] SCP 5

[0202] In some embodiments, the single-chain polypeptide comprises two target cell antigen interaction domains. In some embodiments, the variable domain and the two target cell antigen interaction domains are arranged according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EVH-EVL-EVH-EVL. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment bound to CD3, and EVL comprises a variable light chain of a single-chain variable fragment bound to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody bound to CD33. In some embodiments, the single-chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 56. A summary of the amino acid sequences of the domains of SCP 5 is provided in Table 15.

[0203] Table 15. Summary of SCP 5 sequences

[0204]

[0205]

[0206] SCP 6

[0207] In some embodiments, the single-chain polypeptide comprises two target cell antigen interaction domains. In some embodiments, the variable domain and the two target cell antigen interaction domains are arranged according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EVL-EVH-EVL-EVH. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment bound to CD3, and EVL comprises a variable light chain of a single-chain variable fragment bound to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody bound to CD33. In some embodiments, the single-chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 57. A summary of the amino acid sequences of the domains of SCP6 is provided in Table 16.

[0208] Table 16. Summary of SCP 6 sequences

[0209]

[0210] SCP 7

[0211] In some embodiments, the single-chain polypeptide comprises two target cell antigen interaction domains. In some embodiments, the variable domain and the two target cell antigen interaction domains are arranged according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EVH-EVL-EVH-EVL. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment bound to CD3, and EVL comprises a variable light chain of a single-chain variable fragment bound to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody bound to CD33. In some embodiments, the single-chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 58. A summary of the amino acid sequences of the domains of SCP 7 is provided in Table 17.

[0212] Table 17. Summary of SCP 7 sequences

[0213]

[0214] SCP 8

[0215] In some embodiments, the single-chain polypeptide comprises two target cell antigen interaction domains. In some embodiments, the variable domain and the two target cell antigen interaction domains are arranged according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: TVHH-TVHH-EVL-EVH-EVL-EVH. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment bound to CD3, and EVL comprises a variable light chain of a single-chain variable fragment bound to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody bound to CD33. In some embodiments, the single-chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 59. A summary of the amino acid sequences of the domains of SCP 8 is provided in Table 18.

[0216] Table 18. Summary of SCP 8 sequences

[0217]

[0218]

[0219] SCP 9

[0220] In some embodiments, the single-chain polypeptide comprises a target cell antigen interaction domain. In some embodiments, the variable domain and a target cell antigen interaction domain are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: EVH-EVL-EVH-EVL-TVHH. 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 an amino acid sequence according to SEQ ID NO: 60. A summary of the amino acid sequences of the domains of SCP 9 is provided in Table 19.

[0221] Table 19. Summary of SCP 9 sequences

[0222]

[0223] SCP 10

[0224] In some embodiments, the single-chain polypeptide comprises a target cell antigen interaction domain. In some embodiments, the variable domain and a target cell antigen interaction domain are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: EVL-EVH-EVL-EVH-TVHH. 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 an amino acid sequence according to SEQ ID NO: 61. A summary of the amino acid sequences of the domains of SCP 10 is provided in Table 20.

[0225] Table 20. Summary of SCP 10 sequences

[0226]

[0227] SCP 11

[0228] In some embodiments, the single-chain polypeptide comprises a target cell antigen interaction domain. In some embodiments, the variable domain and a target cell antigen interaction domain are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: EVH-EVL-EVH-EVL-TVHH. 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 an amino acid sequence according to SEQ ID NO: 62. A summary of the amino acid sequences of the domains of SCP 11 is provided in Table 21.

[0229] Table 21. Summary of SCP 11 sequences

[0230]

[0231] SCP 12

[0232] In some embodiments, the single-chain polypeptide comprises a target cell antigen interaction domain. In some embodiments, the variable domain and a target cell antigen interaction domain are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is a linker: EVL-EVH-EVL-EVH-TVHH. 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 an amino acid sequence according to SEQ ID NO: 63. A summary of the amino acid sequences of the domains of SCP 12 is provided in Table 22.

[0233] Table 22. Summary of SCP 12 sequences

[0234]

[0235] Conservative variants of CDR sequences and heavy and light chain domains

[0236] 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 domains that bind to the target antigen or effector cell are similar but not identical to the amino acid sequences depicted in SEQ ID NO:7-SEQ ID NO:63. In some cases, the CDR variant 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 compared to the sequences of SEQ ID NO:13-SEQ ID NO:19, and the sequence is immunologically active.

[0237] In other cases, the CDR variant sequence incorporates 1, 2, 3, 4 or 5 conservative amino acid substitutions. Conservative substitutions include amino acid substitutions that replace a given amino acid with another amino acid of similar characteristics, and further include the exchange of alanine, valine, leucine and isoleucine among aliphatic amino acids; the exchange of hydroxyl residues serine and threonine, the exchange of acidic residues aspartic acid and glutamic acid, the substitution between amide residues asparagine and glutamine, the exchange of basic residues lysine and arginine, and the replacement between aromatic residues phenylalanine and tyrosine.

[0238] In still other cases, the CDR variant sequences incorporate substitutions that enhance the properties of the CDR, such as stability, resistance to proteases, and / or increased binding affinity for the target antigen or CD3.

[0239] In other cases, the CDR variant sequence is modified to change a non-critical residue or a residue in a non-critical region. 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 or alanine scanning mutagenesis.

[0240] In other embodiments, the binding molecule comprises the heavy chain and light chain domains of the immunologically active homologues or variants of the heavy chain and light chain domain sequences provided herein. Therefore, in some embodiments, the binding domain comprises a heavy chain or light chain domain sequence that is similar but not identical to the binding domain provided herein. In some cases, the variant heavy chain or light chain domain sequence has 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 sequence herein, and the sequence is immunologically active.

[0241] In other embodiments, the variant heavy chain or light chain domain sequence incorporates 1, 2, 3, 4 or 5 conservative amino acid substitutions. Conservative substitutions include amino acid substitutions that replace a given amino acid with another amino acid of similar characteristics, and further include the exchange of alanine, valine, leucine and isoleucine among aliphatic amino acids; the exchange of hydroxyl residues serine and threonine, the exchange of acidic residues aspartic acid and glutamic acid, the substitution between amide residues asparagine and glutamine, the exchange of basic residues lysine and arginine, and the substitution between aromatic residues phenylalanine and tyrosine.

[0242] In other cases, the variant heavy or light chain domain sequence incorporates substitutions that enhance the properties of the CDRs, such as stability, resistance to proteases, and / or increased binding affinity for antigen.

[0243] In other cases, the variant heavy chain or light chain domain sequence is modified to change the residue in a non-critical residue or non-critical region. 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 or alanine scanning mutagenesis.

[0244] The percentage of sequence identity (%) relative to the reference polypeptide sequence is the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence after comparing the sequences and introducing gaps when necessary to achieve maximum percentage sequence identity, and not considering any conservative substitutions as a part of sequence identity. For determining the comparison of the purpose of percentage amino acid sequence identity, it can be realized in various known ways (for example, using publicly available computer software (such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software)). Suitable parameters for comparing sequences can be determined, including the algorithm required for maximum comparison on the entire full length of the compared sequences. However, for the purpose of this article, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the US Copyright Office, Washington DC, 20559, together with user documents, where it is registered with US Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and are unchanged.

[0245] Where ALIGN-2 is employed for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A relative to, with or to a given amino acid sequence B (or it may be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity relative to, with or to a given amino acid sequence B) is calculated as follows: 100 multiplied by 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 the program's alignment of A and B, and where 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, then the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A. Unless expressly stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the previous section.

[0246] Treatment

[0247] In certain embodiments, disclosed herein are antibodies for treating cancer or tumors. Treatment refers to a method of trying to improve or alleviate the condition being treated. With respect to cancer, treatment includes, but is not limited to, reducing tumor volume, reducing the growth of tumor volume, increasing progression-free survival or overall life expectancy. In certain embodiments, treatment will affect the relief of the cancer being treated. In certain embodiments, treatment encompasses a preventive or maintenance dose used as a treatment intended to prevent the recurrence or progression of a previously treated cancer or tumor. For the treatment of bacterial or viral diseases, treatment includes, but is not limited to, reducing one or more symptoms associated with viral or bacterial diseases, such as reducing 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 time period during which an individual can infect others, or reduce overall disease or recovery time. Treatment of autoimmune or inflammatory diseases includes, but is not limited to, reducing total antibody levels or autoantibody levels, or reducing total immune response or autocellular immune response. Treatment can also be associated with specific symptoms of autoimmune diseases associated with overzealous antibodies or cellular immune responses. Treatment of fibrotic diseases can reduce or slow the appearance of fibrotic tissue or the deposition of collagen in tissue. Treatment of cardiovascular disease can increase indicators of cardiovascular health, including lowering blood pressure, reducing atherosclerotic lesions, or increasing cardiac function as indicated by the ability to pump blood. Those skilled in the art understand that not all individuals will respond the same or not at all to the administered treatment, even though treatment of such individuals has been considered.

[0248] In certain embodiments, single-chain polypeptides are used to treat viral infections. In certain embodiments, single-chain polypeptides are used to treat bacterial infections. In certain embodiments, single-chain polypeptides are used to treat solid tumor cancers. In certain embodiments, single-chain polypeptides are used to treat hematological cancers. In certain embodiments, single-chain polypeptides are used to treat inflammatory conditions. In certain embodiments, single-chain polypeptides are used to treat autoimmune diseases. In certain embodiments, single-chain polypeptides are used to treat cardiovascular diseases. In certain embodiments, single-chain polypeptides are used to treat fibrotic diseases.

[0249] It is contemplated that the single-chain polypeptide molecules described herein are used as medicines. Administration is implemented in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, intralesional, local or intradermal administration. In some embodiments, the route of administration depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. The dosage of any one patient depends on many factors, including the patient's body shape, body surface area, age, sex, the specific compound to be administered, the time and approach of administration, the type of therapy, general health status and other drugs administered simultaneously.

[0250] An "effective dose" refers to an amount of active ingredient sufficient to affect the course and severity of a disease, leading to a reduction or alleviation of such symptoms. For example, known methods can be used to determine the amount of active ingredient used to treat and / or prevent CD33. + "Effective doses" for cancers such as AML. The maximum tolerated dose (MTD) and the maximum response dose (MRD) can be determined through established animal and human experimental procedures and the examples described herein.

[0251] In some embodiments, the polypeptides herein are administered at dosage levels determined and considered 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 inhibit the symptoms of cancer. The effective amount for this use depends on the severity and course of the cancer, previous therapy, the patient's health, body weight, and response to the drug, and the judgment of the treating physician. The therapeutically effective amount is optionally determined by methods including, but not limited to, dose escalation clinical trials (such as described in the examples).

[0252] Pharmaceutical composition

[0253] In some aspects, pharmaceutical compositions are provided herein, comprising single-chain polypeptide molecules, vectors comprising polynucleotides encoding single-chain polypeptide molecules or host cells transformed by the vectors 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 nontoxic to the patient to whom it is applied. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, etc. Such carriers can be prepared by conventional methods and can be administered to the subject in a suitable dose. Preferably, the composition is sterile. These compositions can also contain adjuvants, such as preservatives, emulsifiers, and dispersants. The effects of microorganisms can be prevented by including a variety of antibacterial and antifungal agents. In other aspects, the pharmaceutical composition comprises excipients for sustained release, such as PLGA nanoparticles, etc. In other aspects, the pharmaceutical composition is coated on a device for insertion into the body for sustained release at a specific site.

[0254] In certain embodiments, the single-chain polypeptide of the present disclosure is included in a pharmaceutical composition, which includes one or more pharmaceutically acceptable excipients, carriers and diluents. In certain embodiments, the antibody of the present disclosure is suspended in a sterile solution for administration. In certain embodiments, the solution includes about 0.9% NaCl. In certain embodiments, the solution includes about 5.0% dextrose. In certain embodiments, the solution further includes 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; polyols / disaccharides / polysaccharides, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose and dextran 40; amino acids, such as glycine or arginine; antioxidants, such as ascorbic acid, methionine; or chelating agents, such as EDTA or EGTA.

[0255] In certain embodiments, before administration, the single-chain polypeptide of the present disclosure is transported / stored freeze-dried and reconstructed. In certain embodiments, the freeze-dried antibody formulation comprises a filler, such as mannitol, sorbitol, sucrose, trehalose, dextran 40 or a combination thereof. The freeze-dried formulation can be contained in a vial consisting of glass or other suitable non-reactive materials. When formulated, whether or not reconstructed, the antibody can be buffered with a certain pH (generally less than 7.0). In certain embodiments, the pH can be 4.5 to 6.5, 4.5 to 6.0, 4.5 to 5.5, 4.5 to 5.0 or 5.0 to 6.0.

[0256] Also described herein are kits comprising, in a suitable container, one or more single-chain polypeptides described herein and one or more additional components selected from the group consisting of: instructions for use; diluents, excipients, carriers, and devices for administration.

[0257] In certain embodiments, described herein are methods of preparing a cancer treatment comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with an antibody of the present disclosure. In certain embodiments, described herein are methods of preparing a cancer treatment for storage or transport comprising lyophilizing one or more antibodies of the present disclosure.

[0258] Protein production

[0259] On the one hand, the present invention provides a single-chain polypeptide. In some cases, the polypeptide is a single-chain fusion protein without branches. For example, multiple antigen binding domains are connected in the polypeptide.

[0260] In some embodiments, the polypeptides described herein are produced by expressing a polynucleotide encoding the polypeptide. Thus, another aspect is a polynucleotide, such as a DNA or RNA, encoding a polypeptide herein.

[0261] The polynucleotide is constructed by known methods, for example, by combining genes encoding at least two or three binding domains into a single gene construct, which are separated by a peptide linker or directly connected by a peptide bond in other embodiments, and the single gene construct is 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 polynucleotide encoding the peptide linker can be suitably a flexible linker (e.g., a Gly-Ser linker; (Gly4Ser) n wherein n is 1, 2, 3, 4, 5 or greater; (Gly3Ser) n Where n is 1, 2, 3, 4, 5 or greater, (Gly3Ser) n Wherein n is 1,2,3,4,5 or greater).Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.The promoter is selected so that it drives the expression of the polynucleotide in the corresponding host cell.

[0262] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment. This recombinant vector can be constructed according to known methods.

[0263] 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.

[0264] Thus, in some embodiments, a polypeptide as described herein is produced by introducing a vector encoding a polypeptide as described above into a host cell and culturing the host cell under conditions whereby the polypeptide chain is expressed, and the polypeptide as described herein can be isolated and, optionally, further purified.

[0265] In some aspects, the single-chain polypeptide described herein has modification. Typical modifications include but are not limited to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moiety, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, drug conjugation, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking formation, cystine formation, pyroglutamic acid formation, formylation, γ carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenization (selenoylation), sulfation, transfer RNA-mediated addition of amino acids (such as arginylation (arginylation)) and ubiquitination to protein. In other embodiments, multivalent antibodies are modified with additional amino acids, such as leading or secretory sequences or sequences for purifying polypeptides.

[0266] In some aspects, the single-chain polypeptide molecules described herein include a half-life extension domain that extends the half-life of the polypeptide. In some embodiments, the half-life extension domain is connected to the N-terminus of the single-chain polypeptide molecule. In some embodiments, the half-life extension domain is connected to the C-terminus of the single-chain polypeptide molecule. In some embodiments, the half-life extension domain replaces the binding domain, such as the VHH domain of the molecule herein. In some embodiments, the half-life extension domain replaces the binding domain, such as the VH and VL pairs of the single-chain polypeptide molecule herein. It is expected that such domains include, but are not limited to, HSA binding domains, pegylation, small molecules, and other half-life extension domains known in the art. Human serum albumin (HSA) (molecular weight of about 67kDa) is the most abundant protein in plasma, present at about 50mg / ml (600μM), and has a half-life of about 20 days in humans. HSA is used to maintain plasma pH, contributes to colloidal blood pressure, acts as a carrier for many metabolites and fatty acids, and acts as the main drug transport protein in plasma. The non-covalent association with albumin extends the elimination half-life of the protein. In some embodiments, the half-life extension domain is a domain that binds to HSA, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single chain variable fragments (scFv), single domain antibodies such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-derived single domain antibodies (VHH), peptides, ligands, or small molecule entities specific for HSA.

[0267] Implementation plan list

[0268] Embodiment 1 comprises a single-chain polypeptide comprising variable domains connected by a linker, folded into a conformation such that the variable domains form two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single-chain variable fragment, and one of the effector cell antigen interaction domains is directly connected to one or more target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH), wherein the single-chain polypeptide does not comprise an antibody constant domain.

[0269] Embodiment 2 comprises the single-chain polypeptide as described in embodiment 1, wherein the two variable domains (EV and EV) of the single-chain variable fragment comprise the variable heavy chain (EVH) of the single-chain variable fragment and the variable light chain (EVL) of the single-chain variable fragment.

[0270] Embodiment 3 comprises a single-chain polypeptide as described in embodiment 1 or 2, wherein one of said effector cell antigen interaction domains linked to said one or more target cell antigen interaction domains is linked to one target cell antigen interaction domain.

[0271] Embodiment 4 comprises the single-chain polypeptide of embodiment 3, wherein said one target cell antigen interaction domain is C-terminally linked to one of said effector cell antigen interaction domains.

[0272] Embodiment 5 comprises the single-chain polypeptide of embodiment 3, wherein said one target cell antigen interaction domain is N-terminally linked to one of said effector cell antigen interaction domains.

[0273] Embodiment 6 comprises the single-chain polypeptide of embodiment 1 or 2, wherein one of said effector cell antigen interaction domains linked to said one or more target cell antigen interaction domains is linked to two target cell antigen interaction domains.

[0274] Embodiment 7 comprises the single-chain polypeptide of embodiment 6, wherein the two target cell antigen interaction domains are C-terminally linked to one of the effector cell antigen interaction domains.

[0275] Embodiment 8 comprises the single-chain polypeptide of embodiment 6, wherein the two target cell antigen interaction domains are N-terminally linked to one of the effector cell antigen interaction domains.

[0276] Embodiment 9 comprises the single-chain polypeptide of embodiment 1 or 2, wherein one of said effector cell antigen interaction domains linked to said one or more target cell antigen interaction domains is linked to three target cell antigen interaction domains.

[0277] Embodiment 10 comprises the single-chain polypeptide of embodiment 9, wherein the three target cell antigen interaction domains are C-terminally linked to one of the effector cell antigen interaction domains.

[0278] Embodiment 11 comprises the single-chain polypeptide of embodiment 9, wherein the three target cell antigen interaction domains are N-terminally linked to one of the effector cell antigen interaction domains.

[0279] Embodiment 12 comprises a single-chain polypeptide as described in embodiment 1 or 2, wherein one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to four target cell antigen interaction domains.

[0280] Embodiment 13 comprises the single-chain polypeptide of embodiment 12, wherein the four target cell antigen interaction domains are C-terminally linked to one of the effector cell antigen interaction domains.

[0281] Embodiment 14 comprises the single-chain polypeptide of embodiment 12, wherein the four target cell antigen interaction domains are N-terminally linked to one of the effector cell antigen interaction domains.

[0282] Embodiment 15 comprises the single-chain polypeptide of embodiment 2, wherein the EVL of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to a TVHH, and the EVH of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to a TVHH.

[0283] Embodiment 16 comprises the single-chain polypeptide of embodiment 2, wherein the EVL of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to one TVHH, and the EVH of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to two TVHHs.

[0284] Embodiment 17 comprises the single-chain polypeptide of embodiment 2, wherein the EVL of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to one TVHH, and the EVH of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to three TVHHs.

[0285] Embodiment 18 comprises the single-chain polypeptide of embodiment 2, wherein the EVH of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to one TVHH, and the EVL of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to two TVHHs.

[0286] Embodiment 19 comprises the single-chain polypeptide of embodiment 2, wherein the EVH of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to one TVHH, and the EVL of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to three TVHHs.

[0287] Embodiment 20 comprises the single-chain polypeptide of embodiment 2, wherein the EVH of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to two TVHHs, and the EVL of one of the effector cell antigen interaction domains connected to the one or more target cell antigen interaction domains is connected to two TVHHs.

[0288] Embodiment 21 comprises the single-chain polypeptide of embodiments 2-20, wherein the variable domains and the one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker:

[0289] TVHH-EV-EV-EV-EV;

[0290] EV-EV-EV-EV-TVHH;

[0291] TVHH-TVHH-EV-EV-EV-EV;

[0292] EV-EV-EV-EV-TVHH-TVHH;

[0293] TVHH-TVHH-TVHH-EV-EV-EV-EV;

[0294] EV-EV-EV-EV-TVHH-TVHH-TVHH;

[0295] TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV;

[0296] EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH;

[0297] TVHH-EV-EV-EV-EV-TVHH;

[0298] TVHH-EV-EV-EV-EV-TVHH-TVHH;

[0299] TVHH-TVHH-EV-EV-EV-EV-TVHH;

[0300] TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH;

[0301] TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH; or

[0302] TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH.

[0303] Embodiment 22 comprises the single-chain polypeptide of embodiment 2-20, wherein the variable domain and the one or more target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker:

[0304] TVHH-EVL-EVH-EVL-EVH;

[0305] TVHH-EVH-EVL-EVH-EVL;

[0306] EVL-EVH-EVL-EVH-TVHH;

[0307] EVH-EVL-EVH-EVL-TVHH;

[0308] TVHH-TVHH-EVL-EVH-EVL-EVH;

[0309] TVHH-TVHH-EVH-EVL-EVH-EVL;

[0310] EVL-EVH-EVL-EVH-TVHH-TVHH;

[0311] EVH-EVL-EVH-EVL-TVHH-TVHH;

[0312] TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH;

[0313] TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL;

[0314] EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH;

[0315] EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH;

[0316] TVHH-TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH;

[0317] TVHH-TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL;

[0318] EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH-TVHH;

[0319] EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH-TVHH;

[0320] TVHH-EVL-EVH-EVL-EVH-TVHH;

[0321] TVHH-EVH-EVL-EVH-EVL-TVHH;

[0322] TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH;

[0323] TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH;

[0324] TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH;

[0325] TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH;

[0326] TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH;

[0327] TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH;

[0328] TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH;

[0329] TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH;

[0330] TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH; or

[0331] TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH.

[0332] Embodiment 23 comprises the single-chain polypeptide of embodiments 1-22, wherein the linker connecting the two effector cell antigen interaction domains (E) to each other comprises an internal linker.

[0333] Embodiment 24 comprises a single-chain polypeptide as described in embodiments 2-23, wherein the effector cell antigen interaction domain that is not directly linked to the one or more target cell antigen interaction domains comprises a terminal linker connecting EVL and EVH.

[0334] Embodiment 25 comprises the single-chain polypeptide of embodiment 24, wherein each of said internal linkers has a length of 6-8 amino acids.

[0335] Embodiment 26 comprises the single-chain polypeptide of embodiment 25, wherein the terminal linker is 15 or 16 amino acids in length.

[0336] Embodiment 27 comprises the single-chain polypeptide of embodiment 2, wherein both of said effector cell antigen interaction domains are directly linked to a target cell antigen interaction domain.

[0337] Embodiment 28 comprises the single-chain polypeptide of embodiment 27, wherein both of said effector cell antigen interaction domains are N-terminally and C-terminally linked to a target cell antigen interaction domain.

[0338] Embodiment 29 comprises the single-chain polypeptide of embodiments 27-28, wherein both of said effector cell antigen interaction domains comprise a terminal linker connecting EVL and EVH.

[0339] Embodiment 30 comprises the single-chain polypeptide of embodiment 29, wherein the terminal linker is 15 or 16 amino acids in length.

[0340] Embodiment 31 comprises the single-chain polypeptide of embodiments 27-30, wherein the variable domain and the target cell antigen interacting domain are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker:

[0341] TVHH-EVH-EVL-TVHH-TVHH-EVH-EVL-TVHH;

[0342] TVHH-EVL-EVH-TVHH-TVHH-EVH-EVL-TVHH;

[0343] TVHH-EVH-EVL-TVHH-TVHH-EVL-EVH-TVHH; and

[0344] TVHH-EVL-EVH-TVHH-TVHH-EVL-EVH-TVHH.

[0345] Embodiment 32 comprises the single-chain polypeptide of embodiments 1-31, wherein each of said effector cell antigen interacting domains binds to an effector cell antigen.

[0346] Embodiment 33 comprises the single-chain polypeptide of embodiment 32, wherein the effector cell antigen comprises CD3, CD16a, or death receptor 5 (DR5).

[0347] Embodiment 34 comprises the single chain polypeptide of embodiment 33, wherein the CD3 is CD3δ, CD3γ or CD3ε.

[0348] Embodiment 35 comprises the single-chain polypeptide of embodiment 32, wherein the effector cell antigen is on an effector cell.

[0349] Embodiment 36 comprises the single-chain polypeptide of embodiment 35, wherein the effector cell comprises a T cell, a NK cell, or a macrophage.

[0350] Embodiment 37 comprises the single-chain polypeptide of embodiments 1-36, wherein the target cell antigen interacting domain (T) that interacts with a target cell binds to a target cell antigen.

[0351] Embodiment 38 comprises the single-chain polypeptide of embodiment 37, wherein the target cell antigen is clustered in lipid rafts when on cells associated with the disease state, and the target cell antigen is not clustered in lipid rafts when on cells not associated with the disease state.

[0352] Embodiment 39 comprises the single-chain polypeptide of embodiment 38, wherein the target cell antigens are clustered when on cells associated with a disease state, and the target cell antigens are not clustered when on cells not associated with a disease state.

[0353] Embodiment 40 comprises the single-chain polypeptide of embodiment 38, wherein when on cells associated with the disease state, the target cell antigen is a dimer, trimer, tetramer or oligomer, and when on cells not associated with the disease state, the target cell antigen is a monomer or dimer.

[0354] Embodiment 41 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 the disease state, and the target cell antigen is not part of the same cell signaling complex when on cells not associated with the disease state.

[0355] 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 the disease state, and the target cell antigen is not part of a cell signaling complex when on cells not associated with the disease state.

[0356] Embodiment 43 comprises the single-chain polypeptide of embodiments 37-40, wherein the target cell antigen comprises CD33, FAP, EGFR, HER2 or EpCAM.

[0357] Embodiment 44 comprises the single chain polypeptide of embodiments 38-41, wherein the cell associated with the disease state is a myeloid cell, a fibroblast, or a cancer cell.

[0358] Embodiment 45 comprises a single-chain polypeptide comprising variable domains connected by a linker, folded into a conformation such that the variable domains form one or more effector cell antigen interaction domains (E), wherein the effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single-chain variable fragment, and the effector cell antigen interaction domain is directly connected to one or more target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH), wherein the single-chain polypeptide does not comprise an antibody constant domain.

[0359] Embodiment 46 comprises a single-chain polypeptide comprising two variable domains (EV and EV) of a single-chain variable fragment connected by a linker, wherein the single-chain polypeptide folds into a conformation to form an effector cell antigen interaction domain (E), wherein the effector cell antigen interaction domain is connected by one or more linkers to two or more target cell antigen interaction domains that interact with a target cell, wherein the target cell antigen interaction domain is a VHH single domain antibody (TVHH), wherein the single-chain polypeptide does not comprise an antibody constant domain.

[0360] Embodiment 47 comprises a single-chain polypeptide as described in embodiment 46, 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.

[0361] Embodiment 48 comprises the single-chain polypeptide of embodiments 46-47, wherein the two or more target cell antigen interaction domains are C-terminally linked to the effector cell antigen interaction domain.

[0362] Embodiment 49 comprises the single-chain polypeptide of embodiments 46-47, wherein the two or more target cell antigen interaction domains are N-terminally linked to the effector cell antigen interaction domain.

[0363] Embodiment 50 comprises the single-chain polypeptide of embodiments 46-47, wherein the two or more target cell antigen interaction domains are N-terminally and C-terminally linked to the effector cell antigen interaction domain.

[0364] Embodiment 51 comprises the single-chain polypeptide of embodiments 46-50, wherein the effector cell antigen interaction domain is linked to two target cell antigen interaction domains.

[0365] Embodiment 52 comprises the single-chain polypeptide of embodiments 46-50, wherein the effector cell antigen interaction domain is linked to three target cell antigen interaction domains.

[0366] Embodiment 53 comprises the single-chain polypeptide of embodiments 46-50, wherein the effector cell antigen interaction domain is linked to four target cell antigen interaction domains.

[0367] Embodiment 54 comprises the single-chain polypeptide of embodiments 46-50, wherein the effector cell antigen interaction domain is linked to five target cell antigen interaction domains.

[0368] Embodiment 55 comprises the single-chain polypeptide of embodiments 46-50, wherein the effector cell antigen interaction domain is linked to six target cell antigen interaction domains.

[0369] Embodiment 56 comprises the single-chain polypeptide of embodiments 46-50, wherein the effector cell antigen interaction domain is linked to two target cell antigen interaction domains and the variable domain and the two target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker:

[0370] TVHH-TVHH-EVL-EVH;

[0371] EVH-EVL-TVHH-TVHH;

[0372] EVL-EVH-TVHH-TVHH;

[0373] TVHH-TVHH-EVH-EVL;

[0374] TVHH-EVL-EVH-TVHH; or

[0375] TVHH-EVH-EVL-TVHH.

[0376] Embodiment 57 comprises the single-chain polypeptide of embodiments 46-50, wherein the effector cell antigen interaction domain is linked to three target cell antigen interaction domains and the variable domain and the three target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker:

[0377] TVHH-TVHH-TVHH-EVL-EVH;

[0378] EVH-EVL-TVHH-TVHH-TVHH;

[0379] EVL-EVH-TVHH-TVHH-TVHH;

[0380] TVHH-TVHH-TVHH-EVH-EVL;

[0381] TVHH-TVHH-EVL-EVH-TVHH;

[0382] TVHH-EVH-EVL-TVHH-TVHH;

[0383] TVHH-EVL-EVH-TVHH-TVHH; or

[0384] TVHH-TVHH-EVH-EVL-TVHH.

[0385] Embodiment 58 comprises the single-chain polypeptide of embodiments 46-50, wherein the effector cell antigen interaction domain is linked to four target cell antigen interaction domains and the variable domain and the four target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker:

[0386] TVHH-TVHH-TVHH-TVHH-EVL-EVH;

[0387] EVH-EVL-TVHH-TVHH-TVHH-TVHH;

[0388] EVL-EVH-TVHH-TVHH-TVHH-TVHH;

[0389] TVHH-TVHH-TVHH-TVHH-EVH-EVL;

[0390] TVHH-TVHH-TVHH-EVL-EVH-TVHH;

[0391] TVHH-EVH-EVL-TVHH-TVHH-TVHH;

[0392] TVHH-EVL-EVH-TVHH-TVHH-TVHH;

[0393] TVHH-TVHH-TVHH-EVH-EVL-TVHH;

[0394] TVHH-TVHH-EVL-EVH-TVHH-TVHH; or

[0395] TVHH-TVHH-EVH-EVL-TVHH-TVHH.

[0396] Embodiment 59 comprises the single-chain polypeptide of embodiments 46-50, wherein the effector cell antigen interaction domain is linked to five target cell antigen interaction domains and the variable domain and the five target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker:

[0397] TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH;

[0398] EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH;

[0399] EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH;

[0400] TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL;

[0401] TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH;

[0402] TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH;

[0403] TVHH-EVL-EVH-TVHH-TVHH-TVHH-TVHH;

[0404] TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH;

[0405] TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH;

[0406] TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH;

[0407] TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH; or TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH.

[0408] Embodiment 60 comprises the single-chain polypeptide of embodiments 46-50, wherein the effector cell antigen interaction domain is linked to six target cell antigen interaction domains and the variable domains and the six target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker:

[0409] TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH;

[0410] EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH;

[0411] EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH;

[0412] TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL;

[0413] TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH;

[0414] TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH;

[0415] TVHH-EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH;

[0416] TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH;

[0417] TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH;

[0418] TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH;

[0419] TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH-TVHH;

[0420] TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH;

[0421] TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH; or

[0422] TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH.

[0423] Embodiment 61 comprises the single-chain polypeptide of embodiments 46-50, wherein the effector cell antigen interaction domain is linked to six target cell antigen interaction domains and the variable domains and the six target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker:

[0424] TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH; or

[0425] EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH.

[0426] Embodiment 62 comprises the single-chain polypeptide of embodiments 47-61, wherein the effector cell antigen interaction domain comprises a terminal linker connecting EVL and EVH.

[0427] Embodiment 63 comprises the single-chain polypeptide of embodiment 62, wherein the terminal linker is 15 or 16 amino acids in length.

[0428] Embodiment 64 comprises the single-chain polypeptide of embodiments 46-63, wherein the one or more linkers connecting the two or more target cell antigen interacting domains comprise an internal linker.

[0429] Embodiment 65 comprises the single-chain polypeptide of embodiment 64, wherein each of said internal linkers is 6-8 amino acids in length.

[0430] Embodiment 66 comprises the single-chain polypeptide of embodiments 46-65, wherein the effector cell antigen interacting domain binds to an effector cell antigen.

[0431] Embodiment 67 comprises the single-chain polypeptide of embodiment 66, wherein the effector cell antigen comprises CD3, CD16a, or death receptor 5 (DR5).

[0432] Embodiment 68 comprises the single chain polypeptide of embodiment 67, wherein the CD3 is CD3δ, CD3γ or CD3ε.

[0433] Embodiment 69 comprises the single-chain polypeptide of embodiment 68, wherein the effector cell antigen is on an effector cell.

[0434] Embodiment 70 comprises the single-chain polypeptide of embodiment 69, wherein the effector cell comprises a T cell, a NK cell, or a macrophage.

[0435] Embodiment 71 comprises a single-chain polypeptide as described in embodiments 46-70, wherein the target cell antigen interacting domain (T) that interacts with the target cell binds to the target cell antigen.

[0436] Embodiment 72 comprises the single-chain polypeptide of embodiment 71, wherein the target cell antigen is clustered in lipid rafts when on cells associated with the disease state, and the target cell antigen is not clustered in lipid rafts when on cells not associated with the disease state.

[0437] Embodiment 73 comprises the single-chain polypeptide of embodiment 71, wherein the target cell antigens are clustered when on cells associated with the disease state, and the target cell antigens are not clustered when on cells not associated with the disease state.

[0438] Embodiment 74 comprises the single-chain polypeptide of embodiment 71, wherein when on cells associated with the disease state, the target cell antigen is a dimer, trimer, tetramer or oligomer, and when on cells not associated with the disease state, the target cell antigen is a monomer or dimer.

[0439] Embodiment 75 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 the disease state, and the target cell antigen is not part of the same cell signaling complex when on cells not associated with the disease state.

[0440] 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 the disease state, and the target cell antigen is not part of a cell signaling complex when on cells not associated with the disease state.

[0441] Embodiment 77 comprises the single-chain polypeptide of embodiments 71-76, wherein the target cell antigen comprises CD33, FAP, EGFR, HER2 or EpCAM.

[0442] Embodiment 78 comprises the single chain polypeptide of embodiments 71-77, wherein the cell associated with the disease state is a myeloid cell, a fibroblast, or a cancer cell.

[0443] Embodiment 79 comprises the single-chain polypeptide of embodiments 46-78, wherein the single-chain polypeptide has one effector cell antigen interaction domain.

[0444] Embodiment 80 comprises a pharmaceutical composition comprising: (a) the single-chain polypeptide of any one of embodiments 1-79; and (b) a pharmaceutically acceptable excipient.

[0445] Embodiment 81 comprises an isolated recombinant nucleic acid encoding the single-chain polypeptide according to any one of embodiments 1-78.

[0446] Embodiment 82 comprises a vector comprising the isolated nucleic acid according to embodiment 81.

[0447] Embodiment 83 comprises a host cell comprising the isolated nucleic acid of embodiment 81 or the vector of embodiment 81.

[0448] Embodiment 84 comprises a method of treating cancer comprising administering a single chain polypeptide according to any one of embodiments 1-79 to a subject in need thereof.

[0449] Embodiment 85 comprises the method of embodiment 84, wherein the cancer is a solid tumor cancer.

[0450] Embodiment 86 comprises the method of embodiment 84, wherein the cancer is a hematological cancer.

[0451] Embodiment 87 comprises 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.

[0452] Embodiment 88 comprises 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.

[0453] Embodiment 89 comprises 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.

[0454] Embodiment 90 comprises 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.

[0455] Embodiment 91 comprises 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.

[0456] Embodiment 92 comprises 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.

[0457] Example

[0458] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0459] Example 1

[0460] Cloning of DNA expression constructs encoding single chain polypeptides specific for CD3 and HER2.

[0461] Generate coding sequences for single-chain polypeptides specific for CD3 and HER2.

[0462] To express the single-chain polypeptides in CHO cells, each coding sequence was cloned into a mammalian expression vector system. The expression construct was designed to contain a C-terminal hexahistidine (6xHis) tag to facilitate antibody secretion and purification, respectively.

[0463] Expression of single-chain peptides in stably transfected CHO cells

[0464] Using CHO cell expression system ( The adherent cells were cultured according to the standard cell culture protocol provided by Life Technologies, 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).

[0465] To adapt to growth in suspension, cells were detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells were cryopreserved in medium with 10% DMSO.

[0466] Recombinant CHO cell lines stably expressing single-chain polypeptides were generated by transfection of suspension-adapted cells. During selection with the antibiotic hygromycin B, viable cell density was measured twice a week and cells were centrifuged and concentrated at 0.1×10 6 The cells are then resuspended in fresh selection medium at a maximum density of 100 viable cells / mL. After 2-3 weeks of selection, the cell pools that stably express the single-chain polypeptide are recovered and the cells are transferred to standard culture medium in a shake flask. The expression of the recombinant secreted protein is confirmed by protein gel electrophoresis or flow cytometry. Stable cell pools are cryopreserved in medium containing DMSO.

[0467] The single-chain polypeptide is produced by secretion into the cell culture supernatant in a 10-day batch feed culture of a stably transfected CHO cell line. After 10 days, the cell culture supernatant is harvested and the culture viability is typically>75%. Samples are collected from the production culture every other day and cell density and viability are assessed. On the harvest day, the cell culture supernatant is removed by centrifugation and vacuum filtration before further use.

[0468] Protein expression titer and product integrity in cell culture supernatants were analyzed by SDS-PAGE.

[0469] Purification of single-chain peptides

[0470] Single-chain polypeptides were purified from CHO cell culture supernatants in a two-step procedure. In the first step, Ni-NTA Superflow chromatography was performed on the His6-tagged construct, followed by preparative size exclusion chromatography (SEC) on Superdex 200 in the second step. The eluted single-chain polypeptides were characterized by size and pooled. The pooled samples were buffer exchanged and concentrated to a typical concentration of >1 mg / mL by ultrafiltration. The purity and homogeneity of the final sample were assessed by SDS-PAGE under reducing and non-reducing conditions, followed by immunoblotting with anti-His tag antibodies and analytical SEC. The purified protein was stored in aliquots at -80 °C until use.

[0471] Table 23. Single chain polypeptide constructs specific for CD3 and HER2

[0472]

[0473]

[0474]

[0475]

[0476]

[0477] Example 2

[0478] Affinity determination of SCP-CD3 / HER2 and huHER2

[0479] The target of this embodiment is to determine the affinity of SCP-CD3 / HER2 construct samples and huHER2-His protein in multi-cycle kinetics (MCK) using Biacore 8K+ by SPR.Constructs are injected into solution for binding to immobilized huHER2-His protein.The equipment used is Biacore 8K+ instrument, CFJB614 CM5 series S sensor chip (Cytivia, Catnr.29104992) with low density (≈1200RU) on flow channel 1 to 4 and high density (≈5000RU) on flow channel 5 to 8 fixed with huHER2-His (AcroBiosystems; Cat.nr.HE2-H5225).1,10 and 100nM are used as construct samples (construct #1,2,3,5 and 6) of the linker with huHER2 and are injected into flow cell 1 and 2 for 2min at 30 μl / min. 5nM control mAb: human anti-HER2 (trastuzumab) (R&D Systems; Cat. nr. MAB9589) was injected into flow cells 1 and 2 at 30μl / min for 2min. 1, 10 and 100nM of an unrelated construct as a linker to huCD33 was injected into flow cells 1 and 2 at 30μl / min for 2min. The dissociation rate was measured for 300 seconds and the baseline level was restored with regeneration solution. The buffer used was 1xHBS-EP + pH 7.4. The data was analyzed using the multi-cycle kinetic predefined evaluation method of Biacore Insight Evaluation software. The kinetic parameters were calculated using a 1:1 binding fit model. The results are shown in Table 24.

[0480] Table 24. Summary of affinity data for SCP-CD3 / HER2 and huHER2

[0481]

[0482] Example 3

[0483] Characterization of single-chain peptides Single chain polypeptide constructs were made according to Tables 25 and 26 and purified according to the protocol outlined in Example 1.

[0484] Table 25. Summary of single-chain polypeptide constructs

[0485]

[0486]

[0487]

[0488]

[0489] Table 26. Sequences of single-chain polypeptides in Table 25

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507] SCP-CD3 / HER2 T cell cytotoxicity assay

[0508] The T cell killing activity of the single-chain polypeptide was evaluated on MCF-7 and SKBR3 cancer cell lines. T cells were negatively selected from PBMC (StemCells; Cat.nr.70025.1, Lot nr.220370304C, donor ID RG2512 (donor 6). The number of cells to be used was adjusted to obtain a T cell to cancer cell (effector to target, E:T) ratio of 10:1 (1.0E+05:1.0E+04). T cells were used for CellTrace Violet (Invitrogen, Cat. nr. C34557), added to a flat-bottom 96-well plate and incubated in a humidified incubator at 5% CO2, 37°C for 48 hours. The single-chain peptide was diluted in complete cell culture medium starting at 30 nM in eleven ten-fold serial dilutions. Negative controls were used at 20 nM in PBMCs alone, SKBR3 or MFC-7 cell lines. Additional controls included unstained cancer cells and T cells, cancer cells incubated alone with the highest concentration of the construct, and co-cultured cells. SKBR3 and MCF-7 cells were harvested for After 48 h of incubation, SKBR3 or MCF7 cells were transferred to opaque plates and incubated at ambient temperature with complete cell culture medium and The mixture was incubated with 5% paraformaldehyde and 1% paraformaldehyde (Promega; Cat.nr. G7571) for 10 min. Cell viability was assessed by luminescence. Controls were unstained cells and Reagents. Luminescence was analyzed in a Pherastar device. T cells were recovered for cell activation assays (CD25 and CD69 staining). The supernatant containing T cell conditioned medium was frozen for further cytotoxicity / activation assays. The buffers used included the following: 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: 1xPBS / 0.5% HI-FBS (heat-inactivated fetal bovine serum) / 1:1000EDTA.

[0509] SCP-CD3 / HER2 T cell activation assay

[0510] T cell activation of single-chain polypeptides was evaluated on MCF-7 and SKBR3 cancer cell lines. T cell activation was measured by FACS at 48 h by evaluating the upregulation of T cell activation markers (CD25 and CD69). After 48 h of incubation, T cells were collected and labeled with CD25 and CD69 markers. A 1:1 detection mixture of mouse anti-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) at a dilution of 1:100 was prepared in FACS buffer and kept on ice for 30 min, protected from light. Controls used included unstained cells, PE detection only, APC detection only, and double detection controls. The analysis was performed in iQue Screener PLUS. CD25 and CD69 expression were read on the BL2 (PE) and RL1 (APC) channels on the single cell population, respectively. Within the single cell gate, the percentage of double positive (CD25+CD69+) cells was determined. The assay buffer included FACS buffer: 1xPBS / 0.5% HI-FBS (heat-inactivated fetal bovine serum) / 1:1000 EDTA.

[0511] T cell proliferation assay

[0512] T cell proliferation of T cells was assessed by Cell Trace Violet dye staining. Cell proliferation was evaluated by FACS 48 hours after co-culture of 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 hours and after 48 hours of incubation with cancer cell lines and HER2 linker. The plates were placed in a 5% CO2, 37°C humidified incubator for 48 hours for co-culture with SKBR3 and MCF7 cancer cell lines. The control used was unstained T cells. Cells were acquired in iQue Screener PLUS and analyzed in Intellicyt Forecyt Software. Cell Trace violet expression was read in the VL1 (Pacific Blue) channel on single cell populations.

[0513] Sequence of Helicobacter pylori BabA_Nb14 / CD3 binder (SEQ ID NO:85): QVQLQESGGGLVQPGGSLRLSCAASGSIYSLIAMGWYRQAPGKEHELVATISSGSTTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAMYYCAAYSDRLTDCSNCEADYWGQGTQVTVSH

[0514] The results of T cell killing on MCF7 cancer cell line are Fig. 7A and Figure 7B Table 27 summarizes the Fig. 7A and Figure 7B IC50 of the graph shown in . Percent dead cancer cells versus log(concentration), nonlinear regression (curve fitting) of log(inhibitor) versus response - variable slope (four parameters) was applied. IC50 values ​​were calculated from the same analysis.

[0515] Table 27. Fig. 7A and Figure 7B IC50 (pM) shown in the graph

[0516]

[0517] ND = Not Determined. Underlined and italicized values ​​are estimates.

[0518] The results of T cell killing on SKBR3 cancer cell line are Fig. 8A and Figure 8B Table 28 summarizes the Fig. 8A and Figure 8B The IC50 values ​​of the graphs shown in . The percentage of dead cancer cells versus log(concentration) was plotted using nonlinear regression (curve fitting) of log(inhibitor) versus response - variable slope (four parameters). IC50 values ​​were calculated from the same analysis. Table 28. Fig. 8A and Figure 8B IC50 (pM) shown in the graph

[0519] Constructs# IC50(pM) SEQ ID NO: 85 ND 7 <![CDATA[ 2.7 ]]> 8 <![CDATA[ 2.82 ]]> 10 <![CDATA[ 2.82 ]]> 11 <![CDATA[ 0.41 ]]> 12 7.34 13 10.0 14 25.66 15 16.32 16 271.3 18 15.02

[0520] ND = Not Determined. Underlined and italicized values ​​are estimates.

[0521] Results of T cell activation assay in MCF7 cancer cell line Fig. 9 Table 29 summarizes the Fig. 9The EC50 of the graph shown in . The graph illustrates the percentage of dead cancer cells versus log(concentration) and was plotted using GraphPadPrism 7, applying nonlinear regression (curve fitting) of log(agonist) versus response - variable slope (four parameters). IC50 values ​​were calculated from the same analysis.

[0522] Table 29. Fig. 9 IC50 (pM) shown in the graph

[0523]

[0524]

[0525] ND = Not Determined. Underlined and italicized values ​​are estimates.

[0526] Results of T cell activation assay in SKBR3 cancer cell line Fig.10 Table 30 summarizes the Fig.10 The EC50 of the graph shown in . The graph illustrates the percentage of dead cancer cells versus log(concentration) and was plotted using GraphPadPrism 7, applying nonlinear regression (curve fitting) of log(agonist) versus response - variable slope (four parameters). IC50 values ​​were calculated from the same analysis.

[0527] Table 30. Fig.10 The IC50 (pM) shown in the graph is that constructs numbers 14 and 15 had the best T cell killing activity on MCF7 cells and construct 11 had the best T cell killing activity on SKBR3 cells.

[0528] Constructs# EC50(pM) SEQ ID NO: 85 ND 7 0.71 8 <![CDATA[ 3.16 ]]> 10 <![CDATA[ 3.88 ]]> 11 <![CDATA[ 3.64 ]]> 12 <![CDATA[ 3.25 ]]> 13 <![CDATA[ 29.02 ]]> 14 <![CDATA[ 3.67 ]]> 15 3.22 16 202.7 18 <![CDATA[ 4.01 ]]>

[0529] ND = Not Determined. Underlined and italicized values ​​are estimates.

[0530] Example 4

[0531] SCP-CD3 / HER2 affinity determination

[0532] The goal of this example is to determine the affinity and avidity of single-chain polypeptide constructs to bio-huCD3, huHER2 and cyHER2 proteins in multi-cycle kinetics (MCK) by SPR using Biacore 8k+.Constructs were injected in solution for binding to immobilized proteins. The device used for affinity determination was a Biacore 8K+ device, and the chip used was a CFJB755 CM5 series S sensor chip (Cytivia, Cat nr. 29104992) immobilized with biotinylated huCD3ε / δ (AcroBiosystems; Cat. nr. CDD-H82W0), huHER2-His (AcroBiosystems; Cat. nr. HE2-H5225-100ug) and cyHER2-His (AcroBiosystems; Cat. nr. HE2-C52Hb-100ug) by amine coupling at low density (1000-3000RU) on channels 1, 3 and 5 and at high density (6200-7000RU) on channels 2, 4 and 6. 0.3, 0.6, 1.3, 2.5 and 5nM single-chain polypeptide construct samples were injected into flow cells 1 and 2 at 30 μL / min for 2 minutes. 10nM control monoclonal antibody was injected into flow cells 1 and 2 at 30 μL / min for 2 minutes. For flow channels 1-2, mouse anti-huCD3 (R&D; Cat.nr.MAB100-100) was used. For flow channels 3-6, human anti-HER2 (R&D; Cat.nr.MAB9589-100) was used. The dissociation rate measurement result was 300 seconds. For regeneration, the regeneration solution was used to restore the baseline level. The buffer used was 1xHBS-EP+pH 7.4. The data was analyzed using the multi-cycle kinetic pre-defined evaluation method of Biacore Insight Evaluation software. The kinetic parameters were calculated using a 1:1 binding fitting model.

[0533] Tables 31A and 31B summarize the huCD3 results. Tables 32A and 32B summarize the huHER2 results. Tables 33A and 33B summarize the cyHER2 results. Table 34 shows that the KD for CD3 binding is more than 100 times higher than the HER2 control (construct #47), and in one case, more than 10,000 times higher, and 3.8nM KD is reported for anti-CD3 2B2 (SEQ ID NO:75 for VH; and SEQ ID NO:76 for VL). In addition, the CD3 configuration of the tested constructs retains binding to cynomolgus CD3. The branched construct with 4 VHHs (construct #18) binds to huHER2 with a 1 / 100 lower KD. HER2 and EGFR constructs with 6, 7, and 8 residue linkers between the CD3 domains are all active.

[0534] Table 31A.

[0535]

[0536] Table 31B

[0537]

[0538] Table 32A.

[0539]

[0540] Table 32B

[0541]

[0542] Table 33A.

[0543]

[0544] Table 33B

[0545]

[0546] Table 34.

[0547]

[0548]

[0549] Example 5

[0550] Efficacy of single-chain peptides in breast cancer PDX models in NSG mice

[0551] NOG mice were treated with 2x10 7 BT-474 tumor cells were inoculated subcutaneously (SC) and 1x10 7huPBMCs were added and treated 2 days later at metabolic tumor volume (MTV) with antibodies at two equimolar doses (e.g., 5 nmol / kg and 10 nmol / kg per day for 3 weeks). Mice were weighed once a week and then sacrificed on day 38 to allow organ collection for analysis by flow cytometry.

[0552] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will now conceive of many changes, variations and substitutions. It should be understood that in practicing the present invention, various alternatives to the embodiments of the present invention described herein may be employed.

[0553] All publications, patent applications, issued patents, and other documents mentioned in this specification are incorporated herein by reference, as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in texts incorporated by reference are excluded to the extent that they conflict with definitions in this disclosure.

Claims

1. A single-chain polypeptide comprising variable domains connected by a linker, folded into a conformation such that the variable domains form two effector cell antigen interaction domains (E), wherein each effector cell antigen interaction domain comprises two variable domains (EV and EV) of a single-chain variable fragment, and at least one of the effector cell antigen interaction domains is connected to one or more target interaction domains.

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

3. The single-chain polypeptide of claim 2, wherein the tumor antigen is on a cell in the tumor microenvironment.

4. The single-chain polypeptide of any one of claims 1-3, wherein the target interaction domain comprises an antibody, an antibody fragment, a ligand or a cofactor.

5. The single-chain polypeptide of claim 4, wherein the antibody or antibody fragment of the target interaction domain comprises Fab, Fab′, F(ab′)2, Fv fragment, single-chain Fv (scFv), tandem single-chain Fv ((scFv)2, dual-affinity retargeting antibody, bispecific antibody, VHH single domain antibody, TriTac, Adnectin, Anticalin, Avimer, Fynomer, Kunitz domain, knottin, affibody or DARPin.

6. The single-chain polypeptide of any one of claims 1-5, wherein the one or more target interaction domains comprise one or more target cell antigen interaction domains that bind to a target cell antigen.

7. The single-chain polypeptide of claim 6, wherein the target cell antigen interacting domain binds to more than one target cell antigen.

8. The single-chain polypeptide of claim 6 or 7, wherein the target cell antigen interaction domain is a bispecific or multispecific antibody or antibody fragment.

9. The single-chain polypeptide of any one of claims 6-8, wherein the target cell antigen interacting domain comprises a VHH single domain antibody (TVHH).

10. The single-chain polypeptide of any one of claims 1 to 9, wherein the single-chain polypeptide does not contain a crystallizable fragment (Fc) domain.

11. The single-chain polypeptide of any one of claims 1-10, 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.

12. The single-chain polypeptide of claim 11, wherein the EVL connected to at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to at least one TVHH, and the EVH connected to the one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to at least one TVHH.

13. The single-chain polypeptide of claim 12, wherein the at least one TVHH attached to the EVL binds to the same target antigen as the at least one TVHH attached to the EVH.

14. The single-chain polypeptide of claim 13, wherein the at least one TVHH attached to the EVL binds to the same target antigen and has the same complementarity determining region (CDR) sequence as the at least one TVHH attached to the EVH.

15. The single-chain polypeptide of claim 13, wherein the at least one TVHH attached to the EVL and the at least one TVHH attached to the EVH bind to the same target antigen and do not have a common complementarity determining region (CDR) sequence.

16. The single-chain polypeptide of claim 12, wherein the at least one TVHH attached to the EVL binds to a different target antigen than the at least one TVHH attached to the EVH.

17. The single-chain polypeptide of any one of claims 11-16, wherein the EVL and the EVH are linked to the same number of TVHHs.

18. The single-chain polypeptide of any one of claims 11-16, wherein the EVL is linked to a different number of TVHHs than the number of TVHHs linked to the EVH.

19. The single-chain polypeptide of claim 17, wherein the EVL connected to at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to a TVHH, and the EVH connected to at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to a TVHH.

20. The single-chain polypeptide of claim 18, wherein the EVL connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to one TVHH, and the EVH connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to two TVHHs.

21. The single-chain polypeptide of claim 18, wherein the EVL connected to the at least one of the effector cell antigen interaction domains of the at least one or more target cell antigen interaction domains is connected to one TVHH, and the EVH connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to three TVHHs.

22. The single-chain polypeptide of claim 18, wherein the EVH connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to one TVHH, and the EVL connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to two TVHHs.

23. The single-chain polypeptide of claim 18, wherein the EVH connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to one TVHH, and the EVL connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to three TVHHs.

24. The single-chain polypeptide of claim 17, wherein the EVH connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to two TVHHs, and the EVL connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to two TVHHs.

25. The single-chain polypeptide of claim 17, wherein the EVH connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to three TVHHs, and the EVL connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to three TVHHs.

26. The single-chain polypeptide of claim 17, wherein the EVH connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to four TVHHs, and the EVL connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to four TVHHs.

27. The single-chain polypeptide of claim 17, wherein the EVH connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to five TVHHs, and the EVL connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to five TVHHs.

28. The single-chain polypeptide of claim 17, wherein the EVH connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to six TVHHs, and the EVL connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to six TVHHs.

29. The single-chain polypeptide of claim 17, wherein the EVH connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to seven TVHHs, and the EVL connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to seven TVHHs.

30. The single-chain polypeptide of claim 17, wherein the EVH connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to eight TVHHs, and the EVL connected to the at least one of the effector cell antigen interaction domains of the one or more target cell antigen interaction domains is connected to eight TVHHs.

31. The single-chain polypeptide of claim 11, wherein said at least one of said effector cell antigen interaction domains linked to said one or more target cell antigen interaction domains is linked to one target cell antigen interaction domain.

32. The single-chain polypeptide of claim 31, wherein the one or more target cell antigen interaction domains are C-terminally linked to the at least one of the effector cell antigen interaction domains.

33. The single-chain polypeptide of claim 31, wherein the one or more target cell antigen interaction domains are N-terminally linked to the at least one of the effector cell antigen interaction domains.

34. The single-chain polypeptide of claim 11, wherein said at least one of said effector cell antigen interaction domains linked to said one or more target cell antigen interaction domains is linked to more than one target cell antigen interaction domain.

35. The single-chain polypeptide of claim 34, wherein all of the more than one target cell antigen interaction domains bind to the same target antigen.

36. The single-chain polypeptide of claim 35, wherein all of the more than one target cell antigen interaction domains bind to the same target antigen and all have the same CDR sequences.

37. The single-chain polypeptide of claim 35, wherein all of the more than one target cell antigen interaction domains bind to the same target antigen and at least two of the more than one target cell antigen interaction domains do not have a common CDR sequence.

38. The single-chain polypeptide of claim 34, wherein at least two of the more than one target cell antigen interaction domains bind to different target antigens.

39. The single-chain polypeptide of claim 11, wherein the at least one of the effector cell antigen interaction domains linked to the one or more target cell antigen interaction domains is linked to two target cell antigen interaction domains.

40. The single-chain polypeptide of claim 39, wherein the two target cell antigen interaction domains are C-terminally linked to the one of the effector cell antigen interaction domains.

41. The single-chain polypeptide of claim 39, wherein the two target cell antigen interaction domains are N-terminally linked to the one of the effector cell antigen interaction domains.

42. The single-chain polypeptide of claim 11, wherein the at least one of the effector cell antigen interaction domains linked to the one or more target cell antigen interaction domains is linked to three target cell antigen interaction domains.

43. The single-chain polypeptide of claim 42, wherein the three target cell antigen interaction domains are C-terminally linked to the one of the effector cell antigen interaction domains.

44. The single-chain polypeptide of claim 42, wherein the three target cell antigen interaction domains are N-terminally linked to the one of the effector cell antigen interaction domains.

45. The single-chain polypeptide of claim 11, wherein the at least one of the effector cell antigen interaction domains linked to the one or more target cell antigen interaction domains is linked to four target cell antigen interaction domains.

46. ​​The single-chain polypeptide of claim 45, wherein the four target cell antigen interaction domains are C-terminally linked to the one of the effector cell antigen interaction domains.

47. The single-chain polypeptide of claim 45, wherein the four target cell antigen interaction domains are N-terminally linked to the one of the effector cell antigen interaction domains.

48. The single-chain polypeptide of any one of claims 9-47, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) 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; 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.

49. The single-chain polypeptide of claim 48, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH; or TVHH-EV-EV-EV-EV-TVHH.

50. The single-chain polypeptide of claim 48, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH.

51. The single-chain polypeptide of claim 48, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EV-EV-EV-EV-TVHH.

52. A single-chain polypeptide as described in claim 48, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH.

53. The single-chain polypeptide of any one of claims 11-52, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the 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-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.

54. The single-chain polypeptide of claim 53, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH; TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH; TVHH-EVL-EVH-EVL-EVH-TVHH; or TVHH-EVH-EVL-EVH-EVL-TVHH.

55. The single-chain polypeptide of claim 53, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH; or TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH.

56. The single-chain polypeptide of claim 53, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EVL-EVH-EVL-EVH-TVHH; or TVHH-EVH-EVL-EVH-EVL-TVHH.

57. The single-chain polypeptide of claim 53, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH.

58. The single-chain polypeptide of claim 53, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH.

59. The single-chain polypeptide of claim 53, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EVL-EVH-EVL-EVH-TVHH.

60. The single-chain polypeptide of claim 53, wherein the variable domains and the one or more target cell antigen interaction domains are ordered according to the following N-terminal to C-terminal arrangement, in which (-) is the linker: TVHH-EVH-EVL-EVH-EVL-TVHH.

61. The single-chain polypeptide of any one of claims 1-60, wherein the linker connecting the two effector cell antigen interaction domains (E) to each other comprises an internal linker.

62. The single-chain polypeptide of any one of claims 11-61, wherein the effector cell antigen interaction domain that is not directly linked to the one or more target cell antigen interaction domains comprises a terminal linker connecting EVL and EVH.

63. The single-chain polypeptide of claim 61 or 62, wherein each of the internal linkers is 6-8 amino acids in length.

64. The single-chain polypeptide of claim 62 or 63, wherein the terminal linker is 15 or 16 amino acids in length.

65. The single-chain polypeptide of any one of claims 1-64, wherein each of said effector cell antigen interacting domains binds to an effector cell antigen.

66. The single-chain polypeptide of claim 65, wherein the effector cell antigen comprises CD3, CD16a or death receptor 5 (DR5).

67. The single-chain polypeptide of claim 66, wherein the CD3 is CD3δ, CD3γ or CD3ε.

68. The single-chain polypeptide of any one of claims 65-67, wherein the effector cell antigen is on an effector cell.

69. The single-chain polypeptide of claim 68, wherein the effector cell comprises a T cell, a NK cell, or a macrophage.

70. The single-chain polypeptide of any one of claims 6-69, wherein the target cell antigen interacting domain (T) that interacts with a target cell binds to the target cell antigen.

71. A single-chain polypeptide as described in claim 70, wherein when on cells associated with a disease state, the target cell antigen is clustered in lipid rafts, and when on cells not associated with a disease state, the target cell antigen is not clustered in lipid rafts.

72. The single-chain polypeptide of claim 70, wherein the target cell antigens are clustered when on cells associated with a disease state and are not clustered when on cells not associated with a disease state.

73. The single-chain polypeptide of claim 70, wherein the target cell antigen is 100 nm or less when on cells associated with a disease state, and the target cell antigen is not within 100 nm when on cells not associated with a disease state.

74. The single-chain polypeptide of claim 70, wherein the target cell antigen is 75 nm or less when on cells associated with a disease state, and the target cell antigen is not within 75 nm when on cells not associated with a disease state.

75. The single-chain polypeptide of claim 70, wherein the target cell antigen is 50 nm or less when on cells associated with a disease state, and the target cell antigen is not within 50 nm when on cells not associated with a disease state.

76. The single-chain polypeptide of claim 70, wherein the target cell antigen is 25 nm or less when on cells associated with a disease state, and the target cell antigen is not within 25 nm when on cells not associated with a disease state.

77. The single-chain polypeptide of claim 70, wherein the target cell antigen is 10 nm or less when on cells associated with a disease state, and the target cell antigen is not within 10 nm when on cells not associated with a disease state.

78. The single-chain polypeptide of claim 70, wherein the target cell antigen is 5 nm or less when on cells associated with a disease state, and the target cell antigen is not within 5 nm when on cells not associated with a disease state.

79. The single-chain polypeptide of claim 70, wherein the target cell antigen is 2 nm or less when on cells associated with a disease state, and the target cell antigen is not within 2 nm when on cells not associated with a disease state.

80. The single-chain polypeptide of claim 70, wherein the target cell antigens are in close proximity when on cells associated with a disease state, and the target cell antigens are not in close proximity when on cells not associated with a disease state.

81. A single-chain polypeptide as described in any one of claims 70-80, wherein when on cells associated with a disease state, the target cell antigen is a dimer, trimer, tetramer or oligomer, and when on cells not associated with a disease state, the target cell antigen is a monomer or dimer.

82. The single-chain polypeptide of any one of claims 70-81, 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 the same cell signaling complex when on cells not associated with a disease state.

83. The single-chain polypeptide of any one of claims 70-82, 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.

84. The single-chain polypeptide of any one of claims 70-83, wherein the target cell antigen comprises CD33, FAP, EGFR, HER2 or EpCAM.

85. The single-chain polypeptide of any one of claims 71-84, wherein the cell associated with the disease state is a myeloid cell, a fibroblast, or a cancer cell.

86. A pharmaceutical composition comprising: (a) the single-chain polypeptide of any one of claims 1 to 85; and (b) a pharmaceutically acceptable excipient.

87. An isolated recombinant nucleic acid encoding the single-chain polypeptide of any one of claims 1-85.

88. A vector comprising the isolated nucleic acid of claim 87.

89. A host cell comprising the isolated nucleic acid of claim 87 or the vector of claim 88.

90. A method for treating cancer, comprising administering the single-chain polypeptide according to any one of claims 1-85 to a subject in need thereof.

91. The method of claim 90, wherein the cancer is a solid tumor cancer.

92. The method of claim 90, wherein the cancer is a hematological cancer.

93. 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 claims 1-85.

94. 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 claims 1-85.

95. 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 claims 1-85.

96. 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 claims 1-85.

97. A method of treating a bacterial infection, comprising administering to a subject in need thereof a single-chain polypeptide according to any one of claims 1-85.

98. A method for treating a viral infection, comprising administering to a subject in need thereof a single-chain polypeptide according to any one of claims 1-85.

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