Multispecific Polypeptide Complexes

Abstract

The present invention provides novel covalent multispecific antibodies and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to novel covalent multispecific antibodies and uses thereof. [Background technology]

[0002] Multispecific antibodies have attracted much attention in the field of antibody engineering and are expected to have a wide range of applications, including anti-tumor immunotherapy.

[0003] Although multispecific antibodies have many advantages, they also pose challenges, for example, in terms of production. Mismatches can occur between heavy chains and / or between heavy and light chains. In bispecific or multispecific antibodies, how to efficiently and effectively remove mismatched by-products is an insurmountable challenge. In addition, because multispecific antibodies bind to at least two targets, balancing the affinities of the multispecific antibodies for these various targets is crucial to achieving the intended regulatory and biological effects on each target.

[0004] Therefore, there is a great need to develop novel constructs that can provide high specificity and affinity for a desired target, high killing efficacy against target cells, and ease of manufacture and downstream purification. Summary of the Invention

[0005] Throughout this disclosure, the articles "a," "an," and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or multiple antibodies.

[0006] The present disclosure provides multispecific polypeptide complexes, nucleotide sequences encoding same, and uses thereof, among others.

[0007] In one embodiment, the multispecific polypeptide complex comprises (a) a first dimerization domain, and (b) a first antigen-binding domain comprising a DICAD domain and a first Fv domain, wherein the DICAD domain comprises i) a first polypeptide fragment comprising a first heavy chain variable domain (VH1) linked to a second light chain variable domain (VL2), and ii) a second polypeptide fragment comprising a second heavy chain variable domain (VH2) linked to a first light chain variable domain (VL1), wherein the VL1 and the VH1 associate to form a first domain capable of binding to a first target, and the VL2 and the VH2 associates to form a second domain capable of binding to a second target, the first Fv domain comprises iii) a third polypeptide fragment comprising a third heavy chain variable domain (VH3), and iv) a fourth polypeptide fragment comprising a third light chain variable domain (VL3), the VH3 and VL3 associate to form a third domain capable of binding to a third target, and one of the termini of the DICAD domain is operably linked to one of the termini of the first Fv domain, and one C-terminus of the first antigen-binding domain is operably linked to the N-terminus of the first dimerization domain.

[0008] In some embodiments, the N-terminus of the VH1 is linked to the C-terminus of the VL2, and the N-terminus of the VH2 is linked to the C-terminus of the VL1, or the N-terminus of the VL1 is linked to the C-terminus of the VH2, and the N-terminus of the VL2 is covalently linked to the C-terminus of the VH1.

[0009] In some embodiments, one of the first domain and the second domain comprises a first non-naturally occurring covalent bond formed between a first pair of two amino acid residues, while the other of the first domain and the second domain does not comprise a non-naturally occurring covalent bond or comprises a second non-naturally occurring covalent bond formed between a different pair of two amino acid residues than the pair of amino acid residues corresponding to the first pair of two amino acid residues.

[0010] In some embodiments, the first non-natural covalent bond is a non-natural disulfide bond. In some embodiments, the non-natural disulfide bond is formed between two non-natural cysteine ​​residues. In some embodiments, the two non-natural cysteine ​​residues are at position 44 in the VH and position 100 in the VL, or at position 105 in the VH and position 43 in the VL, or at position 100 in the VH and position 49 in the VL, or at position 100 in the VH and position 150 in the VL, where numbering is according to the Kabat index.

[0011] In some embodiments, the VL1 and the VH1 are associated by a first non-naturally occurring disulfide bond. In some embodiments, the first non-naturally occurring disulfide bond is formed between two non-naturally occurring cysteine ​​residues in the VL1 and the VH1, respectively. In some embodiments, the first non-naturally occurring disulfide bond is formed between two non-naturally occurring cysteine ​​residues in FR2 of the VL1 and an amino acid residue in FR4 of the VH1, respectively. In some embodiments, the first non-naturally occurring covalent bond is formed between two non-naturally occurring cysteine ​​residues at position 44 in the VH1 and position 100 in the VL1, or at position 105 in the VH1 and position 43 in the VL1, or at position 100 in the VH1 and position 49 in the VL1, or at position 100 in the VH1 and position 150 in the VL1, where numbering is according to the Kabat index.

[0012] In some embodiments, the VL2 and the VH2 are associated by a first non-naturally occurring disulfide bond. In some embodiments, the first non-naturally occurring disulfide bond is formed between two non-naturally occurring cysteine ​​residues in the VL2 and the VH2, respectively. In some embodiments, the first non-naturally occurring disulfide bond is formed between two non-naturally occurring cysteine ​​residues in FR2 of the VL2 and an amino acid residue in FR4 of the VH2, respectively. In some embodiments, the first non-naturally occurring covalent bond is formed between two non-naturally occurring cysteine ​​residues at position 44 in the VH2 and position 100 in the VL2, or at position 105 in the VH2 and position 43 in the VL2, or at position 100 in the VH2 and position 49 in the VL2, or at position 100 in the VH2 and position 150 in the VL2, where numbering is according to the Kabat index.

[0013] In some embodiments, one of the first domain and the second domain that comprises the first non-natural covalent bond further comprises a non-natural pair of two oppositely charged amino acid residues.

[0014] In some embodiments, (a) the two oppositely charged residues are at position 38 in the VL1 and position 39 in the VH1, respectively; (b) the two oppositely charged residues are at position 40 in the VL1 and position 39 in the VH1, respectively; (c) the two oppositely charged residues are at position 37 in the VL1 and position 39 in the VH1, respectively; or (d) the two oppositely charged residues are at position 37 in the VL1 and position 39 in the VH1, respectively, wherein numbering is according to the Kabat index.

[0015] In some embodiments, the VL2 and the VH2 are associated through the second non-native disulfide bond, and the VL1 and the VH1 are associated through either a natural disulfide bond or another non-native disulfide bond formed at a position different from the position of the second non-native disulfide bond. In some embodiments, the second non-native disulfide bond is formed between two non-native cysteine ​​residues in the VL2 and the VH2, respectively, and optionally, the two non-native cysteine ​​residues are Q100C or G100C in the VL2 and G44C or A44C in the VH2, where numbering is according to the Kabat index.

[0016] In some embodiments, the VL2 and the VH2 are further associated by electrostatic interactions between two oppositely charged residues, in some embodiments: (a) the two oppositely charged residues are introduced to replace Q38 in the VL2 and Q39 in the VH2, respectively; (b) the two oppositely charged residues are introduced to replace Q40 in the VL2 and Q39 in the VH2, respectively; (c) the two oppositely charged residues are introduced to replace Q37 in the VL2 and Q39 in the VH2, respectively; or (d) the two oppositely charged residues are introduced to replace Q37 in the VL2 and Q39 in the VH2, respectively, where numbering is according to the Kabat index.

[0017] In some embodiments, the two oppositely charged residues comprise a negatively charged amino acid residue selected from aspartic acid (D) or glutamic acid (E) and a positively charged amino acid residue selected from lysine (K), histidine (H), or arginine (R).

[0018] In some embodiments, the two oppositely charged residues comprise Q38D in said VL1 and Q39K in said VH1, respectively, or Q38D in said VL2 and Q39K in said VH2, respectively, where numbering is according to the Kabat index.

[0019] In some embodiments, the VL1 is linked to the VH2 via a first peptide linker, and the VL2 is linked to the VH1 via a second peptide linker. In some embodiments, the first peptide linker and the second peptide linker each independently comprise 5 to 9 amino acids. In certain embodiments, the peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 95 or 96.

[0020] In some embodiments, the third polypeptide fragment further comprises an antibody heavy chain constant region (CH1) operably linked to the C-terminus of the VH3 domain, and / or the fourth polypeptide fragment further comprises an antibody light chain constant region (CL) operably linked to the C-terminus of the VL3 domain.

[0021] In some embodiments, one of the N-termini of the DICAD domain is operably linked to one of the C-termini of the first Fv domain.

[0022] In some embodiments, one of the N-termini of the DICAD domain is operably linked to the C-terminus of the third polypeptide fragment of the Fv domain.

[0023] In some embodiments, the N-terminus of the second polypeptide fragment of the DICAD domain is operably linked to the C-terminus of the third polypeptide fragment of the first Fv domain.

[0024] In some embodiments, the N-terminus of the first polypeptide fragment of the DICAD domain is operably linked to the C-terminus of the third polypeptide fragment of the first Fv domain.

[0025] In some embodiments, one of the N-termini of the DICAD domain is operably linked to the C-terminus of the fourth polypeptide fragment of the first Fv domain.

[0026] In some embodiments, the N-terminus of the first polypeptide fragment of the DICAD domain is operably linked to the C-terminus of the fourth polypeptide fragment of the first Fv domain.

[0027] In some embodiments, one of the C-termini of the DICAD domain is operably linked to one of the N-termini of the Fv domain.

[0028] In some embodiments, the C-terminus of the second polypeptide fragment of the DICAD domain is operably linked to the N-terminus of the third polypeptide fragment of the first Fv domain.

[0029] In some embodiments, the C-terminus of the first polypeptide fragment of the DICAD domain is operably linked to the N-terminus of the fourth polypeptide fragment of the first Fv domain.

[0030] In some embodiments, the C-terminus of the first polypeptide fragment of the first antigen-binding domain is operably linked to the N-terminus of the first dimerization domain.

[0031] In some embodiments, the C-terminus of the third polypeptide fragment of the first antigen-binding domain is operably linked to the N-terminus of the first dimerization domain.

[0032] In some embodiments, the first dimerization domain comprises an IgG CH3 domain, hi some embodiments, the first dimerization domain further comprises an IgG CH2 domain and / or a hinge region.

[0033] In some embodiments, both the first domain and the second domain comprise a TGFβ targeting domain, and the third domain comprises a PD-1 targeting domain. In some embodiments, the TGFβ targeting domain comprises HCDR1, HCDR2, and HCDR3 identical to the HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence of SEQ ID NO: 38, and LCDR1, LCDR2, and LCDR3 identical to the LCDR1, LCDR2, and LCDR3 contained in the amino acid sequence of SEQ ID NO: 37. In some embodiments, the TGFβ targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequences of SEQ ID NOs: 22 / 23 / 24 / 19 / 20 / 21, respectively. In some embodiments, the first domain comprises a TGFβ targeting domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 50 and a VL domain comprising the amino acid sequence of SEQ ID NO: 49. In some embodiments, the second domain comprises a TGFβ targeting domain wherein the VH2 comprises the amino acid sequence of SEQ ID NO: 38 and the VL2 comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the PD-1 targeting domain comprises HCDR1, HCDR2, and HCDR3 identical to the HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence of SEQ ID NO: 40, and LCDR1, LCDR2, and LCDR3 identical to the LCDR1, LCDR2, and LCDR3 contained in the amino acid sequence of SEQ ID NO: 39. In some embodiments, the PD-1 targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequences of SEQ ID NOs: 28 / 29 / 30 / 25 / 26 / 27, respectively. In some embodiments, the third domain comprises a PD-1 targeting domain wherein the VH3 comprises the amino acid sequence of SEQ ID NO: 40 and the VL3 comprises the amino acid sequence of SEQ ID NO: 39.

[0034] In some embodiments, a) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO:62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO:59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO:53; b) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO:62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO:59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO:56; c) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO:62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO:59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO:58, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO:53; or d) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO:62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO:59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO:61, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO:53.

[0035] In some embodiments, the multispecific polypeptide complex comprises three polypeptide chains having the amino acid sequences of a) SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53; b) SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56; c) SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59; or d) SEQ ID NO:60, SEQ ID NO:61, and SEQ ID NO:59, respectively.

[0036] In some embodiments, at least one or both of the first target and the second target are immunostimulatory targets, hi some embodiments, the third target is a tumor antigen.

[0037] In some embodiments, the first domain and the second domain comprise a CD3 targeting domain and a 4-1BB targeting domain, respectively, and the third domain comprises a CEA targeting domain. In some embodiments, the first domain comprises a 4-1BB targeting domain and the second domain comprises a CD3 targeting domain, while the third domain comprises a CEA targeting domain. In some embodiments, the first domain comprises the 4-1BB targeting domain, and in the 4-1BB targeting domain, the VH1 and the VL1 comprise the first non-natural disulfide bond formed between the two non-natural cysteine ​​residues and further comprise the first non-natural pair of oppositely charged amino acid residues.

[0038] In some embodiments, the 4-1BB targeting domain comprises HCDR1, HCDR2, and HCDR3 identical to the HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence of SEQ ID NO: 32, and LCDR1, LCDR2, and LCDR3 identical to the LCDR1, LCDR2, and LCDR3 contained in the amino acid sequence of SEQ ID NO: 31. In some embodiments, the 4-1BB targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each comprising the amino acid sequence of SEQ ID NO: 4 / 5 / 6 / 1 / 2 / 3, respectively. In some embodiments, the first domain comprises a 4-1BB targeting domain in which the VH1 comprises the amino acid sequence of SEQ ID NO: 48 and the VL1 comprises the amino acid sequence of SEQ ID NO: 47.

[0039] In some embodiments, the CD3 targeting domain comprises HCDR1, HCDR2, and HCDR3 identical to the HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence of SEQ ID NO: 34, and LCDR1, LCDR2, and LCDR3 identical to the LCDR1, LCDR2, and LCDR3 contained in the amino acid sequence of SEQ ID NO: 33. In some embodiments, the CD3 targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequence of SEQ ID NO: 10 / 11 / 12 / 7 / 8 / 9, respectively. In some embodiments, the second domain comprises a CD3 targeting domain in which the VH2 comprises the amino acid sequence of SEQ ID NO: 34 and the VL2 comprises the amino acid sequence of SEQ ID NO: 33.

[0040] In some embodiments, the CEA targeting domain comprises HCDR1, HCDR2, and HCDR3 identical to the HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence of SEQ ID NO: 36, 42, or 44, and LCDR1, LCDR2, and LCDR3 identical to the LCDR1, LCDR2, and LCDR3 contained in the amino acid sequence of SEQ ID NO: 35, 41, or 43. In some embodiments, the CEA targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each comprising the amino acid sequence of SEQ ID NO: 16 / 17 / 18 / 13 / 14 / 15, or SEQ ID NO: 107 / 108 / 18 / 13 / 14 / 15, or SEQ ID NO: 16 / 111 / 18 / 109 / 110 / 15, respectively.

[0041] In some embodiments, the multispecific polypeptide complex comprises: a) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 87, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 75, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 83, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 74; b) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 87, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 75, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 83, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 78; or c) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 87, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 75, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 83, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 82.

[0042] In some embodiments, the multispecific polypeptide complex further comprises a second dimerization domain that associates with the first dimerization domain to form a dimer.

[0043] In some embodiments, the multispecific polypeptide complex further comprises a second antigen-binding domain, wherein one of the C-termini of said second antigen-binding domain is operably linked to the N-terminus of said second dimerization domain.

[0044] In some embodiments, the second antigen-binding domain is identical to the first antigen-binding domain.

[0045] In some embodiments, the second dimerization domain is identical to the first dimerization domain.

[0046] In some embodiments, the second antigen-binding domain is different from the first antigen-binding domain.

[0047] In some embodiments, the second dimerization domain is different from the first dimerization domain and associates with the first dimerization domain to form a heterodimer.

[0048] In some embodiments, the first dimerization domain and / or the second dimerization domain further comprise one or more mutations that promote heterodimerization.

[0049] In some embodiments, both the first dimerization domain and the second dimerization domain comprise an IgG CH3 domain.

[0050] In some embodiments, the first dimerization domain comprises a first mutation and the second dimerization domain comprises a second mutation, wherein a) the first mutation comprises T389W and / or S375C and the second mutation comprises Y438V, T389S, L391A and / or Y370C; b) the first mutation comprises D427K and / or D377K and the second mutation comprises K420D and / or K440D; c) the first mutation comprises D377K, E378K, and / or D427K and the second mutation comprises K393E, K440D, and / or K470E; d) the first mutation comprises S387H and / or F436A and the second mutation comprises Y370T and / or T422F, e) the first mutation comprises S387H and / or T422F and the second mutation comprises Y422T and / or F436A, f) the first mutation comprises K393D and / or K440D and the second mutation comprises E378K and / or D427K, or g) the first mutation comprises L372D and / or L391E and the second mutation comprises L372K or T389K, where numbering is according to the Kabat index. In some embodiments, the first mutation comprises T389W and the second domain comprises T389S, L391A, and Y438V, where numbering is according to the Kabat index.

[0051] In some embodiments, the second antigen-binding domain comprises a second Fv domain. In some embodiments, the second Fv domain is identical to the first Fv domain, and the second Fv domain comprises a fifth polypeptide fragment identical to the third polypeptide fragment comprising the third heavy chain variable domain (VH3), and a sixth polypeptide fragment identical to the fourth polypeptide fragment comprising the third light chain variable domain (VL3). In some embodiments, the C-terminus of the fifth polypeptide fragment of the second Fv domain is operably linked to the N-terminus of the second dimerization domain.

[0052] In some embodiments, the second Fv domain comprises the CEA-targeting domain, wherein a) the VH3 comprises the amino acid sequence of SEQ ID NO: 36 and the VL3 comprises the amino acid sequence of SEQ ID NO: 35, b) the VH3 comprises the amino acid sequence of SEQ ID NO: 42 and the VL3 comprises the amino acid sequence of SEQ ID NO: 41, or c) the VH3 comprises the amino acid sequence of SEQ ID NO: 44 and the VL3 comprises the amino acid sequence of SEQ ID NO: 43.

[0053] In some embodiments, the fifth polypeptide fragment and the sixth polypeptide fragment comprise the amino acid sequences of a) SEQ ID NO: 73 and SEQ ID NO: 74, respectively; b) SEQ ID NO: 77 and SEQ ID NO: 78, respectively; and c) SEQ ID NO: 81 and SEQ ID NO: 82, respectively.

[0054] In some embodiments, the multispecific polypeptide complex comprises polypeptide chains having the amino acid sequences of a) SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74; b) SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78; or c) SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:82, respectively.

[0055] In some embodiments, the multispecific polypeptide complex is linked to one or more conjugate moieties, which in some embodiments comprise an agent for detection or isolation, such as a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or other anti-cancer agent.

[0056] Another aspect of the present disclosure provides an isolated polynucleotide encoding a fusion polypeptide or polypeptide complex described herein. In yet another aspect, the present disclosure provides a vector comprising the isolated polynucleotide described herein. In yet another aspect, the present disclosure provides a host cell comprising the vector described herein.

[0057] In yet another aspect, the present disclosure provides pharmaceutical compositions comprising (i) a multispecific polypeptide complex described herein, or a polynucleotide encoding a multispecific polypeptide complex described herein, and (ii) one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients. In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.

[0058] In yet another aspect, the present disclosure provides a method of expressing a multispecific polypeptide complex described herein, comprising culturing a host cell described herein under conditions in which a vector described herein is expressed.

[0059] In yet another aspect, the present disclosure provides a method of treating, preventing, or alleviating a disease or disorder in a subject, comprising administering to said subject a therapeutically effective amount of a multispecific polypeptide complex described herein.

[0060] In yet another aspect, the present disclosure provides methods of treating, preventing, or alleviating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific polypeptide complex described herein, or a polynucleotide encoding a multispecific polypeptide complex described herein, and / or a pharmaceutical composition described herein. In some embodiments, the subject is a human. In some embodiments, the administration is oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular.

[0061] In yet another aspect, the present disclosure provides the use of a multispecific polypeptide complex described herein, a pharmaceutical composition described herein, and / or said polynucleotide encoding a multispecific polypeptide complex described herein in the manufacture of a medicament for treating, preventing, or ameliorating a disease or disorder. [Brief explanation of the drawings]

[0062] [Figure 1] Schematic representation of the Type A structure of the 2:2:2 symmetric multispecific polypeptide complex 04A25. [Figure 2] 1 shows the purity of the expressed polypeptide complex 04A25 (Type A) as analyzed by SDS-Page. [Figure 3] 1 shows the purity of the expressed polypeptide complex 04A25 (Type A) analyzed by SEC-HPLC. [Figure 4] Schematic representation of the Type B structure of the 2:2:2 symmetric multispecific polypeptide complex 04A26. [Figure 5] 1 shows the purity of the expressed polypeptide complex 04A26 (type B) as analyzed by SDS-Page. [Figure 6] 1 shows the purity of the expressed polypeptide complex 04A26 (type B) analyzed by SEC-HPLC. [Figure 7] Schematic representation of the Type C structure of the 2:2:2 symmetric multispecific polypeptide complex 04A27. [Figure 8]1 shows the purity of the expressed polypeptide complex 04A27 (Type C) as analyzed by SDS-Page. [Figure 9] 1 shows the purity of the expressed polypeptide complex 04A27 (Type C) analyzed by SEC-HPLC. [Figure 10] Schematic representation of the type D structure of the 2:2:2 symmetric multispecific polypeptide complex 04A28. [Figure 11] 1 shows the purity of the expressed polypeptide complex 04A28 (type D) as analyzed by SDS-Page. [Figure 12] 1 shows the purity of the expressed polypeptide complex 04A28 (type D) analyzed by SEC-HPLC. [Figure 13] 1 shows a schematic representation of the TRIAD structure of the 2:1 multispecific polypeptide complex 12A6. [Figure 14] 1 shows a schematic representation of the TRIAD structure of the 1:1:1 multispecific polypeptide complex 12A10. [Figure 15] Schematic representation of the type E structure of the 2:1:1 asymmetric multispecific polypeptide complex 12A14. [Figure 16] The purity of the expressed polypeptide complex 12A14 (type E) analyzed by SDS-Page is shown. [Figure 17] 1 shows the purity of the expressed polypeptide complex 12A14 (type E) analyzed by SEC-HPLC. [Figure 18] FIG. 1 shows a diagram of the Type F structure of the 2:1:1 asymmetric multispecific polypeptide complex 12A15. [Figure 19] The purity of the expressed polypeptide complex 12A15 (type F) analyzed by SDS-Page is shown. [Figure 20] 1 shows the purity of the expressed polypeptide complex 12A15 (type F) analyzed by SEC-HPLC. [Figure 21] FIG. 1 shows a diagram of the type G structure of the 2:1:1 asymmetric multispecific polypeptide complex 12A16. [Figure 22] The purity of the expressed polypeptide complex 12A16 (type G) analyzed by SDS-Page is shown. [Figure 23] The purity of the expressed polypeptide complex 12A16 (type G) analyzed by SEC-HPLC is shown. [Figure 24] Schematic representation of the type H structure of the 2:1:1 asymmetric multispecific polypeptide complex 12A17. [Figure 25] The purity of the expressed polypeptide complex 12A17 (type H) analyzed by SDS-Page is shown. [Figure 26] 1 shows the purity of the expressed polypeptide complex 12A17 (type H) analyzed by SEC-HPLC. [Figure 27A] 1 shows dose-response curves of polypeptide complexes 12A10, 12A14, 12A15, 12A16, and 12A17 in a first test of a reporter assay in HT29 cells; [Figure 27B] 1 shows dose-response curves for polypeptide complexes 12A6, 12A10, 12A15, 12A16, 12A18, 12A19 and 12A20 in a second test of a reporter assay in HT29 cells. [Figure 28A] 1 shows dose-response curves of polypeptide complexes 12A10, 12A14, 12A15, 12A16, and 12A17 in a first test of a reporter assay in KATO3 cells; [Figure 28B] 1 shows dose-response curves for polypeptide complexes 12A6, 12A10, 12A15, 12A16, 12A18, 12A19, and 12A20 in a second test of a reporter assay in KATO3 cells. [Figure 29A] 1 shows dose-response curves of polypeptide complexes 12A10, 12A14, 12A15, 12A16, and 12A17 in a first test of a reporter assay in LS174T cells; [Figure 29B]1 shows dose-response curves for polypeptide complexes 12A6, 12A10, 12A15, 12A16, 12A18, 12A19, and 12A20 in a second test of a reporter assay in KATO3 cells. [Figure 30] 1 shows the results of HT29 cell killing by polypeptide complexes 12A10, 12A14, 12A15, 12A16 and 12A17. [Figure 31] 1 shows the results of killing of LS174T cells by polypeptide complexes 12A10, 12A14, 12A15, 12A16 and 12A17. [Figure 32] FIG. 1 shows a diagram of the 2:1 multispecific polypeptide complex 08B1. [Figure 33] 1 shows dose-response curves of polypeptide complexes 12A6, 12A10, and 08B1 in a reporter assay in KATO3 cells. [Figure 34] 1 shows dose-response curves for polypeptide complexes 12A6 and 12A10 in a reporter assay in the presence and absence of LS174T cells. [Figure 35] 1 shows the effect of polypeptide complexes 08B1, 12A6 and 12A10 on the growth of subcutaneously implanted tumors of LS174T cells. [Figure 36] 1 shows dose-response curves for polypeptide complexes 12A6, 12A14, and 12A19 in a reporter assay in HT29 cells. [Figure 37] 1 shows dose-response curves of polypeptide complexes 12A6, 12A14, and 12A19 in a reporter assay in KATO3 cells. [Figure 38] 1 shows dose-response curves for polypeptide complexes 12A6, 12A14, and 12A19 in a reporter assay in LS174T cells. [Figure 39] 1 shows the effect of polypeptide complexes 12A6 and 12A14 on the growth of subcutaneously implanted tumors of LS174T cells. [Figure 40] Exemplary amino acid sequences of the peptide linker, CH1 region, CL region, hinge region, CH2-CH3 region, CH2-CH3 region (knob) and CH2-CH3 region (hole) are shown. DETAILED DESCRIPTION OF THE INVENTION

[0063] Some aspects of the present invention will be described below with reference to exemplary applications. It should be understood that numerous specific details, relationships, and methods are set forth to provide a thorough understanding of the present invention. However, those skilled in the art will readily recognize that the present invention can be practiced without one or more of the specific details or in other ways. The present invention is not limited to the illustrated order of acts or events, and some acts may occur in a different order and / or simultaneously with other acts or events.

[0064] Moreover, not all illustrated acts or events may be required to implement a methodology in accordance with the present invention.

[0065] I. Definitions and Abbreviations Before describing the present invention in detail, the following will be pointed out and defined.

[0066] All descriptions provided herein are merely for illustrating various embodiments of the present invention provided in this disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments are included in this specification.

[0067] All references cited in this disclosure, including patent applications, issued patents, published articles or other publications, are incorporated by reference in their entirety and are intended to provide methodologies that may be used in connection with the description provided herein. With respect to terms presented in one or more publications that are similar or identical to terms expressly defined in this disclosure, the definitions expressly provided in this disclosure of such terms shall control in all respects.

[0068] Unless expressly defined otherwise, all technical and scientific terms used in this disclosure are generally assumed to have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0069] As used herein, and throughout this disclosure, the terms "a," "an," and "the" are understood to mean "one or more" or "at least one," unless otherwise indicated. By way of example, "a polypeptide complex" means one polypeptide complex or one or more polypeptide complexes.

[0070] As used herein, the terms "about," "approximately," or "approximately" refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% from a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In specific embodiments, the term "about" or "approximately" preceding a numerical value indicates a range of ±15%, 10%, 5%, or 1% of the value.

[0071] As used herein, the terms "comprise," "comprises," "comprising," "include," "includes," "including," "contain," "contains," "containing," and "have," "has," and the like, are synonymous and used in an inclusive and open-ended manner and do not exclude additional elements, features, steps, acts, operations, and the like.

[0072] As used herein, the term "or" is used in an inclusive (rather than exclusive) sense, so that, for example, when used to connect elements of a list, the term "or" means one, some, or all of the elements in the list.

[0073] As used herein, the phrase "at least one" means one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The phrase "at least one of" a list of items is understood to refer to any combination of those items, including single members. As an example, "at least one of A, B, or C" is intended to encompass A, B, C, A and B, A and C, B and C, and A, B, and C. Conjunctions such as "at least one of X, Y, and Z" are generally understood to be used to convey that an item, term, etc., can be at least one of X, Y, or Z, unless otherwise specified, from the context. Thus, such conjunctions are generally not intended to imply that at least one of X, at least one of Y, and at least one of Z, respectively, must be present in a particular embodiment.

[0074] As used herein, reference to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "a particular embodiment," "additional embodiments," "some embodiments," "particular embodiments," or "a further embodiment," or combinations thereof, is understood to mean that the particular feature, structure, or characteristic described in connection with that particular embodiment is included in at least one embodiment of the present disclosure. Thus, the presence or appearance of such a phrase in various places throughout this disclosure do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0075] Conditional language used herein, such as "can," "could," "may," "potential," and "for example," is intended to convey that certain embodiments include certain features, elements, and / or steps, and other embodiments do not, unless expressly stated or understood otherwise within the context in which it is used. Thus, such conditional language is generally not intended to imply that features, elements, and / or steps are in any way required for one or more embodiments.

[0076] In this section, definitions of some general terms are provided. Definitions of other terms may be found in other sections of this disclosure below.

[0077] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a linear series of amino acid residues connected to each other by peptide bonds, and include proteins, polypeptides, oligopeptides, peptides, and fragments thereof. Proteins can be composed of naturally occurring amino acids and / or synthetic (e.g., modified or non-naturally occurring) amino acids. Thus, "amino acid," or "peptide residue," as used herein, refers to both naturally occurring and synthetic amino acids. The terms "polypeptide," "peptide," and "protein" include fusion proteins, including, but not limited to, fusion proteins having heterologous amino acid sequences with or without an N-terminal methionine residue, fusions with heterologous and homologous leader sequences, immunologically tagged proteins, and fusion proteins having a detectable fusion partner, such as fusion proteins that include a fluorescent protein, β-galactosidase, luciferase, etc., as a fusion partner.

[0078] As used herein, the term "amino acid" refers to a building block of a protein, peptide, polypeptide, or amino acid polymer, and further refers to naturally occurring or synthetic amino acids, as well as any amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. As used herein, naturally occurring amino acids encompass the group of naturally occurring carboxy alpha amino acids, including alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0079] As used herein, the term "domain" refers to a globular structure formed by one or more regions of one or more polypeptide chains, including, for example, beta-pleated sheets and / or peptide loops (e.g., containing 3-4 peptide loops) stabilized by intrachain disulfide bonds. Examples may include Fab domains (see below for more details). Note that in this disclosure, the two terms "domain" and "region" may be used interchangeably.

[0080] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleotide," or "polynucleotide," etc., are understood to refer to nucleotide polymers of any length, and can include both DNA and RNA, can be single- or double-stranded, and include analogs of naturally occurring polynucleotides in which one or more nucleotides of the naturally occurring nucleotides have been modified.

[0081] The term "antibody," as used herein, includes any immunoglobulin, monoclonal, polyclonal, chimeric, humanized, multispecific, bispecific, bivalent, or polyvalent antibody that binds to a specific antigen. Antibodies and related terms are described in more detail below.

[0082] In mammals, such as humans, there are five different classes / isotypes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM, corresponding to the five Ig heavy chain types α, δ, ε, γ, and μ, respectively) depending on the different types of heavy chains present in the immunoglobulin. These antibodies typically have different molecular and biological properties, functional distribution, physiological functions, and pathological effects in diseases. A particular antibody class may further include subclasses. For example, in humans, IgA may include the IgA1 and IgA2 subclasses, and IgG may include four subclasses, IgG1, IgG2, IgG3, and IgG4, respectively. With the immunoglobulin monomer as the basic functional unit, mammalian antibodies may exist as monomers (e.g., IgD, IgE, and IgG), dimers (IgA), tetramers (IgM), or pentamers (IgM). In mammals, there are two types of light chains, including kappa (κ) chains and lambda (λ) chains.

[0083] Within the basic immunoglobulin unit, a natural or naturally occurring antibody, such as an IgG, typically contains two identical heavy (H) chains and two identical light (L) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond or linkage formed between a pair of cysteine ​​residues present in each of the light and heavy chains, and the two heavy chains are further linked to each other by several disulfide bonds formed between cysteine ​​residues in each heavy chain. The tetramer thus formed is essentially Y-shaped as an antibody, with the end of each fork arm containing an identical antigen-binding site (i.e., paratope) that specifically interacts with a corresponding epitope on the antigen.

[0084] More specifically, in natural antibodies, from N- to C-terminus, each heavy chain comprises a variable region (VH, or HCVR) followed by three or four constant regions ("CH", where IgA, IgD, and IgG comprise three CH regions, CH1, CH2, and CH3, and IgE and IgM comprise four CH regions, CH1, CH2, CH3, and CH4), and each light chain comprises a variable region (VL, or LCVR) and a constant region (CL). In Y-shaped antibodies, the variable region of each light chain (i.e., VL region) aligns or associates with the variable region of its paired heavy chain (i.e., VH region) to together form the antigen-binding site of the antibody. As used herein, the term antibody is also intended to encompass heavy-chain antibodies having only heavy chains and no light chains.

[0085] The term "variable region" or "VR," as used herein, refers to the region in an antibody heavy or light chain responsible for antigen binding. In a naturally occurring antibody, the heavy chain variable region (VH or HCVR) contains three highly variable loops called "complementarity-determining regions" (CDRs), i.e., the heavy (H) chain CDRs, including HCDR1, HCDR2, and HCDR3, and the light chain variable region (VL or LCVR) contains three light (L) chain CDRs, including LCDR1, LCDR2, and LCDR3. The CDR boundaries of an antibody can be defined or identified according to the Kabat, Chothia, or Al-Lazikani definitions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196,901 (1987); Chothia, C. et al., Nature. Dec 21-28, 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are interposed between flanking stretches known as "framework regions" (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. In native antibodies, each VH and VL comprises four FRs, with the CDRs and FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. However, it should be understood that the term "variable region," as used herein, does not necessarily have to comprise all three CDRs or all four FRs, and should be understood to encompass any variant or derivative of a native variable region derived from a native antibody, so long as such variant or derivative retains antigen-binding activity.

[0086] The term "constant region" or "constant portion," as used herein, refers to a region in an antibody heavy or light chain that is not directly involved in antigen binding. It should be understood that the term "constant region" or "constant portion," as used herein, does not necessarily include the entire natural constant region of a natural antibody, but should be understood to encompass any variant or derivative of such a natural constant region or constant portion, so long as such variant or derivative retains the ability to support the stability of the antigen-binding domain or retains its intended biological function, such as secretion, placental transport, Fc receptor binding, complement binding, and other effector functions.

[0087] The term "CL region" refers to the constant region of an immunoglobulin light chain adjacent to the VL region. The CL region can span from about EU position 108 to about EU position 216 in an immunoglobulin light chain. In natural antibodies, the constant region of each light chain (i.e., the CL region) is associated with the first constant region (i.e., the CH1 region) of the paired heavy chain.

[0088] The term "CH1 region," as used herein, encompasses the first (most amino-terminal) constant region of an immunoglobulin heavy chain extending from about EU position 118 to at least about EU position 220 (and may extend, for example, to EU position 221). The CH1 region is adjacent to the VH region and is amino-terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0089] The term "hinge region," with respect to antibodies, includes the portion of a heavy chain molecule that connects the CH1 region to the CH2 region. The length of the hinge region varies depending on the defined boundaries of the CH1 region and the defined boundaries of the CH2 region. The hinge region is usually flexible, allowing the two N-terminal antigen-binding regions to move independently.

[0090] The term "CH2 region," as used herein, refers to the portion of a heavy chain immunoglobulin molecule extending, for example, from about EU position 231 to EU position 340.

[0091] The term "CH3 region," as used herein, refers to the portion of a heavy chain immunoglobulin molecule extending approximately 110 residues from the N-terminus of the CH2 domain, e.g., from about EU position 341 to EU position 445, 446, or 447. The CH3 domain typically forms the C-terminal portion of antibodies such as IgG, IgA, and IgD. However, in some immunoglobulins, such as IgE and IgM, additional domains may extend from the CH3 domain and form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ chain of IgM and the ε chain of IgE).

[0092] "Fc," as used herein, refers to the portion derived from an antibody, e.g., an IgG, and is composed primarily of the second (CH2) and third (CH3) constant regions of a first heavy chain linked to the CH2 and CH3 of a second heavy chain via one or more covalent bonds that are non-peptide bonds, e.g., disulfide bonds. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0093] "Antigen" or "Ag," as used herein, refers to a compound, composition, peptide, polypeptide, protein, or substance (e.g., a polypeptide, carbohydrate, nucleic acid, lipid, or other naturally occurring or synthetic compound) that can be specifically recognized and bound by a component of the immune system, e.g., an antibody. As used herein, the term "antigen" encompasses antigenic epitopes, e.g., fragments of an antigen that are antigenic epitopes. The terms "antigen" and "target" are used interchangeably in this disclosure.

[0094] "Fv," with respect to antibodies, refers to the smallest antibody fragment containing a complete antigen-binding site. Fv fragments consist of a single light-chain variable domain linked to a single heavy-chain variable domain. Numerous Fv designs are available, including dsFv, in which the association between the two domains is enhanced by an introduced disulfide bond, and scFv, which may be formed by linking the two domains together as a single polypeptide via a peptide linker. Fv constructs containing immunoglobulin heavy or light chain variable domains linked to the corresponding immunoglobulin heavy or light chain variable and constant domains have also been produced. Fvs have also been multimerized to form diabodies and triabodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0095] "Fab," as used herein, refers to a single antigen-binding domain derived from an antibody, which has a single heavy chain fragment associated with a single light chain fragment via one or more covalent bonds that are non-peptide bonds. In some embodiments, the single heavy chain fragment in a Fab domain comprises an HCVR and a CH1 region. In some embodiments, the single light chain fragment in a Fab domain comprises an LCVR and a CL domain. In some embodiments, the CH1 region is associated with the HCVR by a covalent bond, such as a disulfide bond. In a native antibody, the Fab domain substantially corresponds to one arm of the antibody and typically retains the ability to recognize and bind to its corresponding antigen.

[0096] As used herein, the term "multispecific antibody" refers to an artificial or engineered antibody that can simultaneously bind to at least two different epitopes. Bispecific antibodies are essentially one type of multispecific antibody. In addition, other multispecific antibodies may include trispecific antibodies, which have three different antigen-binding specificities, and tetraspecific antibodies, which have four different antigen-binding specificities, etc.

[0097] As used herein, the term "bispecific antibody" refers to an antibody containing two physically separable antigen-binding domains with different antigen specificities. Typically, bispecific antibodies are artificial antibodies that have fragments derived from two different monoclonal antibodies and can bind to two different epitopes. The two epitopes may be on the same antigen or on two different antigens. This is in contrast to naturally occurring antibodies, which have two structurally identical, physically separable antigen-binding portions and therefore have the same antigen specificity.

[0098] Throughout this disclosure, numbers indicating amino acid residue positions in antibody constant regions, e.g., the heavy chain constant region 1 (CH1) and light chain constant region (CL) of the constant portion, are based on EU numbering as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). As indicated, some positions use the IMGT numbering or Kabat index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27: 55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015). These numberings are also available from the IMGT scientific charts accessible from the International ImMunoGeneTics Information System website.

[0099] The term "chimeric," as used herein, refers to an antibody or antigen-binding fragment in which a portion of the heavy and / or light chain is derived from one species and the remaining portion of the heavy and / or light chain is derived from another species. In an illustrative example, a chimeric antibody can contain a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0100] The term "valent," as used herein, refers to the presence of a particular number of antigen-binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen-binding fragment that has only a single antigen-binding site, and the term "multivalent" refers to an antibody or antigen-binding fragment that has multiple antigen-binding sites. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to two, four, and six binding sites, respectively, in an antigen-binding molecule. In some embodiments, an antibody or antigen-binding fragment thereof is bivalent.

[0101] Antibodies and fragments thereof according to the present disclosure include bispecific and multispecific antibodies and fragments thereof. Bispecific or multispecific antibodies may resemble a single antibody (or antibody fragment) but have two or more different antigen-binding sites. Bispecific antibodies may have binding specificities for at least two different epitopes. Bispecific antibodies and fragments may also be in the form of heteroantibodies. Heteroantibodies are two or more antibodies or antibody-binding fragments (e.g., Fabs) linked together, each antibody or fragment having a different specificity.

[0102] The term "specific binding of an antibody" or "antigen-specific antibody" in the context of antibody properties refers to the ability of an antibody to preferentially bind to a particular antigen present in a mixture of different antigens. In certain embodiments, the specific binding interaction distinguishes between desired and undesired antigens (or "target" and "non-target" antigens) in a sample, in some embodiments by about 10-100 fold or more (e.g., about 1000 fold or more than 10,000 fold). In certain embodiments, the affinity of an antibody and an antigen when specifically bound in an antibody-antigen complex is greater than 10 -6 Under M, 10 -7 Under M, 10 -8 Under M, 10 -9 Under M, 10 -9 Under M, 10 -11 Less than M or about 10 -12 K below M D It is characterized by a dissociation constant.

[0103] As used herein, the term "vector" refers to a vehicle into which a genetic element is operably inserted to express the genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. A vector can be used to transform, transduce, or transfect a host cell to express the genetic element carried by the vector within the host cell. Examples of vectors include plasmids, phagemids, cosmids, and artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, a vector can contain an origin of replication. A vector can also contain materials that aid in cell entry, including, but not limited to, viral particles, liposomes, or protein coatings. The vector may be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0104] The term "host cell," as used herein, refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced.

[0105] Antibody conjugates are also provided. The terms "conjugated" and "joining" generally refer to a covalent or non-covalent chemical linkage that brings one molecule into close proximity with a second molecule. A conjugate includes any antibody of the present disclosure and an agent. The agent may be selected from a therapeutic agent, an imaging agent, a labeling agent, or an agent useful for therapeutic and / or labeling purposes.

[0106] As used herein, the term "biological sample" or "sample" refers to a biological composition obtained or derived from a subject of interest that contains cellular and / or molecular entities that can be characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological properties. Biological samples include, but are not limited to, cells, tissues, organs, and / or biological fluids of a subject obtained by any method known to those of skill in the art. In some embodiments, the biological sample is a bodily fluid sample. In some embodiments, the bodily fluid sample is whole blood, plasma, serum, mucus (including nasal secretions and sputum), peritoneal fluid, pleural fluid, pleural effusion, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, intraperitoneal fluid, ascites, or pericardial fluid. In some embodiments, the biological sample is tissue or cells obtained from the heart, liver, spleen, lung, kidney, skin, or blood vessels of a subject.

[0107] II. Multispecific Polypeptide Complexes In one aspect, the present disclosure provides novel multispecific polypeptide complexes that comprise multiple domains capable of binding to one or more targets.

[0108] Multispecific antibodies have attracted considerable interest in the field of antibody engineering and are expected to have a wide range of applications, including antitumor immunotherapy.

[0109] A challenge with multispecific antibodies relates to fine-tuning or differentiating their binding affinities to different target antigens without significantly compromising manufacturability and / or purity. To increase affinity for a desired target, an additional Fab domain targeting that target may be fused to one of the antibody's heavy or light chains. Despite the advantages of tailoring the binding affinity of multispecific antibodies to different targets offered by such modifications, several insurmountable challenges remain in their expression and production. For example, light chain (LC)-heavy chain (HC) bond instability and LC mispairing can occur during assembly of such multispecific antibodies. Because LC mispairing products (i.e., impurities) can vary in molecular weight and physicochemical properties, similar to or indistinguishable from the correctly paired target product, separating or purifying the target product from these impurities can be difficult. This can result in reduced purity and yield of the expressed target product, potentially compromising its therapeutic efficacy and safety profile.

[0110] Another challenge relates to the complexity of designing multispecific antibodies so that different binding domains are positioned and oriented appropriately to enable proper binding to each target. For example, to achieve effective antitumor function, multispecific antibodies are usually configured to target at least one tumor antigen and at least one immunostimulatory target, sometimes at least two immunostimulatory targets. It is desirable to have a configuration that can avoid a decrease in the binding affinity and specificity of the multispecific antibody to different tumor antigens and enable targeted activation of effector cells.

[0111] The present disclosure provides, for example, a multispecific antibody platform with a novel 2:2:2 or 2:1:1 structure, which can not only meet the requirements of effective biological function but also has a much lower production cost than conventional multispecific antibodies. This novel multispecific antibody structure is advantageous because it not only adopts a DICAD structure, which is more stable, has a longer half-life, and is easier for downstream purification, but also contains one or more additional Fab domains linked at appropriate positions, which can adjust the antigen affinity and spatial distribution of antigen-binding fragments to meet various requirements.

[0112] The multispecific polypeptide complexes provided herein comprise two moieties, each comprising a dimerization domain and an antigen-binding domain, wherein the two moieties are dimerized by interaction between the dimerization domains contained within each moiety.

[0113] In certain embodiments, the multispecific polypeptide complexes provided herein comprise (a) a first dimerization domain, and (b) a first antigen-binding domain comprising a DICAD domain and a first Fv domain.

[0114] As used herein, the term "antigen-binding domain" refers to a portion of a multispecific polypeptide complex that is covalently linked to a dimerization domain and is capable of binding to at least one specific target. In some embodiments, the antigen-binding domain comprises two or more polypeptide chains, one of which is covalently linked to the dimerization domain. In certain embodiments, one of the C-terminus of the antigen-binding domain is operably linked to the N-terminus of the dimerization domain.

[0115] In certain embodiments, the antigen-binding domain may comprise one target-binding domain, or alternatively, may comprise a combination or fusion of two or more target-binding domains. In certain embodiments, the antigen-binding domain comprises at least one of a DICAD domain and an Fv domain. In some embodiments, the antigen-binding domain comprises only one targeting domain, which comprises an Fv domain or a DICAD domain. In some embodiments, the antigen-binding domain comprises both a DICAD domain and an Fv domain.

[0116] Multispecific antibodies may be symmetrical and have two identical antigen-binding domains. In certain embodiments, each identical antigen-binding domain has three target-binding domains, resulting in a 2:2:2 structure, where each "2" represents two identical target-binding domains.

[0117] Alternatively, the multispecific antibody may be asymmetric and have two different antigen-binding domains: a first antigen-binding domain and a second antigen-binding domain. In a specific embodiment, the first antigen-binding domain has three target-binding domains, and the second antigen-binding domain has one target-binding domain that is identical to one of the three target-binding domains in the first antigen-binding domain. This results in a 2:1:1 structure, where each "2" represents two identical target-binding domains.

[0118] In certain embodiments, the first antigen-binding domain comprises a DICAD domain and / or an Fv domain. In some embodiments, the second antigen-binding domain comprises only one targeting domain that is an Fv domain or a DICAD domain.

[0119] DICAD Domain As used herein, the term "DICAD" is an abbreviation for "disulfide-charge-modulated diabody" and refers to a diabody structure in which covalent bonds and electrostatic charges are introduced at the VH-VL interface, as disclosed, for example, in PCT Application WO2019 / 120245, which is incorporated herein in its entirety. This DICAD structure has the advantages of (1) retaining the binding activity, affinity, potency, and other properties of each individual targeting domain, (a) being more stable and less prone to aggregation than other diabodies, and (3) being easier to express and purify.

[0120] In some embodiments, the DICAD domain comprises (i) a first polypeptide fragment comprising a first heavy chain variable domain (VH1) linked to a second light chain variable domain (VL2), and (ii) a second polypeptide fragment comprising a second heavy chain variable domain (VH2) linked to the first light chain variable domain (VL1), where VL1 and VH1 associate to form a first domain capable of binding to a first target, and VL2 and VH2 associate to form a second domain capable of binding to a second target.

[0121] In some embodiments, the C-terminus of the VL1 is linked to the N-terminus of the VH2 and the C-terminus of the VL2 is covalently linked to the N-terminus of the VH1. In some embodiments, the C-terminus of the VH1 is linked to the N-terminus of the VL2 and the C-terminus of the VH2 is linked to the N-terminus of the VL1.

[0122] In some embodiments, VH2 and VL1 and / or VH1 and VL2 are directly covalently linked or indirectly covalently linked, for example, via a linker, e.g., a peptide linker. The term "peptide linker," as used herein, refers to any suitable polypeptide capable of linking two entities to form a single molecule or maintaining a sufficiently close association of the two entities without substantially interfering with the biological activity of the two entities. The linker may be composed of amino acid residues linked together by peptide bonds and may optionally further comprise one or more unnatural amino acids. Any suitable polypeptide may be used as a linker. In some embodiments, the polypeptide linker may be composed primarily of sterically unhindered amino acids, such as glycine and alanine. In some embodiments, the linker is polyglycine, polyalanine, a combination of glycine and alanine (e.g., poly(Gly-Ala)), or a combination of glycine and serine (e.g., poly(Gly-Ser)). In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 95 or 96.

[0123] In some embodiments, VL1 is linked to VH2 via a first peptide linker, where VL2 is linked to VH1 via a second peptide linker. In some embodiments, the first peptide linker and the second peptide linker each independently comprise 5 to 9 amino acids.

[0124] a) Disulfide bonds introduced into the VH-VL domain In some embodiments, non-natural covalent bonds may be introduced at the VH1-VL1 interface or the VH2-VL2 interface, and / or electrostatic interactions may be introduced.

[0125] In some embodiments, one of the first domain (formed by the association of VH1 and VL1) and the second domain (formed by the association of VH2 and VL2) comprises a first non-naturally occurring covalent bond formed between two amino acid residues of a first pair.

[0126] In certain of these embodiments, the other of the first domain and the second domain does not include a non-naturally occurring covalent bond. Alternatively, the other of the first domain and the second domain includes a second non-naturally occurring covalent bond that is different from the first non-naturally occurring covalent bond. For example, the second non-naturally occurring covalent bond is formed between a pair of two amino acid residues that is different from the pair of amino acid residues corresponding to the first pair of two amino acid residues.

[0127] In some embodiments, the first non-natural covalent bond can be a non-natural disulfide bond. In such embodiments, at least one of the first and second domains is a disulfide-stabilized Fv. Analysis of antibody crystal structures has revealed that cysteine ​​mutations can be introduced into a relatively conserved sequence at the VL-VH interface to form a disulfide bond between the VL and VH, thereby covalently connecting them. The covalent bond between the VL and VH significantly improved the stability of the antibody. The first dsFv (disulfide Fv) was constructed by introducing a disulfide bond into the VH-VL interface through covalent interactions between cysteine ​​residues in the CDRs of each fragment (see Glockshuber, R., Malia, M., Pfitzinger, I., and Pluckthun, A. A comparison of strategies to stabilize immunoglobulin Fv-fragments. (1990) Biochemistry, 29, 1362-1367). Although this method does not affect the activity of the antibody, the "customized" design requires detailed structural information about the CDRs of the original antibody to avoid interference with the antigen recognition / binding ability of the CDRs, making this method difficult to use as a universal solution for constructing various antibodies. To ensure the wide application of this method, it is important that only amino acids at selected sites in the conserved FRs are involved in the construction of dsFvs.

[0128] Since 1993, several paired sites for VH-VL covalent bond formation have been discovered, such as VH44-VL100, VH105-VL43, VH100b-VL49, VH100-VL50, and VH101-VL46 based on the Kabat index (Reiter, Y., Brinkmann, RJ, Kreitman, RJ, et al. Stabilization of the Fv Fragments in Recombinant Immunotoxins by Disulfide Bonds Engineered Into Conserved Framework Regions. (1994) Biochemistry, 33, 5451-5459; Jung, SH, Pastan, I. and Lee, B. Design of interchain disulfide bonds in the framework region of the Fv fragment of the monoclonal antibody B3. (1994) Proteins, Struc. Func. Genet., 19, (See also Glockshuber, R., Malia, M., Pfitzinger, I., and Plückthun, A. A comparison of strategies to stabilize immunoglobulin Fv-fragments. (1990) Biochemistry, 29, 1362-1367; and Zhu, Z., Presta, LG, Zapata, G., and Carter, P. Remodeling domain interfaces to enhance heterodimer formation. (1997) Prot. Sci., 6, 781-788.) Among these, VH44-VL100 and VH105-VL43 are superior to other sites in many aspects, such as protein expression level, monolayer rate, Tm, and affinity, to different degrees, and are therefore more widely used.

[0129] In some embodiments, the first non-native disulfide bond is formed between two non-native cysteine ​​residues, in some embodiments, at position 44 in VH and position 100 in VL, or at position 105 in VH and position 43 in VL, or at position 100 in VH and position 49 in VL, or at position 100 in VH and position 150 in VL, where numbering is according to the Kabat index.

[0130] In some embodiments, the VL1 and VH1 of the DICAD domain are associated by a first non-natural disulfide bond. In some embodiments, the first non-natural disulfide bond is formed between two non-natural cysteine ​​residues in VL1 and VH1, respectively. In some embodiments, the two non-natural cysteine ​​residues are located in the framework regions (FR) of VL1 and the FR of VH1, respectively. In some embodiments, the two non-natural cysteine ​​residues are located at amino acid residues in FR2 of VL1 and FR4 of VH1, respectively.

[0131] In some embodiments, the two non-native cysteine ​​residues are at position 44 in VH1 and position 100 in VL1, or at position 105 in VH1 and position 43 in VL1, or at position 100 in VH1 and position 49 in VL1, or at position 100 in VH1 and position 150 in VL1. Unless otherwise specified, all position numbering in this disclosure is according to the Kabat index. In some embodiments, the non-native covalent bond is formed between the amino acid residues introduced at position 44 in VH1 and position 100 in VL1, or between the cysteine ​​residues at position 105 in VH1 and position 43 in VL1. In some embodiments, the non-native covalent bond is formed between the cysteine ​​residues at position 44 in VH1 and position 100 in VL1. In some embodiments, the two non-native cysteine ​​residues are 100C in VL1 and 44C in VH1.

[0132] In some embodiments, the VL2 and VH2 of the DICAD domain do not contain any introduced non-native disulfide bonds.

[0133] In some other embodiments, VL2 and VH2 of the DICAD domain are associated by a first non-native disulfide bond. In some embodiments, the first non-native disulfide bond is formed between two non-native cysteine ​​residues in VL2 and VH2, respectively. In some embodiments, the two non-native cysteine ​​residues are in the framework regions (FR) of VL2 and the FR of VH2, respectively. In some embodiments, the two non-native cysteine ​​residues are in amino acid residues in FR2 of VL2 and FR4 of VH2, respectively. In some embodiments, the two non-native cysteine ​​residues are at positions 44 in VH2 and 100 in VL2, or at positions 105 in VH2 and 43 in VL2, or at positions 100 in VH2 and 49 in VL2, or at positions 100 in VH2 and 150 in VL2. Unless otherwise specified, all position numbering in this disclosure is according to the Kabat index. In some embodiments, the non-naturally occurring covalent bond is formed between the introduced amino acid residues at position 44 in VH2 and position 100 in VL2, or between the cysteine ​​residues at position 105 in VH2 and position 43 in VL2. In some embodiments, the non-naturally occurring covalent bond is formed between the cysteine ​​residues at position 44 in VH2 and position 100 in VL2. In some embodiments, the two non-naturally occurring cysteine ​​residues are 100C in VL2 and 44C in VH2. In some of these embodiments, VL1 and VH1 of the DICAD domain do not comprise any introduced non-naturally occurring disulfide bond.

[0134] b) Substitution with a charged amino acid In some embodiments, one of the first and second domains comprises a first non-natural pair of oppositely charged amino acid residues that introduces a non-natural electrostatic interaction to promote or support pairing between the VH and VL in such domains. In some embodiments, the non-naturally charged residue in the VL is in a FR (e.g., FR2). In some embodiments, the non-naturally charged residue in the VH is in a FR (e.g., FR2).

[0135] In some embodiments, the other of the first domain and the second domain does not comprise a non-natural pair of oppositely charged amino acid residues that introduces a non-natural electrostatic interaction.

[0136] In some embodiments, the first domain comprising VH1 and VL1 comprises a first non-natural pair of oppositely charged amino acid residues, wherein: (a) two oppositely charged residues at positions 38 in VL1 and 39 in VH1, respectively; (b) two oppositely charged residues at position 40 in VL1 and position 39 in VH1, respectively; (c) two oppositely charged residues are at position 37 in VL1 and position 39 in VH1, respectively; or (d) two oppositely charged residues at positions 37 in VL1 and 39 in VH1, respectively; Here, the numbering follows the Kabat index.

[0137] In some other embodiments, the second domain comprising VH2 and VL2 comprises a first non-natural pair of oppositely charged amino acid residues, wherein: (a) two oppositely charged residues at positions 38 in VL2 and 39 in VH2, respectively; (b) two oppositely charged residues at position 40 in VL2 and position 39 in VH2, respectively; (c) two oppositely charged residues at positions 37 in VL2 and 39 in VH2, respectively; (d) two oppositely charged residues at position 37 in VL2 and position 39 in VH2, respectively; Here, the numbering follows the Kabat index.

[0138] W103 of VH and P44 of VL are both located in the side chains of the hydrophobic core and are closely spaced. The electrostatic interaction between W103 and P44 was also examined during the development of DICAD and was found to be excellent.

[0139] In some embodiments, the other of the first and second domains comprises a second pair of two oppositely charged residues that pair between the VH and VL in such domain, provided that cross-pairing between the VH in one domain and the VL in the other domain is prevented, e.g., by electrostatic repulsion.

[0140] In some embodiments, the DICAD domains provided herein comprise a first pair of two oppositely charged residues that promote the electrostatic interaction between VL1 and VH1, and a second pair of two oppositely charged residues that promote the electrostatic interaction between VL2 and VH2, whereby pairing between VH1 and VL2 and VH2 and VL1 is prevented, e.g., by electrostatic repulsion. For example, the introduced charged residues in VL2 and VH1 are similarly charged residues and / or the introduced charged residues in VH2 and VL1 are similarly charged residues, thereby preventing mispairing between VL2 and VH1 or VL1 and VH2.

[0141] In some embodiments, a first pair of two oppositely charged residues is introduced to replace Q38 in VL1 and Q39 in VH1, respectively, where numbering is according to the Kabat index. In some embodiments, a second pair of two oppositely charged residues is introduced to replace Q38 in VL2 and Q39 in VH2, respectively, where numbering is according to the Kabat index. In such embodiments, the introduced charged residues in VL2 and VH1 are similarly charged residues and / or the introduced charged residues in VH2 and VL1 are similarly charged residues, thereby preventing mispairing between VL2 and VH1 or VL1 and VH2.

[0142] In some embodiments, the two oppositely charged residues consist of a positively charged residue and a negatively charged residue.

[0143] In certain embodiments, the first pair of two oppositely charged residues comprises a negatively charged residue in VL1 and a positively charged residue in VH1, hi certain embodiments, the second pair of two oppositely charged residues comprises a positively charged residue in VL2 and a negatively charged residue in VH2.

[0144] In some embodiments, the first pair of two oppositely charged residues comprises Q38D in VL1 and Q39K in VH1, respectively, where numbering is according to the Kabat index, hi some embodiments, the second pair of two oppositely charged residues comprises Q39D in VH2 and Q40K in VL2, respectively, where numbering is according to the Kabat index.

[0145] Strategies for enhancing the stable binding of corresponding VH and VL in bispecific antibodies by introducing charged amino acids are known in the art. Tan et al. successfully influenced the stability of scFv (single-chain FV variant) by adjusting amino acids at the VH-VL interface based on electrostatic properties (see Philip H. Tan, Brenda M. Sandmaier, Patrick S. Stayton. Contributions of a Highly Conserved VH VL Hydrogen Bonding Interaction to scFv Folding Stability and Refolding Efficiency. Biophys J. 1998 Sep; 75(3): 1473-1482). Subsequently, Igawa et al. adapted this method to modify scDb. To improve the homogeneity of the product, two pairs of Q39-Q38 in the 4V fragment were replaced with amino acids bearing appropriate electrostatic charges to promote or inhibit specific isoforms (see Igawa T, Tsunoda H, Kikuchi Y, et al. VH / VL interface engineering to promote selective expression and inhibit conformational isomerization of thrombopoietin receptor agonist single-chain diabody. Protein Eng Des Sel. 2010 Aug;23(8):667-77 and WO2006106905A1). Gunasekaran et al. at Amgen further investigated this method and incorporated it into the modification of antibody Fab arms.Modification of the electrostatic steering at the CH1-CL interface, combined with modification of the VH-VL at positions 38-39, promoted specific interactions between CH1-VH and CL-VL (see Gunasekaran K, Pentony M, Shen M, et al. Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. J Biol Chem. 2010 Jun 18;285(25):19637-46 and Liu Z, Leng EC2, Gunasekaran K3, et al. A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism. J Biol Chem. 2015 Mar 20;290(12):7535-62). These strategies enable each HC of the bispecific antibody to interact with the corresponding LC, thereby enabling the bispecific antibody to simultaneously bind to two antigens.

[0146] Methods for introducing positively or negatively charged amino acids into antibodies are also known in the art.

[0147] In some embodiments, the DICAD domains provided herein have modified electrostatic steering of selected regions in addition to the introduction of non-native disulfide bonds, thereby successfully minimizing unwanted non-specific interactions.

[0148] In some embodiments, one of the first domain and the second domain that comprises the first non-natural covalent bond further comprises a first non-natural pair of oppositely charged amino acid residues that introduces a non-natural electrostatic interaction, thereby promoting or supporting pairing between the VH and VL in such domains.

[0149] In some embodiments, the first domain comprising VH1 and VL1 comprises a first non-natural disulfide bond formed between two non-natural cysteine ​​residues and further comprises a first non-natural pair of oppositely charged amino acid residues. In some embodiments, the second domain comprising VH2 and VL2 comprises a second pair of two oppositely charged residues, wherein pairing between VH1 and VL2 and pairing between VH2 and VL1 are prevented, e.g., by electrostatic repulsion. For example, the introduced charged residues in VL2 and VH1 are similarly charged residues and / or the introduced charged residues in VH2 and VL1 are similarly charged residues, thereby preventing mispairing between VL2 and VH1 or VL1 and VH2.

[0150] Alternatively, in some other embodiments, the second domain comprising VH2 and VL2 comprises a first non-naturally occurring disulfide bond formed between two non-naturally occurring cysteine ​​residues and further comprises a first non-naturally occurring pair of oppositely charged amino acid residues. In some embodiments, the first domain comprising VH1 and VL1 comprises a second pair of two oppositely charged residues, wherein pairing between VH2 and VL1 and pairing between VH1 and VL2 are prevented, e.g., by electrostatic repulsion. For example, the introduced charged residues in VL2 and VH1 are similarly charged residues and / or the introduced charged residues in VH2 and VL1 are similarly charged residues, thereby preventing mispairing between VL2 and VH1 or VL1 and VH2.

[0151] In some embodiments, VL1 comprises a non-naturally occurring cysteine ​​residue at position 100 and VH1 comprises a non-naturally occurring cysteine ​​residue at position 44 to form a non-naturally occurring disulfide bond, while VL1 and VH1 further comprise two oppositely charged residues at positions 38 in VL1 and 39 in VH1, respectively, where numbering is according to the Kabat index.

[0152] In some embodiments, VL2 comprises a non-naturally occurring cysteine ​​residue at position 100 and VH2 comprises a non-naturally occurring cysteine ​​residue at position 44 to form a non-naturally occurring disulfide bond, while VL2 and VH2 further comprise two oppositely charged residues at positions 38 in VL2 and 39 in VH2, respectively, where numbering is according to the Kabat index.

[0153] Modifications that introduce electrostatic interactions can improve the stability and homogeneity of polypeptide complexes, help remove bottlenecks in downstream development processes, and increase the probability of successful development of multispecific polypeptide complexes.

[0154] Fv domain As used herein, the term "Fv domain" refers to a domain comprising an antibody heavy chain variable region and an antibody light chain variable region that associate with each other to form a domain capable of binding to an antigen. As used herein, the term Fv domain should be understood to broadly encompass a domain formed by VH and VL, and a Fab domain formed by VH and heavy chain constant region 1 (CH1), and VL and light chain constant region (CL).

[0155] In some embodiments, the Fv domain comprises a third polypeptide fragment comprising a third heavy chain variable domain (VH3), and a fourth polypeptide fragment comprising a third light chain variable domain (VL3), wherein VH3 and VL3 associate to form a third domain capable of binding to a third target.

[0156] In some embodiments, the Fv domain further comprises a scaffold domain fused to VH3 and VL3, respectively. For example, a scaffold region may be fused to VH3, and a paired scaffold region may be fused to VL3. Binding of the scaffold region to the paired scaffold region allows association of the VH3 and VL3 regions, thereby forming an Fv domain capable of binding to a target antigen. Any suitable binding partner may be used as the scaffold region. In certain embodiments, the scaffold region may be selected from an antibody CH1 domain and a CL domain, a paired TCR constant region (TCRα / TCRβ, TCRγ / TCRδ), a PRD (proline-rich domain) and an SH3 domain, or obscurin and titin.

[0157] In some embodiments, the Fv domain comprises a CH1 domain covalently linked to one of VH3 and VL3, and a CL domain covalently linked to the other of VH3 and VL3, hi certain embodiments, the Fv domain comprises a third polypeptide fragment comprising, from N- to C-terminus, the VH3 domain and the CH1 domain, and a fourth polypeptide fragment comprising, from N- to C-terminus, the VL3 domain and the CL domain.

[0158] In some embodiments, the third polypeptide fragment further comprises an antibody heavy chain constant region (CH1) operably linked to the C-terminus of the VH3 domain, and / or the fourth polypeptide fragment further comprises an antibody light chain constant region (CL) operably linked to the C-terminus of the VL3 domain.

[0159] Antigen-binding domain structure The DICAD domain and the Fv domain may be linked to the multispecific polypeptide complex in any suitable manner, as long as these domains are able to substantially retain their antigen-binding activity.

[0160] In some embodiments, the multispecific polypeptide complexes disclosed herein comprise a first antigen-binding domain comprising a DICAD domain and a first Fv domain.

[0161] The DICAD domains provided herein comprise a first polypeptide fragment comprising a first heavy chain variable domain (VH1) linked to a second light chain variable domain (VL2), and a second polypeptide fragment comprising a second heavy chain variable domain (VH2) linked to the first light chain variable domain (VL1), where VL1 and VH1 associate to form a first domain capable of binding to a first target, and VL2 and VH2 associate to form a second domain capable of binding to a second target.

[0162] The first Fv domain provided herein comprises a third polypeptide fragment comprising a third heavy chain variable domain (VH3), and a fourth polypeptide fragment comprising a third light chain variable domain (VL3), wherein VH3 and VL3 associate to form a third domain capable of binding to a third target.

[0163] In some embodiments, one of the N-terminuses of the DICAD domain is operably linked to one of the C-terminuses of the first Fv domain. In some embodiments, one of the N-terminuses of the DICAD domain is operably linked to the C-terminus of the third polypeptide fragment of the Fv domain. In some embodiments, the N-terminus of the second polypeptide fragment of the DICAD domain is operably linked to the C-terminus of the third polypeptide fragment of the first Fv domain. In some embodiments, the N-terminus of the first polypeptide fragment of the DICAD domain is operably linked to the C-terminus of the third polypeptide fragment of the first Fv domain. In some embodiments, one of the N-terminus of the DICAD domain is operably linked to the C-terminus of the fourth polypeptide fragment of the first Fv domain. In some embodiments, the N-terminus of the first polypeptide fragment of the DICAD domain is operably linked to the C-terminus of the fourth polypeptide fragment of the first Fv domain.

[0164] In certain embodiments, one of the C-termini of the first antigen-binding domain is operably linked to the N-terminus of the first dimerization domain. In certain embodiments, the first dimerization domain comprises an IgG CH3 domain. In certain embodiments, the first dimerization domain further comprises an IgG CH2 domain and / or hinge region.

[0165] In a specific embodiment, as shown in the left part of the structure in Figure 1 and the left part of the structure in Figure 15, the C-terminus of the third polypeptide fragment of the first Fv domain is covalently linked to the N-terminus of the second polypeptide fragment of the DICAD domain, and the C-terminus of the first polypeptide fragment of the DICAD domain is covalently linked to the N-terminus of the first dimerization domain.

[0166] In a specific embodiment, as shown in the left part of the structure in Figure 10, the C-terminus of the fourth polypeptide fragment of the first Fv domain is covalently linked to the N-terminus of the first polypeptide fragment of the DICAD domain, and the C-terminus of the third polypeptide fragment of the first Fv domain is covalently linked to the N-terminus of the first dimerization domain.

[0167] In a specific embodiment, as shown in the left part of the structure in Figure 18, the C-terminus of the third polypeptide fragment of the first Fv domain is covalently linked to the N-terminus of the first polypeptide fragment of the DICAD domain, and the C-terminus of the first polypeptide fragment of the DICAD domain is covalently linked to the N-terminus of the first dimerization domain.

[0168] In some embodiments, one of the C-termini of the DICAD domain is operably linked to one of the N-termini of the Fv domain. In some embodiments, the C-terminus of the second polypeptide fragment of the DICAD domain is operably linked to the N-terminus of the third polypeptide fragment of the first Fv domain. In some embodiments, the C-terminus of the first polypeptide fragment of the DICAD domain is operably linked to the N-terminus of the fourth polypeptide fragment of the first Fv domain.

[0169] In a specific embodiment, as shown in the left part of the structure in Figure 4, the C-terminus of the second polypeptide fragment of the DICAD domain is covalently linked to the N-terminus of the third polypeptide fragment of the first Fv domain, and the C-terminus of the first polypeptide fragment of the DICAD domain is covalently linked to the N-terminus of the first dimerization domain.

[0170] In a specific embodiment, as shown in the left part of the structure in Figure 7, the C-terminus of the first polypeptide fragment of the DICAD domain is covalently linked to the N-terminus of the fourth polypeptide fragment of the first Fv domain, and the C-terminus of the third polypeptide fragment of the first Fv domain is covalently linked to the N-terminus of the first dimerization domain.

[0171] In a specific embodiment, as shown in the left part of the structure in Figure 21, the C-terminus of the second polypeptide fragment of the DICAD domain is covalently linked to the N-terminus of the third polypeptide fragment of the first Fv domain, and the C-terminus of the first polypeptide fragment of the DICAD domain is covalently linked to the N-terminus of the first dimerization domain.

[0172] Overall structure of the multispecific polypeptide complex In some embodiments, the multispecific polypeptide complex further comprises a second antigen-binding domain and a second dimerization domain, wherein the second dimerization domain associates with the first dimerization domain to form a dimer, and wherein one of the C-termini of the second antigen-binding domain is operably linked to the N-terminus of the second dimerization domain.

[0173] In some embodiments, the overall structure of the multispecific polypeptide complex is symmetric or asymmetric.

[0174] In some embodiments, the first and second antigen-binding domains are identical, and / or the first and second dimerization domains are identical. Examples of such symmetric structures are shown in Figures 1, 4, 7, and 10.

[0175] In some embodiments, the second antigen-binding domain is different from the first antigen-binding domain.

[0176] In some embodiments, the second dimerization domain is different from the first dimerization domain and associates with the first dimerization domain to form a heterodimer.

[0177] In some embodiments, the second antigen-binding domain comprises a second Fv domain. In certain embodiments, the first and second Fv domains are identical. Examples of such asymmetric structures are shown in Figures 15, 18, and 21.

[0178] In some embodiments, the second Fv domain provided herein comprises a fifth polypeptide fragment identical to a third polypeptide fragment comprising a third heavy chain variable domain (VH3), and a sixth polypeptide fragment identical to a fourth polypeptide fragment comprising a third light chain variable domain (VL3), wherein VH3 and VL3 associate to form a third domain capable of binding to a third target.

[0179] In some embodiments, the C-terminus of the heavy chain of the second Fv domain is operably linked to the N-terminus of the second dimerization domain.

[0180] Dimerization domain In some embodiments, the multispecific polypeptide complexes disclosed herein comprise a first dimerization domain and a second dimerization domain that are associated to form a dimer.

[0181] In some embodiments, the first dimerization domain and the second dimerization domain comprise a CH3 domain of an IgG. In some embodiments, the first dimerization domain and the second dimerization domain further comprise a CH2 domain and / or a hinge region. In some embodiments, the first dimerization domain and / or the first dimerization domain comprises an Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0182] In some embodiments, the multispecific polypeptide complex has a symmetric structure and the first dimerization domain and the second dimerization domain are identical.

[0183] In some embodiments, the first dimerization domain and the second dimerization domain have different amino acid sequences and have at least one arrangement that promotes heterodimerization of the first dimerization domain and the second dimerization domain, and in some embodiments, the first dimerization domain and / or the second dimerization domain further comprise one or more mutations that promote heterodimerization.

[0184] In some embodiments, both the first dimerization domain and the second dimerization domain comprise an IgG CH3 domain.

[0185] In some embodiments, the first dimerization domain comprises a first mutation and the second dimerization domain comprises a second mutation.

[0186] In certain embodiments, the polypeptide complexes disclosed herein comprise one or more amino acid substitutions at the interface of the CH3 domain to promote and / or enhance heterodimerization. These modifications include the introduction of a protrusion into the first CH3 domain and a cavity into the second CH3 domain, where the protrusion may be positioned within the cavity to enhance the interaction of the first and second dimerization domains to form a heterodimer or complex (also called a knob-into-hole structure). Methods for generating antibodies with these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0187] In some embodiments, "knobs" are created by replacing one or more small amino acid side chains from the interface of the first antibody molecule with larger side chains (e.g., tyrosine or tryptophan). Compensatory "holes" of identical or similar size to the large side chains are created on the interface of the second antibody molecule by replacing the large amino acid side chains with smaller side chains (e.g., alanine or threonine).

[0188] In some embodiments, the first dimerization domain comprises a first CH3 domain comprising a first mutation, and / or the second dimerization domain comprises a second CH3 domain comprising a second mutation, wherein a) one of the first and second mutations comprises T389W or S375C, and the other of the first and second mutations comprises Y370C, T389S, L391A, or Y438V; b) one of the first and second mutations comprises D427K or E377K, and the other of the first and second mutations comprises K420D or K440D; or c) one of the first and second mutations comprises E377K, E378K, or D427K, and the other of the first and second mutations comprises K393E, K440D, or comprises K470E; d) one of the first and second mutations comprises S387H or F436A and the other of the first and second mutations comprises Y370T or T422F; e) one of the first and second mutations comprises S387H or T422F and the other of the first and second mutations comprises Y422T or F436A; f) one of the first and second mutations comprises K393D or K440D and the other of the first and second mutations comprises E378K or D427K; or g) one of the first and second mutations comprises L372D or L391E and the other of the first and second mutations comprises L372K or T389K, wherein numbering is according to the Kabat index.

[0189] In some embodiments, the first dimerization domain comprises a first mutation and / or the second dimerization domain comprises a second mutation, wherein a) one of the first and second mutations comprises T389S / L391A / Y438V and the other of the first and second mutations comprises T389W; b) one of the first and second mutations comprises S375C / T389W and the other of the first and second mutations comprises Y370C / T389S / L391A / Y438V; c) one of the first and second mutations comprises T389Y and the other of the first and second mutations comprises Y438T; d) the first mutation a) one of the first and second mutations comprises T389W and the other of the first and second mutations comprises Y438A; b) one of the first and second mutations comprises T422W and the other of the first and second mutations comprises F436A; c) one of the first and second mutations comprises T389Y / F436A and the other of the first and second mutations comprises T422W / Y438T; d) one of the first and second mutations comprises T389W / F436W and the other of the first and second mutations comprises T422S / Y438A; e) one of the first and second mutations comprises F436W and the other of the first and second mutations comprises T389Y / F436A and the other of the first and second mutations comprises T422W / Y438T; the other of the first and second mutations comprises T422S; h) one of the first and second mutations comprises D427C and the other of the first and second mutations comprises K420C; i) one of the first and second mutations comprises T389W / D427C and the other of the first and second mutations comprises T389S / L391A / K420C / Y438V; j) one of the first and second mutations comprises T389W / K420C and the other of the first and second mutations comprises T389S / L391A / D427C / Y438V; k) one of the first and second mutations comprises S375C / T389W and the first and second mutations the other comprises Y370C / T389S / L391A / Y438V; l) one of the first and second mutations comprises Y370C / T389W and the other of the first and second mutations comprises S375C / T389S / L391A / Y438V; m) one of the first and second mutations comprises E377C / T389W and the other of the first and second mutations comprises Y370C / T389S / L391A / Y438V; n) one of the first and second mutations comprises Y370C / T389W and the other of the first and second mutations comprises E377C / T389S / L391A / Y438V;o) one of the first and second mutations comprises E378C / T389W and the other of the first and second mutations comprises Y370C / T389S / L391A / Y438V; p) one of the first and second mutations comprises Y370C / T389W and the other of the first and second mutations comprises E378C / T389S / L391A / Y438V, wherein numbering is according to the Kabat index.

[0190] In some embodiments, the polypeptide complexes disclosed herein comprise a first CH3 region and a second CH3 region, wherein the first CH3 region or the second CH3 region comprises an amino acid sequence that differs from the wild-type human IgG amino acid sequence such that one or more positively charged amino acids (e.g., lysine, histidine, and arginine) in the wild-type human IgG amino acid sequence are replaced with one or more negatively charged amino acids (e.g., aspartic acid and glutamic acid) at corresponding positions in the CH3 region. Alternatively, the first CH3 region or the second CH3 region comprises an amino acid sequence that differs from the wild-type human IgG amino acid sequence such that one or more negatively charged amino acids in the wild-type human IgG amino acid sequence are replaced with one or more positively charged amino acids at corresponding positions in the CH3 region.

[0191] In some embodiments, the first dimerization domain comprises a first mutation and / or the second dimerization domain comprises a second mutation, wherein a) one of the first and second mutations comprises K393E / D427K / K470D and the other of the first and second mutations comprises D377K / E378K / K440D; b) one of the first and second mutations comprises K440D and the other of the first and second mutations comprises D427K; and c) one of the first and second mutations comprises K440E. and the other of the first mutation and the second mutation comprises D427K; d) one of the first mutation and the second mutation comprises K440E and the other of the first mutation and the second mutation comprises D427R; e) one of the first mutation and the second mutation comprises K440D and the other of the first mutation and the second mutation comprises D427R; f) one of the first mutation and the second mutation comprises D427K and the other of the first mutation and the second mutation comprises E377K; g) one of the first mutation and the second mutation comprises E377K / D427K and the first mutation and and the other of the second mutations comprises K420D / K440D; h) one of the first mutation and the second mutation comprises E377K / D427K and the other of the first mutation and the second mutation comprises K440D / K470D; i) one of the first mutation and the second mutation comprises E378K / D427K and the other of the first mutation and the second mutation comprises K393D / K440D; j) one of the first mutation and the second mutation comprises E377K / E378K / D427K and the other of the first mutation and the second mutation comprises K393D / K4 k) one of the first and second mutations comprises E378K / D427K and the other of the first and second mutations comprises K420D / K440D; l) one of the first and second mutations comprises K420D / K440D and the other of the first and second mutations comprises D427K; m) one of the first and second mutations comprises K383D / K440D and the other of the first and second mutations comprises D427K, wherein numbering is according to the Kabat index.

[0192] In some embodiments, the first dimerization domain comprises the amino acid sequence of SEQ ID NO:103 and the second dimerization domain comprises the amino acid sequence of SEQ ID NO:104.

[0193] Target of the Polypeptide Complex In some embodiments, at least one of the first target, the second target, and the third target is a disease-associated antigen. For example, the disease-associated antigen can be a tumor antigen, or an antigen associated with an autoimmune or inflammatory disease, or an antigen associated with an eye disorder, an antigen associated with a central nervous system disease, an antigen associated with an infectious disease, or an antigen associated with a coagulation disorder.

[0194] In some embodiments, the antigen associated with an autoimmune or inflammatory disease is, for example, HSA, TNF, IL6R, IL17A / F, RANKL, IL-13, IL4-IL17, BAFF, ICOSL, IL-17A, NGF, CD32b, CD79b, FGFR1, KLB, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (chain of the IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE Examples include, but are not limited to, Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, LFA-1 (CD11a), myostatin, OX-40, sclerostin, SOST, TGFβ1, TNF-α, or VEGF-A.

[0195] In some embodiments, the antigen associated with an ocular disease includes, for example, age-related macular degeneration (AMD) and diabetic macular edema. In some embodiments, the antigen associated with an ocular disease is, for example, but not limited to, VEGF or ANG-2.

[0196] In certain embodiments, the antigen associated with a central nervous system disease is, for example, neuroblastoma, glioblastoma, or Alzheimer's disease. In some embodiments, the antigen associated with an ocular disease is, for example, but not limited to, GD2, EGFRvIII, Aβ40, or Aβ42.

[0197] In certain embodiments, antigens associated with infectious diseases include, for example, pneumonia, viral infections (such as COVID-19 infection), etc. In some embodiments, antigens associated with autoimmune or inflammatory diseases include, but are not limited to, Psl, Pcrv, or the spike protein of the COVID-19 virus.

[0198] In certain embodiments, the antigen associated with a coagulation disorder includes, for example, hemophilia A. In some embodiments, the antigen associated with hemophilia A is, for example, but not limited to, FIXa or FX.

[0199] Other examples of antigen pairs that may have potential therapeutic benefit when targeted by the multispecific polypeptide complexes provided herein include PD-L1:TGFβ, CD38:EGFR, HER2:VEGF, HER2:EGFR, PD-1:CTLA-4, PD-1:TIM3, OX40:PD-L1, FIXa:FX, CD32B:CD79B, Angiopoietin 2:VEGF, IL13:IL4, TNF:IL17A, DLL4:VEGF, IL1α:IL1β, FAP:DR5, CD30:gpA33, TNF:HSA, IL6R:HSA, IL17A / F:HSA, RANKL:HSA, Aβ40:Aβ42, IL13:IL17, FGFR1:KLB, PsI:PcrV, BAFF:B7RP1, NGF:TNF, and TNF:IL17A (Sedykh SE, et al., Drug Des Devel Ther. 2018; 12: 195-208).

[0200] In certain embodiments, one of the first target, the second target, and the third target is a tumor antigen. Tumor antigens include antigens presented on the surface of tumor cells, antigens present on or within tumor cells, antigens presented only by tumor cells and not by normal cells, i.e., non-tumor cells, antigens representing proteins with one or more tumor-specific mutations compared to non-tumor cells, antigens overexpressed in tumor cells compared to non-tumor cells, antigens available for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to non-tumor tissue, and antigens presented in the tumor vasculature, etc., and refer to antigenic substances produced within tumor cells, i.e., which elicit a host immune response. In some embodiments, tumor antigens are antigens present in tumors but not in normal organs, tissues, and / or cells. In some embodiments, tumor antigens are antigens that are more prevalent in tumors than in normal organs, tissues, and / or cells. In some embodiments, tumor antigens are antigens that are more prevalent in malignant cancer cells than in normal cells.

[0201] Examples of tumor antigens include, but are not limited to, CD19, CD20, CD38, CD30, Her2 / neu / ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, and GD2. Cancer-associated antigens include, for example, 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, and CD44. v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R α, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH Also included are 900105, SDC1, SLAMF7, TAG-72, tenascin-C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin.

[0202] In certain embodiments, in addition to a tumor antigen, at least one of the first target, the second target, and the third target is an immune-related target.

[0203] Immune-related targets provided herein include CD2, CD3, CD7, CD16, CD27, CD30, CD70, CD83, CD28, CD80 (B7-1), CD86 (B7-2), CD40, CD40L (CD154), CD47, CD122, CD137, CD137L, OX40 (CD134), OX40L (CD252), NKG2C, 4-1BB, LIGHT, PVRIG, SLAMF7, HVEM, BAFFR, ICAM-1, 2B4, LFA-1, GITR, ICOS (CD278), ICOS The antigen may be selected from the group consisting of LG (CD275), LAG3 (CD223), A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), BTLA, CD160, CTLA-4 (CD152), IDO1, IDO2, TDO, KIR, LAIR-1, NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC-7 (CD328), TIGIT, PVR (CD155), TGFβ, SIGLEC9 (CD329), and any combination thereof.

[0204] In certain embodiments, the immune-related target is a receptor on cytotoxic T lymphocytes (e.g., CD3 and / or 4-1BB), and the tumor antigen is a cell surface tumor antigen such as CEA, CD19, CD20, CD33, CD123, HER1, HER2, disialoganglioside GD2, PSMA, gpA33, EpCAM, P-cadherin, or B7H3 (Sedykh SE, et al., Drug Des Devel Ther. 2018; 12: 195-208). Thus, the multispecific polypeptide complexes provided herein can be used to bind T cells and tumor cells, allowing the T cells to exert cytotoxic activity against the tumor cells.

[0205] In certain embodiments, the first domain, the second domain, and the third domain comprise an immune-related target.

[0206] In certain embodiments, both the first and second domains comprise a TGFβ targeting domain, and the third domain comprises a PD-1 targeting domain.

[0207] In certain embodiments, one of the first target, the second target, and the third target is a tumor antigen, and the other two are immune-related targets.

[0208] In certain embodiments, the first target and the second target are both immune-related targets. In certain embodiments, the first target and the second target are different immune-related targets.

[0209] In certain embodiments, at least one or both of the first and second targets are immunostimulatory targets, hi certain embodiments, the third target is a first tumor antigen.

[0210] In a specific embodiment, the first and second targets are CD3 and 4-1BB, and the third target is CEA.

[0211] In certain embodiments, one of the first and second targets is CD3 and the other is CEA, hi certain embodiments, the third target is also CEA.

[0212] In some embodiments, in the DICAD domain, the first domain is capable of binding to a first target and the second domain is capable of binding to a second target.

[0213] In certain embodiments, both the first and second domains comprise a TGFβ targeting domain, and the third domain comprises a PD-1 targeting domain.

[0214] In some embodiments, the multispecific polypeptide complexes provided herein have a symmetric structure that is bivalent for each of the first, second, and third targets. In some embodiments, the multispecific polypeptide complexes provided herein have an asymmetric structure that is bivalent for each of the third target and monovalent for each of the first and second targets.

[0215] a) TGFβ targeting domain In certain embodiments, the TGFβ targeting domain comprises an HCDR1, HCDR2, and HCDR3 identical to the HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence of SEQ ID NO: 38, and an LCDR1, LCDR2, and LCDR3 identical to the LCDR1, LCDR2, and LCDR3 contained in the amino acid sequence of SEQ ID NO: 37. In such embodiments, the TGFβ targeting domain comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequences of SEQ ID NOs: 22 / 23 / 24 / 19 / 20 / 21, respectively.

[0216] In such an embodiment, the TGFβ targeting domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO:38, and a VL domain comprising the amino acid sequence of SEQ ID NO:37.

[0217] In certain embodiments, the first domain comprises a TGFβ targeting domain further comprising a first non-naturally occurring covalent bond (e.g., a disulfide bond) formed between a first pair of two amino acid residues (e.g., two non-naturally occurring cysteine ​​residues). In certain embodiments, the first non-naturally occurring covalent bond is formed between two non-naturally occurring cysteine ​​residues at position 44 in the VH domain and position 100 in the VL domain. In certain embodiments, the first domain comprises a TGFβ targeting domain further comprising a non-naturally occurring pair of two oppositely charged amino acid residues. In certain embodiments, the two oppositely charged residues are at position 38 in the VL domain and position 39 in the VH domain, where numbering is according to the Kabat index. In certain embodiments, the first domain comprises a TGFβ targeting domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO:50 and a VL domain comprising the amino acid sequence of SEQ ID NO:49. In certain embodiments, the second domain comprises a TGFβ targeting domain wherein VH2 comprises the amino acid sequence of SEQ ID NO:38 and VL2 comprises the amino acid sequence of SEQ ID NO:37.

[0218] b) PD-1 targeting domain In certain embodiments, the PD-1 targeting domain comprises an HCDR1, HCDR2, and HCDR3 identical to the HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence of SEQ ID NO: 40, and an LCDR1, LCDR2, and LCDR3 identical to the LCDR1, LCDR2, and LCDR3 contained in the amino acid sequence of SEQ ID NO: 39. In such embodiments, the PD-1 targeting domain comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequences of SEQ ID NOs: 28 / 29 / 30 / 25 / 26 / 27, respectively.

[0219] In some embodiments, the third domain comprises a PD-1 targeting domain, wherein VH3 comprises the amino acid sequence of SEQ ID NO:40 and VL3 comprises the amino acid sequence of SEQ ID NO:39.

[0220] In some embodiments, the PD-1 targeting domain further comprises a first non-naturally occurring covalent bond (e.g., a disulfide bond) formed between a first pair of two amino acid residues (e.g., two non-naturally occurring cysteine ​​residues). In certain embodiments, the first non-naturally occurring covalent bond is formed between two non-naturally occurring cysteine ​​residues at position 44 in the VH domain and position 100 in the VL domain. In certain embodiments, the PD-1 targeting domain further comprises a non-naturally occurring pair of two oppositely charged amino acid residues. In certain embodiments, the two oppositely charged residues are at position 38 in the VL domain and position 39 in the VH domain, where numbering is according to the Kabat index.

[0221] In some embodiments, the first and second domains comprise a CD3 targeting domain and a 4-1BB targeting domain, respectively, and the third domain comprises a CEA targeting domain.

[0222] In some embodiments, the first domain comprises a 4-1BB targeting domain and the second domain comprises a CD3 targeting domain, while the third domain comprises a CEA targeting domain.

[0223] c) 4-1BB targeting domain In certain embodiments, the 4-1BB targeting domain comprises HCDR1, HCDR2, and HCDR3 identical to the HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence of SEQ ID NO: 32, and LCDR1, LCDR2, and LCDR3 identical to the LCDR1, LCDR2, and LCDR3 contained in the amino acid sequence of SEQ ID NO: 31. In such embodiments, the 4-1BB targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequences of SEQ ID NOs: 4 / 5 / 6 / 1 / 2 / 3, respectively.

[0224] In some embodiments, the 4-1BB targeting domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO:32 and a VL domain comprising the amino acid sequence of SEQ ID NO:31.

[0225] In some embodiments, the first domain comprises a 4-1BB targeting domain. In some embodiments, the 4-1BB targeting domain further comprises a first non-natural covalent bond (e.g., a disulfide bond) formed between a first pair of two amino acid residues (e.g., two non-natural cysteine ​​residues). In certain embodiments, the first non-natural covalent bond is formed between two non-natural cysteine ​​residues at position 44 in the VH domain and position 100 in the VL domain. In certain embodiments, the 4-1BB targeting domain further comprises a non-natural pair of two oppositely charged amino acid residues. In certain embodiments, the two oppositely charged residues are at position 38 in the VL domain and position 39 in the VH domain, where numbering is according to the Kabat index. In certain embodiments, the first domain comprises a 4-1BB targeting domain, wherein VH1 comprises the amino acid sequence of SEQ ID NO: 48 and VL1 comprises the amino acid sequence of SEQ ID NO: 47.

[0226] d) CD3 targeting domain In certain embodiments, the CD3 targeting domain comprises HCDR1, HCDR2, and HCDR3 identical to the HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence of SEQ ID NO: 34, and LCDR1, LCDR2, and LCDR3 identical to the LCDR1, LCDR2, and LCDR3 contained in the amino acid sequence of SEQ ID NO: 33. In such embodiments, the CD3 targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequences of SEQ ID NOs: 10 / 11 / 12 / 7 / 8 / 9, respectively.

[0227] In some embodiments, the second domain comprises a CD3 targeting domain. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 34 and VL2 comprises the amino acid sequence of SEQ ID NO: 33.

[0228] In some embodiments, the CD3 targeting domain further comprises a first non-naturally occurring covalent bond (e.g., a disulfide bond) formed between a first pair of two amino acid residues (e.g., two non-naturally occurring cysteine ​​residues). In certain embodiments, the first non-naturally occurring covalent bond is formed between two non-naturally occurring cysteine ​​residues at position 44 in the VH domain and position 100 in the VL domain. In certain embodiments, the CD3 targeting domain further comprises a non-naturally occurring pair of two oppositely charged amino acid residues. In certain embodiments, the two oppositely charged residues are at position 38 in the VL domain and position 39 in the VH domain, where numbering is according to the Kabat index.

[0229] e) CEA targeting domain In certain embodiments, the CEA targeting domain comprises HCDR1, HCDR2, and HCDR3 identical to the HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence of SEQ ID NO: 36, 42, or 44, and LCDR1, LCDR2, and LCDR3 identical to the LCDR1, LCDR2, and LCDR3 contained in the amino acid sequence of SEQ ID NO: 35, 41, or 43. In such embodiments, the CEA targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each comprising the amino acid sequence of SEQ ID NO: 16 / 17 / 18 / 13 / 14 / 15, or SEQ ID NO: 107 / 108 / 18 / 13 / 14 / 15, or SEQ ID NO: 16 / 111 / 18 / 109 / 110 / 15, respectively.

[0230] In some embodiments, the third domain comprises a CEA-targeting domain. In some embodiments, VH3 comprises the amino acid sequence of SEQ ID NO: 36, and VL3 comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, VH3 comprises the amino acid sequence of SEQ ID NO: 42, and VL3 comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH3 comprises the amino acid sequence of SEQ ID NO: 44, and VL3 comprises the amino acid sequence of SEQ ID NO: 43.

[0231] In some embodiments, the CEA-targeting domain further comprises a first non-naturally occurring covalent bond (e.g., a disulfide bond) formed between a first pair of two amino acid residues (e.g., two non-naturally occurring cysteine ​​residues). In certain embodiments, the first non-naturally occurring covalent bond is formed between two non-naturally occurring cysteine ​​residues at position 44 in the VH domain and position 100 in the VL domain. In certain embodiments, the CEA-targeting domain further comprises a non-naturally occurring pair of two oppositely charged amino acid residues. In certain embodiments, the two oppositely charged residues are at position 38 in the VL domain and position 39 in the VH domain, where numbering is according to the Kabat index. In certain embodiments, the CEA-targeting domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 46 and a VL domain comprising the amino acid sequence of SEQ ID NO: 45.

[0232] Multispecific polypeptide complex molecule (TGFβ×TGFβ×PD-1) In certain embodiments, the multispecific polypeptide complex has a symmetric structure, as shown in Figures 1, 4, 7, and 10, where both the first and second domains comprise a TGFβ targeting domain, and the third domain comprises a PD-1 targeting domain. In some embodiments, the first domain comprises a TGFβ targeting domain, where VH1 and VL1 comprise a first non-natural disulfide bond formed between two non-natural cysteine ​​residues and further comprise a first non-natural pair of oppositely charged amino acid residues.

[0233] In certain embodiments, the multispecific polypeptide complex has a symmetric structure as shown in Figure 1 (4A25). In such embodiments, the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 67, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 53. In certain embodiments, the multispecific polypeptide complex comprises three polypeptide chains having the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.

[0234] In certain embodiments, the multispecific polypeptide complex has a symmetric structure, as shown in Figure 4 (4A26). In such embodiments, the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 67, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 56. In certain embodiments, the multispecific polypeptide complex comprises three polypeptide chains having the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively.

[0235] In certain embodiments, the multispecific polypeptide complex has a symmetric structure, as shown in Figure 7 (4A27). In such embodiments, the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 58, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 53. In certain embodiments, the multispecific polypeptide complex comprises three polypeptide chains having the amino acid sequences of SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, respectively.

[0236] In certain embodiments, the multispecific polypeptide complex has a symmetric structure, as shown in Figure 10 (4A28). In such embodiments, the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 61, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 53. In certain embodiments, the multispecific polypeptide complex comprises three polypeptide chains having the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 59, respectively.

[0237] Multispecific polypeptide complex molecule (CD3×4-1BB×CEA) In certain embodiments, the multispecific polypeptide complex has an asymmetric structure. In certain embodiments, the multispecific polypeptide complex further comprises a second antigen-binding domain. In certain embodiments, the multispecific polypeptide complex comprises a second Fv domain. In certain embodiments, the second Fv domain is identical to the first Fv domain, and the second Fv domain comprises a fifth polypeptide fragment identical to the third polypeptide fragment comprising a third heavy chain variable domain (VH3), and a sixth polypeptide fragment identical to the fourth polypeptide fragment comprising a third light chain variable domain (VL3). In certain embodiments, the C-terminus of the fifth polypeptide fragment of the second Fv domain is operably linked to the N-terminus of the second dimerization domain.

[0238] In certain embodiments, the second Fv domain comprises a CEA-targeting domain in which a) VH3 comprises the amino acid sequence of SEQ ID NO: 36 and VL3 comprises the amino acid sequence of SEQ ID NO: 35, b) VH3 comprises the amino acid sequence of SEQ ID NO: 42 and VL3 comprises the amino acid sequence of SEQ ID NO: 41, or c) VH3 comprises the amino acid sequence of SEQ ID NO: 44 and VL3 comprises the amino acid sequence of SEQ ID NO: 43.

[0239] In certain embodiments, the fifth polypeptide fragment and the sixth polypeptide fragment comprise the amino acid sequences of a) SEQ ID NO: 73 and SEQ ID NO: 74, respectively; b) SEQ ID NO: 77 and SEQ ID NO: 78, respectively; and c) SEQ ID NO: 81 and SEQ ID NO: 82, respectively.

[0240] In certain embodiments, the multispecific polypeptide complex has an asymmetric structure, as shown in Figure 15 (e.g., 12A14), in which the first and second domains comprise a CD3 targeting domain and a 4-1BB targeting domain, respectively, and the third domain comprises a CEA targeting domain.

[0241] In some embodiments, the first domain comprises a 4-1BB targeting domain, in which VH1 and VL1 comprise a first non-natural disulfide bond formed between two non-natural cysteine ​​residues and further comprise a first non-natural pair of oppositely charged amino acid residues. In some embodiments, the second domain comprises a CD3 targeting domain. In some embodiments, the third domain comprises a CEA targeting domain.

[0242] In such embodiments, the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 87, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 75, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 83, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 74. In certain embodiments, the fifth and sixth polypeptide fragments comprise the amino acid sequences of SEQ ID NO: 73 and SEQ ID NO: 74, respectively. In certain embodiments, the multispecific polypeptide complex comprises four polypeptide chains having the amino acid sequences of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively.

[0243] In certain embodiments, the multispecific polypeptide complex has an asymmetric structure, as shown in Figure 18 (12A15), wherein the first and second domains comprise a CD3 targeting domain and a 4-1BB targeting domain, respectively, and the third domain comprises a CEA targeting domain.

[0244] In such embodiments, the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 87, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 75, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 83, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 78. In certain embodiments, the fifth and sixth polypeptide fragments comprise the amino acid sequences of SEQ ID NO: 77 and SEQ ID NO: 78. In certain embodiments, the multispecific polypeptide complex comprises four polypeptide chains having the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively.

[0245] In certain embodiments, the multispecific polypeptide complex has an asymmetric structure as shown in Figure 21 (12A16), wherein the first and second domains comprise a CD3 targeting domain and a 4-1BB targeting domain, respectively, and the third domain comprises a CEA targeting domain. In such embodiments, the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 87, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 75, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 83, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 82. In certain embodiments, the fifth and sixth polypeptide fragments comprise the amino acid sequences of SEQ ID NO: 81 and SEQ ID NO: 82. In certain embodiments, the multispecific polypeptide complex comprises four polypeptide chains having the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82, respectively.

[0246] TRIAD-based multispecific polypeptide complex molecules In another aspect, the present disclosure provides a TRIAD-based multispecific polypeptide complex molecule.

[0247] As used herein, the term "TRIAD" is an abbreviation for "Tri-specific Adjusted Diabody" and refers to an engineered antibody structure comprising a) a DICAD domain linked to a first dimerization domain and b) an Fv domain linked to a second dimerization domain, where the first dimerization domain and the second dimerization domain associate to form a dimer (e.g., a heterodimer). Examples of TRIAD structures are disclosed, for example, in PCT application WO2019 / 120245, which is incorporated herein in its entirety. By adding a third antigen-recognizing functional domain, the inventors have successfully recognized targets in either an AAB (2:1) or ABC (1:1:1) manner (A, B, and C each represent a target of choice) in target binding, resulting in different mechanisms of action and kinetic properties from DICAD antibodies.

[0248] In certain embodiments, the present disclosure provides multispecific polypeptide complexes having a TRIAD structure as shown in Figure 13 (12A6), Figure 14 (12A10, 12A18 and 12A19) and Figure 24 (12A17).

[0249] In certain embodiments, the TRIAD-based multispecific polypeptide complexes provided herein comprise (a) a DICAD domain comprising a first antigen-targeting domain and a second antigen-targeting domain, and (b) a first Fv domain comprising a third antigen-targeting domain, wherein the DICAD domain is linked to a first dimerization domain and the first Fv domain is linked to a second dimerization domain.

[0250] In some embodiments, the DICAD domain comprises (i) a first polypeptide fragment comprising a first heavy chain variable domain (VH1) linked to a second light chain variable domain (VL2), and (ii) a second polypeptide fragment comprising a second heavy chain variable domain (VH2) linked to the first light chain variable domain (VL1), where VL1 and VH1 associate to form a first antigen-targeting domain and VL2 and VH2 associate to form a second antigen-targeting domain.

[0251] In some embodiments, the C-terminus of VL1 is linked to the N-terminus of VH2 and the C-terminus of VL2 is covalently linked to the N-terminus of VH1, hi some embodiments, the C-terminus of VH1 is linked to the N-terminus of VL2 and the C-terminus of VH2 is linked to the N-terminus of VL1.

[0252] In some embodiments, VH2 and VL1 and / or VH1 and VL2 are covalently linked directly or indirectly, eg, via a linker, such as a peptide linker.

[0253] In some embodiments, non-natural covalent bonds (eg, disulfide bonds) and / or electrostatic interactions may be introduced at the VH1-VL1 interface or the VH2-VL2 interface.

[0254] In some embodiments, the VL1 and VH1 of the DICAD domain are associated by a first non-native disulfide bond, which in some embodiments is formed between two non-native cysteine ​​residues in VL1 and VH1, respectively.

[0255] In some embodiments, the two non-native cysteine ​​residues are at position 44 in VH1 and position 100 in VL1, or at position 105 in VH1 and position 43 in VL1, or at position 100 in VH1 and position 49 in VL1, or at position 100 in VH1 and position 150 in VL1. Unless otherwise specified, all position numbering in this disclosure is according to the Kabat index. In some embodiments, the non-native covalent bond is formed between the amino acid residues introduced at position 44 in VH1 and position 100 in VL1, or between the cysteine ​​residues at position 105 in VH1 and position 43 in VL1. In some embodiments, the non-native covalent bond is formed between the cysteine ​​residues at position 44 in VH1 and position 100 in VL1. In some embodiments, the two non-native cysteine ​​residues are 100C in VL1 and 44C in VH1.

[0256] In some embodiments, the VL2 and VH2 of the DICAD domain do not contain any introduced non-native disulfide bonds.

[0257] In some embodiments, the first Fv domain comprises iii) a third polypeptide fragment comprising a third heavy chain variable domain (VH3), and iv) a fourth polypeptide fragment comprising a third light chain variable domain (VL3), wherein VH3 and VL3 associate to form a third antigen-targeting domain.

[0258] In certain embodiments, the TRIAD-based multispecific polypeptide complexes provided herein further comprise (c) a second Fv domain comprising: iv) a fifth polypeptide fragment comprising a fourth heavy chain variable domain (VH4); and iv) a sixth polypeptide fragment comprising a fourth light chain variable domain (VL4), wherein VH4 and VL4 associate to form a fourth antigen-targeting domain. In certain embodiments, the first Fv domain is identical to the second Fv domain. In certain embodiments, the fifth polypeptide fragment is identical to the third polypeptide fragment, and the sixth polypeptide fragment is identical to the fourth polypeptide fragment.

[0259] In some embodiments, the second Fv domain is operably linked to the first Fv domain. In certain embodiments, the heavy chain of the second Fv domain is operably linked to the heavy chain of the first Fv domain. In certain embodiments, the C-terminus of the heavy chain of the second Fv domain is operably linked to the N-terminus of the heavy chain of the first Fv domain.

[0260] In some embodiments, the Fv domain further comprises a scaffold domain fused to VH3 (or VH4) and VL3 (or VL4), respectively. For example, a scaffold region may be fused to VH3 (or VH4), and a paired scaffold region may be fused to VL3 (or VL4). Binding of the scaffold region to the paired scaffold region allows association of the VH3 (or VH4) region with the VL3 (or VL4) region, thereby forming an Fv domain capable of binding to a target antigen. Any suitable binding partner may be used as the scaffold region. In certain embodiments, the scaffold region may be selected from an antibody CH1 domain and a CL domain, a paired TCR constant region (TCRα / TCRβ, TCRγ / TCRδ), a PRD (proline-rich domain) and an SH3 domain, or obscurin and titin.

[0261] In some embodiments, the first Fv domain comprises a CH1 domain covalently linked to one of VH3 and VL3, and a CL domain covalently linked to the other of VH3 and VL3.

[0262] In some embodiments, the second Fv domain comprises a CH1 domain covalently linked to one of VH4 and VL4, and a CL domain covalently linked to the other of VH4 and VL4.

[0263] In some embodiments, the third polypeptide fragment further comprises an antibody heavy chain constant region (CH1) operably linked to the C-terminus of the VH3 domain, and / or the fourth polypeptide fragment further comprises an antibody light chain constant region (CL) operably linked to the C-terminus of the VL3 domain.

[0264] In some embodiments, the fifth polypeptide fragment further comprises an antibody heavy chain constant region (CH1) operably linked to the C-terminus of the VH4 domain, and / or the sixth polypeptide fragment further comprises an antibody light chain constant region (CL) operably linked to the C-terminus of the VL4 domain.

[0265] In certain embodiments, the first antigen is a tumor-associated antigen (TAA), the second antigen is CD3, and the third antigen is a TAA. In certain embodiments, both the first antigen and the third antigen are the same TAA. In certain embodiments, both the first antigen and the third antigen are CEA.

[0266] In some embodiments, in the TRIAD-based multispecific polypeptide complexes provided herein, VL1 and VH1 associate to form a first CEA-targeting domain, VL2 and VH2 associate to form a CD3-targeting domain, and VL3 and VH3 associate to form a second CEA-targeting domain. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO:46, VL1 comprises the amino acid sequence of SEQ ID NO:45, VH2 comprises the amino acid sequence of SEQ ID NO:34, VL2 comprises the amino acid sequence of SEQ ID NO:33, VH3 comprises the amino acid sequence of SEQ ID NO:36, and VL3 comprises the amino acid sequence of SEQ ID NO:35.

[0267] In a particular embodiment, the multispecific polypeptide complex 12A6 comprises four polypeptide chains having the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, respectively.

[0268] In a specific embodiment, the first antigen is 4-1BB, the second antigen is CD3, and the third antigen is a TAA. In a specific embodiment, the third antigen is CEA.

[0269] In some embodiments, in the TRIAD-based multispecific polypeptide complexes provided herein, VL1 and VH1 associate to form a 4-1BB targeting domain, VL2 and VH2 associate to form a CD3 targeting domain, and VL3 and VH3 associate to form a CEA targeting domain. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO:48, VL1 comprises the amino acid sequence of SEQ ID NO:47, VH2 comprises the amino acid sequence of SEQ ID NO:34, VL2 comprises the amino acid sequence of SEQ ID NO:33, VH3 comprises the amino acid sequence of SEQ ID NO:36, and VL3 comprises the amino acid sequence of SEQ ID NO:35.

[0270] In a particular embodiment, the multispecific polypeptide complex 12A10 comprises four polypeptide chains having the amino acid sequences of SEQ ID NO:75, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70, respectively.

[0271] In some embodiments, in a TRIAD-based multispecific polypeptide complex provided herein, VL1 and VH1 associate to form a 4-1BB targeting domain, VL2 and VH2 associate to form a CD3 targeting domain, and VL3 and VH3 associate to form a CEA targeting domain. In some embodiments, in a TRIAD-based multispecific polypeptide complex provided herein, VH1 comprises the amino acid sequence of SEQ ID NO:48, VL1 comprises the amino acid sequence of SEQ ID NO:47, VH2 comprises the amino acid sequence of SEQ ID NO:34, VL2 comprises the amino acid sequence of SEQ ID NO:33, VH3 comprises the amino acid sequence of SEQ ID NO:42, and VL3 comprises the amino acid sequence of SEQ ID NO:41.

[0272] In certain embodiments, the multispecific polypeptide complex 12A18 comprises polypeptide chains having the amino acid sequences of SEQ ID NO:75, SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90, respectively.

[0273] In some embodiments, in a TRIAD-based multispecific polypeptide complex provided herein, VL1 and VH1 associate to form a 4-1BB targeting domain, VL2 and VH2 associate to form a CD3 targeting domain, and VL3 and VH3 associate to form a CEA targeting domain. In some embodiments, in a TRIAD-based multispecific polypeptide complex provided herein, VH1 comprises the amino acid sequence of SEQ ID NO:48, VL1 comprises the amino acid sequence of SEQ ID NO:47, VH2 comprises the amino acid sequence of SEQ ID NO:34, VL2 comprises the amino acid sequence of SEQ ID NO:33, VH3 comprises the amino acid sequence of SEQ ID NO:44, and VL3 comprises the amino acid sequence of SEQ ID NO:43.

[0274] In certain embodiments, the multispecific polypeptide complex 12A19 comprises polypeptide chains having the amino acid sequences of SEQ ID NO:75, SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, respectively.

[0275] In some embodiments, in the TRIAD-based multispecific polypeptide complexes provided herein, VL1 and VH1 associate to form a 4-1BB targeting domain, VL2 and VH2 associate to form a CD3 targeting domain, VL3 and VH3 associate to form a first CEA targeting domain, and VL4 and VH4 associate to form a second CEA targeting domain. In some embodiments, the first CEA targeting domain and the second CEA targeting domain are identical. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO:48, VL1 comprises the amino acid sequence of SEQ ID NO:47, VH2 comprises the amino acid sequence of SEQ ID NO:34, VL2 comprises the amino acid sequence of SEQ ID NO:33, VH3 comprises the amino acid sequence of SEQ ID NO:36, VL3 comprises the amino acid sequence of SEQ ID NO:35, VH4 comprises the amino acid sequence of SEQ ID NO:36, and VL4 comprises the amino acid sequence of SEQ ID NO:35.

[0276] In certain embodiments, the multispecific polypeptide complex 12A17 comprises polypeptide chains having the amino acid sequences of SEQ ID NO:75, SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, respectively.

[0277] The sequence and mutation information is shown in Tables A, B and C below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 2-1 Table 2-2 [Table 3-1] [Table 3-2] [Table 3-3]

[0278] III. Polynucleotides and Recombinant Methods The present disclosure provides nucleic acids comprising nucleotide sequences encoding the polypeptide complexes provided herein. As used herein, the terms "nucleic acid" or "nucleotide sequence" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985), and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0279] Polynucleotides encoding the polypeptide complexes disclosed herein can be generated by methods known in the art. In certain embodiments, the sequence of the polynucleotide can be obtained based on the amino acid sequence of the polypeptide complex, and the nucleic acid can be generated by synthetic methods. Alternatively, the polynucleotides provided herein can be obtained from another available nucleic acid encoding a polypeptide having a sequence homologous to a polypeptide in the polypeptide complexes disclosed herein. DNA manipulation processes can then be applied to manipulate the sequence of the nucleic acid encoding the parent antibody to introduce, for example, mutations, insertions, deletions, etc., to obtain a nucleic acid encoding the polypeptide complexes disclosed herein.

[0280] The nucleotide sequence encoding the fusion polypeptide or polypeptide complex may be inserted into one or more vectors for further cloning (amplification of the DNA) or expression by recombinant techniques known in the art. Multiple vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), a transcription termination sequence, and one or more other regulatory elements.

[0281] The present disclosure provides a vector comprising a nucleic acid provided herein. In certain embodiments, the nucleic acid provided herein encodes an antibody having at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence and at least one selectable marker. Examples of vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, and M13 phage, as well as plasmids such as pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, and pC Examples of such vectors include, but are not limited to, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, and pEF-Bos.

[0282] Vectors containing nucleotide sequences encoding the fusion polypeptide or polypeptide complex may be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein include the prokaryotes, yeast, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacillus, such as Bacillus subtilis and B. licheniformis, Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces.

[0283] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for vectors encoding polypeptide complexes. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, numerous other genera, species, and strains are commonly available and useful herein, including Schizosaccharomyces pombe, e.g., Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickelamii (ATCC 24,178), Kluyveromyces wartii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906), Kluyveromyces thermotolerans, and others. and Kluyveromyces marxianus; Yarrowia (EP 402,226), Pichia pastoris (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa; Schwanniomyces species such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora crassa, Penicillium, and Tolypocladium; and Aspergillus hosts such as Aspergillus nidulans and Aspergillus niger.

[0284] Suitable host cells for expressing the glycosylated polypeptide complexes provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as the armyworm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of the mosquito virus (NPV) and the Bm-5 strain of the silkworm virus (NPV), are publicly available, and such viruses can be used in accordance with the present invention, particularly for transfection of armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0285] However, vertebrate cells have received the most attention, and propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7, ATCC CRL1651), human embryonic kidney (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat hepatocytes (BRL 3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human hepatocytes (Hep G2, HB8065), mouse mammary tumor (MMT060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and a human hepatocellular carcinoma line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line such as CHO, BHK, NS0, 293, and their derivatives.

[0286] Host cells are transformed with the above-described antibody-producing expression or cloning vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, antibodies may be produced by homologous recombination, as known in the art. In certain embodiments, the host cells are capable of producing the fusion polypeptides or polypeptide complexes provided herein.

[0287] The present disclosure also provides a method for expressing a polypeptide complex provided herein, the method comprising culturing a host cell provided herein under conditions in which a vector of the present disclosure is expressed. The host cells used to produce the antibodies provided herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. Additionally, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO90 / 03430, WO87 / 00195, or U.S. Pat. Reissue No. 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions, such as temperature and pH, will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0288] When using recombinant techniques, polypeptide complexes may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. When fusion polypeptides or polypeptide complexes are produced intracellularly, as a first step, particulate cell debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed for approximately 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When fusion polypeptides or polypeptide complexes are secreted into the medium, supernatants from such expression systems are generally first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0289] The polypeptide complex prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being preferred.

[0290] In certain embodiments, solid-phase-immobilized protein A is used for immunoaffinity purification of fusion polypeptides or polypeptide complexes. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domains present in the polypeptide complex. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than agarose. If the polypeptide complex contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Depending on the antibody recovered, other protein purification techniques can also be used, such as fractional ethanol precipitation on an ion exchange column, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0291] Following any preliminary purification steps, the mixture containing the fusion polypeptide or polypeptide complex of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer of about pH 2.5 to 4.5, preferably at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0292] IV. Pharmaceutical Compositions The present invention further provides pharmaceutical compositions comprising a polypeptide conjugate described herein and a pharmaceutically acceptable carrier.

[0293] As used herein, the term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient, salt, and / or vehicle is generally chemically and / or physiologically compatible with other ingredients, e.g., the active ingredient (i.e., the polypeptide complex or heterodimeric antibody or antigen-binding fragment thereof), comprising the formulation, and physiologically compatible with the subject to which the pharmaceutical composition is administered.

[0294] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is biologically acceptable and non-toxic to a subject. In the context of the present disclosure, pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, or various combinations thereof, known in the art.

[0295] As used herein, suitable "ingredients" may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, coloring agents, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable "antioxidants" may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in the pharmaceutical compositions provided herein reduces oxidation of the polypeptide complex or heterodimeric antibody or antigen-binding fragment thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving protein stability and maximizing shelf life. Thus, in certain embodiments, pharmaceutical compositions are provided that comprise, in addition to the active ingredient (i.e., a polypeptide complex or heterodimeric antibody or antigen-binding fragment thereof disclosed herein), one or more antioxidants, such as methionine.

[0296] Pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifying agents such as polysorbate 80 (TWEEN®-80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antibacterial agents used as carriers may be added to pharmaceutical compositions in multi-dose containers and include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0297] Pharmaceutically acceptable "diluents" may include saline and aqueous buffer solutions.

[0298] Pharmaceutically acceptable "adjuvants" may include preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by both the above-mentioned sterilization procedures and the incorporation of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. In addition, the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin, can prolong the absorption of the injectable dosage form.

[0299] The pharmaceutical compositions may be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0300] In embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition can be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or solid form suitable for preparing a liquid solution, suspension, or emulsion. Preparations for injection can include sterile and / or non-pyrogenic solutions prepared for injection, sterile dry soluble preparations such as lyophilized powders prepared to be combined with a solvent immediately before use, including hypodermic tablets, sterile suspensions prepared for injection, sterile dry insoluble preparations prepared to be combined with a vehicle immediately before use, and sterile and / or non-pyrogenic emulsions. The solution can be aqueous or non-aqueous.

[0301] In certain embodiments, unit dose parenteral preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration shall be sterile and non-pyrogenic, as known and practiced in the art.

[0302] In certain embodiments, a sterile, lyophilized powder is prepared by dissolving a polypeptide conjugate disclosed herein in a suitable solvent. The solvent may contain an excipient that improves the stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer such as citric acid, sodium phosphate, or potassium phosphate, or other such buffer known to those of skill in the art; in one embodiment, the buffer has an approximately neutral pH. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those of skill in the art, provides the desired formulation. In one embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial may contain a single dose or multiple doses of the polypeptide conjugate. Overfilling the vial by a small amount (e.g., about 10%) beyond that required for a dose or set of doses may be acceptable to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0303] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the given selected therapy and can be empirically determined.

[0304] In certain embodiments, further provided are compositions comprising a pharmaceutically acceptable carrier, diluent, or adjuvant and an active ingredient, which may be a polypeptide complex disclosed herein or a conjugate of a polypeptide complex disclosed herein.

[0305] V conjugates The polypeptide conjugates provided herein can be used in unconjugated or conjugated form.

[0306] In the conjugated form, the polypeptide complex is conjugated to one or more desired conjugating moieties, i.e., heterologous moieties, to perform a specific function, for example, to facilitate target detection, or for imaging or therapy.

[0307] The present disclosure provides herein a conjugate comprising a polypeptide complex provided herein and a conjugate moiety (e.g., a payload) conjugated thereto, wherein the payload can be any one of the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification tag, a tracking molecule, an anti-cancer drug, and a cytotoxic molecule.

[0308] A variety of conjugates can be linked to the multispecific polypeptide complexes provided herein by covalent bonding, affinity binding, intercalation, coordinate binding, complex formation, association, blending, or addition (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)).

[0309] In certain embodiments, the polypeptide complexes provided herein may be engineered to contain specific sites on the exterior of the epitope-binding moiety that can be specifically utilized for binding to one or more conjugates. For example, such sites may contain one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent linkage to a conjugate.

[0310] In certain embodiments, the N-terminus and / or C-terminus of the polypeptide conjugates provided herein can also function to provide reactive groups for conjugation, for example, the N-terminus is conjugated to one moiety (such as polyethylene glycol (PEG)) and the C-terminus is conjugated to another moiety (such as biotin).

[0311] In certain embodiments, the polypeptide complexes provided herein may be directly linked to a conjugate or indirectly linked, for example, via another conjugate or linker.

[0312] For example, polypeptide conjugates provided herein having a reactive residue such as cysteine ​​can be linked to thiol-reactive agents in which the reactive group is, for example, maleimide, iodoacetamide, pyridyl disulfide, or other thiol-reactive conjugation partners (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labeling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[0313] As another example, the polypeptide complexes provided herein may be conjugated to biotin and then indirectly conjugated to a second conjugate that is conjugated to avidin. As yet another example, the polypeptide complexes may be linked to a linker that is further linked to the conjugate. Examples of linkers include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), and bifunctional coupling agents such as bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide disulfide linkages.

[0314] In certain embodiments, the conjugate moiety comprises an agent for detection or isolation, such as, for example, a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or other anti-cancer agent.

[0315] The conjugate moiety can be a detectable label, a pharmacokinetic-modifying moiety, a purification moiety, a cytotoxic moiety, or a therapeutic agent. Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 P, other lanthanides, luminescent labels), chromophore moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0316] In certain embodiments, the conjugate moiety may be a pharmacokinetic-modifying moiety such as PEG, which serves to increase the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and the like. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In certain embodiments, the conjugate may be a purification moiety such as a magnetic bead.

[0317] In certain embodiments, the conjugate moiety can be a cytotoxic moiety. A "cytotoxic moiety" can be any agent that is harmful to cells or can damage or kill cells. Examples of cytotoxic moieties include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkyl benzoates, and the like. These include, without limitation, nitrating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). In some embodiments, the conjugate moiety comprises an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Jatropha forsi protein, dianthin protein, Phytolacca americana protein, bitter melon inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0318] Methods for conjugating conjugate moieties to proteins such as antibodies, immunoglobulins or fragments thereof are described, for example, in U.S. Pat. No. 5,208,020, U.S. Pat. No. 6,4411,163, WO2005037992, WO2005081711, and WO2006 / 034488, which are incorporated by reference herein in their entireties.

[0319] In certain embodiments, the polypeptide complexes provided herein are used as the base of a conjugate.

[0320] VI Composition In another aspect, the present invention provides a composition comprising a polypeptide complex or conjugate described herein and a pharmaceutically acceptable carrier.

[0321] VII Medical Use In another aspect, the present invention provides a method for treating a condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide complex of the invention, a pharmaceutical composition described herein, a conjugate described herein, or a composition described herein.

[0322] As used herein, the terms "subject" or "individual" or "animal" or "patient" refer to a human or non-human animal, including a mammal or primate, in need of diagnosis, prognosis, amelioration, prevention, and / or treatment of a disease or disorder. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, and bears. In certain embodiments, the subject is a human.

[0323] As used herein, "treating" a condition may include alleviating the condition, slowing the onset or rate of progression of the condition, slowing the progression of symptoms associated with the condition, reducing or halting symptoms associated with the condition, achieving complete or partial regression of the condition, or any combination thereof.

[0324] As used herein, the terms "disorder," "disease," or "condition," or the like, refer to a condition affecting a subject amenable to treatment with a polypeptide conjugate.

[0325] As used herein, the term "therapeutically effective amount" of a therapeutic agent refers to an amount of the therapeutic agent that, when administered to a subject in an appropriate manner, produces a sufficient therapeutic effect in the subject. As with other therapeutic agents, it is understood that the therapeutically effective amount of the polypeptide conjugates provided above will be affected by various factors known in the art, such as the subject's weight, age, past medical history, current drug treatments, health status and cross-reactions, allergies, hypersensitivity, and potential side effects, as well as the route of administration and the extent of disease onset. Doses can be proportionally increased or decreased by a skilled artisan (e.g., a physician or veterinarian) depending on these and other circumstances or requirements.

[0326] In certain embodiments, the polypeptide conjugates provided herein may be administered in a therapeutically effective amount of about 0.01 mg / kg to about 100 mg / kg. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered, or multiple doses may be administered over time.

[0327] The polypeptide conjugates and methods disclosed herein can be applied to the treatment of a wide variety of diseases. Diseases that are contemplated as treatable by the polypeptide conjugates and methods disclosed herein in humans, and in other primates, may include:

[0328] (1) Cancer and other hyperproliferative disorders, including both benign and malignant tumors, leukemia, and lymphoid tumors. Depending on the type of cell harboring the cancer or hyperproliferative disorder, examples include malignant tumors of neurons, glial cells, astrocytes, hypothalamus, glandular cells, macrophages, epithelial cells, endothelial cells, and stromal cells. Depending on the organ / site affected by the cancer or hyperproliferative disorder, examples include cancers of the head, neck, eye, mouth, throat, esophagus, breast, skin, bone, lung, colon, rectum, colorectum, stomach, spleen, kidney, skeletal muscle, subcutaneous tissue, metastatic melanoma, endometrium, prostate, breast, ovary, testis, thyroid, blood, lymph node, kidney, liver, pancreas, brain, or central nervous system.

[0329] (2) Alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Sjogren's syndrome, psoriasis, atherosclerosis, diabetic retinopathy and other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangioma, thyroid hyperplasia (Graves' disease), corneal transplants and other tissue transplants, and chronic inflammation, suppuration, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion Blood flow injury, sepsis, endotoxin shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock, organ ischemia, reperfusion injury, spinal cord injury and allograft rejection, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac disease-dermatitis, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, Autoimmune and / or inflammatory disorders, including polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, e.g., dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis. Inflammatory disorders may further include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation due to chronic viral or bacterial infection.

[0330] (3) Infectious and parasitic diseases, such as those caused by viruses (e.g., HBV, HCV, HIV, RSV, hMPV, PIV, coronavirus, or influenza virus), fungi (e.g., Naegleria, Aspergillus, Blastomyces, Histoplasma, Candida, or Tinea), eukaryotic microorganisms (e.g., Giardia, Toxoplasma, Plasmodium, Trypanosoma, and Entamoeba), and bacteria (e.g., Staphylococcus, Streptococcus, Pseudomonas, Clostridium, Borrelia, Vibrio, and Neisseria).

[0331] (4) Other diseases or disorders, including those not covered by any of (1) to (3) above, such as cardiovascular disease, neurological disorders, neuropsychiatric conditions, trauma, or coagulation disorders.

[0332] The polypeptide conjugates disclosed herein can be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0333] In some embodiments, the polypeptide conjugates disclosed herein may be administered alone or in combination with one or more additional therapeutic procedures or agents, for example, the polypeptide conjugates disclosed herein may be administered in combination with another therapeutic agent, such as a chemotherapeutic or anti-cancer agent.

[0334] In some of these embodiments, a polypeptide complex disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents; in some of these embodiments, the polypeptide complex and the additional therapeutic agents may be administered as part of the same pharmaceutical composition. However, a polypeptide complex administered "in combination with" another therapeutic agent is not necessarily administered simultaneously with that agent or in the same composition. A polypeptide complex administered before or after another agent is considered to be administered "in combination with" that agent, as that term is used herein, even if the polypeptide complex and the second agent are administered by different routes. Where possible, additional therapeutic agents administered in combination with a polypeptide complex disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to protocols known in the art, such as the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)).

[0335] VIII. Methods of antigen detection In another aspect, the present disclosure provides methods for detecting the presence or amount of an antigen in a sample. In some embodiments, the method comprises contacting a sample suspected of containing the antigen with a polypeptide complex described herein and determining the formation of a complex between the antigen and the polypeptide complex. In some embodiments, the antigen is a disease-associated antigen, e.g., a tumor antigen. In certain embodiments, the polypeptide complexes disclosed herein are used in a method for diagnosing a subject suffering from a disease (e.g., cancer), the method comprising contacting a sample obtained from a subject with a polypeptide complex of the present disclosure and determining the presence or amount of a disease-associated (e.g., tumor-associated) antigen in the sample by detecting the presence of an antigen-binding polypeptide complex.

[0336] Any sample suspected of containing tumor antigens can be used, for example, biological fluids such as blood samples, plasma samples, and urine samples, and biopsies of diseased tissue (eg, tumor tissue).

[0337] The presence or level of a disease-associated (e.g., tumor) antigen in a sample may be determined based on detecting the presence or level of a complex of the disease-associated (e.g., tumor) antigen bound by a polypeptide complex or antigen-binding fragment thereof disclosed herein. Any method suitable for such detection can be used, including, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (e.g., Western blotting), flow cytometry (e.g., 15FACS®), immunoassays such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), and radioimmunoassay (RIA).

[0338] For a review of immunological analysis and immunoassay procedures, see Basic and Clinical Immunology (Stites & Terr eds., 7th ed. 1991). Immunoassays can also be performed in any of several configurations, which are extensively reviewed in Enzyme Immunoassay (Maggio, ed., 1980) and Harlow & Lane, supra. For reviews of immunoassays in general, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993) and Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).

[0339] In certain embodiments, the polypeptide complexes disclosed herein are detectably labeled by a conjugated payload or are unlabeled but capable of reacting with a detectably labeled second molecule (e.g., a detectably labeled secondary antibody).

[0340] In certain embodiments, the polypeptide complexes disclosed herein may be immobilized on a solid substrate. Immobilization can be achieved via covalent or non-covalent attachment (e.g., coating). Examples of solid substrates include porous and non-porous materials, latex particles, magnetic particles, microparticles, strips, beads, membranes, microtiter wells, and plastic tubes. The choice of solid phase material and the method of detectably labeling may be determined based on the performance characteristics of the desired assay format.

[0341] The level of antigen can be determined, for example, by normalizing to a control value or a standard curve. The control value may be predetermined or determined contemporaneously.

[0342] The assays and methods for measuring antigen levels provided herein may be adapted or optimized for use in automated and semi-automated systems or point-of-care assay systems.

[0343] The following examples are provided to better illustrate the present invention and should not be construed as limiting the scope of the present invention. All specific compositions, materials, and methods described below are within the scope of the present invention, in whole or in part. These specific compositions, materials, and methods are not intended to limit the present invention, but are merely intended to illustrate specific embodiments within the scope of the present invention. Those skilled in the art will be able to develop equivalent compositions, materials, and methods without creative effort and without departing from the scope of the present invention. It will be understood that many variations can be made to the procedures described herein within the scope of the present invention. It is the intention of the inventors that such variations are included within the scope of the present invention.

[0344] Example Example 1 1.1 2:2:2 Construction A series of 2:2:2 symmetric triabodies, namely 04A25 (see Figure 1), 04A26 (see Figure 4), 04A27 (see Figure 7), and 04A28 (see Figure 10), were constructed in four different formats or structures. Each triabody is composed of two symmetric halves, with each half composed of three different polypeptide chains. Each polypeptide chain comprises one or more polypeptide fragments, namely a first polypeptide fragment (F1), a second polypeptide fragment (F2), a third polypeptide fragment (F3), and a fourth polypeptide fragment (F4), designated as 1, 2, 3, and 4 in Figures 1, 4, 7, and 10. Each polypeptide fragment is composed of one or more domains, summarized below in Table 1, which provides a more detailed description of the domains in triabodies. [Table 4-1] [Table 4-2]

[0345] 1.2 Gene synthesis, expression, and purification of 2:2:2 symmetric trispecific antibodies Before proceeding to synthesis, the gene sequence encoding the polypeptide chain was codon-optimized using OptimumGene (Genscript). The target gene was first constructed in the PUC57 vector and then subcloned into the pTGE5 vector. DNA for transfection was prepared using Maxiprep (ThermoFisher).

[0346] 0.3 × 10 CHO3E7 cells 6 The cells were cultured and subcultured at a cell density of 1.8–2.5 × 10 6Transfection was performed when the total cell / ml concentration reached 1000 μl. 300 μl of DNA for each of the heavy and light chains (a total of three polypeptide chains) was first added to 50 ml of Freestyle CHO medium (ThermoFisher) and gently mixed. 3 mg of PEI transfection reagent was then added and gently mixed for an additional 3 minutes. The mixture was left to stand at 37°C for 7 minutes before being added to 450 ml of cell suspension for a total volume of 500 ml. 24 hours later, 25 ml of TN1 stock (200 g / L) was added to the mixture.

[0347] One ml of the suspension was taken and tested on days 1, 3, and 5 after transfection. 50 μl of each sample was used for cell counting, and the remainder was centrifuged at 3,000 rpm for 5 minutes, after which the supernatant was kept at -20°C. On day 6, the culture was harvested and centrifuged at 5,500 rpm for 30 minutes. The supernatant was separated, filtered through a 0.22 μm filter, and further purified to obtain the protein.

[0348] Column: 5 ml Monofinity A Resin (GenScript, Cat. No. L00433) column, Equilibration Buffer A: 20 mM PB, 150 mM NaCl, pH 7.2, Wash Buffer B: 50 mM tartaric acid, pH 3.5, Neutralization Buffer C: 1 M Tris-HCl, pH 9.0, Flow Rate: 2 ml / min, Gradient: 100% gradient wash. After fractionation, 0.155 ml of Neutralization Buffer C was added to each 1 ml fraction. The collected protein solution was dialyzed against PBS at pH 7.2 at 4°C for 16 hours.

[0349] 1.3 Expression analysis of each 2:2:2 symmetric trispecific antibody The above samples were analyzed by SDS-PAGE followed by Western blot. Additionally, the samples were purified by SEC to evaluate their properties and activity.

[0350] 1) SDS-PAGE Samples were analyzed by SDS-PAGE followed by Coomassie blue staining.

[0351] 2) Size Exclusion-High Performance Liquid Chromatography (SEC-HPLC) The samples were centrifuged at 10,000 rpm for 5 min at 15°C. The supernatant was collected and analyzed. The relevant SEC chromatography parameters were as follows: Mobile phase A: 100mM PBS pH6.7, Mobile phase B: ultrapure water, Flow rate: 0.35ml / min, Wavelength: 280nm, Column temperature: room temperature, Sample analysis time: 20 min, Injection amount: 20 μg.

[0352] 1.4 Analysis results According to the experimental results shown in Table 2, except for type B (4A26), the four aforementioned symmetric 2:2:2 trispecific antibodies have high purity (Figures 2, 3, 5, 6, 8, 9, 11, 12). [Table 5]

[0353] Example 2 2.1 Construction of 2:1:1 asymmetric trispecific antibodies Based on the above-mentioned types A, B, C, and D structures of the four previously described 2:2:2 symmetric triabodies, asymmetric structures can be applied to construct 2:1:1 triabodies by introducing knob-into-hole mutations into the CH3 domain. Various 2:1:1 asymmetric triabodies structures can be formed according to different construction methods (Table 3).

[0354] A series of 2:1:1 symmetric triabodies, namely 12A6 (see Figure 13), 12A10 (see Figure 14), 12A14 (see Figure 15), 12A15 (see Figure 18), 12A16 (see Figure 21), and 12A17 (see Figure 24), were constructed in five different formats or structures. Each of these triabodies is composed of two asymmetric halves linked via a heterodimerized Fc domain. 12A6 and 12A10 are composed of four distinct polypeptide chains. 12A14, 12A15, 12A16, and 12A7 are composed of five polypeptide chains, two of which are identical, resulting in a total of four distinct polypeptide chains. Each polypeptide chain comprises one or more polypeptide fragments, i.e., a first polypeptide fragment (F1), a second polypeptide fragment (F2), a third polypeptide fragment (F3), and a fourth polypeptide fragment (F4), designated as 1, 2, 3, and 4 in Figures 13, 14, 15, 18, 21, and 24. Each polypeptide fragment is composed of one or more domains, summarized below in Table 3, which provides a more detailed description of the domains in triabodies. [Table 6-1] [Table 6-2] [Table 6-3]

[0355] 2.2 Gene synthesis, expression, and purification of 2:1:1 asymmetric trispecific antibodies The experimental procedure for Example 2.2 is the same as that for Example 1.2, except for the cell transfection step, in which 300 ul of DNA for each of the four polypeptide chains (instead of three) was first added to 50 ml of Freestyle CHO medium and gently mixed.

[0356] 2.3 Expression analysis of each 2:1:1 asymmetric trispecific antibody Both SDS-Page and SEC-HPLC were used to analyze the expression of each 2:1:1 asymmetric trispecific antibody, and the experimental procedures were the same as in Example 1.3.

[0357] 2.4 Analysis results Purity analysis was performed on all previously described 2:1:1 asymmetric trispecific antibodies (Figures 16, 17, 19, 20, 22, 23, 25, and 26), and antibodies constructed with Type E and Type G structures showed high expression purity (Table 4). [Table 7]

[0358] Example 3 The in vitro activity of antibodies 12A14, 12A15, 12A16, and 12A17, which have the aforementioned type E, type F, type G, and type H structures, respectively, was tested and compared with antibody 12A6, constructed using the TRIAD platform, to determine the effect of different structures on in vitro activity.

[0359] 3.1 Reporter assay Experimental Method 1) Jurkat-NFAT cells, obtained by integrating a luciferase gene into the NFAT element of Jurkat cells, were cultured in RPMI-1640 medium supplemented with 10% FBS and 1 μg / ml puromycin, and gastric cancer cell line KATO3 (or colorectal adenocarcinoma cell line HT29, colorectal adenocarcinoma cell line LS147T) cells were cultured in RPMI-1640 medium combined with 10% FBS.

[0360] 2) Jurkat-NFAT, KATO3 (or HT29, LS147T) cells were centrifuged, resuspended in RPMI-1640 medium supplemented with 10% FBS, and plated in a 96-well plate at 2 x 10 cells in a total mixed culture volume of 100 μl. 4 KATO3 (or HT29, LS147T) cells / well and 4*10 4 Jurkat cells / well were counted.

[0361] 3) The antibody was diluted in PBS to different gradients, and 10 μl of the antibody solution was added to each well. After 4 hours of incubation, 100 μl of ONE-Glo™ Reagent (Promega) was added to each well. TCR / CD3 on the surface of Jurkat cells was activated to transmit intracellular signals, which activated the NF-κB response element and stimulated luciferase expression. The generated luminescence was detected and recorded.

[0362] Experimental results The reporter assay experimental data clearly showed that 12A14 (Type E) performed best, followed by 12A17 (Type H), and then 12A15 (Type F) (Figures 27-29). Of all the 2:1:1 asymmetric trispecific antibodies, only 12A14 consistently outperformed 12A10, suggesting the superiority of the 2:1:1 Type E antibody structure over the TRIAD structure.

[0363] 3.2 Killing assay Experimental Method 1) HT29 (or LS147T) cells were cultured under good conditions, and the medium was removed. Then, the cells were digested with trypsin, resuspended in RPMI-1640 medium supplemented with 2% FBS, centrifuged at 1000 rpm for 5 minutes, counted, and seeded into a 96-well cell culture plate at 30,000 cells / 100 μl / well and incubated overnight in a cell incubator.

[0364] 2) Peripheral blood mononuclear cells (PBMCs) were collected and centrifuged at 1000 rpm for 5 minutes. The PBMCs were then resuspended in RPMI-1640 medium supplemented with 2% FBS, counted, and added to HT29 and LS147T cell plates at 30,000 cells / 100 μl / well. Each antibody drug was diluted with PBS, and 10 μl / well of each antibody drug solution was added to the cell culture plate, with two replicate wells per concentration. The cell culture plate was incubated at 37°C in a 5% CO2 incubator for 72 hours.

[0365] 3) The medium was carefully removed from the cell culture plate, 100 μl of PBS was added to each well, and the wells were washed again. Then, 100 μl of RPMI-1640 medium supplemented with 2% FBS and 10 μl of CCK8 assay solution were added to each well, and the cells were incubated for 1 to 2 hours.

[0366] 4) The OD450 value was measured using a microplate reader.

[0367] Experimental results The experimental data from the killing assays reveal that 12A14 and 12A15 performed best, while 12A15 and 12A16 performed worse (Figures 30 and 31). Of all the 2:1:1 asymmetric triabodies, 12A14 and 12A15 consistently outperformed the 1:1:1 TRIAD structure of 12A10.

[0368] Example 4 Based on the results of the preceding examples, 12A14 exhibited higher purity and performed better in in vitro assays, suggesting the advantages of the type E antibody structure. Therefore, a series of 2:1:1 trispecific antibodies based on the type E structure were next constructed for in vivo studies to evaluate the advantages of the TRIAD structure compared to 1:1:1 trispecific antibodies.

[0369] 4.1 In vitro assays 4.1.1 Reporter assay Experimental Method The experimental procedure was the same as the reporter assay described in Example 3.1, except that different antibodies such as 12A6, 12A10 and 08B1 (Figure 32) were used.

[0370] Experimental results Both 8B1 and 12A6 are trivalent bispecific antibodies with two CEA-target binding fractions and one CD3-target binding fraction. However, 8B1 did not exhibit significant CD3 agonist activity in reporter assays at a concentration of 1000 ng / ml, whereas 12A6 and 12A10 clearly demonstrated CD3 activation (Figures 33 and 34). It has been shown that 12A6 or 8B1 alone cannot activate Jurkat-NFAT. Therefore, under the conditions of this experiment, 12A6-mediated Jurkat-NFAT agonism is target cell (KATO3) dependent.

[0371] 4.2 In vivo testing Experimental Method Six-week-old female NCG mice were inoculated into the right dorsum with 0.1 ml of LS174T cells (5 × 10 6 Three days after inoculation, each mouse was subcutaneously inoculated with 0.1 ml of PBMC (1 × 10 7 Ten days after inoculation, the mice were randomly divided into four groups of six mice each, and the average tumor volume in each group was approximately 220 mm. 3 Mice from different groups were administered negative control (PBS), 08B1 (5 mpk), 12A6 (5 mpk) and 12A10 (5 mpk) into the tail vein twice a week for 2 weeks, and then observed for 1 week.

[0372] The effect of the drug on tumor growth was specifically monitored as T / C% or tumor growth inhibition rate (TGI%). Tumor diameters were measured twice a week with a caliper, and tumor volume (V) was calculated using the following formula:

[0373] Metrics

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[0374]

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[0375]

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[0376] Experimental results Tumor-bearing mice were administered a negative control (PBS), 08B1 (5 mg / kg), 12A6 (5 mg / kg), or 12A10 (5 mg / kg) via the tail vein three times a week for two weeks, followed by a one-week cessation of administration. In the negative control group, the mean tumor volume was 2505.8 ± 302.3 mm on day 10. 3 In mice treated with 12A10 (5 mg / kg), tumor volume continued to grow, reaching 1638.7 ± 223.2 mm on day 10. 3 The tumor growth was significantly inhibited in both groups of mice administered 08B1 (5 mg / kg) and 12A6 (5 mg / kg), with tumor volumes reaching 968.6 ± 173.1 mm on day 10, respectively. 3 (TGI: 67.0, P<0.01) and 508.7±119.5mm 3 (TGI: 87.2, P<0.01) (FIG. 35). After drug discontinuation (from day 15), the tumor volume in the 08B1-treated group increased significantly more than that in the 12A6-treated group.

[0377] The results showed that both 08B1 and 12A6 had moderate tumor growth inhibitory effects, with 12A6 showing a stronger and longer-lasting inhibitory effect than 08B1, which is consistent with the results observed in the reporter assay.

[0378] However, although the trispecific antibody 12A10 (CD3 / 4-1BB / CEA) with a triad structure showed a CD3 agonist effect comparable to that of 12A6 in in vitro reporter assays (see Figures 33 and 34), it did not show significant tumor inhibition in in vivo antitumor tests (see Figure 35). In contrast, the 2:1 bispecific antibody 12A6 (CD3 / CEA / CEA) with a triad structure showed significant tumor inhibition (see Figure 35). This difference may be due to the fact that 12A10 was monovalent for the tumor-associated antigen (TAA), i.e., CEA.

[0379] Example 5 To compare whether monovalent binding to TAAs caused the aforementioned differences in drug efficacy, we constructed the 2:1:1 trispecific antibody 12A14 (CEA / CEA / CD3 / 4-1BB) using the preferred Type E structure for comparison.

[0380] 5.1 In vitro assays 5.1.1 Reporter assay Experimental Method The experimental procedure was the same as the reporter assay described in Example 3.1, except that different antibodies such as 12A6, 12A14, and 12A19 (Figures 36-38) were used, of which 12A19 is identical to 12A10 except that the CEA-binding domain is humanized.

[0381] Experimental results Compared with 12A6 and 12A14 (which have two TAA-binding domains), 12A19 (which is monovalent for TAA) exhibits the lowest CD3 agonist activity in both reporter assays using HT29 cells (Figure 36) and LST174 cells (Figure 38), respectively. Furthermore, 12A14 has the best performance in all reporter assays using three different cells (Figures 36-38). Thus, increasing the number of TAA-binding domains appears to increase the efficacy of multispecific antibodies, and a 2:1:1 trispecific antibody constructed with a type E structure containing bivalent TAA-binding domains is superior to a 1:1:1 trispecific antibody constructed with a TRIAD structure containing a single TAA-binding domain.

[0382] 5.1.2 Killing assay Experimental Method The experimental procedure is the same as the killing assay described in Example 3.2, except that different antibodies such as 12A10, 12A14, 12A15, 12A16 and 12A17 (Figures 30, 31) were used.

[0383] Experimental results Of all the 2:1:1 asymmetric trispecific antibodies, 12A14 and 12A15 consistently outperformed the 1:1:1 TRIAD structure 12A10, which is consistent with the reporter assay results in Example 5.1.1, which show that bivalent TAA binding domains perform better than monovalent TAA binding domains.

[0384] 5.2 In vivo assays Experimental Method Six-week-old female NCG mice were inoculated into the right dorsum with 0.1 ml of LS174T cells (5 × 10 6 Three days after inoculation, each mouse was subcutaneously inoculated with 0.1 ml of PBMC (1 × 10 7 Ten days after inoculation, the mice were randomly divided into five groups of six mice each, and the average tumor volume in each group was approximately 200 mm 3 Negative control (PBS), 12A6 (5 mpk) and 12A14 (2 mpk) were administered via the tail vein to mice from different groups twice a week, a total of five times.

[0385] Metrics

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[0386]

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[0387]

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[0388] Experimental results Tumor-bearing rats receiving tail vein injections of the negative control (PBS), 12A6 (5 mpk), and 12A14 (2 mpk) showed good tumor growth inhibition after five consecutive twice-weekly administrations (Figure 39). On day 16, the mean tumor volumes of the negative control (PBS), 12A6 (5 mpk), and 12A14 (2 mpk) were 4696.75 ± 498.91 mm3, 1757.00 ± 502.23 mm3 (TGI: 62.59, P > 0.05), and 2436.44 ± 752.83 mm3 (TGI: 48.12, P < 0.01), respectively.

[0389] The results showed that both 12A6 (5 mpk) and 12A14 (2 mpk) showed significant tumor growth inhibition under the conditions of this experiment. No significant abnormalities were observed in the body weight of each administered animal during the experiment.

[0390] The tumor inhibitory effect of the 2:1:1 trispecific antibody 12A14 was not significantly different from that of 12A6, which indirectly demonstrated the superiority of 12A14 over 12A10 and suggested that the 2:1:1 trispecific antibody constructed with the type E structure was superior to the 1:1:1 trispecific antibody constructed with the TRIAD structure.

Claims

1. (a) a first dimerization domain, and (b) a first antigen-binding domain comprising a DICAD domain and a first Fv domain; 1. A multispecific polypeptide complex comprising: The DICAD domain is i) a first polypeptide fragment comprising a first heavy chain variable domain (VH1) linked to a second light chain variable domain (VL2), and ii) a second polypeptide fragment comprising a second heavy chain variable domain (VH2) linked to a first light chain variable domain (VL1); the VL1 and VH1 associate to form a first domain capable of binding to a first target, and the VL2 and VH2 associate to form a second domain capable of binding to a second target; The first Fv domain comprises: i) a third polypeptide fragment comprising a third heavy chain variable domain (VH3), and ii) a fourth polypeptide fragment comprising a third light chain variable domain (VL3); the VH3 and VL3 associate to form a third domain capable of binding to a third target; and, one of the termini of the DICAD domain is operably linked to one of the termini of the first Fv domain; a multispecific polypeptide complex, wherein one of the C-termini of said first antigen-binding domain is operably linked to the N-terminus of said first dimerization domain.

2. the N-terminus of the VH1 is linked to the C-terminus of the VL2, and the N-terminus of the VH2 is linked to the C-terminus of the VL1; or The multispecific polypeptide complex of claim 1, wherein the N-terminus of VL1 is linked to the C-terminus of VH2 and the N-terminus of VL2 is covalently linked to the C-terminus of VH1.

3. 2. The multispecific polypeptide complex of claim 1, wherein one of the first domain and the second domain comprises a first non-natural covalent bond formed between a first pair of two amino acid residues.

4. 4. The multispecific polypeptide complex of claim 3, wherein the other of the first domain and the second domain does not comprise a non-natural covalent bond or comprises a second non-natural covalent bond formed between a pair of two amino acid residues different from the pair of amino acid residues corresponding to the first pair of two amino acid residues.

5. The multispecific polypeptide complex of claim 3 , wherein the first non-natural covalent bond is a non-natural disulfide bond.

6. The multispecific polypeptide complex of claim 5 , wherein the non-natural disulfide bond is formed between two non-natural cysteine ​​residues.

7. 7. The multispecific polypeptide complex of claim 6, wherein the two non-native cysteine ​​residues are at position 44 in the VH and position 100 in the VL, or at position 105 in the VH and position 43 in the VL, or at position 100 in the VH and position 49 in the VL, or at position 100 in the VH and position 150 in the VL, wherein numbering is according to the Kabat index.

8. The multispecific polypeptide complex of claim 1, wherein the VL1 and the VH1 are associated by a first non-natural disulfide bond.

9. The multispecific polypeptide complex of claim 8, wherein the first non-natural disulfide bond is formed between two non-natural cysteine ​​residues in the VL1 and VH1, respectively.

10. The multispecific polypeptide complex of claim 8, wherein the first non-natural disulfide bond is formed between two non-natural cysteine ​​residues in FR2 of the VL1 and an amino acid residue in FR4 of the VH1, respectively.

11. 9. The multispecific polypeptide complex of claim 8, wherein the first non-natural covalent bond is formed between two non-natural cysteine ​​residues located at position 44 in the VH1 and position 100 in the VL1, or at position 105 in the VH1 and position 43 in the VL1, or at position 100 in the VH1 and position 49 in the VL1, or at position 100 in the VH1 and position 150 in the VL1, wherein numbering is according to the Kabat index.

12. The multispecific polypeptide complex of claim 1, wherein the VL2 and the VH2 are associated by a first non-natural disulfide bond.

13. The multispecific polypeptide complex of claim 12, wherein the first non-natural disulfide bond is formed between two non-natural cysteine ​​residues in the VL2 and VH2, respectively.

14. 13. The multispecific polypeptide complex of claim 12, wherein the first non-natural disulfide bond is formed between two non-natural cysteine ​​residues in FR2 of the VL2 and an amino acid residue in FR4 of the VH2, respectively.

15. 13. The multispecific polypeptide complex of claim 12, wherein the first non-naturally occurring covalent bond is formed between two non-naturally occurring cysteine ​​residues at position 44 in the VH2 and position 100 in the VL2, or at position 105 in the VH2 and position 43 in the VL2, or at position 100 in the VH2 and position 49 in the VL2, or at position 100 in the VH2 and position 150 in the VL2, wherein numbering is according to the Kabat index.

16. 4. The multispecific polypeptide complex of claim 3, wherein one of the first domain and the second domain that comprises the first non-natural covalent bond further comprises a non-natural pair of two oppositely charged amino acid residues.

17. (a) the two oppositely charged residues are at position 38 in the VL1 and position 39 in the VH1, respectively; (b) the two oppositely charged residues are at position 40 in the VL1 and position 39 in the VH1, respectively; (c) the two oppositely charged residues are at position 37 in the VL1 and position 39 in the VH1, respectively; or (d) the two oppositely charged residues are at position 37 in the VL1 and position 39 in the VH1, respectively; 17. The multispecific polypeptide complex of claim 16, wherein the numbering is according to the Kabat index.

18. The multispecific polypeptide complex of claim 4, wherein the VL2 and the VH2 are associated by the second non-native disulfide bond, and the VL1 and the VH1 are associated by either a natural disulfide bond or another non-native disulfide bond formed at a position different from the position of the second non-native disulfide bond.

19. 19. The multispecific polypeptide complex of claim 18, wherein the second non-native disulfide bond is formed between two non-native cysteine ​​residues in the VL2 and the VH2, respectively, and optionally the two non-native cysteine ​​residues are Q100C or G100C in the VL2 and G44C or A44C in the VH2, wherein numbering is according to the Kabat index.

20. 20. The multispecific polypeptide complex of claim 19, wherein the VL2 and the VH2 are further associated by electrostatic interactions between two oppositely charged residues.

21. (a) the two oppositely charged residues are introduced to replace Q38 in the VL2 and Q39 in the VH2, respectively; (b) the two oppositely charged residues are introduced to replace Q40 in the VL2 and Q39 in the VH2, respectively; (c) the two oppositely charged residues are introduced to replace Q37 in the VL2 and Q39 in the VH2, respectively; or (d) the two oppositely charged residues are introduced to replace Q37 in the VL2 and Q39 in the VH2, respectively; 21. The multispecific polypeptide complex of claim 20, wherein the numbering is according to the Kabat index.

22. 21. The multispecific polypeptide complex of claim 16 or 20, wherein the two oppositely charged residues comprise a negatively charged amino acid residue selected from aspartic acid (D) or glutamic acid (E), and a positively charged amino acid residue selected from lysine (K), histidine (H) or arginine (R).

23. 21. The multispecific polypeptide complex of claim 16 or 20, wherein the two oppositely charged residues comprise Q38D in the VL1 and Q39K in the VH1, respectively, or Q38D in the VL2 and Q39K in the VH2, respectively, wherein numbering is according to the Kabat index.

24. The multispecific polypeptide complex of claim 1, wherein the VL1 is linked to the VH2 via a first peptide linker and the VL2 is linked to the VH1 via a second peptide linker.

25. The multispecific polypeptide complex of claim 24, wherein the first peptide linker and the second peptide linker each independently comprise 5 to 9 amino acids.

26. 10. The multispecific polypeptide complex of claim 9, wherein the third polypeptide fragment further comprises an antibody heavy chain constant region (CH1) operably linked to the C-terminus of the VH3 domain, and / or the fourth polypeptide fragment further comprises an antibody light chain constant region (CL) operably linked to the C-terminus of the VL3 domain.

27. The multispecific polypeptide complex of claim 1, wherein one of the N-termini of the DICAD domain is operably linked to one of the C-termini of the first Fv domain.

28. 27. The multispecific polypeptide complex of claim 26, wherein one of the N-termini of the DICAD domain is operably linked to the C-terminus of the third polypeptide fragment of the Fv domain.

29. 27. The multispecific polypeptide complex of claim 26, wherein the N-terminus of the second polypeptide fragment of the DICAD domain is operably linked to the C-terminus of the third polypeptide fragment of the first Fv domain.

30. 27. The multispecific polypeptide complex of claim 26, wherein the N-terminus of the first polypeptide fragment of the DICAD domain is operably linked to the C-terminus of the third polypeptide fragment of the first Fv domain.

31. 27. The multispecific polypeptide complex of claim 26, wherein one of the N-termini of the DICAD domain is operably linked to the C-terminus of the fourth polypeptide fragment of the first Fv domain.

32. 27. The multispecific polypeptide complex of claim 26, wherein the N-terminus of the first polypeptide fragment of the DICAD domain is operably linked to the C-terminus of the fourth polypeptide fragment of the first Fv domain.

33. The multispecific polypeptide complex of claim 1, wherein one of the C-termini of the DICAD domain is operably linked to one of the N-termini of the Fv domain.

34. The multispecific polypeptide complex of claim 33, wherein the C-terminus of the second polypeptide fragment of the DICAD domain is operably linked to the N-terminus of the third polypeptide fragment of the first Fv domain.

35. The multispecific polypeptide complex of claim 33, wherein the C-terminus of the first polypeptide fragment of the DICAD domain is operably linked to the N-terminus of the fourth polypeptide fragment of the first Fv domain.

36. 35. The multispecific polypeptide complex of any one of claims 26 to 30 and 33 to 34, wherein the C-terminus of the first polypeptide fragment of the first antigen-binding domain is operably linked to the N-terminus of the first dimerization domain.

37. 36. The multispecific polypeptide complex of any one of claims 31 to 32 and 35, wherein the C-terminus of the third polypeptide fragment of the first antigen-binding domain is operably linked to the N-terminus of the first dimerization domain.

38. 10. The multispecific polypeptide complex of claim 1, wherein the first dimerization domain comprises an IgG CH3 domain.

39. 39. The multispecific polypeptide complex of claim 38, wherein the first dimerization domain further comprises an IgG CH2 domain and / or hinge region.

40. 10. The multispecific polypeptide complex of claim 1, wherein both the first domain and the second domain comprise a TGFβ targeting domain and the third domain comprises a PD-1 targeting domain.

41. The multispecific polypeptide complex of claim 40, wherein the TGFβ targeting domain comprises HCDR1, HCDR2 and HCDR3 identical to the HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence of SEQ ID NO: 38, and LCDR1, LCDR2 and LCDR3 identical to the LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence of SEQ ID NO:

37.

42. The multispecific polypeptide complex of claim 40, wherein the TGFβ targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, each comprising the amino acid sequence of SEQ ID NO: 22 / 23 / 24 / 19 / 20 / 21, respectively.

43. 43. The multispecific polypeptide complex of any one of claims 40 to 42, wherein the first domain comprises the TGFβ targeting domain, and the TGFβ targeting domain further comprises a first non-natural covalent bond (e.g., a disulfide bond) formed between a first pair of two amino acid residues (e.g., two non-natural cysteine ​​residues) and / or further comprises a non-natural pair of two oppositely charged amino acid residues.

44. 41. The multispecific polypeptide complex of claim 40, wherein the first domain comprises a TGFβ targeting domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 50 and a VL domain comprising the amino acid sequence of SEQ ID NO:

49.

45. 45. The multispecific polypeptide complex of claim 44, wherein the second domain comprises a TGFβ targeting domain wherein the VH2 comprises the amino acid sequence of SEQ ID NO: 38 and the VL2 comprises the amino acid sequence of SEQ ID NO:

37.

46. 46. ​​The multispecific polypeptide complex of any one of claims 40 to 45, wherein the PD-1 targeting domain comprises HCDR1, HCDR2, and HCDR3 identical to the HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence of SEQ ID NO: 40, and LCDR1, LCDR2, and LCDR3 identical to the LCDR1, LCDR2, and LCDR3 contained in the amino acid sequence of SEQ ID NO:

39.

47. 46. ​​The multispecific polypeptide complex of any one of claims 40 to 45, wherein the PD-1 targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each comprising the amino acid sequences of SEQ ID NOs: 28 / 29 / 30 / 25 / 26 / 27, respectively.

48. 46. ​​The multispecific polypeptide complex of any one of claims 40 to 45, wherein the third domain comprises a PD-1 targeting domain, wherein the VH3 comprises the amino acid sequence of SEQ ID NO: 40 and the VL3 comprises the amino acid sequence of SEQ ID NO:

39.

49. a) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO:62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO:59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO:53; b) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 67, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 56; c) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 58, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 53; or d) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 62, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 59, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 61, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO:

53.

50. The multispecific polypeptide complex comprises: a) SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53; b) SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56; c) SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59, or d) SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 59; 41. The multispecific polypeptide complex of claim 40, comprising three polypeptide chains each having the amino acid sequence:

51. 40. The multispecific polypeptide complex of any one of claims 1 to 39, wherein at least one or both of the first target and the second target is an immunostimulatory target.

52. 52. The multispecific polypeptide complex of claim 51 , wherein the third target is a tumor antigen.

53. 53. The multispecific polypeptide complex of claim 52, wherein the first domain and the second domain comprise a CD3 targeting domain and a 4-1BB targeting domain, respectively, and the third domain comprises a CEA targeting domain.

54. 54. The multispecific polypeptide complex of claim 53, wherein the first domain comprises a 4-1BB targeting domain and the second domain comprises a CD3 targeting domain, while the third domain comprises a CEA targeting domain.

55. 55. The multispecific polypeptide complex of claim 54, wherein said first domain comprises the 4-1BB targeting domain, wherein in said 4-1BB targeting domain, said VH1 and said VL1 comprise the first non-natural disulfide bond formed between said two non-natural cysteine ​​residues and further comprise said first non-natural pair of oppositely charged amino acid residues.

56. 55. The multispecific polypeptide complex of claim 54, wherein the 4-1BB targeting domain comprises HCDR1, HCDR2 and HCDR3 identical to the HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence of SEQ ID NO: 32, and LCDR1, LCDR2 and LCDR3 identical to the LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence of SEQ ID NO:

31.

57. The multispecific polypeptide complex of claim 54, wherein the 4-1BB targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, each comprising the amino acid sequence of SEQ ID NO: 4 / 5 / 6 / 1 / 2 / 3, respectively.

58. 55. The multispecific polypeptide complex of claim 54, wherein the first domain comprises a 4-1BB targeting domain, wherein the VH1 comprises the amino acid sequence of SEQ ID NO: 48 and the VL1 comprises the amino acid sequence of SEQ ID NO:

47.

59. 55. The multispecific polypeptide complex of claim 54, wherein the CD3 targeting domain comprises HCDR1, HCDR2 and HCDR3 identical to the HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence of SEQ ID NO: 34, and LCDR1, LCDR2 and LCDR3 identical to the LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence of SEQ ID NO:

33.

60. The multispecific polypeptide complex of claim 54, wherein the CD3 targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each comprising the amino acid sequence of SEQ ID NO: 10 / 11 / 12 / 7 / 8 / 9, respectively.

61. 55. The multispecific polypeptide complex of claim 54, wherein the second domain comprises a CD3 targeting domain wherein the VH2 comprises the amino acid sequence of SEQ ID NO: 34 and the VL2 comprises the amino acid sequence of SEQ ID NO:

33.

62. The multispecific polypeptide complex of claim 54, wherein the CEA targeting domain comprises HCDR1, HCDR2 and HCDR3 identical to the HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence of SEQ ID NO: 36, 42 or 44, and LCDR1, LCDR2 and LCDR3 identical to the LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence of SEQ ID NO: 35, 41 or 43.

63. The multispecific polypeptide complex of claim 54, wherein the CEA targeting domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, each comprising the amino acid sequence of SEQ ID NO: 16 / 17 / 18 / 13 / 14 / 15, or SEQ ID NO: 107 / 108 / 18 / 13 / 14 / 15, or SEQ ID NO: 16 / 111 / 18 / 109 / 110 / 15, respectively.

64. The multispecific polypeptide complex comprises: a) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO:87, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO:75, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO:83, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO:74; b) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 87, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 75, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 83, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 78; or c) the first polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 87, the second polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 75, the third polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 83, and the fourth polypeptide fragment comprises the amino acid sequence of SEQ ID NO: 82; 55. The multispecific polypeptide complex of claim 54, comprising:

65. 10. A multispecific polypeptide complex according to any one of the preceding claims, further comprising a second dimerization domain that associates with the first dimerization domain to form a dimer.

66. 66. The multispecific polypeptide complex of claim 65, further comprising a second antigen-binding domain, wherein one of the C-termini of said second antigen-binding domain is operably linked to the N-terminus of said second dimerization domain.

67. 67. The multispecific polypeptide complex of claim 65 or 66, wherein the second antigen-binding domain is identical to the first antigen-binding domain.

68. 68. The multispecific polypeptide complex of any one of claims 65 to 67, wherein the second dimerization domain is identical to the first dimerization domain.

69. 67. The multispecific polypeptide complex of claim 65 or 66, wherein the second antigen-binding domain is different from the first antigen-binding domain.

70. 70. The multispecific polypeptide complex of claim 69, wherein the second dimerization domain is different from the first dimerization domain and associates with the first dimerization domain to form a heterodimer.

71. 70. The multispecific polypeptide complex of claim 69, wherein the first dimerization domain and / or the second dimerization domain further comprise one or more mutations that promote heterodimerization.

72. 72. The multispecific polypeptide complex of any one of claims 65 to 71, wherein both the first dimerization domain and the second dimerization domain comprise an IgG CH3 domain.

73. the first dimerization domain comprises a first mutation and the second dimerization domain comprises a second mutation; a) the first mutation comprises T389W and / or S375C and the second mutation comprises Y438V, T389S, L391A and / or Y370C; b) the first mutation comprises D427K and / or D377K and the second mutation comprises K420D and / or K440D; c) the first mutation comprises D377K, E378K, and / or D427K, and the second mutation comprises K393E, K440D, and / or K470E; d) the first mutation comprises S387H and / or F436A and the second mutation comprises Y370T and / or T422F; e) the first mutation comprises S387H and / or T422F and the second mutation comprises Y422T and / or F436A; f) the first mutation comprises K393D and / or K440D and the second mutation comprises E378K and / or D427K; or g) the first mutation comprises L372D and / or L391E and the second mutation comprises L372K or T389K; 73. The multispecific polypeptide complex of claim 72, wherein the numbering is according to the Kabat index.

74. 73. The multispecific polypeptide complex of claim 72, wherein the first mutation comprises T389W and the second domain comprises T389S, L391A and Y438V, wherein numbering is according to the Kabat index.

75. 74. The multispecific polypeptide complex of any one of claims 66 to 73, wherein the second antigen-binding domain comprises a second Fv domain.

76. 76. The multispecific polypeptide complex of claim 75, wherein the second Fv domain is identical to the first Fv domain, and the second Fv domain comprises a fifth polypeptide fragment identical to the third polypeptide fragment comprising the third heavy chain variable domain (VH3), and a sixth polypeptide fragment identical to the fourth polypeptide fragment comprising the third light chain variable domain (VL3).

77. 52. The multispecific polypeptide complex of claim 50 or 51, wherein the C-terminus of the fifth polypeptide fragment of the second Fv domain is operably linked to the N-terminus of the second dimerization domain.

78. The second Fv domain comprises the CEA targeting domain, wherein the CEA targeting domain comprises: a) the VH3 comprises the amino acid sequence of SEQ ID NO: 36 and the VL3 comprises the amino acid sequence of SEQ ID NO: 35; b) the VH3 comprises the amino acid sequence of SEQ ID NO: 42 and the VL3 comprises the amino acid sequence of SEQ ID NO: 41; or c) the VH3 comprises the amino acid sequence of SEQ ID NO: 44 and the VL3 comprises the amino acid sequence of SEQ ID NO: 43; 78. The multispecific polypeptide complex of claim 77.

79. 78. The multispecific polypeptide complex of claim 77, wherein the fifth polypeptide fragment and the sixth polypeptide fragment comprise the amino acid sequences of a) SEQ ID NO: 73 and SEQ ID NO: 74, respectively; b) SEQ ID NO: 77 and SEQ ID NO: 78, respectively; and c) SEQ ID NO: 81 and SEQ ID NO: 82, respectively.

80. The multispecific polypeptide complex comprises: a) SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74; b) SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, or c) SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82; 78. The multispecific polypeptide complex of claim 77, comprising polypeptide chains each having the amino acid sequence:

81. 10. A multispecific polypeptide complex according to any one of the preceding claims, linked to one or more conjugate moieties.

82. 82. The multispecific polypeptide complex of claim 81 , wherein the conjugate moiety comprises an agent for detection or isolation such as a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or other anti-cancer agent.

83. 10. An isolated polynucleotide encoding a multispecific polypeptide complex according to any one of the preceding claims.

84. 84. A vector comprising the isolated polynucleotide of claim 83.

85. 85. A host cell comprising the vector of claim 84.

86. (i) a multispecific polypeptide complex according to any one of claims 1 to 82, or a polynucleotide encoding the multispecific polypeptide complex according to any one of claims 1 to 82, and (ii) one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients; A pharmaceutical composition comprising:

87. 87. The pharmaceutical composition of claim 86, further comprising an additional therapeutic agent.

88. 83. A method for expressing a multispecific polypeptide complex according to any one of claims 1 to 82, comprising culturing a host cell according to claim 85 under conditions in which the vector according to claim 84 is expressed.

89. 83. A method of treating, preventing or alleviating a disease or disorder in a subject, comprising administering to said subject a therapeutically effective amount of a multispecific polypeptide complex of any one of claims 1 to 82.

90. 88. A method for treating, preventing or alleviating a disease or disorder in a subject, the method comprising administering to said subject a therapeutically effective amount of a multispecific polypeptide complex of any one of claims 1 to 82, or said polynucleotide encoding the multispecific polypeptide complex of any one of claims 1 to 82, and / or a pharmaceutical composition of claim 86 or 87.

91. 91. The method of any one of claims 88 to 90, wherein the subject is a human.

92. 91. The method of any one of claims 88 to 90, wherein the administration is oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular.

93. 88. Use of a multispecific polypeptide complex according to any one of claims 1 to 82, a pharmaceutical composition according to claim 86 or 87, and / or said polynucleotide encoding a multispecific polypeptide complex according to any one of claims 1 to 82 in the manufacture of a medicament for the treatment, prevention or amelioration of a disease or disorder.

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