JP2025528101A - Multispecific Polypeptide Complexes - Google Patents

Abstract

The present disclosure provides novel covalently linked multispecific antibodies and uses thereof.

Description

[0001] The present invention relates to novel covalently linked multispecific antibodies and uses thereof.

[0002] Bispecific antibodies are artificial antibodies capable of binding to at least two antigens. By simultaneously targeting at least two desired targets, bispecific antibodies can offer advantages over conventional monospecific antibodies through novel and unique mechanisms. For example, blinatumomab (CD3xCD19, Amgen), which targets CD3 and CD19, can efficiently involve T cells in killing CD19-expressing tumor cells through its CD3-recognizing Fv, and has demonstrated superior efficacy compared to conventional antibodies in the treatment of ALL (acute lymphoblastic leukemia). Blinatumomab was approved for sale by the FDA as a treatment for ALL in 2014.

[0003] Numerous bispecific antibody technology platforms have been developed, including BiTE Bi-specific T-cell Engaging (Micromet, acquired by Amgen in 2012), CrossMab (Roche), DVD-Ig (Abbvie), TandAb (Affimed), and DART (Dual Antigen Re-Targeting, Macrogenics).

[0004] However, despite the advantages offered by bispecific or multispecific antibodies, they also pose challenges, for example, in terms of production. Mismatches can occur between heavy chains and/or between heavy and light chains. For bispecific or multispecific antibodies, how to efficiently and effectively remove mismatched by-products is a difficult challenge to overcome. Therefore, there is a strong need for the development of novel structures that not only provide high binding affinity to the target of interest, but are also easy to manufacture and purify downstream.

[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 novel fusion polypeptides, nucleotide sequences encoding the same, and uses thereof.

[0007] In one aspect, the present disclosure provides a fusion polypeptide comprising, from C-terminus to N-terminus, a) a first target-binding fragment A1, b) a polypeptide linker, and c) a second target-binding fragment B2, wherein the polypeptide linker has a length sufficiently short to minimize potential intramolecular interactions between A1 and B2. In some embodiments, A1 is capable of pairing with a first pairing fragment B1 to form a first target-binding domain, and B2 is capable of pairing with a second pairing fragment A2 to form a second target-binding domain. In such embodiments, A1 is configured to have lower binding affinity to B2 compared to B1, and B2 is configured to have lower binding affinity to A1 compared to A2.

[0008] In another aspect, the present disclosure provides a polypeptide complex comprising: a) a fusion polypeptide provided herein; b) a second polypeptide comprising the first pairing fragment B1; c) a third polypeptide comprising the second target-binding fragment B2; and d) a fourth polypeptide and a fifth polypeptide, each comprising the second pairing fragment A2, wherein A1 in the fusion polypeptide pairs with B1 in the second polypeptide to form a first target-binding domain, B2 in the fusion polypeptide pairs with A2 in the fourth polypeptide to form a second target-binding domain, and A2 in the fifth polypeptide pairs with B2 in the third polypeptide to form a second target-binding domain.

[0009] In some embodiments, at least one of the B1 and A1 pair and the B2 and A2 pair includes at least one arrangement capable of preventing mismatching between B1 and A2 and/or B2 and A1.

[0010] In some embodiments, A1 comprises a first antibody variable region VA1 selected from VH1 or VL1. In such embodiments, B1 comprises a first antibody variable region VB1 capable of pairing with VA1 to form the first target-binding domain, where VB1 is selected from VH1 or VL1.

[0011] In some embodiments, B2 comprises a second antibody variable region VB2 selected from VH2 or VL2. In such embodiments, A2 comprises a second antibody variable region VA2 capable of pairing with VB2 to form the second target-binding domain, where VA2 is selected from VH2 or VL2.

[0012] In some embodiments, VB1 comprises VH1, VA1 comprises VL1, VB2 comprises VH2, and VA2 comprises VL2.

[0013] In some embodiments, the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VL2, and the VA2 comprises VH2. In some other embodiments, the VB1 comprises VL1, the VA1 comprises VH1, the VB2 comprises VH2, and the VA2 comprises VL2.

[0014] In some embodiments, A1 further comprises a first scaffold region SR _a operably linked to VA1, and B2 further comprises a second scaffold region SR _b operably linked to VB2, wherein SR _a and SR _b are configured to prevent pairing between SR _a and SR _b .

[0015] In some embodiments, B1 further comprises a first paired scaffold region PSR a operably linked to VB1 and capable of binding to SR _a , and A2 further comprises a second paired scaffold region PSR _b operably linked to VA2 and capable _of binding to SR _b .

[0016] In some embodiments, the SR _a /PSR _a pair or the SR _b /PSR _b pair is selected from the group consisting of: a) a pair of heavy chain constant region 1 (CH1) and light chain constant region (CL), b) a pair of T cell receptor (TCR) constant region α (Calpha) and TCR constant region β (Cbeta), c) a pair of TCR constant region γ (Cgamma) and TCR constant region δ (Cdelta), d) a pair of a ligand-binding domain of a receptor and the ligand, e) a pair of a PRD (proline-rich domain) and an SH3 domain, and f) a pair of obscurin and titin.

In some embodiments, the pair of SR _a and PSR _a is different from the pair of SR _b and PSR _b . In some of these embodiments, the pair of SR _a and PSR _a is a pair of CH1 and CL, and the pair of SR _b and PSR _b is i) a pair of Calpha and Cbeta, ii) a pair of Cgamma and Cdelta, iii) a pair of a ligand-binding domain of a receptor and the ligand, iv) a pair of a PRD (proline-rich domain) and an SH3 domain, or v) a pair of obscurin and titin. In other embodiments, the pair of SR _b and PSR _b is a pair of CH1 and CL, and the pair of SR _a and PSR _a is i) a pair of Calpha and Cbeta, ii) a pair of Cgamma and Cdelta, iii) a pair of a ligand-binding domain of a receptor and the ligand, iv) a pair of a PRD (proline-rich domain) and an SH3 domain, or v) a pair of obscurin and titin.

In some embodiments, the SR _a /PSR _a pair and the SR _b /PSR _b pair are both identical, and the SR _a /PSR _a pair and/or the SR _b /PSR _b pair are configured to prevent mispairing between SR _a /PSR _b or SR _b /PSR _a . In some of these embodiments, the SR _a /PSR _a pair comprises a CH1 domain CH1 a and a CL domain CLa, and the SR _b /PSR _b pair comprises a CH1 domain CH1 b and a CL domain CLb.

In some embodiments, the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VH2, and the VA2 comprises VL2. In some of these embodiments, the CH1 domain and the CL domain pair are interdigitated. For example, the PSR _a is the CL domain CLa, the SR _a is the CH1 domain CH1a, the SR _b is the CH1 domain CH1b, and the PSR _b is the CL domain CLb. Alternatively, the PSR _a is the CH1 domain CH1a, the SR a is the CL domain CLa, _{the SR b is} the CL domain CLb, and the PSR _b is the CH1 domain CH1b.

[0020] In some embodiments, the PSR _a is a CH1 domain CH1 a, the SR _a is a CL domain CLa, the SR _b is a CH1 domain CH1 b, and the PSR _b is a CL domain CLb. In some of these embodiments, the VH and VL domains are interdigitated. For example, the VB1 comprises a VH1, the VA1 comprises a VL1, the VB2 comprises a VL2, and the VA2 comprises a VH2. Alternatively, the VB1 comprises a VL1, the VA1 comprises a VH1, the VB2 comprises a VH2, and the VA2 comprises a VL2.

In certain embodiments, the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VH2, the VA2 comprises VL2, the PSR _a is a CH1 domain CH1a, the SR _a is a CL domain CLa, the SR _b is a CH1 domain CH1b, and the PSR and _b is a CL domain CLb, wherein a) the fusion polypeptide comprises an amino acid sequence of formula (I) VH2-CH1b-Linker-VL1-CLa, b) the second polypeptide comprises an amino acid sequence of formula (II) VH1-CH1a, c) the third polypeptide comprises an amino acid sequence of formula (III) VH2-CH1b, and d) the fourth and fifth polypeptides each comprise an amino acid sequence of formula (IV) VL2-CLb, and the CH1b/CLb pair and/or the CH1a/CLa pair are configured to prevent mispairing between CH1b and CLa and/or between CH1a and CLb.

[0022] In some embodiments, at least one of the CH1b/CLb pair and the CH1a/CLa pair has one or more of the following characteristics: 1) at least one non-native disulfide bond that prevents mispairing between CH1b and CLa and/or between CH1a and CLb; 2) at least one or more introduced charged amino acid residues that prevent mispairing between CH1b and CLa and/or between CH1a and CLb; or 3) one or more introduced amino acid mutations that form an orthogonal CH1-CL interface that prevents mispairing between CH1b and CLa or CH1a and CLb. These characteristics are useful for preventing mispairing between CH1b and CLa and/or between CH1a and CLb.

[0023] In some embodiments, at least one of the CH1b/CLb pair and the CH1a/CLa pair has at least one non-native disulfide bond that prevents mispairing between CH1b and CLa and/or CH1a and CLb. In some of these embodiments, the first CH1/CL pair and the second CH1/CL pair are selected from CH1b/CLb and CH1a/CLa, wherein the first CH1/CL pair is associated by a non-native first disulfide bond, optionally lacking a native disulfide bond or having a native disulfide bond that is disrupted. In some embodiments, the second CH1/CL pair is associated by a second disulfide bond formed at a different position than the first disulfide bond, optionally wherein the second disulfide bond is a native disulfide bond. In some embodiments, the first disulfide bond is formed by two cysteine residues introduced at a series of heavy chain-light chain EU positions selected from the group consisting of: a) heavy chain EU position 126-light chain EU position 121, b) heavy chain EU position 173-light chain EU position 160, and c) heavy chain EU position 128-light chain EU position 118. In some embodiments, the native disulfide bond is between heavy chain EU position 220 and light chain EU position 214. In certain embodiments, the first CH1/CL pair comprises a CH1 comprising a substitution of a cysteine residue at EU position 126 and a non-cysteine residue at EU position 220, and a CL comprising a substitution of a cysteine residue at EU position 121 and a non-cysteine residue at EU position 214.

[0024] In some embodiments, at least one of the CH1b/CLb pair and the CH1a/CLa pair has at least one or more introduced charged amino acid residues that prevent mispairing between CH1b and CLa and/or between CH1a and CLb. In some embodiments, the first CH1/CL pair and the second CH1/CL pair are selected from CH1b/CLb and CH1a/CLa, wherein the first CH1/CL pair includes at least one substitution of an uncharged residue with a charged residue and/or at least one substitution of a charged residue with an oppositely charged residue, such that the first CH1/CL pair includes a first pair of oppositely charged residues that favors pairing of the first CH1/CL pair. In some embodiments, the second CH1/CL pair comprises at least one substitution of an uncharged residue with a charged residue and/or at least one substitution of a charged residue with an oppositely charged residue, whereby the second CH1/CL pair comprises a second pair of oppositely charged residues that favors pairing of the second CH1/CL pair, and optionally the first pair of oppositely charged residues and the second pair of oppositely charged residues prevent pairing of CH1a with CLb or CH1b with CLa. In some embodiments, the first pair of oppositely charged residues and/or the second pair of oppositely charged residues are configured such that CH1a and CLb both have positively or negatively charged residues, and/or CH1b and CLa both have positively or negatively charged residues. In certain embodiments, the first pair of oppositely charged residues and/or the second pair of oppositely charged residues are introduced at heavy chain-light chain EU positions selected from the group consisting of: a) heavy chain EU position 183:light chain EU position 176; b) heavy chain EU position 183:light chain EU position 133; c) heavy chain EU position 147:light chain EU position 176; d) heavy chain EU position 141:light chain EU position 116; e) heavy chain EU position 126:light chain EU position 121; and f) heavy chain EU position 218:light chain EU position 122. In certain embodiments, the pair of oppositely charged residues comprises a positively charged amino acid residue and a negatively charged amino acid residue, wherein the positively charged amino acid residue is selected from the group consisting of lysine (K), histidine (H), and arginine (R), and/or the negatively charged amino acid residue is selected from the group consisting of aspartic acid (D) and glutamic acid (E).

[0025] In some embodiments, at least one of the CH1b/CLb pair and the CH1a/CLa pair has one or more introduced amino acid mutations that form an orthogonal CH1-CL interface that prevents mispairing between CH1b and CLa or CH1a and CLb. In some embodiments, the first CH1/CL pair and the second CH1/CL pair are selected from CH1b/CLb and CH1a/CLa, respectively, and the first CH1/CL pair includes one or more introduced amino acid mutations that form an orthogonal CH1-CL interface. In some embodiments, the orthogonal CH1-CL interface is introduced at a series of heavy chain-light chain EU positions, including heavy chain EU positions H168A and F170G, and light chain EU positions L135Y and S176W. In some embodiments, the first CH1/CL pair comprises one or more introduced amino acid mutations forming an orthogonal Fab design at a set of heavy chain-light chain EU positions selected from the group consisting of: a) substitutions at heavy chain EU positions A141I, F170S, S181M, S183A, and V185A, and substitutions at light chain EU positions F116A, A235V, S174A, S176F, and T178V.

[0026] In some embodiments, the VH1/VL1 pair and/or the VH2/VL2 pair are configured to prevent mispairing between VH1 and VL2 and/or VH2 and VL1. In some of these embodiments, the first VH/VL pair and the second VH/VL pair are selected from VH1/VL1 and VH2/VL2, wherein the first VH/VL pair has at least one substitution of an uncharged residue with a charged residue and/or at least one substitution of a charged residue with an oppositely charged residue, such that the first VH/VL includes a third pair of oppositely charged residues that favors pairing of the first VH/VL pair. In some embodiments, the second VH/VL pair has at least one substitution of an uncharged residue for a charged residue and/or at least one substitution of a charged residue for an oppositely charged residue, and the second VH/VL includes a fourth pair of oppositely charged residues that favors pairing of the second VH/VL pair, and optionally the third pair of oppositely charged residues and the fourth pair of oppositely charged residues prevent pairing of VH1 with VL2 or VH2 with VL1. In some of these embodiments, the third pair of oppositely charged residues and the fourth pair of oppositely charged residues are configured such that VH1 and VL2 both have positively or negatively charged residues and/or VH2 and VL1 both have positively or negatively charged residues. In some embodiments, the third pair of oppositely charged residues and/or the fourth pair of oppositely charged residues are introduced at heavy chain-light chain EU positions selected from the group consisting of: a) heavy chain EU position 39:light chain EU position 38, b) heavy chain EU position 105:light chain EU position 43, and c) heavy chain EU position 62:light chain EU position 1, or any combination thereof. In some embodiments, the pair of oppositely charged residues comprises a positively charged amino acid residue and a negatively charged amino acid residue, wherein the positively charged amino acid residue is selected from the group consisting of lysine (K), histidine (H), and arginine (R), and/or the negatively charged amino acid residue is selected from the group consisting of aspartic acid (D) and glutamic acid (E).

[0027] In some embodiments, the second polypeptide and the third polypeptide each further comprise an operably linked first dimerization domain and an operably linked second dimerization domain that associate to form a dimer, and optionally, the first dimerization domain comprises a first Fc region and/or the second dimerization domain comprises a second Fc region. In some embodiments, the first Fc region and/or the second Fc region of the polypeptide complex are derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the first Fc region and the second Fc region of the polypeptide complex have different amino acid sequences and at least one arrangement that promotes heterodimerization of the first Fc region and the second Fc region.

[0028] In some embodiments, the first Fc region of the polypeptide complex comprises a first Fc mutation, and/or the second Fc region of the polypeptide complex comprises a second Fc mutation, wherein: a) the first Fc mutation comprises T366W or S354C and the second Fc mutation comprises Y349C, T366S, L368A, or Y407V; b) the first Fc mutation comprises D399K or E356K and the second Fc mutation comprises K392D or K409D; or c) the first Fc mutation comprises E356K, E357K, or D399K and the second Fc mutation comprises K370 d) the first Fc mutation comprises S364H or F405A and the second Fc mutation comprises Y349T or T394F; e) the first Fc mutation comprises S364H or T394F and the second Fc mutation comprises Y394T or F405A; f) the first Fc mutation comprises K370D or K409D and the second Fc mutation comprises E357K or D399K; or g) the first Fc mutation comprises L351D or L368E and the second Fc mutation comprises L351K or T366K, wherein numbering is according to the EU index.

[0029] In another embodiment, at least one of the first target binding domain and the second target binding domain is a chimeric domain, a humanized domain, or a fully human domain.

In some embodiments, the first target binding domain and the second target binding domain bind to different targets. In some embodiments, at least one of the first target binding domain and the second target binding domain binds to a tumor-associated antigen or an immune-related target. In some embodiments, one of the first target binding domain and the second target binding domain binds to a tumor-associated antigen and the other binds to an immune-related target. In some embodiments, at least one of the first target binding domain and the second target binding domain binds to a disease-associated antigen or an immune-related target, and optionally the disease-associated antigen is a tumor-associated antigen, 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.
[0031] Another aspect of the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide or polypeptide complex described herein.

[0032] In yet another aspect, the present disclosure provides a vector comprising the nucleic acid described herein.

[0033] In yet another aspect, the present disclosure provides host cells comprising the nucleic acids and vectors described herein.

[0034] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide complex described herein and a pharmaceutically acceptable carrier.

[0035] In yet another aspect, the present disclosure provides a conjugate comprising a polypeptide complex described herein and a payload conjugated thereto, wherein the payload is selected from the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification tag, a tracking molecule, an anticancer drug, an immune-related molecule, and a cytotoxic molecule.

[0036] In yet another aspect, the present disclosure provides a composition comprising a polypeptide complex or conjugate described herein and a pharmaceutically acceptable carrier.

[0037] In yet another aspect, the present disclosure provides methods for treating or preventing a disease, condition, or symptom. In some embodiments, the methods comprise administering to a subject in need of treatment a therapeutically effective amount of a polypeptide complex described herein, a pharmaceutical composition described herein, a conjugate described herein, or a composition described herein.

[0038] In some embodiments, the disease is selected from the group consisting of cancer, an inflammatory disease, an infectious or parasitic disease, a cardiovascular disease, an eye disease, a central nervous system (CNS) disease, trauma, a metabolic disease, an autoimmune disease, or a coagulation disorder. In some embodiments, the CNS disease is a neurological disorder, a neuropsychiatric condition, neuroblastoma, glioblastoma, or Alzheimer's disease.

[0039] In yet another aspect, the present disclosure provides methods for detecting the presence or level of an antigen. In some embodiments, the methods include 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.

[0040] Figure 1 shows each polypeptide fragment of an exemplary polypeptide complex constructed from two pairs of monoclonal antibodies and one pair of Fc regions. [0041] Figure 2 shows a schematic representation of the structure of an exemplary polypeptide complex constructed by the method of the present invention (Method B). [0042] Figure 3 further shows the structure of an exemplary polypeptide complex using Fab fragments from both monoclonal antibodies subdivided into VH/VL and CH1/CL pairs constructed by the method of the present invention (Method B). [0043] Figure 4A further shows the structure of an exemplary polypeptide complex using defined VH/VL and CH1/CL pairs of Fab fragments from both monoclonal antibodies constructed by the method of the present invention (Method B). FIG. 4B further shows the structure of an exemplary polypeptide complex with defined VH/VL and CH1/CL pairs of Fab fragments from both monoclonal antibodies constructed by the method of the present invention (Method B). 4C-E show antibody structures with a B1-A2 mismatch (FIG. 4C), a B2-A1 mismatch (FIG. 4D), and both a B1-A2 and a B2-A1 mismatch (FIG. 4E), respectively. 4C-E show antibody structures with a B1-A2 mismatch (FIG. 4C), a B2-A1 mismatch (FIG. 4D), and both a B1-A2 and a B2-A1 mismatch (FIG. 4E), respectively. 4C-4E show antibody structures with a B1-A2 mismatch (FIG. 4C), a B2-A1 mismatch (FIG. 4D), and both a B1-A2 and a B2-A1 mismatch (FIG. 4E), respectively. [0044] Figure 5A shows a structural diagram of an exemplary bispecific antibody constructed by a conventional approach (Method A). FIG. 5B shows a structural diagram of an exemplary bispecific antibody constructed by the conventional approach (Method A). 5C-5E show antibody structures with a B1-A2 mismatch (FIG. 5C), a B2-A1 mismatch (FIG. 5D), and both a B1-A2 and a B2-A1 mismatch (FIG. 5E), respectively. 5C-5E show antibody structures with a B1-A2 mismatch (FIG. 5C), a B2-A1 mismatch (FIG. 5D), and both a B1-A2 and a B2-A1 mismatch (FIG. 5E), respectively. 5C-5E show antibody structures with a B1-A2 mismatch (FIG. 5C), a B2-A1 mismatch (FIG. 5D), and both a B1-A2 and a B2-A1 mismatch (FIG. 5E), respectively. [0045] Figure 6A shows the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SDS-Page. FIG. 6B shows the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SDS-Page. [0046] Figures 7A-7P show expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. 7A-7P show the expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMAT NEW1-8) analyzed by SEC-HPLC. [0047] Figure 8 shows the purity of the expressed polypeptide complex anti-CD20xCD3 (FORMAT NEW7) as analyzed by SDS-Page. [0048] Figure 9 shows the purity of the expressed polypeptide complex anti-CD20xCD3 (FORMAT NEW7) analyzed by CE-SDS. [0049] Figure 10 shows the purity of the expressed polypeptide conjugate anti-CD20xCD3 (FORMAT NEW7) as analyzed by SEC-HPLC. [0050] Figure 11 shows the purification profile of the expressed polypeptide complex anti-CD20xCD3 (FORMAT NEW7) by affinity chromatography. [0051] Figure 12 shows SEC-HPLC purity analysis of the expressed polypeptide conjugate anti-CD20xCD3 (FORMAT NEW7) purified by affinity chromatography. [0052] Figure 13 shows an SDS-Page purity analysis of the expressed polypeptide complex anti-CD20xCD3 (FORMAT NEW7) purified by affinity chromatography. [0053] Figure 14 shows the purification profile of the expressed polypeptide complex anti-CD20xCD3 (FORMAT NEW7) with linear gradient elution by CEX. [0054] Figure 15 shows SDS-Page purity analysis of the expressed polypeptide complex anti-CD20xCD3 (fractions C01-C04) purified by CEX. [0055] Figure 16 shows SEC-HPLC purity analysis of the expressed polypeptide conjugate anti-CD20xCD3 (fractions C01-C04) purified by CEX. [0056] Figure 17 shows the sequences of exemplary scaffold region pairs used in bispecific antibodies. FIG. 17 shows the sequences of exemplary scaffold region pairs used in bispecific antibodies. FIG. 17 shows the sequences of exemplary scaffold region pairs used in bispecific antibodies.

[0057] Several aspects of the present invention are 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, one skilled in the art will readily recognize that the present invention can be practiced without one or more of the specific details or by other methods. The present invention is not limited by the illustrated order of acts or events, and some acts may occur in a different order and/or simultaneously with other acts or events.

[0058] Furthermore, not all illustrated acts or events are required to implement the methodologies of the present invention.

I. Definitions and Abbreviations [0059] Prior to describing the present invention in detail, the following will be pointed out and defined.

[0060] All descriptions provided herein are merely illustrative of various embodiments of the present invention provided in this disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of the disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the disclosure, and such equivalent embodiments are understood to be included herein.

[0061] 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 descriptions 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 definition of such terms expressly provided in this disclosure shall control in all respects.

[0062] 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.

[0063] 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 specified. By way of example, "a polypeptide complex" means one polypeptide complex or one or more polypeptide complexes.

[0064] 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.

[0065] As used herein, the terms "comprise," "comprises," "comprising," "include," "includes," "including," "contain," "contains," "containing," and "have," "has," "having," etc. are synonymous and are used in an inclusive and open-ended manner and do not exclude additional elements, features, steps, actions, operations, etc.

[0066] 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 that list.

[0067] 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" items in a list 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 convey that an item, term, etc. may 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.

[0068] 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 "further embodiments," 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 an expression 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.

[0069] 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 generally is not intended to imply that features, elements, and/or steps are in any way required for one or more embodiments.

[0070] This section provides definitions of some general terms. Definitions of other terms may be found in other sections of this disclosure below.

[0071] 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" also 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.

[0072] 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).

[0073] 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). It should be noted that in this disclosure, the terms "domain" and "region" may be used interchangeably.

[0074] 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 may include both DNA and RNA, may be single- or double-stranded, and include analogs of naturally occurring polynucleotides in which one or more nucleotides are modified relative to the naturally occurring nucleotides.

[0075] The term "antibody," as used herein, includes any immunoglobulin, monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, multispecific antibody, bispecific antibody, bivalent antibody, or polyvalent antibody that binds to a specific antigen. Antibodies and related terms are described in more detail below.

[0076] 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 disease. Particular antibody classes 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: kappa (κ) chains and lambda (λ) chains.

[0077] 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.

[0078] More specifically, in natural antibodies, from the N-terminus to the C-terminus, each heavy chain comprises a variable region (VH, or HCVR) followed by three or four constant regions ("CHs"; IgA, IgD, and IgG comprise three CH regions, CH1, CH2, and CH3, while 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., the VL region) aligns or associates with the variable region (i.e., the VH region) of its paired heavy chain to together form the antigen-binding site of the antibody.

[0079] 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 naturally occurring antibodies, 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 by the Kabat, Chothia, or Al-Lazikani definitions (Al-Lazikani, B., Chothia, C., Lesk, A. M., 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, A. M., J. Mol. Biol., 196,901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83 (1989), Kabat E. A. et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are interposed between adjacent segments 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 contains four FRs, and the CDRs and FRs are arranged from the amino terminus to the 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 need to include all three CDRs or all four FRs, but 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.

[0080] 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 native constant region of a native antibody, but should be understood to encompass any variant or derivative of such a native 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 mobility, Fc receptor binding, complement binding, and other effector functions.

[0081] 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 native antibodies, the constant region of each light chain (i.e., the CL region) associates with the first constant region (i.e., the CH1 region) of the paired heavy chain.

[0082] 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 amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0083] The term "hinge region," with respect to antibodies, includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. The length of the hinge region varies depending on the defined boundaries of the CH1 domain and the CH2 domain. The hinge region is typically flexible, allowing the two N-terminal antigen-binding domains to move independently.

[0084] 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.

[0085] 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).

[0086] "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.

[0087] "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" includes antigenic epitopes, e.g., fragments of an antigen that are antigenic epitopes. The terms "antigen" and "target" are used interchangeably in this disclosure.

[0088] "Fv," with respect to antibodies, refers to the smallest antibody fragment containing an intact 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)).

[0089] "Fab," as used herein, refers to a single antigen-binding domain derived from an antibody, which domain 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 the Fab domain comprises an HCVR and a CH1 region. In some embodiments, the single light chain fragment in the 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.

[0090] 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.

[0091] As used herein, the term "bispecific antibody" refers to an antibody that contains 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 are capable of binding 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.

[0092] 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, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). As indicated, some positions are numbered according to the EU numbering system 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) IMGT numbering or Kabat index is used. These numbering schemes are also available from the IMGT scientific atlas, accessible from the International ImMunoGeneTics Information System website.

[0093] 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.

[0094] 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 having only a single antigen-binding site, and the term "multivalent" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to two binding sites, four binding sites, and six binding sites, respectively, in an antigen-binding molecule. In some embodiments, an antibody or antigen-binding fragment thereof is bivalent.

[0095] 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., Fab) linked together, with each antibody or fragment having a different specificity.

[0096] 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 the antibody and antigen when specifically bound in an antibody-antigen complex is characterized by a KD (dissociation constant) of less than ¹⁰ M, less than 10 M, less than ¹⁰ M, less than ¹⁰ M, less than ¹⁰ M, less than ¹⁰ M, less than ¹⁰ M, less than 10 _M , or less than about ¹⁰ M or less.

[0097] As used herein, the term "vector" refers to a vehicle into which a genetic element is operably inserted to express the genetic element, e.g., 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, 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.

[0098] 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.

[0099] 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 and a drug of the present disclosure. The drug may be selected from a therapeutic agent, an imaging agent, a labeling agent, or a drug useful for therapeutic and/or labeling purposes.

[0100] 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.

II. Novel Multispecific (or Bispecific) Polypeptide Conjugates [0101] In one aspect, the present disclosure provides novel multispecific (or bispecific) polypeptide conjugates that can be conveniently produced and purified, and thus provided in high purity and yield.

[0102] Multispecific (or bispecific) antibodies have attracted considerable interest in the field of antibody engineering and are expected to have a wide range of applications, including anti-tumor immunotherapy. Various forms of bispecific antibodies have been developed, including IgG-like bispecific antibodies. IgG-like bispecific antibodies are bivalent and contain two pairs of heavy chains (HC) and light chains (LC) derived from two different antibodies.

[0103] IgG-like bispecific antibodies provide two monovalent Fabs that each bind to a different antigen. However, compared to conventional IgG antibodies, which provide bivalent binding to a single antigen, this can result in reduced affinity for each antigen and therefore loss of specific function.

[0104] Another challenge relates to determining the binding affinity of an IgG-like bispecific antibody for two target antigens. Depending on the intended mechanism of action (i.e., MOA) of the two target antigens, the requirements for binding affinity to the two target antigens may differ. For example, for a CD3/CD20 bispecific antibody, nanomolar affinity for CD20 is desired, while submicromolar affinity (e.g., 30-100 nm) for CD3 is desired. To increase affinity for a target of interest, an additional Fab domain targeting that target may be fused to one of the heavy chains of the IgG-like bispecific antibody (see Figure 5A). Such a bispecific, trivalent antibody structure is referred to herein as a 2:1 structure; that is, binding to one of the antigens of interest is bivalent, while binding to the other antigen remains monovalent. Such bivalent binding can restore binding affinity to the antigen target while allowing for different affinities between the two antigen targets.

[0105] Despite the advantages offered by 2:1 bispecific antibodies, their expression and production present several insurmountable challenges. For example, LC-HC bond instability and LC mismatching can occur during assembly of such 2:1 bispecific antibodies. Because a variety of LC mismatched products (i.e., impurities) can be similar to or indistinguishable from the target product (i.e., correctly matched) in terms of molecular weight and physicochemical properties, 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.

[0106] The present disclosure provides a novel 2:1 bispecific antibody platform that can not only satisfy the requirement for affinity difference between two antigen targets, but also can be purified significantly more easily than conventional 2:1 bispecific antibodies. This novel 2:1 structure is advantageous because potential mismatched products (if any) are sufficiently different from correctly matched products, e.g., in molecular weight and physicochemical properties, that they can be more easily removed. Therefore, bispecific antibodies constructed according to this novel 2:1 structure platform can achieve higher purity and yield with a relatively simple purification process.

III. NOVEL 2:1 STRUCTURES OF MULTISPECIFIC ANTIBODIES [0107] The novel 2:1 structures provided herein are based on an IgG-like bispecific antibody fused with an additional Fab domain that binds to one of two targets. In particular, unlike conventional 2:1 structure bispecific antibodies in which an additional Fab domain is fused to the heavy chain of the Ig-like bispecific antibody (see second polypeptide chain in Figure 5A), the novel 2:1 structures provided herein have an additional Fab domain fused to the light chain of the IgG-like bispecific antibody (see first polypeptide chain in Figure 2).

[0108] Specifically, as shown in FIG. 2, the novel 2:1 bispecific antibody comprises a polypeptide complex composed of five polypeptide chains: 1) a first polypeptide chain comprising a fusion polypeptide comprising a first target-binding fragment A1 and a second target-binding fragment B2; 2) a second polypeptide comprising a first pairing fragment B1; 3) a third polypeptide comprising a second target-binding fragment B2; 4) a fourth polypeptide comprising a second pairing fragment A2; and 5) a fifth polypeptide identical to the fourth polypeptide, i.e., comprising a second target-binding fragment A2. A1 in the fusion polypeptide pairs with B1 in the second polypeptide to form a first target-binding domain; B2 in the fusion polypeptide pairs with A2 in the fourth polypeptide to form a second target-binding domain; and A2 in the fifth polypeptide pairs with B2 in the third polypeptide to form a separate second target-binding domain.

[0109] The uniqueness of the polypeptide complexes provided herein depends, at least in part, on the fusion polypeptide (i.e., first polypeptide chain) they contain, which is novel and not contained in any prior 2:1 bispecific antibody. This fusion polypeptide allows any mismatched products to have significantly different molecular weights and physicochemical properties from the target bispecific product.

[0110] In some embodiments, the fusion polypeptide comprises, from C-terminus to N-terminus, a first target-binding fragment A1, a polypeptide linker, and a second target-binding fragment B2, wherein the polypeptide linker has a length sufficiently short to minimize potential intramolecular interactions between A1 and B2, A1 is capable of pairing with a first pairing fragment B1 to form a first target-binding domain, B2 is capable of pairing with a second pairing fragment A2 to form a second target-binding domain, A1 is configured to have reduced binding affinity to B2 relative to B1, and B2 is configured to have reduced binding affinity to A1 relative to A2.

[0111] In the novel 2:1 structure shown in Figure 2, the two light chains are identical, which helps reduce unwanted mismatching. In the exemplary novel 2:1 structure provided herein (see Figure 4A), a total of three different mismatched products (see Figures 4C-4E) are expected, estimated to have molecular weights of approximately 150 KDa, 250 KDa, and 200 KDa, respectively (see Figure 4B). Therefore, most potential mismatched products have molecular weights different from the correctly matched product. Furthermore, these potential mismatched products have been shown to have different physicochemical properties from the correctly matched bispecific product, and therefore can be easily removed by conventional purification methods.

[0112] In contrast, conventional 2:1 bispecific antibodies, as shown in Figure 5A, have two different light chains that can potentially mispair with different heavy chains, potentially generating a total of five different mispaired products (see Figures 5C-5E), all of which have the same molecular weight (i.e., 200 KDa) as the correctly paired product (see Figure 5B). These similar mispaired products are difficult to separate from the target product, reducing both the purity and yield of the target product.

[0113] The novel 2:1 polypeptide complex provided herein binds to a first target and a second target. It has two antigen-binding domains that both bind to the second target, and a third antigen-binding domain that binds to the first target. In other words, it is called "2:1" because it is bivalent for the second target and monovalent for the first target. The two antigen-binding domains for the second target are located in different arms of the polypeptide complex provided herein.

[0114] In some embodiments, the target binding domain in the polypeptide complexes provided herein is formed by pairing of two polypeptide fragments. In certain embodiments, the first target binding domain is formed by pairing of a first target binding fragment A1 with a first paired fragment B1 (i.e., A1/B1 pair). In certain embodiments, the second target binding domain is formed by pairing of a second target binding fragment A2 with a second paired fragment B2 (i.e., A2/B2 pair).

[0115] In some embodiments, the first target-binding fragment A1 and the first paired fragment B1 (i.e., A1/B1) can be paired, for example, by a disulfide bond, hydrogen bond, electrostatic interaction, salt bridge, or hydrophobic-hydrophilic interaction formed between A1 and B1, or more specifically, between at least two amino acid residues from A1 and B1, a connector, or a combination thereof.
Similarly, in some embodiments, the second target-binding fragment B2 and the second pairing fragment A2 (i.e., A2/B2) can be paired, for example, by a disulfide bond, hydrogen bond, electrostatic interaction, salt bridge, or hydrophobic-hydrophilic interaction formed between A2 and B2, or more specifically, between at least two amino acid residues from A2 and B2, a connector, or a combination thereof.

[0117] "Disulfide bond" refers to a covalent bond having the structure R-S-S-R'. The amino acid cysteine contains a thiol group that can form a disulfide bond with a second thiol group from another cysteine residue, for example. Disulfide bonds can form between the thiol groups of two cysteine residues present on two polypeptide chains, respectively, thereby forming interchain disulfide bonds. To name just a few examples, disulfide bonds can form between known scaffold polypeptide fragments, such as antibody CH1 domains and CL domains, or between TCR constant regions (TCRα/TCRβ, TCRγ/TCRδ, etc.).

[0118] Electrostatic interactions are non-covalent interactions that are important for protein folding, stability, flexibility, and function and include ionic interactions, hydrogen bonds, and halogen bonds. Electrostatic interactions can form in a polypeptide, for example, between Lys and Asp, Lys and Glu, Glu and Arg, or between Glu, Trp on a first polypeptide chain and Arg, Val, or Thr on a second polypeptide chain.

[0119] Salt bridges are short-range electrostatic interactions resulting from spatially adjacent pairs of oppositely charged residues in the native protein structure: the anionic carboxylate of Asp or Glu and the cationic ammonium of Lys or the guanidinium of Arg. Charged and polar residues at the predominantly hydrophobic interface can act as binding hot spots. Residues with ionizable side chains, such as His, Tyr, and Ser, among others, can also participate in salt bridge formation.

[0120] Hydrophobic interactions can form between one or more of Val, Tyr, and Ala on one polypeptide chain and Val, Leu, and Trp on a second chain, or between His and Ala on a first chain and Thr and Phe on another polypeptide chain.

[0121] Hydrogen bonds are formed by electrostatic attraction between two polar groups when a hydrogen atom is covalently bonded to a highly electronegative atom such as nitrogen, oxygen, or fluorine. Hydrogen bonds can form, for example, between the nitrogen group in Asn and the oxygen group in His, or between the oxygen group in Asn and the nitrogen group in Lys.

[0122] In some embodiments, the cognate pairing of A1 and B1 shown in the novel 2:1 structure of Figure 4A is facilitated by a natural or introduced non-natural disulfide bond, whereas the mismatched product (mismatched A1 and B2, or mismatched A2 and B1) lacks such a disulfide bond. Without wishing to be bound by any particular theory, it is expected that such a disulfide bond will be particularly advantageous in allowing the mismatched product to be readily identified and/or characterized by conventional methods such as, for example, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This is at least in part due to the following reasons: While disulfide-linked cognate pairing products can withstand SDS-PAGE, mismatched pairs lacking such disulfide bonds are susceptible to dissociation of the mismatched polypeptide chains, resulting in different molecular weights for the cognate and mismatched pairs observed by SDS-PAGE, as shown in Figures 4B-4E. Furthermore, the novel 2:1 structure of Figure 4A allows for the mismatched pairing products to be easily separated from the cognate pairing products using relatively conventional purification methods, such as affinity purification and/or ion exchange purification, thereby achieving highly pure cognate pairing products that would otherwise be impossible or require cumbersome purification processes.

[0123] In certain embodiments, the A1/B1 pair and/or the A2/B2 pair comprise antibody Fv domains.

[0124] In some embodiments, in the fusion polypeptide or polypeptide complex provided herein, A1 comprises a first antibody variable region VA1 selected from VH1 or VL1, and B1 comprises a first paired antibody variable region VB1 capable of pairing with VA1 to form a first target binding domain, where VB1 is selected from VH1 or VL1. In other words, the first target binding domain comprises a first Fv domain comprising a co-assembled VH1 and VL1 capable of binding to a first target.

[0125] In some embodiments, in the fusion polypeptide or polypeptide complex provided herein, B2 comprises a second antibody variable region VB2 selected from VH2 or VL2, and A2 comprises a second paired antibody variable region VA2 capable of pairing with VB2 to form a second target binding domain, where VA2 is selected from VH2 or VL2. In other words, the second target binding domain comprises a second Fv domain comprising a co-assembled VH2 and VL2 capable of binding to a second target.

[0126] In some embodiments, VB1 comprises VH1, VA1 comprises VL1, VB2 comprises VH2, and VA2 comprises VL2. In some embodiments, VB1 comprises VL1, VA1 comprises VH1, VB2 comprises VL2, and VA2 comprises VH2.

[0127] In some embodiments, the first Fv domain or the second Fv domain intersects. In some embodiments, VB1 comprises VH1, VA1 comprises VL1, VB2 comprises VL2, and VA2 comprises VH2. In some embodiments, VB1 comprises VL1, VA1 comprises VH1, VB2 comprises VH2, and VA2 comprises VL2.

[0128] The Fv domains may be associated by any suitable means known in the art, including, without limitation, non-native disulfide bonds introduced into the Fv domains or non-native electrostatic interactions introduced into the Fv domains. In some other embodiments, the Fv domains may be associated by a scaffold domain fused to the Fv domains. For example, a scaffold region may be fused to one chain of the Fv domain, and a paired scaffold region may be fused to the other chain of the Fv domain. Binding of the scaffold region to the paired scaffold region allows the VH and VL regions to associate, thereby forming an Fv domain capable of binding to a target antigen.

[0129] In some embodiments, in a polypeptide complex disclosed herein, A1 further comprises a first scaffold region R _a operably linked to VA1, and B2 further comprises a second scaffold region R _b operably linked to VB2, wherein SR _a and SR _b are configured to prevent pairing between SR _a and SR _b . In some embodiments, in a polypeptide complex disclosed herein, B1 further comprises a first pairing scaffold region PSR a operably linked to VB1 and capable of binding to SR _a , and A2 further comprises a second pairing scaffold region PSR _b operably linked to VA2 and capable of binding to SR _b . Binding of SR _a and PSR _a forms a first scaffold domain, and binding of SR _b and PSR _b forms _a second scaffold domain. An exemplary diagram is shown in Figure 3.

[0130] 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 CL domain, a paired TCR constant region (TCRα/TCRβ, TCRγ/TCRδ), a PRD (proline-rich domain) and an SH3 domain, or obscurin and titin.

[0131] TCRs belong to the immunoglobulin superfamily and resemble half antibodies, possessing a single heavy chain and a single light chain. Naturally occurring TCRs consist of two polypeptide chains and are broadly classified into two types: one type consisting of an α chain and a β chain (i.e., α/β TCR), and another type consisting of a γ chain and a δ chain (i.e., γ/δ TCR). The two TCR chains are linked by a disulfide bond formed between the constant regions in the extracellular portions of the TCR chains. In other words, a disulfide bond is formed between the TCR constant region α and the TCR constant region β, or between the TCR constant region γ and the TCR constant region δ.

[0132] The SH3 domain (short for SRC homology 3 domain) is a small protein domain of approximately 60 amino acid residues, originally described as a conserved sequence in the viral adaptor protein v-Crk. Conventional SH3 domains are commonly found in proteins that interact with other proteins and typically mediate the assembly of specific protein complexes by binding to proline-rich domains (i.e., PRDs) in their respective binding partners (Liubov V Gushchina et al., J Biomol Struct Dyn. 2011 Dec;29(3):485-95).

[0133] Titin (also known as connectin) is a human protein encoded by the TTN gene. Titin is a large protein, >1 μm in length, that functions as a molecular spring responsible for the passive elasticity of muscle. The newest member of the titin family, obscurin (~800 kDa), is primarily localized to the sarcomere M-band in mature muscle and was initially identified as a ligand for the Zdisk portion of titin. The giant muscle proteins titin and obscurin bind to each other at the Zdisk during muscle development. This interaction helps stabilize and organize sarcomeres. Ablation of this bond leads to muscular dystrophy (Allyn G Letourneau et al., Protein Pept Lett. 2018;25(11):973-979). In particular, the titin Ig-like 152 domain of titin interacts with the obscurin Ig-like 1 domain of obscurin, and they can form a complex. In several previously reported studies, the titin T chain and obscurin O chain can be used to replace the CH1 and CL regions of an antibody, where the titin T chain is a peptide containing the titin Ig-like 152 domain, or a functional variant thereof, and the obscurin O chain is a peptide containing the obscurin Ig-like 1 domain, or a functional variant thereof, with the titin T chain being 78-118 amino acids in length (see, e.g., WO 2021/139757 A, incorporated herein in its entirety).

[0134] Suitable pairs of scaffold domains useful in the present invention (i.e., SR _a /PSR _a pair or SR _b /PSR _b pair) include a) a pair of heavy chain constant region 1 (CH1) and light chain constant region (CL), b) a pair of T cell receptor (TCR) constant region α (Calpha) and TCR constant region β (Cbeta), c) a pair of TCR constant region γ (Cgamma) and TCR constant region δ (Cdelta), d) a pair of a ligand-binding domain of a receptor and said ligand, e) a pair of a PRD (proline-rich domain) and an SH3 domain, or f) a pair of obscurin and titin. The expressions "pair of X and Y" or "X/Y pair" used throughout this disclosure are not intended to limit the order. For example, specifying that "the pair SR _a /PSR _a may be a pair of CH1 and CL" is intended to mean that SR _a may be CH1 or CL, and accordingly, if SR _a is CH1, PSR _a may be CL, or if SR _a is CL, PSR _a may be CH1.

In some embodiments, SR _a and PSR _a are paired by a first disulfide bond. In such embodiments, mispairing of SR _a and PSR _b or SR _b and PSR _a lacks the first disulfide bond. In some embodiments, SR _b and PSR _b are paired by a second disulfide bond. In such embodiments, mispairing of SR _a and PSR _b or SR _b and PSR _a lacks the second disulfide bond. Any suitable pair of scaffold regions capable of pairing by a disulfide bond can be used as the SR _a and PSR _a pair or the SR _b /PSR _b pair. For example, antibody CH1 domain and CL domain can be paired by a disulfide bond, TCR constant regions (e.g., Calpha and Cbeta, or Cgamma and Cdelta) can be paired by a disulfide bond, and obscurin and titin can be paired by a disulfide bond.

[0136] In some embodiments, the SR _a /PSR _a pair comprises a pair of CH1 domain CH1 a and CL domain CLa, and the SR _b /PSR _b pair comprises a pair of CH1 domain CH1 b and CL domain CLb. An exemplary diagram is shown in Figure 4A. In certain embodiments, the first target binding domain (formed by the A1/B1 pair) and the second target binding domain (formed by the A2/B2 pair) comprise antibody Fab domains.

In certain embodiments, the pairing between A1 and B1 may be formed between two amino acid residues in VA1 and VB1, VH1 and VL1, _SRa and _PSRa , or CH1a and CLa. The pairing between A2 and B2 may be formed between two amino acid residues in VA2 and VB2, VH2 and VL2, _SRb and _PSRb , or CH1b and CLb.

[0138] IV. Configurations to Avoid Mismatches [0139] Although a target-binding polypeptide fragment is intended to bind to its matching polypeptide fragment to form a target-binding domain (i.e., a cognate pairing), mismatches can occur between polypeptide fragments that are not intended to pair. For example, a first target-binding fragment A1 can mismatch with a second target-binding fragment B2, or a first target-binding fragment A2 can mismatch with a second target-binding fragment B1, resulting in failure to form the intended target-binding domain.

[0140] For example, it is intended that VH1 pairs with VL1 to form a first target binding domain, and VH2 pairs with VL2 to form a second target binding domain, and that such cognate pairings are promoted. However, mispairing of VH1 with VL2 and mispairing of VH2 with VL1 is possible, and it is preferable that such mispairings be prevented or reduced.

[0141] To promote cognate pairing and possibly reduce mispairing, polypeptide fragments can be designed or engineered to contain specific arrangements that aid in achieving this goal. In some embodiments, in the fusion polypeptides or polypeptide complexes disclosed herein, at least one of the pair A1 and B1 (referred to as A1/B1) and the pair A2 and B2 (referred to as A2/B2) contains at least one arrangement that can a) promote cognate pairing between A1 and A2 and/or B1 and B2, and/or b) prevent mispairing between B1 and A2 and/or B2 and A1.

[0142] The polypeptide complexes disclosed herein can be engineered to promote cognate pairing and/or reduce mispairing by any suitable method known in the art. Several strategies have been applied to design orthogonal interfaces that promote cognate pairing. For example, Roche swapped the CH1 and CL domains to create the CrossMab platform (Schaefer et al. Proceedings of the National Academy of Sciences of the United States of America, 108(27), pp.11187-11192 (2011)), MedImmune introduced a non-native disulfide bond (Mazor et al. mAbs, 7(2), pp.377-389 (2015)), and Amgen introduced additional electrostatic interactions in the CH1-CL region (Liu et al. Journal of Biological Chemistry, 290(12), pp. 7535-7562 (2015)), Eli Lilly (Lewis et al. Nature Biotechnology, 32(2), pp. 191-198 (2014)), Genentech (Dillon et al. mAbs, 9(2), pp. 213-230 (2017)) introduced mutations into both the variable and constant domains, and Wuxi Biologics replaced one pair of CH1-CL regions with a T cell receptor (TCR) constant region (Guo et al., Protein Expr Purif., 173:105647 (2020)). These strategies have been shown to be useful in promoting cognate pairing and may be useful in the present disclosure.

[0143] In certain embodiments, in the fusion polypeptide or polypeptide complex disclosed herein, the SR _a /PSR _a pair and the SR _b /PSR _b pair are both the same or of the same type, and the SR _a /PSR _a pair and/or the SR _b /PSR _b pair are configured to prevent mispairing between SR _a /PSR _b or SR _b /PSR _a .

[0144] Any suitable means for preventing or reducing mismatching may be used. For example, the A1/B1 and A2/B2 pairs may be designed to incorporate crossed VA1/VB1 configurations (e.g., CrossMab), to incorporate different paired scaffold regions, to incorporate crossed paired scaffold regions, or to introduce at least one mutation into the same paired scaffold region. These are discussed in more detail below.

[0145] A) Crossed VA1/VB1 Configurations [0146] In certain embodiments, the B1/A1 pair and the B2/A2 pair comprise antibody Fv domains, and the Fv domains are crossed to prevent or reduce mispairing between B2 and A1, or B1 and A2. The Fv domains comprise a heavy chain variable domain, VH, and a light chain variable domain, VL. The two Fv domains can be in a crossed configuration, so that if mispairing between B2 and A1 occurs and/or if mispairing between B1 and A2 occurs, pairing of the two VH domains or pairing of the two VL domains will occur, but these are less likely to form than pairings between VH and VL, as they are less stable.

[0147] In some embodiments, VB1 comprises VH1, VA1 comprises VL1, VB2 comprises VL2, and VA2 comprises VH2. In some embodiments, VB1 comprises VL1, VA1 comprises VH1, VB2 comprises VH2, and VA2 comprises VL2. In these crossover VA/VB1 configurations, if B2 mispairs with A1 or if B1 mispairs with A2, pairing of VH1 with VH2 or pairing of VL1 with VL2 will occur, which is thought to be unlikely to form a stable product.

In some of these embodiments, VB1 is operably linked to a first paired scaffold region, PSR _a , and VA1 is operably linked to SR _a , where SR _a binds to PSR _a to form a first paired scaffold domain. In some of these embodiments, VB2 is operably linked to a second paired scaffold region, R _b , and VA2 is operably linked to PSR _b , where SR _b binds to PSR _b to form a second paired scaffold domain.

[0149] In some embodiments, PSR _a is a CH1 domain CH1 a, SR _a is a CL domain CLa, SR _b is a CH1 domain CH1 b, and PSR _b is a CL domain CLb, wherein a) VB1 comprises VH1, VA1 comprises VL1, VB2 comprises VL2, and VA2 comprises VH2, or b) VB1 comprises VL1, VA1 comprises VH1, VB2 comprises VH2, and VA2 comprises VL2.

[0150] B) Different Paired Scaffold Regions [0151] In certain embodiments, the B1/A1 pair comprises a first antibody Fv domain and a first pair of scaffold regions (i.e., SR _a /PSR _a ), and the B2/A2 pair comprises a second antibody Fv domain and a second pair of scaffold regions (i.e., SR _b /PSR _b ), where the first pair of scaffold regions and the second scaffold regions are different or are different types of scaffold regions. Thus, mispairing between A2 and B1, or mispairing between A1 and B2, results in incompatible scaffold regions that do not naturally bind to each other.

In some embodiments, the pair of SR _a and PSR _a is different from the pair of SR _b and PSR _b . In certain embodiments, the pair of SR _a /PSR _a or the pair of SR _b /PSR _b is selected from the group consisting of: a) a pair of heavy chain constant region 1 (CH1) and light chain constant region (CL), b) a pair of T cell receptor (TCR) constant region α (Calpha) and TCR constant region β (Cbeta), c) a pair of TCR constant region γ (Cgamma) and TCR constant region δ (Cdelta), d) a pair of a ligand-binding domain of a receptor and its ligand, e) a pair of a PRD (proline-rich domain) and an SH3 domain, and f) a pair of obscurin and titin. In some embodiments, the TCR constant region alpha (ie, Calpha) comprises the amino acid sequence of SEQ ID NO:97 and the TCR constant region beta (ie, Cbeta) comprises the amino acid sequence of SEQ ID NO:98.

In some embodiments, the pair of SR _a and PSR _a is a pair of CH1 and CL, and the pair of SR _b and PSR _b is a pair of i) Calpha and Cbeta, ii) Cgamma and Cdelta, iii) a ligand-binding domain of a receptor and the ligand, iv) a proline-rich domain (PRD) and an SH3 domain, or v) an obscurin and titin. In some embodiments, the pair of SR _b and PSR _b is a pair of CH1 and CL, and the pair of SR _a and PSR _a is a pair of i) Calpha and Cbeta, ii) Cgamma and Cdelta, iii) a ligand-binding domain of a receptor and the ligand, iv) a proline-rich domain (PRD) and an SH3 domain, or v) an obscurin and titin.

In some embodiments, the pair of SR _a and PSR _a is a pair of Calpha and Cbeta, and the pair of SR _b and PSR _b is a pair of i) CH1 and CL, ii) Cgamma and Cdelta, iii) a ligand-binding domain of a receptor and the ligand, iv) a proline-rich domain (PRD) and an SH3 domain, or v) an obscurin and titin. In some embodiments, the pair of SR _b and PSR _b is a pair of Calpha and Cbeta, and the pair of SR _a and PSR _a is a pair of i) CH1 and CL, ii) Cgamma and Cdelta, iii) a ligand-binding domain of a receptor and the ligand, iv) a proline-rich domain (PRD) and an SH3 domain, or v) an obscurin and titin.

In some embodiments, the pair of SR _a and PSR _a is a pair of Cgamma and Cdelta, and the pair of SR _b and PSR _b is a pair of i) CH1 and CL, ii) Calpha and Cbeta, iii) a ligand-binding domain of a receptor and the ligand, iv) a proline-rich domain (PRD) and an SH3 domain, or v) an obscurin and titin. In some embodiments, the pair of SR _b and PSR _b is a pair of Cgamma and Cdelta, and the pair of SR _a and PSR _a is a pair of i) CH1 and CL, ii) Calpha and Cbeta, iii) a ligand-binding domain of a receptor and the ligand, iv) a proline-rich domain (PRD) and an SH3 domain, or v) an obscurin and titin.

In some embodiments, the pair of SR _a and PSR _a is a pair of a ligand-binding domain of a receptor and the ligand, and the pair of SR _b and PSR _b is a pair of i) CH1 and CL, ii) Calpha and Cbeta, iii) Cgamma and Cdelta, iv) PRD (proline-rich domain) and SH3 domain, or v) obscurin and titin. In some embodiments, the pair of SR _b and PSR _b is a pair of a ligand-binding domain of a receptor and the ligand, and the pair of SR _a and PSR _a is a pair of i) CH1 and CL, ii) Calpha and Cbeta, iii) Cgamma and Cdelta, iv) PRD (proline-rich domain) and SH3 domain, or v) obscurin and titin.

In some embodiments, the pair of SR _a and PSR _a is a pair of a PRD (proline-rich domain) and an SH3 domain, and the pair of SR _b and PSR _b is a pair of i) CH1 and CL, ii) Calpha and Cbeta, iii) Cgamma and Cdelta, iv) a ligand-binding domain of a receptor and the ligand, or v) obscurin and titin. In some embodiments, the pair of SR _b and PSR _b is a pair of a PRD (proline-rich domain) and an SH3 domain, and the pair of SR _a and PSR _a is a pair of i) CH1 and CL, ii) Calpha and Cbeta, iii) Cgamma and Cdelta, iv) a ligand-binding domain of a receptor and the ligand, or v) obscurin and titin.

In some embodiments, the pair of SR _a and PSR _a is a pair of obscurin and titin, and the pair of SR _b and PSR _b is a pair of i) CH1 and CL, ii) Calpha and Cbeta, iii) Cgamma and Cdelta, iv) a ligand-binding domain of a receptor and the ligand, or v) a pair of a PRD (proline-rich domain) and an SH3 domain. In some embodiments, the pair of SR _b and PSR _b is a pair of obscurin and titin, and the pair of SR _a and PSR _a is a pair of i) CH1 and CL, ii) Calpha and Cbeta, iii) Cgamma and Cdelta, iv) a ligand-binding domain of a receptor and the ligand, or v) a pair of a PRD (proline-rich domain) and an SH3 domain.

[0159] In some embodiments, cognate pairing of the scaffold region is facilitated by the introduction of a non-native disulfide bond. In such embodiments, the scaffold region is CH1 and CL, Calpha and Cbeta, or Cgamma and Cdelta.

[0160] C) Cross-Scaffold Regions [0161] In certain embodiments, the first pair of scaffold regions and the second pair of scaffold regions are the same type of scaffold domain, but are configured to prevent mismatching between the first pair of scaffold regions and the second pair of scaffold regions.

In some embodiments, in the fusion polypeptide or polypeptide complex provided herein, the SR _a /PSR _a pair and the SR _b /PSR _b pair are both of the same type, e.g., both are CH1/CL domains, or both are Calpha/Cbeta domains, or both are Cgamma and Cdelta domains, etc., with the proviso that the SR _a /PSR _a pair and/or the SR _b /PSR _b pair are configured to prevent mispairing between SR _a /PSR _b or SR _b /PSR _a . Any suitable means may be used to prevent mispairing between the two pairs of scaffold regions, for example, the SR _a /PSR _a pair and the SR _b /PSR _b pair may be configured to cross each other.

In certain embodiments, for example, the SR _a /PSR _a pair and the SR _b /PSR _b pair are both antibody constant domains formed by CH1/CL pairing. In certain embodiments, the A1/B1 pair and the A2/B2 pair comprise antibody Fab domains, and one of the VH/VL pairs in the Fab domain is crossed to prevent or reduce mispairing between B2 and A1, or B1 and A2.
In some embodiments, the SR _a /PSR _a pair comprises a CH1 domain CH1 a and a CL domain CLa, and the SR _b /PSR _b pair comprises a CH1 domain CH1 b and a CL domain CLb. In some embodiments, the CH1 a/CLa pair and the CH1 b/CLb pair are configured to cross over each other, so that if mispairing between B2 and A1 occurs and/or mispairing between B1 and A2 occurs, pairing of two unpaired scaffold regions, such as CH1 a and CH1 b, or CLa and CLb, will occur, but is less likely to form because it is less stable than mispairing between CH1 and CL.

In some embodiments, VB1 comprises VH1, VA1 comprises VL1, VB2 comprises VH2, and VA2 comprises VL2, wherein a) PSR _a is the CL domain CLa, SR _a is the CH1 domain CH1a, SR _b is the CH1 domain CH1b, and PSR b is the CL domain CLb; or b) PSR _a is the CH1 domain CH1a, SR _a is the CL domain CLa, SR _b is the CL domain CLb, and _{PSR b} is the CH1 domain CH1b.

[0166] D) Mutations Introduced into the Same Paired Scaffold Region [0167] In some embodiments, the first pair of scaffold regions and the second pair of scaffold regions are both the same type of scaffold domain, with one or more mutations introduced to reduce or prevent mispairing.

In some embodiments, VB1 comprises VH1, VA1 comprises VL1, VB2 comprises VH2, VA2 comprises VL2, PSR _a is a CH1 domain CH1a, SR _a is a CL domain CLa, SR _b is a CH1 domain CH1b, and PSR and _b is a CL domain CLb, wherein a) the fusion polypeptide comprises the amino acid sequence of formula (I) VH2-CH1b-Linker-VL1-CLa, b) the second polypeptide comprises the amino acid sequence of formula (II) VH1-CH1a, c) the third polypeptide comprises the amino acid sequence of formula (III) VH2-CH1b, and d) the fourth and fifth polypeptides each comprise the amino acid sequence of formula (IV) VL2-CLb, wherein the CH1b/CLb pair and/or the CH1a/CLa pair are configured to prevent mispairing between CH1b and CLa and/or between CH1a and CLb.

[0169] In some embodiments, one or more mutations are introduced into the CH1b/CLb pair and/or the CH1a/CLa pair to prevent mispairing between CH1b and CLa and/or between CH1a and CLb. In certain embodiments, the one or more mutations introduce a non-natural binding interaction, e.g., a non-natural covalent bond, a non-natural electrostatic interaction, a non-natural salt bridge, a non-natural hydrophobic-hydrophilic interaction, a non-natural connector, or any combination thereof. Preferably, the non-natural binding interaction is introduced into the CH1b/CLb pair or the CH1a/CLa pair, thereby preventing mispairing between the CH1b/CLb pair and the CH1a/CLa pair.

[0170] In certain embodiments, at least one of the CH1b/CLb pair and the CH1a/CLa pair has one or more of the following characteristics: 1) having at least one non-native disulfide bond that prevents mispairing between CH1b and CLa and/or between CH1a and CLb; 2) including one or more introduced amino acid mutations that form at least one or more introduced charged amino acid residues that prevent mispairing between CH1b and CLa and/or between CH1a and CLb; or 3) having one or more introduced amino acid mutations that form an orthogonal CH1-CL interface that prevents mispairing between CH1b and CLa or CH1a and CLb.

[0171] i) Disulfide bond introduced into CH1/CL region
[0172] In certain embodiments, at least one of the CH1b/CLb pair and the CH1a/CLa pair has at least one non-native disulfide bond that prevents mispairing between CH1b and CLa and/or CH1a and CLb.

In some embodiments, the first CH1/CL pair and the second CH1/CL pair are selected from CH1b/CLb and CH1a/CLa. For example, the first CH1/CL pair can be _CH1b / _CLb and the second CH1/CL pair can be CH1a/ _CLa . Alternatively, the first CH1/CL pair can be _CH1a / _CLa and the second _CH1 /CL pair can be _CH1b / _CLb . In some embodiments, the first CH1/CL pair is associated by a non-native first disulfide bond. In some embodiments, the first CH1/CL pair lacks a native disulfide bond or has a native disulfide bond disrupted, e.g., by mutation of the cysteine residue that forms the native disulfide bond.

[0174] In some embodiments, the second CH1/CL pair is associated by a second disulfide bond formed at a different position than the first disulfide bond. In some embodiments, the second disulfide bond formed in the second CH1/CL pair is a native disulfide bond or a non-native disulfide bond.

[0175] In some embodiments, the first disulfide bond is formed by two cysteine residues introduced at a series of heavy chain-light chain EU positions selected from the group consisting of: a) heavy chain position 134 and light chain position 116, b) heavy chain position 141 and light chain position 116, c) heavy chain position 128 and light chain position 118, d) heavy chain position 126 and light chain position 121, e) heavy chain position 127 and light chain position 121, f) heavy chain position 126 and light chain position 124, g) heavy chain position 170 and light chain position 162, h) heavy chain position 171 and light chain position 162, and i) heavy chain position 173 and light chain position 162.

[0176] In some embodiments, the first disulfide bond is formed by two cysteine residues introduced at a series of heavy chain-light chain EU positions selected from the group consisting of: j) heavy chain position 133 and light chain position 209; k) heavy chain position 131 and light chain position 119; l) heavy chain position 133 and light chain position 207; m) heavy chain position 170 and light chain position 176; n) heavy chain position 173 and light chain position 160; o) heavy chain position 133 and light chain position 117; and p) heavy chain position 129 and light chain position 121.

[0177] In some embodiments, the first disulfide bond is formed by two cysteine residues introduced into a series of heavy chain-light chain EU positions selected from the group consisting of: a) heavy chain EU position 126 and light chain EU position 121; b) heavy chain EU position 173 and light chain EU position 160; and c) heavy chain EU position 128-light chain EU position 118.

[0178] In some embodiments, the native disulfide bond is between a heavy chain EU position selected from 131, 219, and 220 and light chain EU position 214. In some embodiments, the native disulfide bond is between heavy chain EU position 220 and light chain EU position 214.

[0179] In some embodiments, the first CH1/CL pair comprises a CH1 that includes a substitution of a cysteine residue at EU position 126 and a substitution of a non-cysteine residue at EU position 220, and a CL that includes a substitution of a cysteine residue at EU position 121 and a substitution of a non-cysteine residue at EU position 214.

[0180] In some embodiments, CH1b comprises a substitution of a cysteine residue at EU position 126 and a substitution of a non-cysteine residue at EU position 220, and CLb comprises a substitution of a cysteine residue at EU position 121 and a substitution of a non-cysteine residue at EU position 214. In some embodiments, CH1b comprises the amino acid sequence of SEQ ID NO: 77 and/or CLb comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, CH1a comprises a substitution of a cysteine residue at EU position 126 and a substitution of a non-cysteine residue at EU position 220, and CLa comprises a substitution of a cysteine residue at EU position 121 and a substitution of a non-cysteine residue at EU position 214. In some embodiments, CH1a comprises the amino acid sequence of SEQ ID NO: 77 and/or CLa comprises the amino acid sequence of SEQ ID NO: 79.

[0181] ii) Substitution with Charged Amino Acids
[0182] In certain embodiments, at least one of the CH1b/CLb pair and the CH1a/CLa pair has at least one or more introduced charged amino acid residues that prevent mispairing between CH1b and CLa and/or CH1a and CLb.
In some embodiments, the first CH1/CL pair and the second CH1/CL pair are selected from CH1b/CLb and CH1a/CLa. For example, the first CH1/CL pair can be _CH1b / _CLb , and the second CH1/CL pair can be _CH1a / _CLa . Alternatively, the first CH1/CL pair can be _CH1a / _CLa , and the second CH1/CL pair can be _CH1b / _CLb . In some embodiments, the first CH1/CL pair includes at least one substitution of an uncharged residue with a charged residue and/or at least one substitution of a charged residue with an oppositely charged residue, such that the first CH1/CL pair contains a first pair of oppositely charged residues that favor pairing of the first CH1/CL pair. In some embodiments, the first CH1/CL pair may contain a combination of substitutions that collectively provide a first pair of oppositely charged residues that favors pairing of the first CH1/CL pair.

[0184] In other words, a pair of oppositely charged residues may be introduced into the first CH1/CL pair to promote cognate pairing between the CH1 domain and the CL domain in the first CH1/CL pair. For example, a charged residue may be introduced to replace an uncharged residue at a position in CH1 (or CL), whereby the introduced charged residue forms an electrostatic interaction with another oppositely charged residue already present or to be introduced in the CL (or CH1), promoting pairing of the first CH1/CL pair. As another example, an existing charged residue at a certain position in CH1 (or CL) may be replaced with an oppositely charged residue, whereby the substituted charged residue forms an electrostatic interaction with another oppositely charged residue already present or to be introduced in the CL (or CH1), promoting pairing of the first CH1/CL pair. In certain embodiments, an existing charged residue in CH1 or CL may be replaced with an uncharged residue to reduce potential interference with electrostatic interactions between the first CH1/CL pair.

[0185] In some embodiments, the second CH1/CL pair comprises at least one substitution of an uncharged residue with a charged residue and/or at least one substitution of a charged residue with an oppositely charged residue, whereby the second CH1/CL pair contains a second pair of oppositely charged residues that favors pairing of the second CH1/CL pair, and optionally, the first pair of oppositely charged residues and the second pair of oppositely charged residues prevent pairing of CH1a with CLb or CH1b with CLa.

[0186] In some embodiments, the first pair of oppositely charged residues and the second pair of oppositely charged residues are configured such that CH1a and CLb both have positively or negatively charged residues at corresponding positions in the first and second pairs of oppositely charged residues, and/or CH1b and CLa both have positively or negatively charged residues at corresponding positions in the first and second pairs of oppositely charged residues.

[0187] In some embodiments, the first pair of oppositely charged residues and/or the second pair of oppositely charged residues are introduced at heavy chain-light chain EU positions selected from the group consisting of: a) heavy chain EU position 183 and light chain EU position 176, b) heavy chain EU position 183 and light chain EU position 133, c) heavy chain EU position 147 and light chain EU position 176, d) heavy chain EU position 141 and light chain EU position 116, e) heavy chain EU position 126 and light chain EU position 121, and f) heavy chain EU position 218 and light chain EU position 122.

[0188] In some embodiments, the first pair of oppositely charged residues and/or the second pair of oppositely charged residues are introduced at heavy chain-light chain EU positions selected from the group consisting of: g) heavy chain EU position 147 and light chain EU position 131; h) heavy chain EU position 168 and light chain EU position 174; i) heavy chain EU positions 147 and 168, and light chain EU positions 131 and 174.

[0189] In some embodiments, the pair of oppositely charged residues comprises a positively charged amino acid residue and a negatively charged amino acid residue, wherein the positively charged amino acid residue is selected from the group consisting of lysine (K), histidine (H), and arginine (R), and/or the negatively charged amino acid residue is selected from the group consisting of aspartic acid (D) and glutamic acid (E).

[0190] In some embodiments, CH1b comprises a lysine substitution at EU position 147 for a negatively charged residue, CLb comprises a serine substitution at EU position 176 for a positively charged residue, CH1a comprises a serine substitution at EU position 183 for a positively charged residue, and CLa comprises a serine substitution at EU position 176 for a negatively charged residue. In some embodiments, CH1b comprises the substitution K147D, CLb comprises the substitution S176K, CH1a comprises the substitution S183K, and CLa comprises the substitution S176D. In some embodiments, CH1b comprises the amino acid sequence of SEQ ID NO:88, CLb comprises the amino acid sequence of SEQ ID NO:90, CH1a comprises the amino acid sequence of SEQ ID NO:87, and CLa comprises the amino acid sequence of SEQ ID NO:92.

[0191] In some embodiments, CH1a comprises a lysine substitution at EU position 147, a negatively charged residue; CLa comprises a serine substitution at EU position 176, a positively charged residue; CH1b comprises a serine substitution at EU position 183, a positively charged residue; and CLb comprises a serine substitution at EU position 176, a negatively charged residue. In some embodiments, CH1a comprises a substitution K147D, CLa comprises a substitution S176K, CH1b comprises a substitution S183K, and CLb comprises a substitution S176D.

[0192] In some embodiments, CH1a comprises the amino acid sequence of SEQ ID NO: 88, CLa comprises the amino acid sequence of SEQ ID NO: 90, CH1b comprises the amino acid sequence of SEQ ID NO: 87, and CLb comprises the amino acid sequence of SEQ ID NO: 92.

[0193] In some embodiments, the first CH1/CL pair comprises CH1b and CLb.

[0194] In some embodiments, with respect to CH1b, the amino acid residue at EU position 173 is replaced with a cysteine residue, the amino acid residue at EU position 183 is replaced with a positively charged residue, and the amino acid residue at EU position 220 is replaced with a non-cysteine residue; and with respect to CLb, the amino acid residue at EU position 160 is replaced with a cysteine residue, the amino acid residue at EU position 176 is replaced with a negatively charged residue, and the amino acid residue at EU position 214 is replaced with a non-cysteine residue.

[0195] In some embodiments, with respect to CH1b, the amino acid residue at EU position 173 is replaced with a cysteine residue, the amino acid residue at EU position 183 is replaced with a negatively charged residue, and the amino acid residue at EU position 220 is replaced with a non-cysteine residue; and with respect to CLb, the amino acid residue at EU position 160 is replaced with a cysteine residue, the amino acid residue at EU position 176 is replaced with a positively charged residue, and the amino acid residue at EU position 214 is replaced with a non-cysteine residue.

[0196] In some embodiments, CH1b comprises the substitutions V173C, S183K, and C220S, and CLb comprises the substitutions Q160C, S176D, and C214S. In some embodiments, CH1b comprises the amino acid sequence of SEQ ID NO: 103, and CLb comprises the amino acid sequence of SEQ ID NO: 104.

[0197] In some embodiments, the first CH1/CL pair comprises a combination of at least one non-native disulfide bond and a non-native electrostatic interaction.

[0198] In some embodiments, the first CH1/CL pair comprises CH1a and CLa.

[0199] In some embodiments, with respect to CH1a, the amino acid residue at EU position 173 is replaced with a cysteine residue, the amino acid residue at EU position 183 is replaced with a positively charged residue, and the amino acid residue at EU position 220 is replaced with a non-cysteine residue; with respect to CLa, the amino acid residue at EU position 160 is replaced with a cysteine residue, the amino acid residue at EU position 176 is replaced with a negatively charged residue, and the amino acid residue at EU position 214 is replaced with a non-cysteine residue. In some embodiments, CH1a comprises V173C, S183K, and C220S substitutions, and CLa comprises Q160C, S176D, and C214S substitutions. In some embodiments, CH1a comprises the amino acid sequence of SEQ ID NO: 103, and CLa comprises the amino acid sequence of SEQ ID NO: 104.

[0200] In some embodiments, for CH1a, the amino acid residue at EU position 173 is replaced with a cysteine residue, the amino acid residue at EU position 183 is replaced with a negatively charged residue, and the amino acid residue at EU position 220 is replaced with a non-cysteine residue; and for CLa, the amino acid residue at EU position 160 is replaced with a cysteine residue, the amino acid residue at EU position 176 is replaced with a positively charged residue, and the amino acid residue at EU position 214 is replaced with a non-cysteine residue.

[0201] iii) Formation of Orthogonal CH1/CL Interface
[0202] In certain embodiments, at least one of the CH1b/CLb pair and the CH1a/CLa pair has one or more introduced amino acid mutations that form an orthogonal CH1-CL interface that prevents mispairing between CH1b and CLa or CH1a and CLb.

[0203] In certain embodiments, the first CH1/CL pair and the second CH1/CL pair are selected from CH1b/CLb and CH1a/CLa, wherein the first CH1/CL pair includes one or more introduced amino acid mutations that form an orthogonal CH1-CL interface.

[0204] In some embodiments, at least one modification comprises one or more introduced amino acid mutations that form an orthogonal CH1-CL interface that favors pairing of CH1b and CLb, and optionally prevents pairing of CH1b and CLa or CH1a and CLb. In some embodiments, at least one modification comprises one or more introduced amino acid mutations that form an orthogonal CH1-CL interface that favors pairing of CH1a and CLa, and optionally prevents pairing of CH1a and CLb or CH1b and CLa.

[0205] In some embodiments, the orthogonal CH1-CL interface comprises mutations at heavy chain EU positions H168A, F170G, and light chain EU positions L135Y, S176W. In some embodiments, the orthogonal CH1-CL interface comprises mutations at heavy chain EU positions H168A and F170G, and light chain EU positions L135Y and S176W, wherein the heavy chain CH1 domain comprises the amino acid sequence of SEQ ID NO:83 and the light chain CL domain comprises the amino acid sequence of SEQ ID NO:95.

[0206] In some embodiments, CH1a comprises a S183E substitution, CLa comprises a V133K substitution, CH1b comprises A141I, F170S, S181M, S183A, and V185A substitutions, and CLb comprises F116A, L235V, S174A, S176F, and T178V substitutions. In some embodiments, CH1b comprises the amino acid sequence of SEQ ID NO: 68, and/or CLb comprises the amino acid sequence of SEQ ID NO: 71.

[0207] In some embodiments, CH1b comprises a S183E substitution, CLb comprises a V133K substitution, CH1a comprises A141I, F170S, S181M, S183A, and V185A substitutions, and CLa comprises F116A, L235V, S174A, S176F, and T178V substitutions. In some embodiments, CH1a comprises the amino acid sequence of SEQ ID NO: 68, and/or CLa comprises the amino acid sequence of SEQ ID NO: 71.

[0208] In some embodiments, the first CH1/CL pair comprises one or more introduced amino acid mutations at a set of heavy chain-light chain EU positions selected from the group consisting of substitutions at heavy chain EU positions A141I, F170S, S181M, S183A, and V185A, and substitutions at light chain EU positions F116A, A235V, S174A, S176F, and T178V, forming an orthogonal Fab design.

[0209] In some embodiments, the first CH1/CL pair comprises introduced amino acid mutations at a series of heavy chain-light chain EU positions selected from the group consisting of substitutions at heavy chain EU positions A141I, F170S, S181M, S183A, and V185A, and substitutions at light chain EU positions F116A, A235V, S174A, S176F, and T178V, forming an orthogonal Fab design; and the second CH1/CL pair comprises introduced amino acid mutations forming a pair of oppositely charged residues comprising S183E in CH1 and V133K in CL. In some embodiments, CH1a comprises the amino acid sequence of SEQ ID NO:68, CLa comprises the amino acid sequence of SEQ ID NO:71, CH1b comprises the amino acid sequence of SEQ ID NO:66, and/or CLb comprises the amino acid sequence of SEQ ID NO:73.

[0210] E) Mutations introduced into Fab variable regions to promote specific pairing [0211] In addition to the scaffold region, mutations can also be introduced into the variable regions in either VA1/VB1 and VA2/VB2 to prevent mispairing of B1/A2 or B2/A1.

[0212] In some embodiments, in a fusion polypeptide or polypeptide complex disclosed herein, the first VH/VL pair and the second VH/VL pair are selected from VH1/VL1 and VH2/VL2, wherein the first VH/VL pair has at least one substitution of an uncharged residue for a charged residue and/or at least one substitution of a charged residue for an oppositely charged residue, such that the first VH/VL includes a third pair of oppositely charged residues that favors pairing of the first VH/VL pair.

[0213] In some embodiments, the second VH/VL pair has at least one substitution of an uncharged residue for a charged residue and/or at least one substitution of a charged residue for an oppositely charged residue, whereby the second VH/VL includes a fourth pair of oppositely charged residues that favors pairing of the second VH/VL pair, and optionally a third pair of oppositely charged residues and a fourth pair of oppositely charged residues that prevent pairing of VH1 with VL2 or VH2 with VL1.

[0214] In some embodiments, the third pair of oppositely charged residues and the fourth pair of oppositely charged residues are configured such that VH1 and VL2 both have positively or negatively charged residues and/or VH2 and VL1 both have positively or negatively charged residues.

[0215] In some embodiments, the third pair of oppositely charged residues and/or the fourth pair of oppositely charged residues are introduced at heavy chain-light chain EU positions selected from the group consisting of: a) heavy chain EU position 39:light chain EU position 38; b) heavy chain EU position 105:light chain EU position 43; c) heavy chain EU position 62:light chain EU position 1; or d) any combination thereof.

[0216] In some embodiments, the pair of oppositely charged residues comprises a positively charged amino acid residue and a negatively charged amino acid residue, wherein the positively charged amino acid residue is selected from the group consisting of lysine (K), histidine (H), and arginine (R), and/or the negatively charged amino acid residue is selected from the group consisting of aspartic acid (D) and glutamic acid (E).

[0217] F) Modifications in the Fc Region [0218] In some embodiments, in the polypeptide complexes disclosed herein, the second polypeptide and the third polypeptide each further comprise an operably linked first dimerization domain and an operably linked second dimerization domain that associate to form a dimer.

[0219] In some embodiments, the first dimerization domain and the second dimerization domain comprise an IgG CH3 domain. In some embodiments, the first dimerization domain and the second dimerization domain further comprise a hinge region.

[0220] In some embodiments, in a polypeptide complex disclosed herein, the second polypeptide comprises an operably linked first dimerization domain and/or the third polypeptide further comprises an operably linked second dimerization domain. In some embodiments, in a polypeptide complex disclosed herein, the second polypeptide comprises an operably linked second dimerization domain and/or the third polypeptide further comprises an operably linked first dimerization domain.

[0221] In some embodiments, in the polypeptide complexes disclosed herein, the first dimerization domain comprises an operably linked first Fc region and/or the second dimerization domain comprises an operably linked second Fc region. In some embodiments, the first Fc region and/or the second Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

[0222] In some embodiments, the first Fc region and the second Fc region have different amino acid sequences and at least one configuration that promotes heterodimerization of the first Fc region and the second Fc region.

[0223] In certain embodiments, the polypeptide complexes disclosed herein contain one or more amino acid substitutions at the interface of the Fc region to promote and/or enhance heterodimerization. These modifications include the introduction of a protrusion in the first Fc region and a cavity in the second Fc region, where the protrusion may be positioned within the cavity (also referred to as a knob-in-hole structure) to enhance the interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies with these modifications are known in the art, for example, as described in U.S. Pat. No. 5,731,168.

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).
[0225] In some embodiments, the first Fc region comprises a first Fc mutation, and/or the second Fc region comprises a second Fc mutation, wherein a) the first Fc mutation comprises T366W or S354C and the second Fc mutation comprises Y349C, T366S, L368A, or Y407V; b) the first Fc mutation comprises D399K or E356K and the second Fc mutation comprises K392D or K409D; c) the first Fc mutation comprises E356K, E357K, or D399K and the second Fc mutation comprises K370E, K409D, or K 439E; d) the first Fc mutation comprises S364H or F405A and the second Fc mutation comprises Y349T or T394F; e) the first Fc mutation comprises S364H or T394F and the second Fc mutation comprises Y394T or F405A; f) the first Fc mutation comprises K370D or K409D and the second Fc mutation comprises E357K or D399K; or g) the first Fc mutation comprises L351D or L368E and the second Fc mutation comprises L351K or T366K, numbering according to the EU index.
[0226] In some embodiments, the first Fc region comprises a first Fc mutation, and/or the second Fc region comprises a second Fc mutation, wherein a) the first Fc mutation comprises T366S/L368A/Y407V and the second Fc mutation comprises T366W; b) the first Fc mutation comprises S354C/T366W and the second Fc mutation comprises Y349C/T366S/L368A/Y407V; c) the first Fc mutation comprises T366Y and the second Fc mutation comprises Y407T; or d) the first Fc mutation comprises T366W and the second Fc mutation comprises Y407A; e) the first Fc mutation comprises T394W and the second Fc mutation comprises F405A; f) the first Fc mutation comprises T366Y/F405A and the second Fc mutation comprises T394W/Y407T; g) the first Fc mutation comprises T366W/F405W and the second Fc mutation comprises T394S/Y407A; g) the first Fc mutation comprises F405W and the second Fc mutation comprises T394S; h) the first Fc mutation comprises D399C and the second Fc mutation comprises K392C; i) the first Fc mutation comprises T366W/D and the second Fc mutations comprise T366S/L368A/K392C/Y407V; j) the first Fc mutations comprise T366W/K392C and the second Fc mutations comprise T366S/L368A/D399C/Y407V; k) the first Fc mutations comprise S354C/T366W and the second Fc mutations comprise Y349C/T366S/L368A/Y407V; l) the first Fc mutations comprise Y349C/T366W and the second Fc mutations comprise S354C/T366S/L368A/Y407V; m) the first Fc mutations comprise comprises E356C/T366W and the second Fc mutations comprise Y349C/T366S/L368A/Y407V; n) the first Fc mutations comprise Y349C/T366W and the second Fc mutations comprise E356C/T366S/L368A/Y407V; o) the first Fc mutations comprise E357C/T366W and the second Fc mutations comprise Y349C/T366S/L368A/Y407V; p) the first Fc mutations comprise Y349C/T366W and the second Fc mutations comprise E357C/T366S/L368A/Y407V.

[0227] 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 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 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.

[0228] In some embodiments, the first Fc region comprises a first Fc mutation, and/or the second Fc region comprises a second Fc mutation, wherein a) the first Fc mutation comprises K370E/D399K/K439D and the second Fc mutation comprises D356K/E357K/K409D; b) the first Fc mutation comprises K409D and the second Fc mutation comprises D399K; c) the first Fc mutation comprises K409E and the second Fc mutation comprises D399K; d) the first Fc mutation comprises K409E and the second Fc mutation comprises D399R; e) the first Fc mutation comprises K409D and the second Fc mutation comprises D399R; f) the first Fc mutation comprises D339K and the second Fc mutation comprises E356K; g) the first Fc mutation comprises E356K/ a) the first Fc mutation comprises E356K/D399K and the second Fc mutation comprises K392D/K409D; b) the first Fc mutation comprises E356K/D399K and the second Fc mutation comprises K409D/K439D; c) the first Fc mutation comprises E357K/D399K and the second Fc mutation comprises K370D/K409D; and d) the first Fc mutation comprises E356K/E357K/D399K and the second Fc mutation comprises K409D/K439D. k) the first Fc mutation comprises E357K/D399K and the second Fc mutation comprises K392D/K409D; l) the first Fc mutation comprises K392D/K409D and the second Fc mutation comprises D399K; m) the first Fc mutation comprises K360D/K409D and the second Fc mutation comprises D399K.

[0229] V Target of Polypeptide Complex [0230] In some embodiments, in the polypeptide complexes disclosed herein, at least one of the first target binding domain and the second target binding domain is a chimeric domain, a humanized domain, or a fully human domain.

[0231] In some embodiments, a "chimeric target binding domain" herein refers to a recombinant protein having variable domains comprising the complementarity determining regions (CDRs) of an antibody derived from one species, such as a rodent or murine antibody, and the constant domains of the antibody molecule derived from the complementarity determining regions of an antibody of another species, such as a human antibody. For veterinary applications, the constant domains of the chimeric antibody may be derived from the complementarity determining regions of other species, such as a subhuman primate, cat, or dog.

[0232] In some embodiments, the term "humanized target binding domain" herein refers to a recombinant protein in which CDRs derived from an antibody from one species, e.g., a rodent antibody, have been introduced into the framework regions of human heavy and light variable domains. The constant domains of the antibody molecule are derived from the CDRs of a human antibody. In some embodiments, certain residues in the framework regions of the humanized antibody, particularly those contacting or close to the CDR sequences, can be modified, e.g., so that they are replaced with corresponding residues from the rodent, subhuman primate, or other antibody of origin.

In some embodiments, at least one of the first target binding domain and the second target binding domain is fully human and can be generated by any suitable method known in the art, for example, from transgenic mice that have been genetically engineered to produce specific human antibodies in response to antigenic challenge. Methods for obtaining human antibodies from transgenic mice are described in Green et al., Nature Genet. 7: 13 (1994), Lonberg et al., Nature 368: 856 (1994), and Taylor et al., Int. Immun. 6: 579 (1994). Fully human target binding domains can also be constructed by genetic or chromosomal transfection methods and phage display technology, all of which are known in the art. For in vitro production of human antibodies and fragments thereof from immunoglobulin variable domain gene repertoires from unimmunized donors, see, e.g., McCafferty et al., Nature 348: 552-553 (1990). Phage display can be performed in a variety of formats, for reviews of which see, e.g., Johnson and Chiswell, Current Opinion in Structural Biology 3: 5564-571 (1993). Human target binding domains can also be generated by in vitro activated B cells. See U.S. Patent Nos. 5,567,610 and 5,229,275, which are incorporated herein by reference in their entireties.

[0234] The polypeptide complexes provided herein are based on a novel 2:1 structure, which is bivalent to a second target and monovalent to a first target. In particular, an additional Fab region is introduced based on an IgG-like bispecific antibody, thereby making one of the two target binding sites bivalent and restoring its target binding affinity, which effectively acts to compensate for the affinity difference between the two target binding sites without any adverse effects on antibody specificity or safety risks. Such affinity difference can be useful because bivalent binding to the second target confers avidity and enhances differentiation between high and low expressing cells of the second target. Furthermore, relatively low affinity binding to the first target can be useful to reduce or avoid undesirable effects of strong binding to the first target (e.g., nonspecific activation of the first target, which can cause unwanted biological effects).

[0235] In some embodiments, the first target binding domain and the second target binding domain bind to different targets. In some embodiments, at least one of the first target and the second target is a disease-associated antigen. For example, the disease-associated antigen can be a tumor-associated antigen, an antigen associated with an autoimmune or inflammatory disease, 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.

[0236] In certain embodiments, one of the first target and the second target is a tumor-associated antigen. In some embodiments, the second target is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is an antigen that is present in tumors but not in normal organs, tissues, and/or cells. In some embodiments, the tumor-associated antigen is an antigen that is more prevalent in tumors than in normal organs, tissues, and/or cells. In some embodiments, the tumor-associated antigen is an antigen that is more prevalent in malignant cancer cells than in normal cells.

[0237] In some embodiments, the second target binding domain binds to a tumor-associated antigen. Tumor-associated antigens include antigens presented on the surface of tumor cells, antigens located 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, and refer to antigenic substances produced within tumor cells, i.e., which elicit a host immune response.

[0238] Examples of tumor-associated 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 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.

[0239] In certain embodiments, one of the first target and the second target is a tumor-associated antigen and the other is an immune-related target. In some embodiments, the first target binding domain binds to the immune-related target. In some embodiments, the second target is a tumor-associated antigen and the first target binding domain binds to the immune-related target.

[0240] 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), I It may be selected from the group consisting of COSLG (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.

[0241] In some preferred embodiments of the bispecific polypeptide complexes provided herein, the second target is associated with a receptor on a cytotoxic T lymphocyte (e.g., CD3), and the first target is associated with a cell surface tumor antigen, such as CD19, CD20, CD33, CD123, HER1, HER2, CEA, disialoganglioside GD2, PSMA, gpA33, EpCAM, P-cadherin, or B7H3 (Sedykh SE, et al., Drug Des Devel Ther. 2018; 12: 195-208). Thus, the bispecific polypeptide complexes provided herein can be used to form a link between a T cell and a tumor cell, allowing the T cell to exert cytotoxic activity against the tumor cell.

[0242] In certain embodiments, at least one of the first target and the second target is an antigen associated with an autoimmune or inflammatory disease, such as, for example, rheumatoid arthritis (RA), psoriasis, osteoporosis, idiopathic pulmonary fibrosis, asthma, Sjogren's syndrome, or type 2 diabetes. 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.

[0243] In certain embodiments, at least one of the first target and the second target is an antigen associated with an ocular disease, such as, 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.

[0244] In certain embodiments, at least one of the first target and the second target is an antigen associated with a central nervous system disease, such as, 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.

[0245] In certain embodiments, at least one of the first target and the second target is an antigen associated with an infectious disease, such as, for example, pneumonia, a viral infection (e.g., COVID-19 infection), etc. In some embodiments, the antigen associated with an autoimmune disease or inflammatory disease is, for example, but not limited to, Psl, Pcrv, or the spike protein of the COVID-19 virus.

[0246] In certain embodiments, at least one of the first target and the second target is an antigen associated with a coagulation disorder, such as, for example, hemophilia A. In some embodiments, the antigen associated with hemophilia A is, for example, but not limited to, FIXa or FX.

[0247] Other examples of antigen pairs that can be targeted by the bispecific polypeptide complexes provided herein and thus have potential therapeutic effects 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:IL 4, 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, but are not limited thereto (Sedykh SE, et al., Drug Des Devel Ther. 2018; 12: 195-208).

[0248] In certain embodiments, the first target is CD3 and the second target is CD20.

[0249] In certain embodiments, the first target binding domain is capable of binding to CD3 and comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 113, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 114, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 115, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 116, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 117, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 118.

[0250] In certain embodiments, the second target binding domain is capable of binding to CD20 and comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 107, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 108, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 109, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 110, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 111, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 112.

[0251] Exemplary Polypeptide Conjugates
[0252] In certain embodiments of the polypeptide complexes provided herein, a) the fusion polypeptide comprises an amino acid sequence of Formula (I) VH2-CH1b-Linker-VL1-CLa; b) the second polypeptide comprises an amino acid sequence of Formula (II) VH1-CH1a; c) the third polypeptide comprises an amino acid sequence of Formula (III) VH2-CH1b; and d) the fourth and fifth polypeptides each comprise an amino acid sequence of Formula (IV) VL2-CLb, wherein the CH1b/CLb pair and/or the CH1a/CLa pair are configured to prevent mispairing between CH1b and CLa and/or between CH1a and CLb.

[0253] FORMAT NEW1
[0254] In certain embodiments of the polypeptide complexes provided herein, CH1a has substitutions at heavy chain EU positions A141I, F170S, S181M, S183A, and V185A, CLa has substitutions at light chain EU positions F116A, A235V, S174A, S176F, and T178V, CH1b has a substitution at heavy chain EU position S183E, and CLb has a substitution at light chain EU position V133K. In certain embodiments, _CH1a comprises the amino acid sequence of SEQ ID NO:68, _CLa comprises the amino acid sequence of SEQ ID NO:71, _CH1b comprises the amino acid sequence of SEQ ID NO:66, and _CLb comprises the amino acid sequence of SEQ ID NO:73.

[0255] In certain embodiments, VH1 has a substitution at heavy chain EU position Q39E and VL1 has a substitution at light chain EU position Q38K. In certain embodiments, VH2 has a substitution at heavy chain EU position Q39K and VL1 has a substitution at light chain EU position Q38E. In certain embodiments, VH1 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:113, SEQ ID NO:114, and SEQ ID NO:115, respectively, and/or VL1 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118, respectively. In certain embodiments, VH2 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively, and/or VL2 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively.

[0256] In certain embodiments of the polypeptide complexes provided herein, VH1 comprises the amino acid sequence of SEQ ID NO: 67, VL1 comprises the amino acid sequence of SEQ ID NO: 70, VH2 comprises the amino acid sequence of SEQ ID NO: 65, and VL2 comprises the amino acid sequence of SEQ ID NO: 72.

[0257] In some of these embodiments, the first Fc and second Fc comprise the amino acid sequences of SEQ ID NOs: 69 and 84. In some of these embodiments, the fusion polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide comprise the amino acid sequences of SEQ ID NOs: 6, 5, 7, and 8, respectively. This polypeptide complex is also referred to as anti-CD20 x CD3 FORMAT NEW1.

[0258] FORMAT NEW2
[0259] In certain embodiments of the polypeptide complexes provided herein, CH1a has a substitution of a cysteine residue at EU position 126 and a substitution of a non-cysteine residue at EU position 220, and CLa has a substitution of a cysteine residue at EU position 121 and a substitution of a non-cysteine residue at EU position 214. In some of these embodiments, CH1a comprises the amino acid sequence of SEQ ID NO:77, CLa comprises the amino acid sequence of SEQ ID NO:79, CH1b comprises the amino acid sequence of SEQ ID NO:75, and CLb comprises the amino acid sequence of SEQ ID NO:81.

[0260] In certain embodiments, VH1 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:113, SEQ ID NO:114, and SEQ ID NO:115, respectively, and/or VL1 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118, respectively. In certain embodiments, VH2 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:107, SEQ ID NO:108, and SEQ ID NO:109, respectively, and/or VL2 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:112, respectively.

[0261] In certain embodiments of the polypeptide complexes provided herein, VH1 comprises the amino acid sequence of SEQ ID NO: 76, VL1 comprises the amino acid sequence of SEQ ID NO: 78, VH2 comprises the amino acid sequence of SEQ ID NO: 74, and VL2 comprises the amino acid sequence of SEQ ID NO: 80. In some of these embodiments, the first Fc and second Fc comprise the amino acid sequences of SEQ ID NOs: 69 and 84. In some of these embodiments, the fusion polypeptide, second polypeptide, third polypeptide, and fourth polypeptide comprise the amino acid sequences of SEQ ID NOs: 14, 13, 15, and 16, respectively. This polypeptide complex is also referred to as anti-CD20xCD3 FORMAT NEW2.

[0262] FORMAT NEW3
[0263] In certain embodiments of the polypeptide complexes provided herein, CH1a has a substitution at heavy chain EU position K147D, CLa has a substitution at light chain EU position S176K, CH1b has a substitution at heavy chain EU position S183K, and CLb has a substitution at light chain EU position S176D. In some of these embodiments, _CH1a comprises the amino acid sequence of SEQ ID NO:88, _CLa comprises the amino acid sequence of SEQ ID NO:90, _CH1b comprises the amino acid sequence of SEQ ID NO:87, and _CLb comprises the amino acid sequence of SEQ ID NO:92.

[0264] In certain embodiments, VH1 has substitutions at heavy chain EU positions Q39K and Q105K, and VL1 has substitutions at light chain EU positions Q38D and A43D. In certain embodiments, VH2 has substitutions at heavy chain EU positions Q39D and Q105D, and VL2 has substitutions at light chain EU positions Q38K and A43K. In certain embodiments, VH1 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:113, SEQ ID NO:114, and SEQ ID NO:115, respectively, and/or VL1 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118, respectively. In certain embodiments, VH2 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively, and/or VL2 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively.

[0265] In certain embodiments of the polypeptide complexes provided herein, VH1 comprises the amino acid sequence of SEQ ID NO: 85, VL1 comprises the amino acid sequence of SEQ ID NO: 89, VH2 comprises the amino acid sequence of SEQ ID NO: 86, and VL2 comprises the amino acid sequence of SEQ ID NO: 91. In some of these embodiments, the first Fc and second Fc comprise the amino acid sequences of SEQ ID NOs: 69 and 84. In some of these embodiments, the fusion polypeptide, second polypeptide, third polypeptide, and fourth polypeptide comprise the amino acid sequences of SEQ ID NOs: 22, 21, 23, and 24, respectively. This polypeptide complex is also referred to as anti-CD20xCD3 FORMAT NEW3.

[0266] FORMAT NEW4
[0267] In certain embodiments of the polypeptide complexes provided herein, CH1a has substitutions at heavy chain EU positions H168A and F170G, and CLa has substitutions at light chain EU positions L135Y and S176W. In some of these embodiments, _CH1a comprises the amino acid sequence of SEQ ID NO:83, _CLa comprises the amino acid sequence of SEQ ID NO:95, _CH1b comprises the amino acid sequence of SEQ ID NO:75, and _CLb comprises the amino acid sequence of SEQ ID NO:81.

[0268] In certain embodiments, VH1 has substitutions at heavy chain EU positions 62E and Q39K, and VL1 has substitutions at light chain EU positions D1R and Q38D. In certain embodiments, VH2 has substitutions at heavy chain EU position Q39Y, and VL2 has substitutions at light chain EU position Q38R. In certain embodiments, VH1 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:113, SEQ ID NO:114, and SEQ ID NO:115, respectively, and/or VL1 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118, respectively. In certain embodiments, VH2 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively, and/or VL2 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively.

[0269] In certain embodiments of the polypeptide complexes provided herein, VH1 comprises the amino acid sequence of SEQ ID NO:93, VL1 comprises the amino acid sequence of SEQ ID NO:94, VH2 comprises the amino acid sequence of SEQ ID NO:82, and VL2 comprises the amino acid sequence of SEQ ID NO:96. In some of these embodiments, the first Fc and second Fc comprise the amino acid sequences of SEQ ID NOs:69 and 84. In some of these embodiments, the fusion polypeptide, second polypeptide, third polypeptide, and fourth polypeptide comprise the amino acid sequences of SEQ ID NOs:30, 29, 31, and 32, respectively. This polypeptide complex is also referred to as anti-CD20xCD3 FORMAT NEW4.

[0270] FORMAT NEW7
[0271] In certain embodiments of the polypeptide complexes provided herein, CH1a has a substitution of a cysteine residue at EU position 173, a substitution of a non-cysteine residue at EU position 220, and a substitution at EU position S183K, and CLa has a substitution of a cysteine residue at EU position 160, a substitution of a non-cysteine residue at EU position 214, and a substitution at EU position S176D. In some of these embodiments, CH1a comprises the amino acid sequence of SEQ ID NO:103, CLa comprises the amino acid sequence of SEQ ID NO:104, CH1b comprises the amino acid sequence of SEQ ID NO:75, and CLb comprises the amino acid sequence of SEQ ID NO:81.

[0272] In certain embodiments, VH1 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:113, SEQ ID NO:114, and SEQ ID NO:115, respectively, and/or VL1 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118, respectively. In certain embodiments, VH2 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:107, SEQ ID NO:108, and SEQ ID NO:109, respectively, and/or VL2 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:112, respectively.

[0273] In certain embodiments of the polypeptide complexes provided herein, VH1 comprises the amino acid sequence of SEQ ID NO: 76, VL1 comprises the amino acid sequence of SEQ ID NO: 78, VH2 comprises the amino acid sequence of SEQ ID NO: 74, and VL2 comprises the amino acid sequence of SEQ ID NO: 80. In some of these embodiments, the first Fc and second Fc comprise the amino acid sequences of SEQ ID NOs: 69 and 84. In some of these embodiments, the fusion polypeptide, second polypeptide, third polypeptide, and fourth polypeptide comprise the amino acid sequences of SEQ ID NOs: 54, 53, 55, and 56, respectively. This polypeptide complex is also referred to as anti-CD20 x CD3 FORMAT NEW7.

[0274] FORMAT NEW5
[0275] In certain embodiments of the polypeptide complexes provided herein, a) the fusion polypeptide comprises an amino acid sequence of formula (I) VH2-CH1 _b -Linker-VL1-TCRβ; b) the second polypeptide comprises an amino acid sequence of formula (II) VH1-TCRα; c) the third polypeptide comprises an amino acid sequence of formula (III) VH2-CH1b; and d) the fourth and fifth polypeptides each comprise an amino acid sequence of formula (IV) VL2-CLb.

[0276] In some of these embodiments, TCRα comprises the amino acid sequence of SEQ ID NO:97, TCRβ comprises the amino acid sequence of SEQ ID NO:98, CH1b comprises the amino acid sequence of SEQ ID NO:75, and CLb comprises the amino acid sequence of SEQ ID NO:81.

[0277] In certain embodiments, VH1 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:113, SEQ ID NO:114, and SEQ ID NO:115, respectively, and/or VL1 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118, respectively. In certain embodiments, VH2 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:107, SEQ ID NO:108, and SEQ ID NO:109, respectively, and/or VL2 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:112, respectively.

[0278] In certain embodiments of the polypeptide complexes provided herein, VH1 comprises the amino acid sequence of SEQ ID NO: 76, VL1 comprises the amino acid sequence of SEQ ID NO: 78, VH2 comprises the amino acid sequence of SEQ ID NO: 74, and VL2 comprises the amino acid sequence of SEQ ID NO: 80. In some of these embodiments, the first Fc and second Fc comprise the amino acid sequences of SEQ ID NOs: 69 and 84. In some of these embodiments, the fusion polypeptide, second polypeptide, third polypeptide, and fourth polypeptide comprise the amino acid sequences of SEQ ID NOs: 38, 37, 39, and 40, respectively. This polypeptide complex is also referred to as anti-CD20 x CD3 FORMAT NEW5.

[0279] FORMAT NEW6
[0280] In certain embodiments of the polypeptide complexes provided herein, a) the fusion polypeptide comprises an amino acid sequence of Formula (I) VH2-CH1 _b -Linker-VH1-CL _a ; b) the second polypeptide comprises an amino acid sequence of Formula (II) VL1-CH1 _a ; c) the third polypeptide comprises an amino acid sequence of Formula (III) VH2-CH1 b; and d) the fourth and fifth polypeptides each comprise an amino acid sequence of Formula (IV) VL2-CLb.

[0281] In certain embodiments of the polypeptide complexes provided herein, CH1 _a comprises the amino acid sequence of SEQ ID NO: 100, CL _a comprises the amino acid sequence of SEQ ID NO: 101, CH1 _b comprises the amino acid sequence of SEQ ID NO: 99, and CL _b comprises the amino acid sequence of SEQ ID NO: 102.

[0282] In certain embodiments, VH1 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:113, SEQ ID NO:114, and SEQ ID NO:115, respectively, and/or VL1 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118, respectively. In certain embodiments, VH2 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:107, SEQ ID NO:108, and SEQ ID NO:109, respectively, and/or VL2 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:112, respectively.

[0283] In certain embodiments of the polypeptide complexes provided herein, VH1 comprises the amino acid sequence of SEQ ID NO: 76, VL1 comprises the amino acid sequence of SEQ ID NO: 78, VH2 comprises the amino acid sequence of SEQ ID NO: 74, and VL2 comprises the amino acid sequence of SEQ ID NO: 80. In some of these embodiments, the first Fc and second Fc comprise the amino acid sequences of SEQ ID NOs: 69 and 84. In some of these embodiments, the fusion polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide comprise the amino acid sequences of SEQ ID NOs: 46, 45, 47, and 48, respectively. This polypeptide complex is also referred to as anti-CD20xCD3 FORMAT NEW6.

[0284] FORMAT NEW8
[0285] In certain embodiments of the polypeptide complexes provided herein, a) the fusion polypeptide comprises an amino acid sequence of Formula (I) VH2-CH1b-Linker-VL1-CH1a; b) the second polypeptide comprises an amino acid sequence of Formula (II) VH1-CLa; c) the third polypeptide comprises an amino acid sequence of Formula (III) VH2-CH1b; and d) the fourth and fifth polypeptides each comprise an amino acid sequence of Formula (IV) VL2-CLb.

[0286] In certain embodiments of the polypeptide complexes provided herein, CH1 _a comprises the amino acid sequence of SEQ ID NO: 105, CLa comprises the amino acid sequence of SEQ ID NO: 106, CH1 _b comprises the amino acid sequence of SEQ ID NO: 75, and CLb comprises the amino acid sequence of SEQ ID NO: 81.

[0287] In certain embodiments, VH1 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:113, SEQ ID NO:114, and SEQ ID NO:115, respectively, and/or VL1 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118, respectively. In certain embodiments, VH2 comprises heavy chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:107, SEQ ID NO:108, and SEQ ID NO:109, respectively, and/or VL2 comprises light chain CDR1/CDR2/CDR3 comprising the amino acid sequences of SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:112, respectively.

[0288] In certain embodiments of the polypeptide complexes provided herein, VH1 comprises the amino acid sequence of SEQ ID NO: 76, VL1 comprises the amino acid sequence of SEQ ID NO: 78, VH2 comprises the amino acid sequence of SEQ ID NO: 74, and VL2 comprises the amino acid sequence of SEQ ID NO: 80. In some of these embodiments, the first Fc and second Fc comprise the amino acid sequences of SEQ ID NOs: 69 and 84. In some of these embodiments, the fusion polypeptide, second polypeptide, third polypeptide, and fourth polypeptide comprise the amino acid sequences of SEQ ID NOs: 62, 61, 63, and 64, respectively. This polypeptide complex is also referred to as anti-CD20 x CD3 FORMAT NEW8.

[0289] VI. Polynucleotides and Recombinant Methods [0290] The present disclosure provides nucleic acids comprising nucleotide sequences encoding the fusion polypeptides or polypeptide complexes provided herein. The terms "nucleic acid" or "nucleotide sequence," as used herein, refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either 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)).

[0291] Polynucleotides encoding the polypeptide complexes disclosed herein can be generated by methods known in the art. In certain embodiments, the sequence of the polynucleotide may be obtained based on the amino acid sequence of the polypeptide complex, or the nucleic acid may 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 a polypeptide complex disclosed herein.

[0292] 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. 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.

[0293] 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, herpes viruses (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, and pQ. D-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pM ONO, 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, pEF-Bos, etc., but are not limited to these.

[0294] A vector containing a nucleotide sequence encoding a fusion polypeptide or polypeptide complex may be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein are the prokaryotes, yeast, or higher eukaryotic cells described above. Prokaryotes suitable for this purpose include eubacteria, such as gram-negative or gram-positive organisms, e.g., enterobacteria such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as bacilli, e.g., Bacillus, e.g., Bacillus subtilis and B. licheniformis, Pseudomonas, e.g., Pseudomonas aeruginosa, and Streptomyces.

[0295] 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, for example, 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 the like. and Kluyveromyces marxianus; Yarrowia (EP 402,226), Pichia pastoris (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa, Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, and Tolypocladium; and Aspergillus hosts such as Aspergillus nidulans and Aspergillus niger.

[0296] 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 golden borer NPV and the Bm-5 strain of the silkworm NPV, are publicly available, and such viruses can be used in accordance with the invention herein, particularly as viruses for transfection of armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0297] 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 line (COS-7, ATCC CRL1651), human embryonic kidney line (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), dog 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 N.Y. 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 derivatives thereof.

[0298] 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.

[0299] The present disclosure also provides a method for expressing a fusion polypeptide or 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. 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. In addition, see Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90/03430, WO 87/00195, or U.S. Patent No. 30,985 can be used as a culture medium for host cells. Any of these media can 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 (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 can also be included at appropriate concentrations known to those skilled 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 skilled in the art.

[0300] When using recombinant techniques, the fusion polypeptide or polypeptide complex may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the fusion polypeptide or polypeptide complex is 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. If the fusion polypeptide or polypeptide complex is secreted into the medium, the supernatant from such expression systems is typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. 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.

[0301] The fusion polypeptide or 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.

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 (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Depending on the antibody to be recovered, other protein purification techniques can also be used, such as fractionation on an ion exchange column, ethanol precipitation, 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.

[0303] 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 with a pH of about 2.5 to 4.5, preferably at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0304] VII Pharmaceutical Compositions [0305] The present invention further provides pharmaceutical compositions comprising a polypeptide conjugate described herein and a pharmaceutically acceptable carrier.

[0306] 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.

[0307] "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 may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonicity agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, or other ingredients known in the art, or various combinations thereof.

[0308] 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 contain, in addition to the active ingredient (i.e., the polypeptide complex or heterodimeric antibody or antigen-binding fragment thereof disclosed herein), one or more antioxidants, such as methionine.

[0309] 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 utilized as carriers may be added to pharmaceutical compositions in multidose 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.

[0310] Pharmaceutically acceptable "diluents" may include saline and aqueous buffer solutions.

[0311] Pharmaceutically acceptable "adjuvants" may include preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of presence of microorganisms can be ensured both by sterilization procedures, supra, and by the inclusion 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. Additionally, prolonged absorption of the injectable dosage form may be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0312] 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.

[0313] In embodiments, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as, for example, a liquid solution, suspension, emulsion, or solid form suitable for making a liquid solution, suspension, or emulsion. Preparations for injection can include sterile and/or non-pyrogenic solutions ready for injection, sterile dry soluble preparations such as lyophilized powders ready to be combined with a solvent immediately before use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble preparations ready to be combined with a vehicle immediately before use, and sterile and/or non-pyrogenic emulsions. Solutions can be aqueous or non-aqueous.

[0314] In certain embodiments, unit-dose parenteral preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration should be sterile and non-pyrogenic, as known and practiced in the art.

[0315] 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 buffers known to those of skill in the art, in one embodiment at about 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 with a small amount (e.g., about 10%) beyond that required for the dose or set of doses may be acceptable to facilitate accurate sample draws and accurate dosing. The lyophilized powder may be stored under appropriate conditions, such as at about 4°C to room temperature.

[0316] 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 will depend on the given selected therapy and can be determined empirically.

[0317] In certain embodiments, the present invention further provides a composition comprising a pharmaceutically acceptable carrier, diluent, or adjuvant and an active ingredient. The active ingredient may be a polypeptide complex disclosed herein or a conjugate of a polypeptide complex disclosed herein.

[0318] VIII Conjugates [0319] The polypeptide conjugates provided herein can be used in unconjugated or conjugated form.

[0320] In the conjugated form, the polypeptide complex is conjugated to one or more desired conjugation moieties, i.e., heterologous moieties, to perform a specific function, e.g., to facilitate target detection, or for imaging or therapy.

[0321] The present disclosure provides herein a conjugate comprising a polypeptide complex provided herein and a conjugate moiety (e.g., a payload) conjugated thereto. 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 anticancer drug, and a cytotoxic molecule.

[0322] Various conjugates can be linked to the engineered antibodies provided herein by covalent bonding, affinity binding, intercalation, coordinate bonding, complex formation, association, blending, or addition (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)).

[0323] In certain embodiments, the polypeptide complexes provided herein may be engineered to contain specific sites outside 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.

[0324] 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 (e.g., polyethylene glycol (PEG)) and the C-terminus is conjugated to another moiety (e.g., biotin).

[0325] 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.

[0326] For example, polypeptide conjugates provided herein having a reactive residue, such as cysteine, can be linked to a thiol-reactive agent, where the reactive group is, for example, maleimide, iodoacetamide, pyridyl disulfide, or other thiol-reactive conjugation partner (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).

[0327] As another example, a polypeptide conjugate 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 conjugate 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.

[0328] 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 another anti-cancer agent.

[0329] 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.

[0330] 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.

[0331] 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. Anti-inflammatory drugs include, without limitation, anti-inflammatory drugs (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 antimitotics (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, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Jatropha forsi protein, dianthin protein, Phytolacca americana protein, bitter melon inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0332] 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.

[0333] In certain embodiments, the polypeptide complexes provided herein are used as bases in conjugates.

[0334] IX Compositions [0335] In another aspect, the present invention provides compositions comprising a polypeptide complex or conjugate described herein and a pharmaceutically acceptable carrier.

[0336] X Medical Uses
[0337] 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.

[0338] 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, sports, 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.

[0339] As used herein, "treatment" of 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.

[0340] As used herein, the terms "disorder," "disease," or "condition," etc., refer to a condition affecting a subject amenable to treatment with a polypeptide complex.

[0341] As used herein, the term "therapeutically effective amount" of a therapeutic agent refers to an amount of the therapeutic agent capable of producing a sufficient therapeutic effect in a subject when administered to the subject in an appropriate manner. 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 potential for cross-reactivity, allergies, hypersensitivity, and side effects, as well as the route of administration and the extent of disease onset. Doses can be proportionally increased or decreased by one skilled in the art (e.g., a physician or veterinarian) depending on these and other circumstances or requirements.

[0342] 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., therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0343] The polypeptide conjugates and methods disclosed herein can be applied to the treatment of a wide variety of diseases. Diseases that may be treatable in humans and other primates using the polypeptide conjugates and methods disclosed herein include the following:

[0344] (1) Cancer and other hyperproliferative disorders, including both benign and malignant tumors, leukemias, 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.

[0345] (2) Alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal glands, 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 Disease, reperfusion 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 agglutination 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 mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, rheumatoid arthritis, polymyalgia ... Autoimmune and/or inflammatory disorders, including pain, 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.

[0346] (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).

[0347] (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.

[0348] In some embodiments, the disease is selected from the group consisting of cancer, an inflammatory disease, an infectious or parasitic disease, a cardiovascular disease, an eye disease, a central nervous system (CNS) disease, trauma, a metabolic disease, an autoimmune disease, or a coagulation disorder. In some embodiments, the CNS disease is a neurological disorder, a neuropsychiatric condition, neuroblastoma, glioblastoma, or Alzheimer's disease.

[0349] The polypeptide conjugates disclosed herein can be administered by any route known in the art, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous infusion, intramuscular injection, or intravenous, including intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0350] 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, e.g., a chemotherapeutic agent or anti-cancer agent.

[0351] 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, and 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 that is administered "in combination with" another therapeutic agent is not necessarily administered simultaneously with or in the same composition as that agent. A polypeptide complex that is administered before or after another agent is considered to be administered "in combination with" that agent, as that term "combination" is used herein, even if the polypeptide complex and the second agent are administered by different routes. When possible, additional therapeutic agents administered in combination with the polypeptide complexes disclosed herein are administered according to the schedule listed on the product information sheet of the additional therapeutic agent, or according to protocols described in the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)), or known in the art.

[0352] XI. Methods of Antigen Detection [0353] 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-associated 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 the subject with a polypeptide complex of the present disclosure and determining the presence or amount of the disease-associated (e.g., tumor-associated) antigen in the sample by detecting the presence of an antigen-binding polypeptide complex.

[0354] Any sample suspected of containing tumor-associated antigens can be used, including biological fluids such as blood samples, plasma samples, and urine samples, as well as biopsies of diseased tissue (e.g., tumor tissue).

[0355] The presence or level of a disease-associated (e.g., tumor-associated) antigen in a sample may be determined based on detecting the presence or level of a complex of the disease-associated (e.g., tumor-associated) 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).

[0356] 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 extensively reviewed in Enzyme Immunoassay (Maggio, ed., 1980) and Harlow & Lane, supra. For reviews of general immunoassays, 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).

[0357] 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).

[0358] 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 method of detectably labeling may be determined based on the performance characteristics of the desired assay format.

[0359] The level of antigen can be determined, for example, by normalizing to a control value or a standard curve. The control value can be predetermined or determined simultaneously.

[0360] 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.

[0361] 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, in whole or in part, are within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the present invention, but are merely illustrative of 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 inventive effort and without departing from the scope of the present invention. It will be understood that many variations can be made in the procedures described herein within the scope of the present invention. It is the inventor's intention that such variations be included within the scope of the present invention.

Example 1
[0362] 1.1 Antibody Construction
[0363] Bispecific antibodies were constructed using both the novel 2:1 format disclosed in this invention (i.e., hereinafter designated FORMAT NEW and shown in Figure 5A) and an existing 2:1 format known in the art (i.e., hereinafter designated FORMAT EX and shown in Figure 4A). To demonstrate the advantages of FORMAT NEW over FORMAT EX, eight pairs of bispecific antibodies were constructed and expressed, which are identical except for the use of FORMAT EX or FORMAT NEW, in order to directly compare the two formats.

[0364] Both FORMAT NEW and FORMAT EX are IgG-like bispecific antibodies, primarily composed of three Fab domains derived from two monoclonal antibodies, one Fab domain derived from the first antibody and the other two Fab domains derived from the second antibody (Figures 4A and 5A). The first antibody heavy chain is designated B1 (comprising VH1 and CH1a), and the first antibody light chain is designated A1 (comprising VL1 and CLa), making the bispecific antibody monovalent. The second antibody heavy chain is designated B2 (comprising VH2 and CH1b), and the second antibody light chain is designated A2 (comprising VL2 + CLb), making the bispecific antibody bivalent. A linker (GGGGS) ₂ was used in the bispecific antibody to link the different functional domains.

[0365] Two monoclonal antibodies targeting CD20 and CD3, respectively, were selected to construct the bispecific antibody used in the examples. In the exemplary bispecific antibody constructed, the anti-CD20 antibody was made bivalent (i.e., corresponding to A2 and B2 in Figures 4A and 5A), and the anti-CD3 antibody was made monovalent (i.e., corresponding to A1 and B1 in Figures 4A and 5A). The anti-CD20 antibody used in this example is 2F2, previously reported in US20040167319. The anti-CD3 antibody used in this example is SP34, previously described in EMBO Journal vol. 4 no. 2 pp. 337-344, 1985.

[0366] To reduce mismatching, mutations or crossover configurations were introduced into the Fab regions of the first and second antibodies, respectively, as detailed in Table 1 below.

[0367]

[0368] Mutations were also introduced into the CH3 regions to enhance heterodimerization of the first and second Fc regions. Specifically, T366S, L368A, and Y407V were introduced into one of the CH3 regions, and a T366W mutation was introduced into the other. The CH3 region mutations in the following examples are provided for knobs-in-hole design in bispecific antibodies.

[0369] Based on the bispecific antibody structures shown above (Table 1), eight pairs of bispecific antibodies were constructed and expressed using the novel 2:1 format of the present invention, FORMAT NEW (shown in Figure 4A), and the conventional 2:1 format, FORMAT EX (shown in Figure 5A). These eight pairs of bispecific antibodies (i.e., a total of 16 bispecific antibodies) were recombinantly expressed and designated FORMAT NEW1 to FORMAT NEW8 and FORMAT EX1 to FORMAT EX8, respectively. For each antibody, the full-length amino acid sequence of each of the polypeptide chains is listed below, with functional domains marked with different underlines as noted below the sequence.

[0370] FORMAT EX1
a) FORMAT EX1 chain 2 (AH): CD20HC + CD3HC (knob) SEQ ID NO: 1
b) FORMAT EX1 chain 1 (AL): CD3 LC SEQ ID NO: 2
c) FORMAT EX1 chain 3 (BH): CD20 HC (Whole) SEQ ID NO: 3
d) FORMAT EX1 chain 4 (BL): CD20 LC SEQ ID NO: 4

[0371] FORMAT NEW1
a) FORMAT NEW1 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID NO: 5
b) FORMAT NEW1 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID NO: 6
c) FORMAT NEW1 Chain 3 (B2): Anti-CD20 Heavy Chain (Whole) SEQ ID NO: 7
d) FORMAT NEW1 chain 4 (A2): anti-CD20 light chain SEQ ID NO: 8

[0372] FORMAT EX2
a) FORMAT EX2 chain 2 (AH): CD20HC + CD3HC (knob) SEQ ID NO: 9
b) FORMAT EX2 chain 1 (AL): CD3 LC SEQ ID NO: 10
c) FORMAT EX2 chain 3 (BH): CD20 HC (Whole) SEQ ID NO: 11
d) FORMAT EX2 chain 4 (BL): CD20 LC SEQ ID NO: 12

[0373] FORMAT NEW2
a) FORMAT NEW2 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID NO: 13
b) FORMAT NEW2 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID NO: 14
c) FORMAT NEW2 chain 3 (B2): anti-CD20 heavy chain (hole) SEQ ID NO: 15
d) FORMAT NEW2 chain 4 (A2): anti-CD20 light chain SEQ ID NO: 16

[0374] FORMAT EX3
a) FORMAT EX3 chain 2 (AH): CD20HC + CD3HC (knob) SEQ ID NO: 17
b) FORMAT EX3 chain 1 (AL): CD3LC SEQ ID NO: 18
c) FORMAT EX3 strand 3 (BH): CD20HC (Whole) SEQ ID NO: 19
d) FORMAT EX3 chain 4 (BL): CD20LC SEQ ID NO: 20

[0375] FORMAT NEW3
a) FORMAT NEW3 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID NO: 21
b) FORMAT NEW3 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID NO: 22
c) FORMAT NEW3 Chain 3 (B2): Anti-CD20 heavy chain (Whole) SEQ ID NO: 23
d) FORMAT NEW3 chain 4 (A2): anti-CD20 light chain SEQ ID NO: 24

[0376] FORMAT EX4
a) FORMAT EX4 chain 2 (AH): CD20HC + CD3HC (knob) SEQ ID NO: 25
b) FORMAT EX4 chain 1 (AL): CD3 LC SEQ ID NO: 26
c) FORMAT EX4 chain 3 (BH): CD20HC (Whole) SEQ ID NO: 27
d) FORMAT EX4 chain 4 (BL): CD20LC SEQ ID NO: 28

[0377] FORMAT NEW4
a) FORMAT NEW4 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID NO: 29
b) FORMAT NEW4 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID NO: 30
c) FORMAT NEW4 chain 3 (B2): anti-CD20 heavy chain (hole) SEQ ID NO: 31
d) FORMAT NEW4 Chain 4 (A2): Anti-CD20 light chain SEQ ID NO: 32

[0378] FORMAT EX5
a) FORMAT EX5 chain 2 (AH): CD20HC + CD3HC (knob) SEQ ID NO: 33
b) FORMAT EX5 chain 1 (AL): CD3LC SEQ ID NO: 34
c) FORMAT EX5 strand 3 (BH): CD20HC (Whole) SEQ ID NO: 35
d) FORMAT EX5 chain 4 (BL): CD20LC SEQ ID NO: 36

[0379] FORMAT NEW5
a) FORMAT NEW5 chain 2 (B1): anti-CD3 heavy chain (knob) SEQ ID NO: 37
b) FORMAT NEW5 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID NO: 38
c) FORMAT NEW5 chain 3 (B2): anti-CD20 heavy chain (hole) SEQ ID NO: 39
d) FORMAT NEW5 chain 4 (A2): anti-CD20 light chain SEQ ID NO: 40

[0380] FORMAT EX6
a) FORMAT EX6 chain 2 (AH): CD20HC + CD3HC (knob) SEQ ID NO: 41
b) FORMAT EX6 chain 1 (AL): CD3LC SEQ ID NO: 42
c) FORMAT EX6 chain 3 (BH): CD20HC (Whole) SEQ ID NO: 43
d) FORMAT EX6 chain 4 (BL): CD20LC SEQ ID NO: 44

[0381] FORMAT NEW6
a) FORMAT NEW6 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID NO: 45
b) FORMAT NEW6 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID NO: 46
c) FORMAT NEW6 chain 3 (B2): anti-CD20 heavy chain (hole) SEQ ID NO: 47
d) FORMAT NEW6 chain 4 (A2): anti-CD20 light chain SEQ ID NO: 48

[0382] FORMAT EX7
a) FORMAT EX7 chain 2 (AH): CD20HC + CD3HC (knob) SEQ ID NO: 49
b) FORMAT EX7 chain 1 (AL): CD3LC SEQ ID NO: 50
c) FORMAT EX7 chain 3 (BH): CD20 HC (Whole) SEQ ID NO: 51
d) FORMAT EX7 chain 4 (BL): CD20 LC SEQ ID NO: 52

[0383] FORMAT NEW7
a) FORMAT NEW7 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID NO: 53
b) FORMAT NEW7 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID NO: 54
c) FORMAT NEW7 chain 3 (B2): anti-CD20 heavy chain (hole) SEQ ID NO: 55
d) FORMAT NEW7 chain 4 (A2): anti-CD20 light chain SEQ ID NO: 56

[0384] FORMAT EX8
a) FORMAT EX8 chain 2 (AH): CD20HC+CD3VHLC (knob) SEQ ID NO: 57
b) FORMAT EX8 chain 1 (AL): CD3LC SEQ ID NO: 58
c) FORMAT EX8 chain 3 (BH): CD20HC (Whole) SEQ ID NO: 59
d) FORMAT EX8 chain 4 (BL): CD20LC SEQ ID NO: 60

[0385] FORMAT NEW8
a) FORMAT NEW8 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID NO: 61
b) FORMAT NEW8 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID NO: 62
c) FORMAT NEW8 Chain 3 (B2): Anti-CD20 Heavy Chain (Whole) SEQ ID NO: 63
d) FORMAT NEW8 chain 4 (A2): anti-CD20 light chain SEQ ID NO: 64

[0386] 1.2 Gene synthesis, expression and purification of bispecific antibodies
[0387] Plasmids were constructed to encode each polypeptide chain of each of the above bispecific antibodies. The plasmids encoding each bispecific antibody were transfected into ExpiCHO-S cells to allow expression of the polypeptide chains, which were then incorporated into the respective bispecific antibodies. The expressed bispecific antibodies were collected and purified with protein A. Specific experimental procedures are described below.

[0388] Prior to proceeding with synthesis, the gene sequence encoding the polypeptide chain was codon-optimized using OptimumGene. The gene sequence was constructed in a pcDNA3.4 vector, and the confirmed recombinant vector DNA was prepared for transfection using PureLink® Hipure.

[0389] Cells were cultured in a _CO2 shaker with ExpiCHO Expression Medium. When the ExpiCHO- ^S cell density reached ^7x10-10x10 viable cells/ml and the viability was >95%, 25 ml of cell suspension was transfected with approximately 25 ul of ExpiFectamine® CHO/DNA complexes containing DNA heavy and light chains.

[0390] On day 8-9 after transfection, the culture was harvested, and the cell suspension was collected, centrifuged, and further purified by chromatography. Chromatography column: 1 ml Mab Select Sure LX (GE) pre-assembled column; equilibration buffer A: 50 mM acetic acid-sodium acetate, pH 6.0; elution buffer B: 50 mM acetic acid-sodium acetate, pH 3.6; neutralization buffer C: 1 M Tris-HCl, pH 8.5; flow rate: 1 ml/min; gradient: linear gradient elution from 0 to 100% B. After separation, the eluate was collected in a volume of 1 ml per tube, and 0.1 ml of neutralization buffer C was added to each 1 ml fraction, resulting in a pH of approximately 6-7.

[0391] 1.3 Expression results of each bispecific antibody
[0392] The purified antibodies were analyzed and identified by SEC-HPLC (see Figures 7A-7P) and SDS-PAGE (see Figures 6A and 6B), respectively.

[0393] 1) SDS-PAGE : The sample was mixed with 4X sample buffer (15 μl + 5 μl) in an Eppendorf tube, heated at 100°C for 5-10 min, centrifuged, and the supernatant was collected. Five μg of the treated sample was then carefully pipetted into each well of the gel in turn, and a marker was added to one of the wells. The electrode chamber was placed in the electrophoresis tank, powered on, and the positive and negative electrodes were aligned, setting the voltage to 150 V. After 5 min of electrophoresis, the voltage was adjusted to 200 V. Electrophoresis was again carried out for approximately 35 min, after which the power was turned off and the gel was stopped. Unstained SDS-PAGE gels were scanned and photographed using a Bio-Rad gel imager, and the photographs were analyzed using Image Lab 5.2.1 to calculate protein purity.

[0394] 2) Capillary Electrophoresis Sodium Dodecyl Sulfate ( CE-SDS) : Samples were diluted to 2.5 mg/ml with ultrapure water and added to 55 μL of sample buffer, followed by 2 μL of 10 kDa IS and 5 μL of 250 mM IAM and mixed thoroughly. Samples were then heated in a metal bath at 70 ± 2°C for 10 ± 2 min, then cooled to room temperature in a water bath, transferred to the provided sample tubes, labeled, and prepared for sampling.

[0395] 3) 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 taken and analyzed. The relevant SEC chromatography parameters were as follows:
Mobile phase A: 200mM PBS pH6.9,
Mobile phase B: ultrapure water,
Flow rate: 0.7ml/min,
Wavelength: 280nm,
Column temperature: 25°C,
Sample analysis time: 25 min,
Injection amount: 20 μg.

[0396] 1.4 Analysis results
[0397] Conventional 2:1 Format (FORMAT EX)
[0398] According to the SEC-HPLC results (Figures 7A, 7C, 7E, 7G, 7I, 7K, 7M, and 7O), all of the samples except for the FORMAT EX5 construct appeared to show a clear, single major peak at 200 kDa in size (Figure 7I), suggesting that almost all samples were highly pure. However, this result was inconsistent with the SDS-PAGE results, which showed multiple protein bands within the samples, indicating that the samples contained small molecular weight impurities and therefore required further purification (see Figures 6A and 6B).

[0399] The reason why the SEC-HPLC results did not match the SDS-PAGE results is as follows: Most of the A1-B2 and A2-B1 mismatch products in the FORMAT EX antibody samples (FORMAT EX1 to FORMAT EX8) have molecular weights of approximately 200 kDa, which is identical to or very close to the target product, and therefore the SEC-HPLC method cannot identify these mismatch products present in the samples. In contrast, such mismatch products can be revealed by the SDS-PAGE method because the A1-B2 and A2-B1 mismatch products in Figure 5B are not covalently linked by a disulfide bond and dissociate during the SDS-PAGE process. In a recent study, SDS-PAGE results showed that 175 kDa and even 150 kDa products close to the 200 kDa band were observed in FORMAT EX1, FORMAT EX2, FORMAT EX3, FORMAT EX4, FORMAT EX5, FORMAT EX7, and FORMAT EX8, but not in FORMAT EX6. These mismatched products were not observed in SEC-HPLC results, and due to their similarity to the physicochemical properties of the corresponding cognate matched target products, they may be very difficult to remove using conventional purification processes.

[0400] NEW 2:1 FORMAT (FORMAT NEW)
[0401] As shown in the SDS-PAGE results (see Figures 6A and 6B), the impurities far from the target band were mostly at 150 kDa on SDS-PAGE. These results are more consistent with the SEC-HPLC results (see Figures 7B, 7D, 7F, 7H, 7J, 7L, 7N, and 7P), i.e., the major peak at 200 kDa on the SEC-HPLC profile is the single target product, and the A1-B2 and A2-B1 mismatch products (see Figure 4B) are more easily separated by SEC-HPLC due to their significantly different molecular weights from the target product, and the final product is also easier to purify due to significant differences in physicochemical properties.

Example 2
[0402] The anti-CD20xCD3 bispecific molecule FORMAT New7 was constructed and purified by conventional methods, demonstrating the convenience of the present invention in terms of purification.

[0403] 2.1 Antibody Construction
[0404] Chain 1 (AL): B2-Linker-A1 (MW: approx. 50 kDa)
[0405] Chain 2 (AH): B1-1st Fc (MW: about 50 kDa)
[0406] Chain 3 (BH): B2-2nd Fc (MW: about 50 kDa)
[0407] Chain 4 (BL): A2 (MW: about 25 kDa)

[0408] Mutations such as V173C, S183K, and C220S were introduced into CH1 of B1.

[0409] Mutations such as Q160C, S176D, and C214S were introduced into the CL of A1.

[0410] 2.2 Expression of anti-CD20 x CD3 FORMAT New7 bispecific antibody
[0411] Anti-CD20xCD3 FORMAT New7 is a bispecific antibody consisting of two pairs of different light and heavy chains. Two stable transfer vectors were used to co-transfect host cells with CHO-K1 cells. After transfection, the cells were seeded at a density of 2 x 10e4 cells/well and cultured under selective pressure in a _CO2 incubator. After approximately 3 weeks of culture, supernatants were collected from the cell cultures and tested for binding to CD20 and CD3, respectively. Supernatants showing high binding activity to CD20 and CD3 were identified, and the corresponding cell clones were expanded in 24-well cell culture plates. After 4-6 days, the supernatants were tested for binding activity to CD20 and CD3. Cell clones with higher binding activity to CD20 and CD3 were expanded, and finally, stably transfected cell lines were selected and expanded. Stable cell lines were subcultured in shake flasks for 3 days, tested for binding activity to CD20 and CD3, and subjected to fed-batch evaluation and cryopreservation based on cell growth and expression. Expression and quality of fed-batch samples were tested (SEC, non-reducing CE-SDS, CEX, SDS-PAGE) to evaluate growth, metabolism, productivity, and product quality of stable cell pools, and stable cell pools were finally selected for subsequent monoclonal cell line screening.

[0412] The above cell lines were seeded at one cell per well into 96-well plates using a single cell sorter, cultured in a _CO2 incubator, and imaged by scanning the plates with a Solentim Cell Metric. Monoclonal cells were selected for fed-batch evaluation and cell cryopreservation. Expression and quality analyses (SEC, nrCE-SDS, iCIEF, N-Glycan, molecular weight) were performed to evaluate the growth, metabolism, productivity, and product quality of stable cell pools, and six monoclonal cell lines were selected for RCB banking.

[0413] Selected monoclonal cell lines were regenerated, expanded, seeded, and cultured in shaker flasks for 14 days. Supernatants were collected for purification and analysis.

[0414] 2.2.1 Analysis method
[0415] 1) SDS-PAGE : The supernatant sample was mixed with 4X sample buffer (15 μl + 5 μl) in an Eppendorf tube, heated at 100°C for 5-10 min, centrifuged, and the supernatant was collected. Five μg of the treated sample was then carefully pipetted into each well of the gel in turn, and a marker was added to one of the wells. The electrode chamber was placed in the electrophoresis tank, the power was turned on, the positive and negative electrodes were aligned, and the voltage was set to 150 V. After 5 min of electrophoresis, the voltage was adjusted to 200 V. Electrophoresis was again carried out for approximately 35 min, after which the power was turned off and the gel was stopped. Unstained SDS-PAGE gels were scanned and photographed using a Bio-Rad gel imager, and the photographs were analyzed using ImageLab 5.2.1 to calculate protein purity.

[0416] 2) CE-SDS : Samples were diluted to 2.5 mg/ml with ultrapure water and added to 55 μL of sample buffer, followed by 2 μL of 10 kDa IS and 5 μL of 250 mM IAM and mixed thoroughly. Samples were then heated at 70±2°C for 10±2 min, then cooled to room temperature, transferred to the provided sample tubes, labeled, and prepared for sampling.

[0417] 3) SEC-HPLC : Samples were centrifuged at 10,000 rpm for 5 min at 15°C. The supernatant was collected and analyzed. The relevant SEC chromatography parameters are as follows:
Mobile phase A: 200mM PBS pH6.9,
Mobile phase B: ultrapure water,
Flow rate: 0.7ml/min,
Wavelength: 280nm,
Column temperature: room temperature
Sample analysis time: 20 min,
Injection amount: 20 μg.

[0418] 2.2.2 Analysis results
[0419] SDS-PAGE analysis showed that the purity of the expression product of the anti-CD20xCD3 FORMAT New7 bispecific antibody was approximately 84.1% (Figure 8).

[0420] CE-SDS analysis revealed that the purity of the expression product of the anti-CD20 x CD3 FORMAT New7 bispecific antibody was approximately 82.91% (see peak #10 in Figure 9).

[0421] SEC-HPLC analysis showed that the purity of the expressed anti-CD20xCD3 FORMAT New7 bispecific antibody was approximately 90.23% (see Peak No. 4 in Figure 10).

[0422] In addition to the target protein, the expression product still contains a small amount of low molecular weight (LMW) fragments (see Figures 8, 9, and 10) and high molecular weight (HMW, see Figure 10) aggregates. Related products can be more clearly distinguished by SDS-PAGE, CE-SDS, and SEC-HPLC. The results are shown in Figures 8-10.

[0423] 2.3 Antibody purification
[0424] 2.3.1 Experimental Methods
[0425] 1) Affinity Chromatography (AC) : Supernatant samples containing anti-CD20xCD3 FORMAT New7 bispecific antibody harvested from cell cultures were eluted by affinity chromatography at pH 4.0 and the elution peaks were subsequently analyzed.

[0426] Chromatography column: 1 ml Mab Select Sure LX (GE) pre-assembled column, equilibration buffer A: 50 mM acetic acid-sodium acetate, pH 6.0, elution buffer B: 50 mM acetic acid-sodium acetate, pH 3.6, neutralization buffer C: 1 M Tris-HCl, pH 8.5, flow rate: 1 ml/min, gradient: linear gradient elution from 0 to 100% B. After separation, the eluate was collected in a volume of 1 ml per tube, and 0.1 ml of neutralization buffer C was added to each 1 ml fraction, resulting in a pH of approximately 6-7.

[0427] 2) Cation Exchange Chromatography (CEX) : The eluates from the previous step were combined and the pH was adjusted to 6.1. The combined eluates were then filtered and used as the sample CEX-Load. Chromatography column: Capto S Impact was applied with the following buffers:
EQ/Wash/Elution Buffer A: 50mM NaAc-HAc, pH 6.0
Elution buffer B: 50mM NaAc-HAc+1M NaCl, pH 6.0
A linear gradient elution is performed: Gradient: 0→50% B, 60 CV.

[0428] 2.3.2 Purification results
[0429] 1) Affinity Chromatography (AC)
[0430] The purification profile is shown in Figure 11. As shown in Figure 11, the target product was successfully eluted from the affinity chromatography column by the elution buffer, and a single high peak was detected by UV signal (see arrow in Figure 11), indicating that the purified product has high purity.

[0431] The purity of the eluate was analyzed by SEC-HPLC and was found to be 92.55% (see peak #3 in Figure 12), indicating that AC reduced the LMW content.

[0432] The purity of the eluate was also analyzed by SDS-PAGE and was found to be 85.7% (see Figure 13), indicating that LMW was still present after purification in this step (Figure 13).

[0433] Compared to the results observed for the unpurified sample in Example 1, the SEC-HPLC and SDS-PAGE results clearly reflected the improved purification achieved by the AC process, which also indicated that the impurities were LMW, providing a clear direction for the subsequent purification process.

[0434] 2) Cation Exchange Chromatography (CEX)
[0435] To further remove LMW in the AC purified sample, the eluate from the AC process was collected and further purified by CEX. The elution profile after CEX purification is shown in Figure 14. A large single peak was detected by the UV signal (see the peak in the eluate from 100 ml to 120 ml), indicating that the target product was successfully eluted by the elution buffer with a linear gradient elution.

[0436] Three eluate samples (i.e., C01-C03, see the three arrows in Figure 14) were collected and analyzed for purity by SDS-PAGE and SEC, respectively.

[0437] SDS-PAGE showed purity results for C01, C02, and C03 of 94.6%, 95.0%, and 91.0%, respectively (Figure 15). SEC-HPLC showed purity results for C01, C02, and C03 of 93.89%, 99.74%, and 97.73%, respectively (see Table 2 below). The SEC-HPLC trace for C02 is shown in Figure 16.

[0438]
[0439] Notably, after purification by CEX, the purity of the CO2 fraction was over 99% by SEC-HPLC (Figure 16) and over 95% by SDS-PAGE (Figure 15), significantly improving the purity of the CEX load sample compared to before CEX purification, and removing most of the LMW and HMW from the AC purified product.

[0440] SEC-HPLC and SDS-PAGE clearly reflected the improved purity achieved by CEX, which also demonstrated high homogeneity of the purified final product.

[0441] The above results demonstrate that the novel 2:1 bispecific antibody structure (FORMAT NEW) of the present invention can be easily purified using conventional purification methods such as AC and CEX. A highly purified sample with a purity of over 95% can be obtained through a simple two-step purification process, demonstrating the significant advantages of FORMAT NEW antibodies over FORMAT EX antibodies in antibody purification and also suggesting the ease of use of the FORMAT NEW antibody structure of the present invention.

[0442] While the examples demonstrate convenient purification and high purity using an anti-CD20 x CD3 bispecific antibody, the inventors have also tested other bispecific antibodies constructed using the novel bispecific antibody format provided in the present disclosure, and similar results were obtained with regard to convenient purification and high purity. Therefore, it should be understood that the novel format provided in the present disclosure can be applied to a variety of targets and can provide convenient purification and high purity for any bispecific antibody.

Claims (52)

From the C-terminus to the N-terminus,
a) a first target-binding fragment A1; and
b) a polypeptide linker; and
c) a second target-binding fragment B2; and
Including,
the polypeptide linker has a length short enough to minimize potential intramolecular interactions between A1 and B2;
A1 is capable of pairing with a first pairing fragment B1 to form a first target-binding domain;
the B2 is capable of pairing with a second pairing fragment A2 to form a second target-binding domain; and the A1 is configured to have a lower binding affinity to B2 compared to B1, and the B2 is configured to have a lower binding affinity to the A1 compared to A2. a) a fusion polypeptide according to claim 1;
b) a second polypeptide comprising the first paired fragment B1;
c) a third polypeptide comprising the second target-binding fragment B2; and
d) a fourth polypeptide and a fifth polypeptide, each of which comprises the second paired fragment A2; and
Including,
said A1 in said fusion polypeptide pairs with said B1 in said second polypeptide to form a first target binding domain;
the B2 in the fusion polypeptide pairs with the A2 in the fourth polypeptide to form a second target binding domain, and the A2 in the fifth polypeptide pairs with the B2 in the third polypeptide to form another second target binding domain. The fusion polypeptide of claim 1 or the polypeptide complex of claim 2, wherein at least one of the pair of B1 and A1 and the pair of B2 and A2 includes at least one arrangement capable of preventing mispairing between B1 and A2 and/or B2 and A1. A1 comprises a first antibody variable region VA1 selected from VH1 or VL1,
4. The fusion polypeptide of claim 1 or 3, or the polypeptide complex of claim 2 or 3, wherein B1 comprises a first antibody variable region VB1 capable of pairing with VA1 to form the first target-binding domain, wherein VB1 is selected from VH1 or VL1; B2 comprises a second antibody variable region VB2 selected from VH2 or VL2; and A2 comprises a second antibody variable region VA2 capable of pairing with VB2 to form the second target-binding domain, wherein VA2 is selected from VH2 or VL2. The fusion polypeptide of claim 4 or the polypeptide complex of claim 4, wherein VB1 comprises VH1, VA1 comprises VL1, VB2 comprises VH2, and VA2 comprises VL2. a) the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VL2, and the VA2 comprises VH2, or
b) A fusion polypeptide according to claim 4 or a polypeptide complex according to claim 4, wherein VB1 comprises VL1, VA1 comprises VH1, VB2 comprises VH2, and VA2 comprises VL2. A fusion polypeptide described in any one of claims 1 and 3 _{to 6} or a polypeptide complex described in any one of claims 2 to 6, wherein A1 further comprises a first scaffold region R a operably linked to VA1, and B2 further comprises a second scaffold region R _b operably linked to VB2, and SR _a and SR _b are configured to prevent pairing between SR _a and SR _b . The fusion polypeptide of claim 7 or the polypeptide complex of claim 7, wherein B1 further comprises a first pairing scaffold region PSR _a operably linked to VB1 and capable of binding to SR _a , and A2 further comprises a second pairing scaffold region PSR _b operably linked to VA2 and capable of binding to SR _b , and optionally SR _a and PSR _a are paired by a first disulfide bond and/or SR _b and PSR _b are paired by a second disulfide bond. The SR _a /PSR _a pair or the SR _b /PSR _b pair is
a) a pair of heavy chain constant region 1 (CH1) and light chain constant region (CL);
b) a pair of T cell receptor (TCR) constant region α (Calpha) and TCR constant region β (Cbeta);
c) a pair of TCR constant region γ (Cgamma) and TCR constant region δ (Cdelta);
d) a pair of the ligand-binding domain of a receptor and said ligand;
e) a pair of a PRD (proline-rich domain) and an SH3 domain;
f) the pair obscurin and titin;
9. A fusion polypeptide according to claim 7 or 8, or a polypeptide complex according to claim 7 or 8, selected from the group consisting of: A fusion polypeptide described in any one of claims 7 to 9 or a polypeptide complex described in any one of claims 7 to 9, wherein the pair of SR _a and PSR _a is different from the pair of SR _b and PSR _b . a) The pair of the SR _a and the PSR _a is a pair of CH1 and CL, and the pair of the SR _b and the PSR _b is
i) Calpha and Cbeta,
ii) Cgamma and Cdelta;
iii) the ligand-binding domain of a receptor and said ligand;
iv) a PRD (proline-rich domain) and an SH3 domain, or
v) obscurin and titin, a pair
Or,
b) The pair of SR _b and PSR _b is a pair of CH1 and CL, and the pair of SR _a and PSR _a is
i) Calpha and Cbeta,
ii) Cgamma and Cdelta;
iii) the ligand-binding domain of a receptor and said ligand;
iv) a PRD (proline-rich domain) and an SH3 domain, or
v) A fusion polypeptide according to any one of claims 7 to 10 or a polypeptide complex according to any one of claims 7 to 10, which is a pair of obscurin and titin. A fusion polypeptide described in any one _of claims 7 to ₉ or a polypeptide complex described in any one of claims 7 to ₉ , wherein the SR _{a /} PSR _a pair and the SR _b /PSR _b pair are both the same, and the SR _a /PSR _a pair and/or the SR _b /PSR _b pair are configured to prevent mispairing between SR a /PSR b or SR b /PSR a. 13. The fusion polypeptide of claim 12 or the polypeptide complex of claim 12, wherein the SR _a /PSR _a pair comprises a CH1 domain CH1a and a CL domain CLa, and the SR _b /PSR _b pair comprises a CH1 domain CH1b and a CL domain CLb. said VB1 comprises VH1, said VA1 comprises VL1, said VB2 comprises VH2, and said VA2 comprises VL2;
a) the PSR _a is a CL domain CLa, the SR _a is a CH1 domain CH1 a, the SR _b is a CH1 domain CH1 b, and the PSR _b is a CL domain CLb, or
b) The fusion polypeptide of claim 13 or the polypeptide complex of claim 13, wherein PSR _a is a CH1 domain CH1a, SR _a is a CL domain CLa, SR _b is a CL domain CLb, and PSR _b is a CH1 domain CH1b. the PSR _a is a CH1 domain CH1a, the SR _a is a CL domain CLa, the SR _b is a CH1 domain CH1b, and the PSR _b is a CL domain CLb;
a) the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VL2, and the VA2 comprises VH2, or
b) A fusion polypeptide according to claim 13 or a polypeptide complex according to claim 13, wherein VB1 comprises VL1, VA1 comprises VH1, VB2 comprises VH2, and VA2 comprises VL2. the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VH2, the VA2 comprises VL2, the PSR _a is a CH1 domain CH1a, the SR _a is a CL domain CLa, the SR _b is a CH1 domain CH1b, and the PSR _b is a CL domain CLb;
a) the fusion polypeptide comprises an amino acid sequence of formula (I) VH2-CH1b-Linker-VL1-CLa;
b) the second polypeptide comprises the amino acid sequence of formula (II) VH1-CH1a;
c) the third polypeptide comprises the amino acid sequence of formula (III) VH2-CH1b;
d) the fourth and fifth polypeptides each comprise an amino acid sequence of formula (IV) VL2-CLb;
The fusion polypeptide of claim 13 or the polypeptide complex of claim 13, wherein the CH1b/CLb pair and/or the CH1a/CLa pair are configured to prevent mispairing between CH1b and CLa and/or between CH1a and CLb. At least one of the CH1b/CLb pair and the CH1a/CLa pair is
1) having at least one non-native disulfide bond that prevents mispairing between CH1b and CLa and/or CH1a and CLb;
2) containing one or more introduced amino acid mutations that result in at least one or more introduced charged amino acid residues that prevent mispairing between CH1b and CLa and/or CH1a and CLb; or
3) have one or more introduced amino acid mutations that form an orthogonal CH1-CL interface that prevents mispairing between CH1b and CLa or between CH1a and CLb;
17. The fusion polypeptide of claim 16 or the polypeptide complex of claim 16, having one or more of the following properties: 18. The fusion polypeptide of claim 17 or the polypeptide complex of claim 17, wherein the first CH1/CL pair and the second CH1/CL pair are selected from CH1b/CLb and CH1a/CLa, and the first CH1/CL pair is associated by a non-native first disulfide bond, optionally lacking a native disulfide bond or having a disrupted native disulfide bond. The fusion polypeptide of claim 18 or the polypeptide complex of claim 18, wherein the second CH1/CL pair is associated by a second disulfide bond formed at a position different from the first disulfide bond, and optionally the second disulfide bond is a native disulfide bond. The first disulfide bond is
a) heavy chain EU position 126 - light chain EU position 121;
b) heavy chain EU position 173 - light chain EU position 160;
c) heavy chain EU position 128 - light chain EU position 118;
20. The fusion polypeptide of claim 18 or 19 or the polypeptide complex of claim 18 or 19, which is formed by introducing two cysteine residues into a series of heavy chain-light chain EU positions selected from the group consisting of: A fusion polypeptide described in any one of claims 18 to 20, or a polypeptide complex described in any one of claims 18 to 20, wherein the native disulfide bond is between heavy chain EU position 220 and light chain EU position 214. 22. The fusion polypeptide of any one of claims 18 to 21, or the polypeptide complex of any one of claims 18 to 21, wherein the first CH1/CL pair comprises a CH1 comprising a substitution of a cysteine residue at EU position 126 and a substitution of a non-cysteine residue at EU position 220, and a CL comprising a substitution of a cysteine residue at EU position 121 and a substitution of a non-cysteine residue at EU position 214. 23. The fusion polypeptide of any one of claims 17 to 22, or the polypeptide complex of any one of claims 17 to 22, wherein the first CH1/CL pair and the second CH1/CL pair are selected from CH1b/CLb and CH1a/CLa, and the first CH1/CL pair comprises at least one substitution of an uncharged residue with a charged residue and/or at least one substitution of a charged residue with an oppositely charged residue, whereby the first CH1/CL pair comprises a first pair of oppositely charged residues that favors pairing of the first CH1/CL pair. 24. The fusion polypeptide of claim 23, or the polypeptide complex of claim 23, wherein the second CH1/CL pair comprises at least one substitution of an uncharged residue with a charged residue and/or at least one substitution of a charged residue with an oppositely charged residue, whereby the second CH1/CL pair comprises a second pair of oppositely charged residues that favors pairing of the second CH1/CL pair, and optionally the first pair of oppositely charged residues and the second pair of oppositely charged residues prevent pairing of CH1a with CLb or CH1b with CLa. 25. The fusion polypeptide of claim 24, or the polypeptide complex of claim 24, wherein the first pair of oppositely charged residues and/or the second pair of oppositely charged residues are configured such that CH1a and CLb both have positively or negatively charged residues, and/or CH1b and CLa both have positively or negatively charged residues. The first pair of oppositely charged residues and/or the second pair of oppositely charged residues are
a) heavy chain EU position 183:light chain EU position 176;
b) heavy chain EU position 183:light chain EU position 133;
c) heavy chain EU position 147:light chain EU position 176; and
d) heavy chain EU position 141:light chain EU position 116;
e) heavy chain EU position 126:light chain EU position 121;
f) heavy chain EU position 218:light chain EU position 122;
26. The fusion polypeptide of any one of claims 23 to 25, or the polypeptide complex of any one of claims 23 to 25, wherein a heavy chain-light chain EU position selected from the group consisting of: The fusion polypeptide of any one of claims 22 to 26, or the polypeptide complex of any one of claims 22 to 26, wherein the pair of oppositely charged residues comprises a positively charged amino acid residue and a negatively charged amino acid residue, the positively charged amino acid residue being selected from the group consisting of lysine (K), histidine (H) and arginine (R), and/or the negatively charged amino acid residue being selected from the group consisting of aspartic acid (D) and glutamic acid (E). The fusion polypeptide of any one of claims 17 to 27, or the polypeptide complex of any one of claims 17 to 27, wherein the first CH1/CL pair and the second CH1/CL pair are selected from CH1b/CLb and CH1a/CLa, and the first CH1/CL pair contains one or more introduced amino acid mutations that form an orthogonal CH1-CL interface. The fusion polypeptide of claim 28 or the polypeptide complex of claim 28, wherein the orthogonal CH1-CL interface is introduced at a series of heavy chain-light chain EU positions including heavy chain EU positions H168A and F170G, and light chain EU positions L135Y and S176W. The first CH1/CL pair is
30. The fusion polypeptide of any one of claims 17 to 29, or the polypeptide complex of any one of claims 17 to 29, comprising one or more introduced amino acid mutations at a series of heavy chain-light chain EU positions selected from the group consisting of: a) substitutions at heavy chain EU positions A141I, F170S, S181M, S183A, and V185A, and substitutions at light chain EU positions F116A, A235V, S174A, S176F, and T178V, forming an orthogonal Fab design. 31. The fusion polypeptide of any one of claims 16 to 30, or the polypeptide complex of any one of claims 16 to 30, wherein the first VH/VL pair and the second VH/VL pair are selected from VH1/VL1 and VH2/VL2, and the first VH/VL pair has at least one substitution of an uncharged residue with a charged residue and/or at least one substitution of a charged residue with an oppositely charged residue, whereby the first VH/VL includes a third pair of oppositely charged residues that favors pairing of the first VH/VL pair. The fusion polypeptide of claim 31 or the polypeptide complex of claim 31, wherein the second VH/VL pair has at least one substitution of an uncharged residue with a charged residue and/or at least one substitution of a charged residue with an oppositely charged residue, whereby the second VH/VL includes a fourth pair of oppositely charged residues that favors pairing of the second VH/VL pair, and optionally the third pair of oppositely charged residues and the fourth pair of oppositely charged residues prevent pairing of VH1 with VL2 or VH2 with VL1. 33. The fusion polypeptide of claim 32 or the polypeptide complex of claim 32, wherein the third pair of oppositely charged residues and the fourth pair of oppositely charged residues are configured such that VH1 and VL2 both have positively or negatively charged residues and/or VH2 and VL1 both have positively or negatively charged residues. the third pair of oppositely charged residues and/or the fourth pair of oppositely charged residues are
a) heavy chain EU position 39: light chain EU position 38;
b) heavy chain EU position 105:light chain EU position 43, and c) heavy chain EU position 62:light chain EU position 1, or
A fusion polypeptide according to any one of claims 31 to 33 or a polypeptide complex according to any one of claims 31 to 33, wherein a series of heavy chain-light chain EU positions selected from the group consisting of any combination thereof are introduced. The fusion polypeptide of any one of claims 31 to 34 or the polypeptide complex of any one of claims 31 to 34, wherein the pair of oppositely charged residues comprises a positively charged amino acid residue and a negatively charged amino acid residue, the positively charged amino acid residue being selected from the group consisting of lysine (K), histidine (H) and arginine (R), and/or the negatively charged amino acid residue being selected from the group consisting of aspartic acid (D) and glutamic acid (E). The polypeptide complex of any one of the preceding claims, wherein the second polypeptide and the third polypeptide further comprise an operably linked first dimerization domain and an operably linked second dimerization domain that associate to form a dimer, and optionally, the first dimerization domain comprises a first Fc region and/or the second dimerization domain comprises a second Fc region. The polypeptide complex of claim 36, wherein the first Fc region and/or the second Fc region is derived from IgG1, IgG2, IgG3, or IgG4. The polypeptide complex of claim 36, wherein the first Fc region and the second Fc region have different amino acid sequences and at least one arrangement that promotes heterodimerization of the first Fc region and the second Fc region. said first Fc region comprises a first Fc variant, and/or said second Fc region comprises a second Fc variant;
a) said first Fc mutation comprises T366W or S354C and said second Fc mutation comprises Y349C, T366S, L368A, or Y407V;
b) said first Fc mutation comprises D399K or E356K and said second Fc mutation comprises K392D, or K409D;
c) said first Fc mutation comprises E356K, E357K, or D399K and said second Fc mutation comprises K370E, K409D, or K439E;
d) the first Fc mutation comprises S364H, or F405A, and the second Fc mutation comprises Y349T, or T394F;
e) the first Fc mutation comprises S364H, or T394F, and the second Fc mutation comprises Y394T, or F405A;
f) the first Fc mutation comprises K370D, or K409D, and the second Fc mutation comprises E357K, or D399K; or
g) said first Fc mutation comprises L351D, or L368E, and said second Fc mutation comprises L351K, or T366K;
39. The polypeptide complex of claim 38, wherein the numbering is according to the EU index. The fusion polypeptide of any one of the preceding claims or the polypeptide complex of any one of the preceding claims, wherein the first target binding domain and the second target binding domain bind to different targets. The fusion polypeptide of any one of the preceding claims or the polypeptide complex of any one of the preceding claims, wherein at least one of the first target binding domain and the second target binding domain is a chimeric domain, a humanized domain, or a fully human domain. The fusion polypeptide of any one of the preceding claims or the polypeptide complex of any one of the preceding claims, wherein at least one of the first target binding domain and the second target binding domain binds to a disease-associated antigen or an immune-related target, and optionally the disease-associated antigen is a tumor-associated antigen, an antigen associated with an autoimmune or inflammatory disease, 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. The fusion polypeptide of any one of the preceding claims or the polypeptide complex of any one of the preceding claims, wherein one of the first target-binding domain and the second target-binding domain binds to a tumor-associated antigen, and the other binds to an immune-related target. A nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide described in any one of the preceding claims or a polypeptide complex described in any one of the preceding claims. A vector comprising the nucleic acid of claim 44. A host cell comprising the nucleic acid of claim 44 or the vector of claim 45. A pharmaceutical composition comprising the polypeptide complex of any one of claims 1 to 43 and a pharmaceutically acceptable carrier. A conjugate comprising the polypeptide complex of any one of claims 1 to 43 and a payload conjugated thereto, wherein the payload is selected from the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification tag, a tracking molecule, an anticancer drug, and a cytotoxic molecule. A composition comprising the polypeptide complex of any one of claims 1 to 43 or the conjugate of claim 48 and a pharmaceutically acceptable carrier. A method for treating or preventing a disease, condition, or symptom, comprising administering to a subject in need of treatment a therapeutically effective amount of a polypeptide complex described in any one of claims 1 to 43, a pharmaceutical composition described in claim 47, a conjugate described in claim 48, or a composition described in claim 49. The method of claim 50, wherein the disease is selected from the group consisting of cancer, an inflammatory disease, an infectious or parasitic disease, a cardiovascular disease, an eye disease, a central nervous system (CNS) disease, trauma, a metabolic disease, an autoimmune disease, or a coagulation disorder. A method for detecting the presence or level of an antigen, comprising contacting a sample suspected of containing the antigen with a polypeptide complex described in any one of claims 1 to 43, and determining the formation of a complex between the antigen and the polypeptide complex.