Multi-specific polypeptide complexes

Abstract

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

Description

Technical Field

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

[0002] Bispecific antibodies are artificial antibodies that can bind to at least two antigens. By simultaneously engaging at least two targets of interest, bispecific antibodies can provide benefits over conventional monospecific antibodies through novel and unique mechanisms. For example, blinatumomab (CD3×CD19, Amgen), which targets CD3 and CD19, can effectively engage T cells in killing tumor cells expressing CD19 through its Fv that recognizes CD3, and has shown efficacy superior to conventional antibodies in the treatment of ALL (acute lymphocytic leukemia). Binatumomab was approved by the FDA for the treatment of ALL in 2014.

[0003] Many bispecific antibody technology platforms have been developed, to name a few: BiTE bispecific T cell engager (Micromet, acquired by Amgen in 2012), CrossMab (Roche), DVD-Ig (AbbVie), TandAb (Affimed), and DART (dual antigen retargeting, Macrogenics).

[0004] However, despite the advantages of bispecific or multispecific antibodies, they also bring challenges, such as in preparation. Mismatches may occur between heavy chains and / or between heavy chains and light chains. For bispecific or multispecific antibodies, how to efficiently and effectively remove mismatched byproducts is an insurmountable challenge. Therefore, it is highly desirable to develop new constructs that can provide good binding affinity to the target of interest and are easy to prepare and purify downstream. Summary of the invention

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

[0006] The present disclosure provides, inter alia, novel fusion polypeptides, nucleotide sequences encoding such polypeptides, and uses thereof.

[0007] On the one hand, the present disclosure provides a fusion polypeptide, which comprises from the C-terminus to the N-terminus: a) a first target binding fragment A1; b) a polypeptide linker; c) a second target binding fragment B2, wherein the length of the polypeptide linker is short enough to minimize potential intramolecular interactions between A1 and B2. In some embodiments, the A1 can be paired with the first paired fragment B1 to form a first target binding domain; the B2 can be paired with the second paired fragment A2 to form a second target binding domain. In such embodiments, the A1 is configured to exhibit less binding to the B2 relative to the binding to the B1, and the B2 is configured to exhibit less binding to the A1 relative to the binding to the A2.

[0008] On the other hand, 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 the A1 in the fusion polypeptide is paired with the B1 in the second polypeptide to form a first target binding domain; the B2 in the fusion polypeptide is paired with the A2 in the fourth polypeptide to form the second target binding domain; and the A2 in the fifth polypeptide is paired with the B2 in the third polypeptide to form another second target binding domain.

[0009] In some embodiments, at least one of the pair of B1 and A1 and the pair of B2 and A2 contains at least one configuration that can hinder mismatching between B1 and A2 and / or between B2 and A1.

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

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

[0012] In some embodiments, the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VH2, and the 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, the A1 further comprises a first scaffold region SR operably linked to the VA1. a , and the B2 further comprises a second scaffold region SR operably connected to the VB2 b , where the SR a and the SR b is configured so as to hinder the SR a With the SR b The pairing between.

[0015] In some embodiments, the B1 further comprises a first paired scaffold region PSR a , the first paired scaffold region PSR a is operably connected to the VB1 and is capable of communicating with the SR a Combination, and wherein said A2 further comprises a second paired scaffold region PSR b , the second paired scaffold region PSR b is operably connected to the VA2 and is capable of communicating with the SR b Combine.

[0016] In some embodiments, SR a / PSR a Yes or SR b / PSR b The pair is selected from the group consisting of: a) a heavy chain constant region 1 (CH1) and a light chain constant region (CL); b) a T cell receptor (TCR) constant region α (C α ) and TCR constant region β (C β ) right; c) TCR constant region γ (C γ ) and TCR constant region δ (C δ ) pairs; d) ligand binding domain of a receptor and a ligand pair; and e) PRD (proline rich domain) and SH3 domain pair; f) obscurin and titin pair.

[0017] In some embodiments, the SR a and PSR a Different from the SR b and PSR b In some of these embodiments, the SR a and PSRa pairs are the CH1 and CL pairs, and the SR b and PSR b Yes, it is the following: i) C α and C β Yes, ii) C γ and C δ iii) a ligand binding domain of a receptor and a ligand; iv) a PRD (proline-rich domain) and an SH3 domain; or v) an obscurin and a titin. In some other embodiments of these embodiments, the SR b and PSR b pairs are the CH1 and CL pairs, and the SR a and PSR a The following are true: i) C α and C β Yes, ii) C γ and C δ pairs, iii) the ligand binding domain of a receptor and a ligand; iv) the PRD (proline rich domain) and SH3 domain pairs, or v) the obscurin and titin pairs.

[0018] In some embodiments, the SR a / PSR a To and the SR b / PSR b is the same for both, and the SR a / PSR a To and / or the SR b / PSR b The pair is configured to block SR a / PSR b Between or SR b / PSR a In some of these embodiments, the SR a / PSR a The pair comprises a CH1 domain CH1a and a CL domain CLa, and the SR b / PSR b The pair comprises a CH1 domain CH1b and a CL domain CLb.

[0019] 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, a pair of CH1 domains and CL domains are crossed. For example, the PSR a is the CL domain CLa, the SR ais the CH1 domain CH1a, the SR b is the CH1 domain CH1b, 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, the PSR b It is the CH1 domain CH1b.

[0020] In some embodiments, the PSR a is the CH1 domain CH1a, the SR a is the CL domain CLa, the SR b is the CH1 domain CH1b, the PSR b is a CL domain CLb. In some of these embodiments, a pair of VH domains and VL domains are crossed. For example, the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VL2, and the VA2 comprises VH2. Alternatively, the VB1 comprises VL1, the VA1 comprises VH1, the VB2 comprises VH2, and the VA2 comprises VL2.

[0021] In certain embodiments, the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VH2, and the VA2 comprises VL2, the PSR a is the CH1 domain CH1a, the SR a is the CL domain CLa, the SR b is the CH1 domain CH1b, the PSR b It is a CL domain CLb, and 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; d) the fourth polypeptide and the fifth polypeptide each comprise the amino acid sequence of formula (IV): VL2-CLb; and wherein the CH1b / CLb pair and / or the CH1a / CLa pair are configured to hinder mismatching 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 properties: 1) having at least one non-native disulfide bond that hinders mispairing between CH1b and CLa and / or between CH1a and CLb; 2) having one or more introduced charged amino acid residues that hinder 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 hinders mispairing between CH1b and CLa or between CH1a and CLb. These properties are useful for hindering 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-natural disulfide bond that prevents mispairing between CH1b and CLa and / or between 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, and wherein the first CH1 / CL pair is associated through a non-natural first disulfide bond and optionally lacks a natural disulfide bond or has a disrupted natural disulfide bond. In some embodiments, the second CH1 / CL pair is associated through a second disulfide bond, the second disulfide bond is formed at a position different from the first disulfide bond, and optionally the second disulfide bond is a natural disulfide bond. In some embodiments, the first disulfide bond is formed by two cysteine ​​residues introduced at a set of heavy chain-light chain EU positions, the set of heavy chain-light chain EU positions being 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 located between heavy chain EU position 220 and light chain EU position 214. In certain embodiments, the first CH1 / CL pair comprises CH1 and CL, wherein CH1 comprises a substitution at EU position 126 with a cysteine ​​residue and a substitution at EU position 220 with a non-cysteine ​​residue; and CL comprises a substitution at EU position 121 with a cysteine ​​residue and a substitution at EU position 214 with a non-cysteine ​​residue.

[0024] In some embodiments, at least one of the CH1b / CLb pair and the CH1a / CLa pair has at least one or more charged amino acid residues that hinder the introduction of mismatches 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, and wherein the first CH1 / CL pair contains at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of a residue with an opposite charge for a charged residue, so that the first CH1 / CL pair contains a first pair of oppositely charged residues that are conducive to the pairing of the first CH1 / CL pair. In some embodiments, the second CH1 / CL pair contains at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of an oppositely charged residue for a charged residue, so that the second CH1 / CL pair contains a second pair of oppositely charged residues that is conducive to the pairing of the second CH1 / CL pair, and optionally, the first pair of oppositely charged residues and the second pair of oppositely charged residues hinder the pairing between CH1a and CLb or hinder the pairing between CH1b and CLa. In some embodiments, the first pair of oppositely charged residues and / or the second pair of oppositely charged residues are configured so that CH1a and CLb both have positively charged residues or both have negatively charged residues, and / or CH1b and CLa both have positively charged residues or both have negatively charged residues. In certain embodiments, wherein the first pair of oppositely charged residues and / or the second pair of oppositely charged residues are introduced at a set of heavy chain-light chain EU positions, the 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 oppositely charged residue pairs comprise 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 hinders mismatching between CH1b and CLa 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, respectively, and wherein the first CH1 / CL pair comprises 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 set of heavy chain-light chain EU positions, and the set of heavy chain-light chain EU positions includes heavy chain EU positions H168A, F170G and light chain EU positions L135Y, S176W. 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 that form an orthogonal Fab design, wherein the one or more introduced amino acid mutations are 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 hinder mispairing between VH1 and VL2 and / or between 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, and wherein the first VH / VL pair has at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of a residue with an opposite charge for a charged residue, such that the first VH / VL contains a third pair of residues with an opposite charge that facilitates pairing of the first VH / VL pair. In some embodiments, the second VH / VL pair has at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of an oppositely charged residue for a charged residue, such that the second VH / VL contains a fourth pair of oppositely charged residues that facilitates pairing of the second VH / VL pair, and optionally, the third pair of oppositely charged residues and the fourth pair of oppositely charged residues hinder pairing between VH1 and VL2 or hinder pairing between VH2 and 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 charged residues or both have negatively charged residues, and / or VH2 and VL1 both have positively charged residues or both have 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 a set of heavy chain-light chain EU positions, the 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 oppositely charged residue pairs comprise 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 further comprise an operably linked first dimerization domain and an operably linked second dimerization domain, respectively, the first dimerization domain and the second dimerization domain associate to form a dimer; 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 have at least one configuration 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; c) the first Fc mutation comprises E356K, E357K or D399K, and the 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) 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 aspect, at least one of the first target binding domain and the second target binding domain is chimeric, humanized or fully human.

[0030] 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-associated target, optionally, the disease-associated antigen is a tumor-associated antigen, an antigen associated with an autoimmune disease or an 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 disease.

[0031] Another aspect of the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide or polypeptide complex as 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 a host cell comprising the nucleic acid described herein and a vector.

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

[0035] In yet another aspect, the present disclosure provides a conjugate comprising the polypeptide complex described herein and a payload conjugated to the polypeptide complex, 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 tracer molecule, an anti-cancer drug, an immune-related molecule, and a cytotoxic molecule.

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

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

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

[0039] In yet another aspect, the present disclosure provides a method for detecting the presence or level of an antigen. In some embodiments, the method comprises: contacting a sample suspected of containing the antigen with a polypeptide complex described herein; and determining that a complex is formed between the antigen and the polypeptide complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Each polypeptide fragment of an exemplary polypeptide complex constructed from two pairs of monoclonal antibodies and a pair of Fc regions is depicted.

[0041] Figure 2 The structure of an exemplary polypeptide complex constructed by the method of the invention (Method B) is schematically depicted.

[0042] Figure 3 Further depicted is the structure of an exemplary polypeptide complex constructed by the method of the present invention (Method B), in which Fab fragments from two monoclonal antibodies are subdivided into VH / VL pairs and CH1 / CL pairs.

[0043] Figure 4A and 4B Further depicted is the structure of an exemplary polypeptide complex constructed by the method of the invention (Method B) having defined VH / VL pairs and CH1 / CL pairs from Fab fragments of two monoclonal antibodies. Figure 4C-4E The B1-A2 mismatch ( Figure 4C )、B2-A1 mismatch ( Figure 4D ) and B1-A2 and B2-A1 mismatches ( Figure 4E ) antibody structure.

[0044] Figure 5A and 5B Depicted are structural diagrams of exemplary bispecific antibodies constructed by conventional methods (Method A). Figure 5C-5E The B1-A2 mismatch ( Figure 5C )、B2-A1 mismatch ( Figure 5D ) and B1-A2 and B2-A1 mismatches ( Figure 5E ) antibody structure.

[0045] Fig. 6A and 6BThe expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMATNEW1-8) analyzed by SDS-Page are indicated.

[0046] Figures 7A-7P The expression results of exemplary polypeptide complexes (FORMAT EX1-8 and FORMATNEW1-8) analyzed by SEC-HPLC are indicated.

[0047] Figure 8 Depicted is the purity of the expressed polypeptide complex anti-CD20 x CD3 (FORMATNEW7) analyzed by SDS-Page.

[0048] Fig. 9 Depicted is the purity of the expressed polypeptide complex anti-CD20 x CD3 (FORMAT NEW7) analyzed by CE-SDS.

[0049] Fig.10 Depicted is the purity of the expressed polypeptide complex anti-CD20 x CD3 (FORMATNEW7) analyzed by SEC-HPLC.

[0050] Fig.11 Depicted is the purification curve of the expressed polypeptide complex anti-CD20 x CD3 (FORMATNEW7) by affinity chromatography.

[0051] Fig.12 Shown is the SEC-HPLC purity analysis of the expressed polypeptide complex anti-CD20 x CD3 (FORMATNEW7) purified by affinity chromatography.

[0052] Fig.13 Shown is the SDS-Page purity analysis of the expressed polypeptide complex anti-CD20 x CD3 (FORMATNEW7) purified by affinity chromatography.

[0053] Fig.14 Depicted is the purification curve of the expressed polypeptide complex anti-CD20 x CD3 (FORMAT NEW7) under linear gradient elution by CEX.

[0054] Fig.15 Shown is the SDS-Page purity analysis of the expressed polypeptide complex anti-CD20 x CD3 (fractions C01-C04) purified by CEX.

[0055] Fig.16 Shown is the SEC-HPLC purity analysis of the expressed polypeptide complex anti-CD20 x CD3 (fractions C01-C04) purified by CEX.

[0056] Fig.17 Sequences of exemplary scaffold region pairs used in bispecific antibodies are shown. DETAILED DESCRIPTION

[0057] Several aspects of the present invention are described below with reference to the example applications used for illustration. It should be understood that many specific details, relationships and methods are set forth to provide a full understanding of the present invention. However, those of ordinary skill in the relevant art will readily recognize that the present invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the order of the activities or events shown, because some activities can occur in different orders and / or simultaneously with other activities or events.

[0058] Furthermore, not all of the illustrated activities or events are required to implement the methods according to the present invention.

[0059] I. Definitions and Abbreviations

[0060] Before providing a detailed description of the present invention, the following are noted and defined.

[0061] All descriptions provided herein are intended only to illustrate the various embodiments of the invention provided in the present disclosure. Thus, the specific modifications discussed should not be construed as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes and modifications may be made without departing from the scope of the present disclosure, and it should be understood that such equivalent embodiments will be included herein.

[0062] All references cited in this disclosure, including patent applications, issued patents, published articles or other publications, are incorporated by reference in their entirety for the purpose of providing methods that can be used in conjunction with the descriptions provided herein. For any terms that appear in one or more publications that are similar or identical to terms explicitly defined in this disclosure, the definitions of terms explicitly provided in this disclosure shall prevail in all respects.

[0063] Unless explicitly defined otherwise, all technical and scientific terms used in the present disclosure are generally considered to have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0064] As used herein, i.e. throughout the entire disclosure, unless otherwise indicated, the articles "a", "an", and "the" are to be interpreted as meaning "one or more" or "at least one". For example, "a polypeptide complex" means one polypeptide complex peptide or more than one polypeptide complex.

[0065] As used herein, the terms "about", "approximately", "around", etc. refer to a certain quantity, level, value, number, frequency, percentage, size, size, amount, weight or length that varies by up to 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference quantity, level, value, number, frequency, percentage, size, size, amount, weight or length. In specific embodiments, the term "about" or "approximately" when preceding a numerical value indicates a range of plus or minus 15%, 10%, 5% or 1% of the value.

[0066] As used herein, the terms "comprise", "comprises", "comprising", "include", "includes", "including", "contain", "contains", "containing", "have", "has", "having", etc. are synonymous and are used in an open-ended manner and do not exclude other elements, features, steps, actions, operations, etc.

[0067] As used herein, the term "or" is used in its inclusive sense (and not in its exclusive sense), so that, for example, when used to connect a list of elements, the term "or" means one, some, or all of the elements in the list.

[0068] As used herein, the phrase "at least one / kind" means one or more / one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. The phrase "at least one / kind" referring to a series of items is interpreted as referring to any combination of these items, including a single member. As an example, "at least one of A, B, or C" is intended to cover: A, B, C; A and B; A and C; B and C; and A, B and C. Unless otherwise specifically stated, connecting language such as the phrase "at least one of X, Y, and Z" is understood according to the context that it is usually used to express that an item, item, etc. may be at least one of X, Y, or Z. Therefore, such connecting language is generally not intended to imply that certain embodiments require each of at least one of X, at least one of Y, and at least one of Z to exist.

[0069] As used herein, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," "some embodiments," "certain embodiments," or "another embodiment," or combinations thereof, are understood to mean that a particular feature, structure, or characteristic described in conjunction with the particular embodiment is included in at least one embodiment of the present disclosure. Thus, the presence or occurrence of the aforementioned phrases in various places throughout the present disclosure are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0070] Conditional language used herein, such as "can, could, might, may", "eg", etc., unless otherwise expressly stated or otherwise understood in the context of use, is generally intended to convey that certain embodiments include certain features, elements, and / or steps, while other embodiments do not include certain features, elements, and / or steps. Therefore, such conditional language is generally not intended to imply that features, elements, and / or steps are in any way essential to one or more embodiments.

[0071] In this section, definitions of some general terms are provided. Definitions of other terms can be found in other sections of this disclosure that follow.

[0072] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a series of linear amino acid residues interconnected by peptide bonds, including 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. Therefore, as used herein, "amino acid" or "peptide residue" means both naturally occurring amino acids and synthetic amino acids. The terms "polypeptide", "peptide" and "protein" include fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences; fusion proteins with heterologous and homologous leader sequences with or without N-terminal methionine residues; immunolabeled proteins; fusion proteins with detectable fusion partners, for example, fusion proteins including fluorescent proteins, β-galactosidase, luciferase, etc. as fusion partners; and the like.

[0073] As used herein, the term "amino acid" refers to the building blocks of proteins, peptides, polypeptides, or amino acid polymers, and the term "amino acid" 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 later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. As used in this application, naturally occurring amino acids include the group of naturally occurring carboxy α-amino acids comprising: 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).

[0074] As used herein, the term "domain" refers to a globular structure formed by one or more regions of one or more polypeptide chains including peptide loops (e.g., including 3 to 4 peptide loops), which are stabilized, for example, by β-pleated sheets and / or intrachain disulfide bonds. Examples may include Fab domains (see below for more details). Note that in the present disclosure, the two terms "domain" and "region" can be used interchangeably.

[0075] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleotide," "polynucleotide," and the like are interpreted to refer to nucleotide polymers of any length, can include both DNA and RNA, can be single-stranded or double-stranded, and include analogs of naturally occurring polynucleotides in which one or more nucleotides are modified relative to naturally occurring nucleotides.

[0076] As used herein, the term "antibody" encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, multispecific antibody, bispecific antibody, bivalent antibody or multivalent antibody that binds to a specific antigen. Hereinafter, a description of antibodies and terms associated therewith is provided in more detail.

[0077] In mammals such as humans, there are five different classes / isotypes (i.e., IgA, IgD, IgE, IgG, and IgM, corresponding to five Ig heavy chain types α, δ, ε, γ, and μ) of antibodies, which generally have different molecular and biological properties, functional positions, physiological functions, and pathological significance of diseases, according to the different types of heavy chains present in immunoglobulins. Some antibody classes may further include subclasses. For example, in humans, IgA may include IgA1 and IgA2 subclasses, and IgG may include four subclasses represented as IgG1, IgG2, IgG3, and IgG4, respectively. With immunoglobulin monomers as their basic functional units, mammalian antibodies may exist as monomers (e.g., IgD, IgE, and IgG), dimers (IgA), tetramers (IgM), or pentamers (IgM). In mammals, there are two types of light chains, including kappa (κ) chains and lambda (λ) chains.

[0078] In the basic immunoglobulin unit, natural or naturally occurring antibodies such as IgG generally include two identical heavy (H) chains and two identical light (L) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond or a connection formed between a pair of cysteine ​​residues present in each light and heavy chain, respectively, and the two heavy chains are further interconnected by several disulfide bonds formed between cysteine ​​residues in each heavy chain. The tetramer thus formed is essentially a Y-like antibody shape, wherein the end of each fork arm contains an identical antigen binding site (i.e., a paratope) that specifically interacts with the corresponding epitope of the antigen.

[0079] More specifically, in a natural antibody, in the direction from N-terminal to C-terminal, each heavy chain includes a variable region (VH or HCVR), followed by three or four constant regions ("CH", IgA, IgD, IgG contain three CH regions CH1, CH2 and CH3; and IgE and IgM contain four CH regions CH1, CH2, CH3 and CH4), and each light chain includes a variable region (VL or LCVR) and a constant region (CL). In a Y-shaped antibody, the variable region of each light chain (i.e., VL region) is aligned or associated with the variable region of its paired heavy chain (i.e., VH region) to jointly form the antigen binding site of the antibody.

[0080] As used herein, the term "variable region" or "VR" means the region of an antibody heavy or light chain that is responsible for antigen binding. In native 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, 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 may be defined or determined by the Kabat, Chothia, or Al-Lazikani numbering conventions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are inserted between flanking extensions called "framework regions" (FRs), which are more highly conserved than CDRs and form a scaffold to support hypervariable loops. In natural antibodies, each VH and VL includes four FRs, and CDRs and FRs are arranged in order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. However, it should be understood that, as used herein, the term "variable region" does not necessarily need to include all three CDRs or all four FRs, and should be interpreted as covering any variant or derivative of the natural variable region from a natural antibody, as long as such variant or derivative retains antigen binding activity.

[0081] The term "constant region" or "constant portion" as used herein means a region of an antibody heavy chain 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 need to include the natural constant region of a full-length native antibody, and should be interpreted as covering any variant or derivative of such a natural constant region or constant portion, as long as such variant or derivative retains, for example, the ability to support the stability of the antigen binding domain, or retains the expected biological function, such as effector function, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc.

[0082] The term "CL region" refers to the constant region of an immunoglobulin light chain adjacent to the VL region. The CL region can extend from about EU position 108 to about EU position 216 in an immunoglobulin light chain. In a native antibody, the constant region of each light chain (i.e., CL region) is associated with the first constant region (i.e., CH1 region) of the paired heavy chain.

[0083] As used herein, the term "CH1 region" encompasses the first (most amino-terminal) constant region of an immunoglobulin heavy chain, which extends from about EU position 118 to at least about EU position 220 (e.g., can extend to EU position 221, etc.). The CH1 region is adjacent to the VH region and is located amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0084] In the case of antibodies, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 region to the CH2 region. The length of the hinge region can vary depending on the defined boundaries of the CH1 region and the CH2 region. The hinge region is generally flexible, thus allowing the two N-terminal antigen binding regions to move independently.

[0085] 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.

[0086] As used herein, the term "CH3 region" refers to the portion of a heavy chain immunoglobulin molecule extending from the N-terminus of the CH2 domain for about 110 residues, for example 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 to 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).

[0087] As used herein, "Fc" refers to a portion derived from an antibody (e.g., IgG), which is primarily composed of the CH2 and CH3 of a first heavy chain bound to a second constant region (CH2) and a third constant region (CH3) of a second heavy chain by one or more covalent bonds as non-peptide bonds (e.g., by disulfide bonding). The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), etc., but does not play a role in antigen binding.

[0088] As used herein, "antigen" or "Ag" refers to a compound, composition, peptide, polypeptide, protein or substance (e.g., polypeptide, carbohydrate, nucleic acid, lipid or other naturally occurring or synthetic compound) that can be specifically recognized and bound by components of the immune system (e.g., antibodies). As used herein, the term "antigen" encompasses antigenic epitopes, such as antigenic fragments that are antigenic epitopes. The terms "antigen" and "target" are used interchangeably in this disclosure.

[0089] "Fv" about antibodies refers to the smallest fragment of an antibody that carries a complete antigen binding site. The Fv fragment consists of the variable domain of a single light chain combined with the variable domain of a single heavy chain. Many Fv designs have been provided, including dsFv, in which the association between the two domains is enhanced by the disulfide bond introduced; and a peptide linker can be used to combine the two domains together as a single polypeptide to form scFv. Fv constructs containing the variable domains of the heavy or light immunoglobulin chains associated with the variable domains and constant domains of the corresponding immunoglobulin heavy or light chains have been produced. Fv has also been polymerized to form bifunctional antibodies and trifunctional antibodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0090] As used herein, "Fab" refers to a single antigen-binding domain derived from an antibody, wherein the domain has a single heavy chain fragment associated with a single light chain fragment by one or more covalent bonds that are non-peptide bonds. In some embodiments, the single heavy chain fragment in the Fab domain includes the HCVR and CH1 regions. In some embodiments, the single light chain fragment in the Fab domain includes the LCVR and CL domains. In some embodiments, the CH1 region is associated with the HCVR by covalent bonds such as disulfide bonds. In natural antibodies, the Fab domain essentially corresponds to one arm of the antibody, typically retaining the ability to recognize and bind to its corresponding antigen.

[0091] As used herein, the term "multispecific antibody" refers to an artificial or engineered antibody that can bind to at least two different epitopes simultaneously. Bispecific antibodies are essentially a type of multispecific antibody. In addition, other multispecific antibodies may include trispecific antibodies with three different antigen binding specificities, tetraspecific antibodies with four different antigen binding specificities, and the like.

[0092] As used herein, the term "bispecific antibody" refers to an antibody comprising two physically separable antigen-binding domains that differ from each other in their antigenic specificity. Typically, a bispecific antibody is an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens. It contrasts with naturally occurring antibodies, which have two physically separable antigen-binding moieties that are structurally identical and therefore have the same antigenic specificity.

[0093] Throughout the disclosure, numbers indicating amino acid residue positions in antibody constant regions, such as those in the heavy chain constant region 1 (CH1) and the light chain constant region (CL) in the constant portion, are based on EU numbering as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). As described above, some positions use IMGT numbering or the Kabat index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Chapter 26, 481-514, (2015). These numbers can also be obtained from the IMGT scientific chart, which can be accessed from the website of the international ImMunoGeneTics information system.

[0094] As used herein, the term "chimeric" means an antibody or antigen-binding fragment having a portion of a heavy chain and / or light chain derived from one species and the remainder of a heavy chain and / or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise 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.

[0095] As used herein, the term "valence" refers to the presence of a specified number of antigen binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen binding fragment having only one 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 the presence of two binding sites, four binding sites and six binding sites in an antigen binding molecule, respectively. In some embodiments, the antibody or its antigen binding fragment is bivalent.

[0096] Antibodies and fragments thereof according to the present disclosure encompass bispecific and multispecific antibodies and fragments thereof. Bispecific or multispecific antibodies may be similar to single antibodies (or antibody fragments), but have two or more different antigen binding sites. Bispecific antibodies may have binding specificity for at least two different epitopes. Bispecific antibodies and fragments may also be in the form of heterologous antibodies. Heterologous antibodies are two or more antibodies or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having different specificities.

[0097] In the context of properties of antibodies, the term "specific binding of an antibody" or "antigen-specific antibody" 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 will discriminate between desired and undesired antigens (or "target" and "non-target" antigens) in a sample, in some embodiments, by more than about 10-100 times or more (e.g., more than about 1000 times or 10,000 times). In certain embodiments, when an antibody and antigen specifically bind in an antibody-antigen complex, the affinity between them is characterized by a K. D (Dissociation constant) less than 10 -6 M, less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -9 M, less than 10 -11 M or less than about 10 -12 M or smaller.

[0098] As used herein, the term "vector" refers to a medium into which a genetic element can be operably inserted so that the genetic element is expressed, so as to produce a protein, RNA or DNA encoded by the genetic element or to replicate the genetic element. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. Examples of vectors include plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages, such as lambda phages or M13 phages; and animal viruses. The vector can contain a variety of elements for controlling expression, including promoter sequences, transcription start sequences, enhancer sequences, selectable elements and reporter genes. In addition, the vector can also contain a replication origin. The vector can also include materials that assist it in entering the cell, including but not limited to viral particles, liposomes or protein coatings. The vector can be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0099] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced.

[0100] Antibody conjugates are also provided. The terms "conjugated" and "linked" generally refer to a covalent or non-covalent chemical bond that tightly associates one molecule with a second molecule. The conjugate includes any antibody disclosed herein and an agent. The agent can be selected from a therapeutic agent, an imaging agent, a labeling agent, or an agent for therapeutic and / or labeling purposes.

[0101] As used herein, the term "biological sample" or "sample" refers to a biological composition obtained from or derived from a subject of interest, the biological composition containing, for example, cells and / or other molecular entities to be characterized and / or identified based on 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 skilled in the art. In some embodiments, the biological sample is a body fluid sample. In some embodiments, the body fluid sample is whole blood, plasma, serum, mucus (including nasal drainage and sputum), peritoneal fluid, pleural fluid, pleural effusion, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, peritoneal fluid, ascites or pericardial fluid. In some embodiments, the biological sample is a tissue or cell obtained from the heart, liver, spleen, lung, kidney, skin or blood vessel of a subject.

[0102] II. Novel multispecific (or bispecific) polypeptide complexes

[0103] In one aspect, the present disclosure provides a novel multispecific (or bispecific) polypeptide complex that is easy to prepare and purify and can therefore be provided in high purity and high yield.

[0104] Multispecific (or bispecific) antibodies are an important research direction in the field of antibody engineering and have broad application prospects in areas such as anti-tumor immunotherapy. Different 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.

[0105] IgG-like bispecific antibodies provide two monovalent Fabs, each binding to a different antigen. However, when compared to conventional IgG antibodies that provide bivalent binding to a single antigen, this can reduce the affinity for each antigen and even lose some functions.

[0106] Another challenge involves differentiating the binding affinity of an IgG-like bispecific antibody to two target antigens. Depending on the intended mechanism of action (i.e., MOA) of the two target antigens, different binding affinities may be required for the two target antigens. For example, for a CD3 / CD20 bispecific antibody, nanomolar affinity is desired for CD20, but submicromolar affinity (e.g., 30-100 nm) for CD3. To increase affinity for a target of interest, an additional Fab domain targeting such a target can be fused to one of the heavy chains of an IgG-like bispecific antibody (see Figure 5A ). The structure of such bispecific but trivalent antibodies is referred to herein as a 2:1 structure, i.e., the binding to one antigen of interest is bivalent, while the binding to the other antigen remains monovalent. Such bivalent binding restores the binding affinity to the antigen target and can simultaneously confer a difference in affinity between the two antigen targets.

[0107] Although 2:1 bispecific antibodies provide benefits, there are some insurmountable challenges in their expression and production. For example, LC-HC binding instability and LC mismatching may occur during the assembly of such 2:1 bispecific antibodies. There may be different LC mismatch products (i.e., impurities) that are similar or indistinguishable from the target product (i.e., correctly paired) in terms of molecular weight and physical and chemical properties, making it difficult to separate or purify the target product from these impurities. This may result in low purity and reduced yield of the expressed target product.

[0108] The present disclosure provides a novel 2:1 structural bispecific antibody platform, which can not only meet the requirements of affinity differences between two antigen targets, but also is easier to purify than conventional 2:1 structural bispecific antibodies. This novel 2:1 structure is advantageous because potential mismatch products (if any) are sufficiently different from the correctly paired products in terms of, for example, molecular weight and physical and chemical properties, which makes it easier to remove mismatch products. Therefore, bispecific antibodies constructed according to this novel 2:1 structural platform can obtain higher purity and yield with a relatively simple purification process.

[0109] III. Novel 2:1 structure of multispecific antibodies

[0110] The novel 2:1 structure provided herein is based on the fusion of an IgG-like bispecific antibody with an additional Fab domain, which binds to one of the two targets. Specifically, compared with conventional 2:1 structure bispecific antibodies (i.e., the additional Fab domain is fused to the heavy chain of an Ig-like bispecific antibody, see Figure 5A Unlike the second polypeptide chain in the present invention, the novel 2:1 structure provided herein fuses the additional Fab region to the light chain of the IgG-like bispecific antibody (see Figure 2 The first polypeptide chain in

[0111] Specifically, Figure 2 As shown, the novel 2:1 structure bispecific antibody comprises a polypeptide complex composed of five polypeptide chains, the polypeptide complex comprising: 1) a first polypeptide chain comprising a fusion polypeptide, the fusion polypeptide comprising a first target binding fragment A1 and a second target binding fragment B2; 2) a second polypeptide comprising a first paired fragment B1; 3) a third polypeptide comprising a second target binding fragment B2; 4) a fourth polypeptide comprising a second paired fragment A2; and 5) a fifth polypeptide identical to the fourth polypeptide, i.e., also 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 another second target binding domain.

[0112] The uniqueness of the polypeptide complex provided herein lies at least in part in that the fusion polypeptide (i.e., the first polypeptide chain) contained therein is novel and is not contained in any conventional 2:1 structural bispecific antibody. Such fusion polypeptides make it possible for any mismatched product to have a molecular weight and physical and chemical properties that are significantly different from the target bispecific product.

[0113] In some embodiments, the fusion polypeptide comprises, from C-terminus to N-terminus: a first target binding fragment A1; a polypeptide linker; a second target binding fragment B2; wherein the length of the polypeptide linker is short enough to minimize potential intramolecular interactions between A1 and B2, and A1 is capable of pairing with the first pairing fragment B1 to form a first target binding domain; B2 is capable of pairing with the second pairing fragment A2 to form a second target binding domain; and relative to the binding with B1, A1 is configured to exhibit less binding with B2, and relative to the binding with A2, B2 is configured to exhibit less binding with A1.

[0114] In such Figure 2 In the novel 2:1 structure shown, the two light chains are identical and thus help reduce unwanted mispairing. In the illustrative novel 2:1 structure provided herein (see Figure 4A ), a total of 3 different mismatch products are expected (see Figure 4C-4E ), the molecular weights of the mismatch products were estimated to be approximately 150 KDa, 250 KDa and 200 KDa, respectively (see Figure 4B ), and therefore the molecular weight of most potential mispairing products is different from that of the correctly paired products. In addition, these potential mispairing products also show different physical and chemical properties compared to the correctly paired bispecific products, and therefore can be easily removed by conventional purification methods.

[0115] In contrast, Figure 5A As shown, a conventional 2:1 bispecific antibody has two different light chains that may mispair with different heavy chains, resulting in a total of five different mispairing products (see Figure 5C-5E ), it is estimated that the mismatched products all have the same molecular weight as the correctly matched products (i.e., 200 KDa) (see Figure 5B ). These similar mismatched products are difficult to separate from the target product, thus affecting not only the purity of the target product but also its yield.

[0116] The novel 2:1 polypeptide complex provided herein binds to a first target and a second target. The polypeptide complex has two antigen binding domains that are both bound to the second target and a third antigen binding domain that is bound to the first target. In other words, the polypeptide complex is divalent for the second target and monovalent for the first target, and is therefore referred to as "2:1". The two antigen binding domains for the second target are located on different arms of the polypeptide complex provided herein.

[0117] In some embodiments, the target binding domain in the polypeptide complex provided herein is formed by pairing of two polypeptide fragments. In certain embodiments, the first target binding domain is formed by pairing of the first target binding fragment A1 and the first pairing fragment B1 (i.e., A1 / B1 pair). In certain embodiments, the second target binding domain is formed by pairing of the second target binding fragment A2 and the second pairing fragment B2 (i.e., A2 / B2 pair).

[0118] In some embodiments, the first target binding fragment A1 and the first pairing fragment B1 (i.e., A1 / B1) can be paired by, for example, disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges or hydrophobic-hydrophilic interactions, connectors or a combination thereof, wherein the pairing is formed between A1 and B1, or more specifically between at least two amino acid residues from A1 and B1.

[0119] Similarly, in some embodiments, the second target binding fragment B2 and the second pairing fragment A2 (i.e., A2 / B2) can be paired by, for example, disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges or hydrophobic-hydrophilic interactions, connectors or a combination thereof, wherein the pairing is formed between A2 and B2, or more specifically between at least two amino acid residues from A2 and B2.

[0120] "Disulfide bond" refers to a covalent bond having the structure RSS-R'. The amino acid cysteine ​​includes a thiol group that can form a disulfide bond with, for example, a second thiol group from another cysteine ​​residue. A disulfide bond can be formed between the sulfhydryl groups of two cysteine ​​residues, respectively, located on two polypeptide chains, thereby forming an interchain disulfide bond. Disulfide bonds can be formed between known scaffold polypeptide fragments, such as antibody CH1 domains and CL domains, or TCR constant regions (e.g., TCRα / TCRβ, TCRγ / TCRδ), to name a few).

[0121] Electrostatic interactions are non-covalent interactions and are important in protein folding, stability, flexibility and function, and include ionic interactions, hydrogen bonding and halogen bonding. Electrostatic interactions can form in polypeptides, for example, between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu, Trp on a first polypeptide chain and Arg, Val or Thr on a second polypeptide chain.

[0122] "Salt bridge" is a close-range electrostatic interaction, mainly generated by the anionic carboxylates of Asp or Glu and the cationic ammonium of the guanidinium of Lys or Arg, which are spatially adjacent pairs of oppositely charged residues in the native protein structure. Most of the charged and polar residues in the hydrophobic interface may act as hot spots for binding. Among them, residues with ionizable side chains such as His, Tyr and Ser can also participate in the formation of salt bridges.

[0123] Hydrophobic interactions can be formed between one or more of Val, Tyr and Ala on one polypeptide chain and one or more of 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.

[0124] When a hydrogen atom covalently bonds to a highly electronegative atom such as nitrogen, oxygen, or fluorine, a hydrogen bond is formed between the two polar groups through electrostatic attraction. Hydrogen bonds can be formed, 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.

[0125] In some embodiments, Figure 4A As shown in the novel 2: 1 structure of , natural or introduced non-natural disulfide bonds favor homologous pairing of A1 and B1, while mismatched products (such as mismatched A1 and B2, or mismatched A2 and B1) will lack such disulfide bonds. Without wishing to be bound by any theory, it is expected that such disulfide bonds will be particularly advantageous in allowing the presence of mismatched products to be easily identified and / or characterized, for example, by using conventional methods such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This is at least in part because disulfide-bonded homologous paired products can withstand SDS-PAGE treatment, while mismatched products lacking such disulfide bonds will suffer from the dissociation of mismatched polypeptide chains, resulting in molecular weight differences between homologous paired products and mismatched products observed by SDS-PAGE, such as Figure 4B-4E In addition, Figure 4A The novel 2:1 structure allows the mismatched products to be easily separated from the homologous paired products using relatively conventional purification methods, such as affinity purification and / or ion exchange purification, thereby obtaining highly pure homologous paired products that would otherwise be impossible or require a lengthy purification process.

[0126] In certain embodiments, the A1 / B1 pair and / or the A2 / B2 pair comprise an antibody Fv domain.

[0127] In some embodiments, in the fusion polypeptide or polypeptide complex provided herein, the A1 comprises a first antibody variable region VA1 selected from VH1 or VL1, and the B1 comprises a first paired antibody variable region VB1 capable of pairing with VA1 to form a first target binding domain, wherein the VB1 is selected from VH1 or VL1. In other words, the first target binding domain comprises a first Fv domain, which comprises VH1 and VL1 associated together and capable of binding to a first target.

[0128] In some embodiments, in the fusion polypeptide or polypeptide complex provided herein, the B2 comprises a second antibody variable region VB2 selected from VH2 or VL2, and the A2 comprises a second paired antibody variable region VA2 capable of pairing with VB2 to form a second target binding domain, wherein the VA2 is selected from VH2 or VL2. In other words, the second target binding domain comprises a second Fv domain, which comprises VH2 and VL2 associated together and capable of binding to a second target.

[0129] In some embodiments, the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VH2, and the VA2 comprises VL2. In some embodiments, the VB1 comprises VL1, the VA1 comprises VH1, the VB2 comprises VL2, and the VA2 comprises VH2.

[0130] In some embodiments, the first Fv domain or the second Fv domain is crossed. In some embodiments, the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VL2, and the VA2 comprises VH2. In some embodiments, the VB1 comprises VL1, the VA1 comprises VH1, the VB2 comprises VH2, and the VA2 comprises VL2.

[0131] The Fv domains can be associated by any suitable means known in the art (e.g., but not limited to, by non-native disulfide bonds introduced into the Fv domains or by non-native electrostatic interactions introduced into the Fv domains). In some other embodiments, the Fv domains can be associated by a scaffold domain fused to the Fv domains. For example, a scaffold region can be fused to one chain of the Fv domain, and a paired scaffold region can be fused to another chain of the Fv domain. The combination of a scaffold region and a paired scaffold region can allow the association of the VH region and the VL region, thereby forming an Fv domain capable of binding to a target antigen.

[0132] In some embodiments, in the polypeptide complex disclosed herein, the A1 further comprises a first scaffold region SR operably linked to the VA1. a, and the B2 further comprises a second scaffold region SR operably connected to the VB2 b , where the SR a and the SR b is configured so as to hinder the SR a With the SR b In some embodiments, in the polypeptide complex disclosed herein, the B1 further comprises a first pairing scaffold region PSR a , the first paired scaffold region PSR a is operably connected to the VB1 and is capable of communicating with the SR a Combination, and wherein said A2 further comprises a second paired scaffold region PSR b , the second paired scaffold region PSR b is operably connected to the VA2 and is capable of communicating with the SR b Combined. SR a and PSR a The combination of SR b and PSR b The combination of forms the second scaffold domain. Figure 3 An illustrative diagram is shown in FIG.

[0133] Any suitable binding partner can be used as the scaffold region. In certain embodiments, the scaffold region can be selected from antibody CH1 domain and CL domain, paired TCR constant region (such as TCRα / TCRβ, TCRγ / TCRδ), or PRD (proline-rich domain) and SH3 domain or shielding protein and titin.

[0134] TCR belongs to the immunoglobulin superfamily, and is similar to a half antibody with a single heavy chain and a single light chain. Natural TCR consists of two polypeptide chains, and there are generally two types: one is composed of an α chain and a β chain (i.e., α / βTCR), and the other is composed of a γ chain and a δ chain (i.e., γ / δTCR). Two TCR chains are connected by the disulfide bonds formed between the constant regions in the extracellular part of the TCR chains. In other words, a disulfide bond is formed between TCR constant region α and TCR constant region β or between TCR constant region γ and TCR constant region δ.

[0135] SH3 domain, short for SRC homology 3 domain, is a small protein domain of about 60 amino acid residues that was originally described as a conserved sequence in the viral adaptor protein v-Crk. Classical SH3 domains are often found in proteins that interact with other proteins and mediate the assembly of specific protein complexes, usually by binding to proline-rich domains (i.e., PRDs) in their corresponding binding partners (Liubov V Gushchina et al., J Biomol Struct Dyn. 2011 Dec; 29(3): 485-95.).

[0136] Titin (also known as connexin) is a protein encoded by the TTN gene. Titin is a giant protein that is more than 1 μm in length and acts as a molecular spring responsible for the passive elasticity of muscle. Shieldin (about 800 kDa), the newest member of the titin family, was initially identified as a ligand for the Zdisk portion of titin, although it is mainly localized to the sarcomere M band of mature muscle. During muscle development, the giant muscle protein titin and shieldin bind to each other at Zdisk. This interaction helps to stabilize and organize the sarcomere; the elimination of this binding leads to muscular dystrophy (Allyn GLetourneau et al., Protein Pept Lett. 2018; 25 (11): 973-979.). Specifically, the titin Ig-like 152 domain of titin interacts with the shieldin Ig-like 1 domain of shieldin, and they can form a complex. In some previously reported studies, titin T chain and shielding protein O chain can be used to replace the CH1 region and CL region of an antibody, wherein the titin T chain is a peptide containing a titin Ig-like 152 domain or a functional variant thereof with a length of 78-118 amino acids, and the shielding protein O chain is a peptide containing a shielding protein Ig-like 1 domain or a functional variant thereof with a length of 87-117 amino acids (see, for example, WO2021 / 139757A, which is incorporated herein in its entirety).

[0137] The scaffold region pairs suitable for use in the present invention (ie, SR a / PSR a Yes or SR b / PSR bPairs) include: a) a heavy chain constant region 1 (CH1) and a light chain constant region (CL) pair; b) a T cell receptor (TCR) constant region α (Cα) and a TCR constant region β (Cβ) pair; c) a TCR constant region γ (Cγ) and a TCR constant region δ (Cδ) pair; d) a receptor ligand binding domain and a ligand pair; and e) a PRD (proline-rich domain) and an SH3 domain pair; f) a shielding protein and a titin pair, etc. It should be understood that as used throughout the disclosure herein, the expression "a pair of X and Y" or "X / Y pair" is not intended to limit the sequential order. For example, by stating "SR a / PSR a A pair may be a CH1 and CL pair", which is intended to mean a SR a can be CH1 or CL, and therefore, when SR a When it is CH1, PSR a Can be CL, or SR a When it is CL, then PSR a It can be CH1.

[0138] In some embodiments, SR a and PSR a Paired via a first disulfide bond. In such embodiments, SR a and PSR b or SR b and PSR a A mismatch of SR will lack the first disulfide bond. b and PSR b Paired via a second disulfide bond. In such embodiments, SR a and PSR b or SR b and PSR a Any suitable pair of scaffold regions that can pair via disulfide bonds can be used as SR a and PSR a Yes or SR b / PSR b For example, an antibody CH1 domain and a CL domain can be paired via a disulfide bond; TCR constant regions (such as Cα and Cβ, or Cγ and Cδ) can be paired via a disulfide bond; and shielding protein and titin can be paired via a disulfide bond.

[0139] In some embodiments, SR a / PSR a The pair contains a CH1 domain CH1a and a CL domain CLa pair, and SR b / PSR b The pair comprises a CH1 domain CH1b and a CL domain CLb pair. Figure 4A An illustrative diagram is shown in 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.

[0140] In some embodiments, the pairing between A1 and B1 can be between VA1 and VB1, VH1 and VL1, SR a and PSR a or between two amino acid residues in CH1a and CLa. Pairing between A2 and B2 can be formed between VA2 and VB2, VH2 and VL2, SR b and PSR b Or formed between two amino acid residues in CH1b and CLb.

[0141] IV. Configurations for Avoiding Mismatching

[0142] Although the target binding polypeptide fragment is intended to bind to its paired polypeptide fragment to form a target binding domain (i.e., homologous pairing), mismatches may still occur between polypeptide fragments that are not intended to be paired. For example, the first target binding fragment A1 may mismatch with the second target binding fragment B2, or the first target binding fragment A2 may mismatch with the second target binding fragment B1, thereby failing to form the intended target binding domain.

[0143] For example, VH1 is intended to pair with VL1 to form a first target binding domain, and VH2 is intended to pair with VL2 to form a second target binding domain, and such homologous pairing is intended to be promoted. However, VH1 may mispair with VL2, and VH2 may mispair with VL1, and such mispairing is preferably hindered or reduced.

[0144] In order to promote homologous pairing and hopefully reduce mispairing, polypeptide fragments can be designed or engineered to contain certain configurations that help achieve the above purpose. In some embodiments, in the fusion polypeptides or polypeptide complexes disclosed herein, at least one of the pair of A1 and B1 (referred to as A1 / B1) and the pair of A2 and B2 (referred to as A2 / B2) contains at least one configuration that can: a) promote homologous pairing between A1 and A2 and / or B1 and B2, and / or b) hinder mispairing between B1 and A2 and / or between B2 and A1.

[0145] Any suitable method known in the art can be used for the polypeptide complex disclosed herein to promote homologous pairing and / or reduce mispairing. Several strategies have been applied to design orthogonal interfaces to promote homologous pairing. For example, Roche exchanged the CH1 and CL domains and created 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 non-natural disulfide bonds (Mazor et al., mAbs, 7(2), pp. 377-389 (2015)), Amgen further introduced electrostatic interactions in the CH1-CL region (Liu et al., Journal of Biological Chemistry, 290(12), pp. 7535-7562 (2015)), Eli Lilly and Company (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 in both the variable and constant domains, and Wuxi Biologics replaced a 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 for promoting homologous pairing and can be used in the present disclosure.

[0146] In certain embodiments, in the fusion polypeptide or polypeptide complex disclosed herein, the SRa / PSR a To and the SR b / PSR b are the same or of the same type, and the SR a / PSR a To and / or the SR b / PSR b The pair is configured to block SR a / PSR b Between or SR b / PSR a Mismatch between.

[0147] Any suitable means can be used to hinder or reduce mispairing. For example, the A1 / B1 pair and the A2 / B2 pair can be designed to incorporate a crossed VA1 / VB1 configuration (e.g., CrossMab), or to incorporate different paired scaffold regions, or to incorporate crossed paired scaffold regions, or to introduce at least one mutation in otherwise identical paired scaffold regions. More details will be discussed below.

[0148] A). Crossover VA1 / VB1 configuration

[0149] 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 between B1 and A2. The Fv domain comprises a heavy chain variable domain VH and a light chain variable domain VL. The two Fv domains may be in a crossed configuration, so that when a mispairing occurs between B2 and A1 and / or a mispairing occurs between B1 and A2, it will result in pairing of two VH domains or pairing of two VL domains, which is considered less stable than pairing between VH and VL, and is therefore less likely to form.

[0150] In some embodiments, the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VL2, and the VA2 comprises VH2. In some embodiments, the VB1 comprises VL1, the VA1 comprises VH1, the VB2 comprises VH2, and the VA2 comprises VL2. In these crossover VA / VB1 configurations, when B2 is mispaired with A1 or when B1 is mispaired with A2, it will result in pairing of VH1 with VH2 or pairing of VL1 with VL2, which is considered unlikely to form a stable product.

[0151] In certain of these embodiments, VB1 and the first paired scaffold region PSR a operably connected, and VA1 and SR a operably connected, wherein SR a With PSR a In some embodiments of these embodiments, VB2 and the second paired scaffold region SR b operably connected, and VA2 and PSR b operably connected, wherein SR b With PSR b Combine to form a second paired scaffold domain.

[0152] In some embodiments, PSR a CH1 domain CH1a, SR a It is the CL domain CLa, SR bCH1 domain CH1b, PSR b is a CL domain CLb, and 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.

[0153] B). Different paired scaffold regions

[0154] In certain embodiments, the B1 / A1 pair comprises a first antibody Fv domain and a first pair of scaffold regions (ie, 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 ), wherein the first pair of scaffold regions and the second scaffold region are different, or are scaffold regions of different types. Therefore, a mismatch between A2 and B1 or a mismatch between A1 and B2 will produce incompatible scaffold regions that cannot naturally bind to each other.

[0155] In some embodiments, the SR a and PSR a Different from the SR b and PSR b Yes. In certain embodiments, SR a / PSR a Yes or SR b / PSR b The pair is selected from the group consisting of: a) a heavy chain constant region 1 (CH1) and a light chain constant region (CL) pair; b) a T cell receptor (TCR) constant region α (Cα) and a TCR constant region β (Cβ) pair; c) a TCR constant region γ (Cγ) and a TCR constant region δ (Cδ) pair; d) a receptor ligand binding domain and a ligand pair; and e) a PRD (proline-rich domain) and an SH3 domain pair; and f) an obscurin and a titin pair. In some embodiments, the TCR constant region α (i.e., Cα) comprises the amino acid sequence of SEQ ID NO: 97; and the TCR constant region β (i.e., Cβ) comprises the amino acid sequence of SEQ ID NO: 98.

[0156] In some embodiments, the SR a and PSR a pairs are the CH1 and CL pairs, and the SR b and PSR bThe pair is one of the following: i) a Cα and Cβ pair, ii) a Cγ and Cδ pair, iii) a ligand binding domain of a receptor and a ligand pair; iv) a PRD (proline-rich domain) and an SH3 domain pair, or v) an obscurin and a titin pair. In some embodiments, the SR b and PSR b pairs are the CH1 and CL pairs, and the SR a and PSR a The pairs are the following: i) a Cα and Cβ pair, ii) a Cγ and Cδ pair, iii) a ligand binding domain of a receptor and a ligand pair, iv) a PRD (proline rich domain) and a SH3 domain pair, or v) an obscurin and titin pair.

[0157] In some embodiments, the SR a and PSR a The pair is a Cα and Cβ pair, and the SR b and PSR b The pair is one of the following: i) a CH1 and CL pair, ii) a Cγ and Cδ pair, iii) a receptor ligand binding domain and a ligand pair; iv) a PRD (proline-rich domain) and an SH3 domain pair, or v) an obscurin and a titin pair. In some embodiments, the SR b and PSR b The pair is a Cα and Cβ pair, and the SR a and PSR a The pairs are the following: i) CH1 and CL pair, ii) Cγ and Cδ pair, iii) ligand binding domain of a receptor and ligand pair, iv) PRD (proline rich domain) and SH3 domain pair, or v) obscurin and titin pair.

[0158] In some embodiments, the SR a and PSR a The pair is a Cγ and Cδ pair, and the SR b and PSR b The pair is the following: i) CH1 and CL pair, ii) Cα and Cβ pair, iii) a ligand binding domain of a receptor and a ligand pair; iv) a PRD (proline-rich domain) and an SH3 domain pair, or v) an obscurin and a titin pair. In some embodiments, the SR b and PSR b The pair is a Cγ and Cδ pair, and the SR a and PSR aThe pairs are the following: i) CH1 and CL pair, ii) Ca and Cβ pair, iii) ligand binding domain of a receptor and ligand pair, iv) PRD (proline rich domain) and SH3 domain pair, or v) obscurin and titin pair.

[0159] In some embodiments, the SR a and PSR a The pair is the ligand binding domain of the receptor and the ligand pair, and the SR b and PSR b The pair is one of the following: i) CH1 and CL, ii) Cα and Cβ, iii) Cγ and Cδ; iv) PRD (proline-rich domain) and SH3 domain, or v) shielding protein and titin. In some embodiments, the SR b and PSR b The pair is the ligand binding domain of the receptor and the ligand pair, and the SR a and PSR a The pairs are the following: i) a CH1 and CL pair, ii) a Ca and Cβ pair, iii) a Cγ and Cδ pair, iv) a PRD (proline rich domain) and SH3 domain pair, or v) an obscurin and titin pair.

[0160] In some embodiments, the SR a and PSR a The pair is a PRD (proline-rich domain) and an SH3 domain pair, and the SR b and PSR b The pair is one of the following: i) a CH1 and CL pair, ii) a Cα and Cβ pair, iii) a Cγ and Cδ pair; iv) a ligand binding domain of a receptor and a ligand pair, or v) an obscurin and titin pair. In some embodiments, the SR b and PSR b The pair is a PRD (proline-rich domain) and an SH3 domain pair, and the SR a and PSR a The pairs are the following: i) a CH1 and CL pair, ii) a Cα and Cβ pair, iii) a Cγ and Cδ pair; iv) a ligand binding domain of a receptor and a ligand pair, or v) an obscurin and titin pair.

[0161] In some embodiments, the SR a and PSR a The pair is the opaque protein and titin pair, and the SR b and PSR bThe pair is the following: i) CH1 and CL pair, ii) Cα and Cβ pair, iii) Cγ and Cδ pair; iv) a ligand binding domain of a receptor and a ligand pair, or v) a PRD (proline-rich domain) and an SH3 domain pair. In some embodiments, the SR b and PSR b The pair is the opaque protein and titin pair, and the SR a and PSR a The pairs are the following: i) a CH1 and CL pair, ii) a Cα and Cβ pair, iii) a Cγ and Cδ pair, iv) a ligand binding domain of a receptor and a ligand pair, or v) a PRD (proline rich domain) and SH3 domain pair.

[0162] In some embodiments, homologous pairing of scaffold regions is promoted by introducing non-native disulfide bonds. In such embodiments, the scaffold regions are CH1 and CL, Ca and Cβ, or cγ and cδ.

[0163] C). Cross-bracket area

[0164] In certain embodiments, the first pair of scaffold regions and the second pair of scaffold regions are of the same type of scaffold domains, but are configured to discourage mismatching between the first pair of scaffold regions and the second pair of scaffold regions.

[0165] In some embodiments, in the fusion polypeptide or polypeptide complex provided herein, the SR a / PSR a To and the SR b / PSR b For two of the same type, for example, both are CH1 / CL domains, or Cα / Cβ domains, or Cγ / Cδ domains, but the SR a / PSR a To and / or the SR b / PSR b The pair is configured to block SR a / PSR b Between or SR b / PSR a Any suitable means can be used to prevent mismatches between two scaffold region pairs, for example, SR a / PSR a For and SR b / PSR b Pairs can be configured to be interleaved.

[0166] In certain embodiments, for example, SR a / PSR a For and SR b / PSR bBoth pairs are 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 domains is crossed to prevent or reduce mispairing between B2 and A1 or between B1 and A2.

[0167] In some embodiments, SR a / PSR a The pair contains the CH1 domain CH1a and the CL domain CLa, and the SR b / PSR b The pair comprises a CH1 domain CH1b and a CL domain CLb. In some embodiments, the CH1a / CLa pair and the CH1b / CLb pair are configured to be crossed so that when a mismatch occurs between B2 and A1 and / or a mismatch occurs between B1 and A2, it will result in a pairing of two non-paired scaffold regions, such as CH1a and CH1b, or CLa and CLb, which are less stable than a mismatch between CH1 and CL and are therefore less likely to form.

[0168] In some embodiments, VB1 comprises VH1, VA1 comprises VL1, VB2 comprises VH2, and VA2 comprises VL2, and wherein: a) PSR a It is the CL domain CLa, SR a CH1 domain CH1a, SR b CH1 domain CH1b, PSR b is the CL domain CLb; or b) PSR a CH1 domain CH1a, SR a It is the CL domain CLa, SR b It is the CL domain CLb, PSR b It is the CH1 domain CH1b.

[0169] D) Mutations introduced in otherwise identical paired scaffold regions

[0170] In some embodiments, both the first pair of scaffold regions and the second pair of scaffold regions are the same type of scaffold domains with one or more mutations introduced to reduce or prevent mispairing.

[0171] In some embodiments, the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VH2, and the VA2 comprises VL2, the PSRa is a CH1 domain CH1a, the SRa is a CL domain CLa, the SRb is a CH1 domain CH1b, the PSRb is a CL domain CLb, and 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, d) the fourth polypeptide and the fifth polypeptide 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 hinder mispairing between CH1b and CLa and / or between CH1a and CLb.

[0172] In some embodiments, one or more mutations are introduced into the CH1b / CLb pair and / or the CH1a / CLa pair, thereby hindering mispairing between CH1b and CLa and / or between CH1a and CLb. In certain embodiments, the one or more mutations introduce non-natural binding interactions, such as, for example, non-natural covalent bonds, non-natural electrostatic interactions, non-natural salt bridges, non-natural hydrophobic-hydrophilic interactions, non-natural linkers, or any combination thereof. Preferably, non-natural binding interactions are introduced into the CH1b / CLb pair or the CH1a / CLa pair, so as to hinder mispairing between the CH1b / CLb pair and the CH1a / CLa pair.

[0173] In certain embodiments, at least one of the CH1b / CLb pair and the CH1a / CLa pair has one or more of the following properties: 1) having at least one non-native disulfide bond that hinders mispairing between CH1b and CLa and / or between CH1a and CLb; 2) having one or more introduced amino acid mutations that form one or more introduced charged amino acid residues that hinder 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 hinders mispairing between CH1b and CLa or between CH1a and CLb.

[0174] i) Disulfide bonds introduced into the CH1 / CL region

[0175] 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 hinders mispairing between CH1b and CLa and / or between CH1a and CLb.

[0176] 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 CH1 b / CL b , and the second CH1 / CL pair can be CH1 a / CL a ; or alternatively, the first CH1 / CL pair may be CH1 a / CL a , and the second CH1 / CL pair can be CH1 b / CL b In some embodiments, the first CH1 / CL pair is associated via a non-natural first disulfide bond. In some embodiments, and the first CH1 / CL pair lacks a natural disulfide bond or has a disrupted natural disulfide bond, for example, by mutation of a cysteine ​​residue that forms a natural disulfide bond.

[0177] In some embodiments, the second CH1 / CL pair is associated via a second disulfide bond, the second disulfide bond being formed at a position different from the first disulfide bond. In some embodiments, the second disulfide bond formed in the second CH1 / CL pair is a natural disulfide bond or a non-natural disulfide bond.

[0178] In some embodiments, the first disulfide bond is formed by two cysteine ​​residues introduced at a set 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.

[0179] In some embodiments, the first disulfide bond is formed by two cysteine ​​residues introduced at a set 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.

[0180] In some embodiments, the first disulfide bond is formed by two cysteine ​​residues introduced at a set of heavy chain-light chain EU positions, wherein the set of heavy chain-light chain EU positions is 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 and light chain EU position 118.

[0181] In some embodiments, the native disulfide bond is located between a heavy chain EU position selected from 131, 219, and 220 and the light chain EU position 214. In some embodiments, the native disulfide bond is located between heavy chain EU position 220 and light chain EU position 214.

[0182] In some embodiments, the first CH1 / CL pair comprises CH1 and CL, wherein the CH1 comprises a substitution at EU position 126 with a cysteine ​​residue and a substitution at EU position 220 with a non-cysteine ​​residue; and the CL comprises a substitution at EU position 121 with a cysteine ​​residue and a substitution at EU position 214 with a non-cysteine ​​residue.

[0183] In some embodiments, the CH1b comprises a substitution at EU position 126 with a cysteine ​​residue and at EU position 220 with a non-cysteine ​​residue; and the CLb comprises a substitution at EU position 121 with a cysteine ​​residue and at EU position 214 with a non-cysteine ​​residue. 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, the CH1a comprises a substitution at EU position 126 with a cysteine ​​residue and at EU position 220 with a non-cysteine ​​residue; the CLa comprises a substitution at EU position 121 with a cysteine ​​residue and at EU position 214 with a non-cysteine ​​residue. In some embodiments, the CH1a comprises the amino acid sequence of SEQ ID NO: 77, and / or CLa comprises the amino acid sequence of SEQ ID NO: 79.

[0184] ii) Substitution with charged amino acids

[0185] In certain embodiments, at least one of the CH1b / CLb pair and the CH1a / CLa pair has at least one or more charged amino acid residues that hinder the introduction of mismatches between CH1b and CLa and / or between CH1a and CLb.

[0186] 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 CH1 b / CL b , and the second CH1 / CL pair can be CH1 a / CL a ; or alternatively, the first CH1 / CL pair may be CH1 a / CL a , and the second CH1 / CL pair can be CH1 b / CL b In some embodiments, the first CH1 / CL pair contains at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of an oppositely charged residue for a charged residue, such that the first CH1 / CL pair contains a first pair of oppositely charged residues that are favorable for pairing of the first CH1 / CL pair. In some embodiments, the first CH1 / CL pair may contain a combination of substitutions that together provide a first pair of oppositely charged residues that are favorable for pairing of the first CH1 / CL pair.

[0187] In other words, a pair of oppositely charged residues can be introduced into the first CH1 / CL pair to promote homologous pairing between the CH1 domain and the CL domain in the first CH1 / CL pair. For example, a charged residue can be introduced to replace an uncharged residue at a certain position in CH1 (or CL), so that the introduced charged residue will form an electrostatic interaction with another oppositely charged residue that already exists or will be introduced in CL (or CH1), thereby facilitating the pairing of the first CH1 / CL pair. For another example, an existing charged residue at a certain position in CH1 (or CL) can be replaced by an oppositely charged residue, so that the replaced charged residue will form an electrostatic interaction with another oppositely charged residue that already exists or will be introduced in CL (or CH1), thereby facilitating the pairing of the first CH1 / CL pair. In certain embodiments, an existing charged residue in CH1 or CL can be replaced by an uncharged residue to reduce potential interference with the electrostatic interaction between the first CH1 / CL pair.

[0188] In some embodiments, the second CH1 / CL pair contains at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of an oppositely charged residue for a charged residue, such that the second CH1 / CL pair contains a second pair of oppositely charged residues that facilitate pairing of the second CH1 / CL pair, and optionally, the first pair of oppositely charged residues and the second pair of oppositely charged residues hinder pairing between CH1a and CLb or hinder pairing between CH1b and CLa.

[0189] 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 charged residues or both have negatively charged residues at corresponding positions of the first pair of oppositely charged residues and the second pair of oppositely charged residues, and / or CH1b and CLa both have positively charged residues or both have negatively charged residues at corresponding positions of the first pair of oppositely charged residues and the second pair of oppositely charged residues.

[0190] In some embodiments, the first pair of oppositely charged residues and / or the second pair of oppositely charged residues are introduced at a set of 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.

[0191] In some embodiments, the first pair of oppositely charged residues and / or the second pair of oppositely charged residues are introduced at a set of heavy chain-light chain EU positions, wherein the heavy chain-light chain EU positions are 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.

[0192] 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).

[0193] In some embodiments, CH1b comprises a substitution of a negatively charged residue with lysine at EU position 147; CLb comprises a substitution of a positively charged residue with serine at EU position 176; CH1a comprises a substitution of a positively charged residue with serine at EU position 183; and CLa comprises a substitution of a negatively charged residue with serine at EU position 176. 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.

[0194] In some embodiments, CH1a comprises a substitution of a negatively charged residue with lysine at EU position 147; CLa comprises a substitution of a positively charged residue with serine at EU position 176; CH1b comprises a substitution of a positively charged residue with serine at EU position 183; and CLb comprises a substitution of a negatively charged residue with serine at EU position 176. In some embodiments, CH1a comprises substitution K147D; CLa comprises substitution S176K; CH1b comprises substitution S183K; and CLb comprises substitution S176D.

[0195] 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.

[0196] In some embodiments, the first CH1 / CL pair includes CH1b and CLb.

[0197] In some embodiments, for CH1b, the amino acid residue at EU position 173 is replaced by a cysteine ​​residue, the amino acid residue at EU position 183 is replaced by a positively charged residue, and the amino acid residue at EU position 220 is replaced by a non-cysteine ​​residue, and for CLb, the amino acid residue at EU position 160 is replaced by a cysteine ​​residue, the amino acid residue at EU position 176 is replaced by a negatively charged residue, and the amino acid residue at EU position 214 is replaced by a non-cysteine ​​residue.

[0198] In some embodiments, for CH1b, the amino acid residue at EU position 173 is replaced by a cysteine ​​residue, the amino acid residue at EU position 183 is replaced by a negatively charged residue, and the amino acid residue at EU position 220 is replaced by a non-cysteine ​​residue, and for CLb, the amino acid residue at EU position 160 is replaced by a cysteine ​​residue, the amino acid residue at EU position 176 is replaced by a positively charged residue, and the amino acid residue at EU position 214 is replaced by a non-cysteine ​​residue.

[0199] In some embodiments, CH1b comprises V173C, S183K and C220S substitutions, and CLb comprises Q160C, S176D and C214S substitutions. 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.

[0200] 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.

[0201] In some embodiments, the first CH1 / CL pair comprises CH1a and CLa.

[0202] In some embodiments, for CH1a, the amino acid residue at EU position 173 is replaced by a cysteine ​​residue, the amino acid residue at EU position 183 is replaced by a positively charged residue, and the amino acid residue at EU position 220 is replaced by a non-cysteine ​​residue, and for CLa, the amino acid residue at EU position 160 is replaced by a cysteine ​​residue, the amino acid residue at EU position 176 is replaced by a negatively charged residue, and the amino acid residue at EU position 214 is replaced by 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.

[0203] In some embodiments, for CH1a, the amino acid residue at EU position 173 is replaced by a cysteine ​​residue, the amino acid residue at EU position 183 is replaced by a negatively charged residue, and the amino acid residue at EU position 220 is replaced by a non-cysteine ​​residue, and for CLa, the amino acid residue at EU position 160 is replaced by a cysteine ​​residue, the amino acid residue at EU position 176 is replaced by a positively charged residue, and the amino acid residue at EU position 214 is replaced by a non-cysteine ​​residue.

[0204] iii) Formation of orthogonal CH1 / CL interface

[0205] 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 hinders mispairing between CH1b and CLa or between CH1a and CLb.

[0206] In certain embodiments, the first CH1 / CL pair and the second CH1 / CL pair are selected from CH1b / CLb and CH1a / CLa, and wherein the first CH1 / CL pair comprises one or more introduced amino acid mutations that form an orthogonal CH1-CL interface.

[0207] In some embodiments, the at least one modification comprises one or more introduced amino acid mutations that form an orthogonal CH1-CL interface that facilitates pairing of CH1b and CLb and optionally hinders pairing between CH1b and CLa or hinders pairing between CH1a and CLb. In some embodiments, the at least one modification comprises one or more introduced amino acid mutations that form an orthogonal CH1-CL interface that facilitates pairing of CH1a and CLa and optionally hinders pairing between CH1a and CLb or hinders pairing between CH1b and CLa.

[0208] 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.

[0209] In some embodiments, CH1a comprises an S183E substitution, and CLa comprises a V133K substitution, CH1b comprises an A141I, F170S, S181M, S183A, and V185A substitution, and CLb comprises F116A, L235V, S174A, S176F, and T178V substitutions. In some embodiments, CH1b comprises an amino acid sequence of SEQ ID NO: 68, and / or CLb comprises an amino acid sequence of SEQ ID NO: 71.

[0210] In some embodiments, CH1b comprises an S183E substitution, and CLb comprises a V133K substitution, CH1a comprises an A141I, F170S, S181M, S183A, and V185A substitution, 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.

[0211] 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 that form an orthogonal Fab design, wherein the one or more introduced amino acid mutations are 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.

[0212] In some embodiments, the first CH1 / CL pair comprises introduced amino acid mutations at a set of heavy chain-light chain EU positions forming an orthogonal Fab design, the introduced amino acid mutations 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; the second CH1 / CL pair comprises introduced amino acid mutations, the introduced amino acid mutations forming a pair of oppositely charged residues, including 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.

[0213] E). Mutations introduced into the Fab variable region promote specific pairing

[0214] In addition to the scaffold regions, mutations can also be introduced in the variable regions of either VA1 / VB1 and VA2 / VB2 to prevent mispairing of B1 / A2 or B2 / A1.

[0215] In some embodiments, in the 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, and wherein the first VH / VL pair has at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of an oppositely charged residue for a charged residue, such that the first VH / VL contains a third pair of oppositely charged residues that facilitates the pairing of the first VH / VL pair.

[0216] In some embodiments, the second VH / VL pair has at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of an oppositely charged residue for a charged residue, such that the second VH / VL contains a fourth pair of oppositely charged residues that facilitate pairing of the second VH / VL pair, and optionally, the third pair of oppositely charged residues and the fourth pair of oppositely charged residues hinder pairing of VH1 with VL2 or VH2 with VL1.

[0217] 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 charged residues or both have negatively charged residues, and / or VH2 and VL1 both have positively charged residues or both have negatively charged residues.

[0218] In some embodiments, the third pair of oppositely charged residues and / or the fourth pair of oppositely charged residues are introduced at a set of heavy chain-light chain EU positions, wherein the heavy chain-light chain EU positions are 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.

[0219] 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).

[0220] F) Modifications in the Fc region

[0221] In some embodiments, in the polypeptide complex disclosed herein, the second polypeptide and the third polypeptide further comprise an operably linked first dimerization domain and an operably linked second dimerization domain, respectively, and the first dimerization domain and the second dimerization domain associate to form a dimer.

[0222] In some embodiments, the first dimerization domain and the second dimerization domain comprise the CH3 domain of IgG.In some embodiments, the first dimerization domain and the second dimerization domain further comprise a hinge region.

[0223] In some embodiments, in the 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 the 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.

[0224] In some embodiments, in the polypeptide complex 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 are derived from IgG1, IgG2, IgG3 or IgG4.

[0225] In some embodiments, the first Fc region and the second Fc region have different amino acid sequences and have at least one configuration that promotes heterodimerization of the first Fc region and the second Fc region.

[0226] In certain embodiments, the polypeptide complexes disclosed herein comprise one or more amino acid substitutions in the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications comprise introducing a protrusion into the first Fc region and introducing a cavity into the second Fc region, wherein the protrusion can be positioned in the cavity to promote the interaction of the first Fc polypeptide with the second Fc polypeptide to form a heterodimer or complex (also referred to as a knob-and-hole configuration). Methods for producing antibodies with these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0227] In some embodiments, a "knob" is created by replacing one or more small amino acid side chains on 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 chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[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 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 K409D; 39E; 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.

[0229] 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; 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 / D399C, and the second Fc mutation includes T366S / L368A / K392C / Y407V; j) the first Fc mutation includes T366W / K392C, and the second Fc mutation includes T366S / L368A / D399C / Y407V; k) the first Fc mutation includes S354C / T366W, and the second Fc mutation includes Y349C / T366S / L368A / Y407V; l) the first Fc mutation includes Y349C / T366W, and the second Fc mutation includes S354C / T366S / L368A / Y407V; m) the first Fc mutation includes E356C / T366W, and the second Fc mutation includes Y349C / T366S / L368A / Y407V; n) the first Fc mutation includes Y349C / T366W, and the second Fc mutation includes E356C / T366S / L368A / Y407V; o) the first Fc mutation includes E357C / T366W, and the second Fc mutation includes Y349C / T366S / L368A / Y407V; p) the first Fc mutation includes Y349C / T366W, and the second Fc mutation includes E357C / T366S / L368A / Y407V.

[0230] In some embodiments, the polypeptide complex disclosed herein comprises a first CH3 region and a second CH3 region, wherein the first CH3 region or the second CH3 region comprises an amino acid sequence different 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 by 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 a van amino acid sequence different 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 by one or more positively charged amino acids at corresponding positions in the CH3 region.

[0231] 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 / D399K, and the second Fc mutation includes K392D / K409D; h) the first Fc mutation includes E356K / D399K, and the second Fc mutation includes K409D / K439D; i) the first Fc mutation includes E357K / D399K, and the second Fc mutation includes K370D / K409D; j) the first Fc mutation includes E356K / E357K / D399K, and the second Fc mutation includes K370D / K392D / K409D; k) the first Fc mutation includes E357K / D399K, and the second Fc mutation includes K392D / K409D; l) the first Fc mutation includes K392D / K409D, and the second Fc mutation includes D399K; m) the first Fc mutation includes K360D / K409D, and the second Fc mutation includes D399K.

[0232] V. Targets of the Peptide Complex

[0233] In some embodiments, in the polypeptide complex disclosed herein, at least one of the first target binding domain and the second target binding domain is chimeric, humanized, or fully human.

[0234] In some embodiments, the "chimeric target binding domain" herein refers to a recombinant protein having a variable domain, wherein the variable domain includes a complementary determining region (CDR) derived from an antibody of one species (such as a rodent antibody or a mouse antibody), and the constant domain of the antibody molecule is derived from a constant domain of an antibody of a different species (such as a human antibody). For veterinary applications, the constant domain of the chimeric antibody can be derived from other species, such as a constant domain of a non-human primate, a cat or a dog.

[0235] In some embodiments, "humanized target binding domain" herein means a recombinant protein in which the CDR of an antibody from one species (e.g., a rodent antibody) is transferred to the framework region of human heavy and light chain variable domains. The constant domains of the antibody molecule are derived from the constant domains of human antibodies. In some embodiments, specific residues in the framework region of the humanized antibody, particularly those that contact or approach the CDR sequence, can be modified, for example, by replacing the corresponding residues from the original rodent, non-human primate or other antibody.

[0236] In some embodiments, at least one of the first target binding domain and the second target binding domain is fully human, which can be produced using 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 attack. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994) and Taylor et al., International Immunopharmacology 6:579 (1994). Fully human target binding domains can also be constructed by genetic or chromosomal transfection methods and phage display techniques, all of which are known in the art. For in vitro production of human antibodies and fragments thereof from immunoglobulin variable domain gene libraries of unimmunized donors, see, for example, McCafferty et al., Nature 348:552-553 (1990). Phage display can be performed in a variety of formats, for their review, 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. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated herein by reference in their entirety.

[0237] The polypeptide complex provided herein is based on a new 2:1 structure, and is divalent for the second target, and is monovalent for the first target. Specifically, an additional Fab region is introduced on the basis of an IgG-like bispecific antibody so that one of the two target binding sites will be divalent, and its target binding affinity is regained, which effectively regulates the affinity difference between the two target binding sites without any adverse effects or any safety risks to antibody specificity. Such affinity differences may be useful because the divalent binding to the second target confer affinity, and allows the differentiation between the high expression and low expression cells of the second target to be enhanced. In addition, the relatively low affinity binding to the first target can be used to reduce or avoid the undesirable effects of the strong binding to the first target (e.g., the nonspecific activation of the first target, which can lead to undesirable biological effects).

[0238] 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, or an antigen associated with an autoimmune disease or an 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.

[0239] 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, a tumor-associated antigen is an antigen present in a tumor but not in a normal organ, tissue and / or cell. In some embodiments, a tumor-associated antigen is an antigen that is more prevalent in a tumor than in a normal organ, tissue and / or cell. In some embodiments, a tumor-associated antigen is an antigen that is more prevalent in a malignant cancer cell than in a normal cell.

[0240] In some embodiments, the second target binding domain binds to a tumor-associated antigen. Tumor-associated antigens include antigens that are presented on the surface of tumor cells, located on or in tumor cells, presented only by tumor cells and not by normal cells (i.e., non-tumor cells), represent proteins with one or more tumor-specific mutations compared to non-tumor cells, are overexpressed in tumor cells when compared to non-tumor cells, are easily bound to antibodies in tumor cells due to the less compact structure of tumor tissues compared to non-tumor tissues, and are presented on the vasculature of tumors, etc., meaning antigenic substances produced in tumor cells, i.e., it triggers an immune response in the host.

[0241] 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, GD2, etc. Cancer-associated antigens also 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, 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, glycolylneuraminic acid, NPC-1C, PDGF-Rα, 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, etc.

[0242] 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.

[0243] The immune-related targets provided herein can be selected from the group consisting of: 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, IC OS (CD278), ICOSLG (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.

[0244] In some preferred embodiments of the bispecific polypeptide complexes provided herein, the second target involves a receptor on a cytotoxic T lymphocyte (e.g., CD3), and the first target involves 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.). Therefore, the bispecific polypeptide complexes provided herein can be used to form a connection between T cells and tumor cells, which may result in T cells exerting cytotoxic activity against tumor cells.

[0245] In certain embodiments, at least one of the first target and the second target is an antigen associated with an autoimmune disease or an inflammatory disease (such as, for example, rheumatoid arthritis (RA), psoriasis, osteoporosis, idiopathic pulmonary fibrosis, asthma, Sjögren's syndrome, type II diabetes). In some embodiments, antigens associated with autoimmune or inflammatory diseases are, for example but not limited to, 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 (basic glycoprotein (basigin)), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (IL-2 receptor chain), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE 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), myosin, OX-40, scleroscin, SOST, TGFβ1, TNF-α or VEGF-A.

[0246] In certain embodiments, at least one of the first target and the second target is an antigen associated with an eye disease, such as, for example, age-related macular degeneration (AMD) and diabetic macular edema. In some embodiments, the antigen associated with an eye disease is, for example, but not limited to, VEGF or ANG-2.

[0247] 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, Alzheimer's disease). In some embodiments, the antigen associated with an eye disease is, for example, but not limited to, GD2, EGFRvIII, Aβ40, or Aβ42.

[0248] 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, such as a COVID-19 infection. In some embodiments, the antigen associated with an autoimmune disease or an inflammatory disease is, for example, but not limited to, a spike protein of Psl, Pcrv, or a COVID-19 virus.

[0249] 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.

[0250] Other examples of antigen pairs that can be targeted by the bispecific polypeptide complexes provided herein and thus have potential therapeutic effects can include, but are not limited to, PD-L1:TGFβ, CD38:EGFR, HER2:VEGF, HER2:EGFR, PD-1:CTLA-4, PD-1:TIM3, OX40:PD-L1, FIXa:FX, CD32B:CD79B, Angiopoietin 2:VEGF, IL13:IL4, TNF:IL17A, DLL4:VEGF, IL1α:IL1β, FAP:DR5, CD30:gpA33, TNF:HSA, IL6R:HSA, IL17A / F:HSA, RANKL:HSA, Aβ40:Aβ42, IL13:IL17, FGFR1:KLB, PsI:PcrV, BAFF:B7RP1, NGF:TNF, and TNF:IL17A (Sedykh SE et al., Drug Design Dev Therapeutics 2018;12:195-208).

[0251] In certain embodiments, the first target is CD3 and the second target is CD20.

[0252] 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.

[0253] 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.

[0254] Exemplary polypeptide complexes

[0255] In certain embodiments, the polypeptide complex provided herein comprises: 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, d) the fourth polypeptide and the fifth polypeptide 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 hinder mismatching between CH1b and CLa and / or between CH1a and CLb.

[0256] FORMAT NEW 1

[0257] In certain embodiments of the polypeptide complexes provided herein, CH1a has substitutions at heavy chain EU positions A141I, F170S, S181M, S183A, and V185A, and CLa has substitutions at light chain EU positions F116A, A235V, S174A, S176F, and T178V; and CH1b has a substitution at heavy chain EU position S183E, and CLb has a substitution at light chain EU position V133K. In certain embodiments, CH1 a Comprising the amino acid sequence of SEQ ID NO:68, CL a Comprising the amino acid sequence of SEQ ID NO:71, CH1 b comprising the amino acid sequence of SEQ ID NO: 66, and CL b Comprising the amino acid sequence of SEQ ID NO:73.

[0258] In certain embodiments, VH1 has a substitution at EU position Q39E of the heavy chain, and VL1 has a substitution at EU position Q38K of the light chain. In certain embodiments, VH2 has a substitution at EU position Q39K of the heavy chain, and VL1 has a substitution at EU position Q38E of the light chain. In certain embodiments, VH1 comprises a 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 a 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.

[0259] In certain embodiments of the polypeptide complex 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.

[0260] In some of these embodiments, the first Fc and the 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-CD20xCD3FORMAT NEW1.

[0261] FORMAT NEW 2

[0262] In certain embodiments of the polypeptide complexes provided herein, the CH1a has a substitution of a cysteine ​​residue at EU position 126 and a substitution of a non-cysteine ​​residue at EU position 220; the 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 certain embodiments 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; CLb comprises the amino acid sequence of SEQ ID NO: 81.

[0263] 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.

[0264] 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 certain embodiments, the first Fc and the second Fc comprise the amino acid sequences of SEQ ID NO: 69 and 84. In certain embodiments of these embodiments, the fusion polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide comprise the amino acid sequences of SEQ ID NO: 14, 13, 15, and 16, respectively. This polypeptide complex is also referred to as anti-CD20 xCD3 FORMAT NEW2.

[0265] FORMAT NEW 3

[0266] In certain embodiments of the polypeptide complexes provided herein, CH1a has a substitution at heavy chain EU position K147D, and CLa has a substitution at light chain EU position S176K; and CH1b has a substitution at heavy chain EU position S183K, and CLb has a substitution at light chain EU position S176D. In certain embodiments of these embodiments, CH1 a Comprising the amino acid sequence of SEQ ID NO:88, CL a Comprising the amino acid sequence of SEQ ID NO:90, CH1 b Comprising the amino acid sequence of SEQ ID NO: 87, CL b Comprising the amino acid sequence of SEQ ID NO:92.

[0267] 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 a 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 a 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.

[0268] 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 certain embodiments, the first Fc and the second Fc comprise the amino acid sequences of SEQ ID NO: 69 and 84. In certain embodiments of these embodiments, the fusion polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide comprise the amino acid sequences of SEQ ID NO: 22, 21, 23, and 24, respectively. This polypeptide complex is also referred to as anti-CD20 xCD3 FORMAT NEW3.

[0269] FORMAT NEW 4

[0270] In certain embodiments of the polypeptide complexes provided herein, CH1a has substitutions at EU positions H168A and F170G in the heavy chain, and CLa has substitutions at EU positions L135Y and S176W in the light chain. In certain embodiments of these embodiments, CH1 a Comprising the amino acid sequence of SEQ ID NO:83, CL a Comprising the amino acid sequence of SEQ ID NO:95, CH1 b comprising the amino acid sequence of SEQ ID NO: 75, and CL b Comprising the amino acid sequence of SEQ ID NO:81.

[0271] In certain embodiments, VH1 has a substitution at heavy chain EU position 62E and Q39K, and VL1 has a substitution at light chain EU position D1R and Q38D. In certain embodiments, VH2 has a substitution at heavy chain EU position Q39Y, and VL2 has a substitution at light chain EU position Q38R. In certain embodiments, VH1 comprises a 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 a 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.

[0272] 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 certain embodiments, the first Fc and the second Fc comprise the amino acid sequences of SEQ ID NO: 69 and 84. In certain embodiments of these embodiments, the fusion polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide comprise the amino acid sequences of SEQ ID NO: 30, 29, 31, and 32, respectively. This polypeptide complex is also referred to as anti-CD20 xCD3 FORMAT NEW4.

[0273] FORMAT NEW 7

[0274] In certain embodiments of the polypeptide complexes provided herein, the CH1a has a substitution at EU position 173 to a cysteine ​​residue, a substitution at EU position 220 to a non-cysteine ​​residue, and a substitution at EU position S183K; the CLa has a substitution at EU position 160 to a cysteine ​​residue, a substitution at EU position 214 to a non-cysteine ​​residue, and a substitution at EU position S176D. In certain embodiments 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; CLb comprises the amino acid sequence of SEQ ID NO: 81.

[0275] 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.

[0276] 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 certain embodiments, the first Fc and the second Fc comprise the amino acid sequences of SEQ ID NO: 69 and 84. In certain embodiments of these embodiments, the fusion polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide comprise the amino acid sequences of SEQ ID NO: 54, 53, 55, and 56, respectively. This polypeptide complex is also referred to as anti-CD20 xCD3 FORMAT NEW7.

[0277] FORMAT NEW 5

[0278] In certain embodiments, the polypeptide complex provided herein comprises: a) a fusion polypeptide comprising an amino acid sequence of formula (I): VH2-CH1 b -Linker-VL1-TCRβ; b) a second polypeptide comprising an amino acid sequence of formula (II): VH1-TCRα; c) a third polypeptide comprising an amino acid sequence of formula (III): VH2-CH1 b ; d) a fourth polypeptide and a fifth polypeptide, each of which comprises an amino acid sequence of formula (IV): VL2-CL b .

[0279] In certain 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.

[0280] 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.

[0281] 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 certain embodiments, the first Fc and the second Fc comprise the amino acid sequences of SEQ ID NO: 69 and 84. In certain embodiments of these embodiments, the fusion polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide comprise the amino acid sequences of SEQ ID NO: 38, 37, 39, and 40, respectively. This polypeptide complex is also referred to as anti-CD20 xCD3 FORMAT NEW5.

[0282] FORMAT NEW 6

[0283] In certain embodiments, the polypeptide complex provided herein comprises: a) a fusion polypeptide comprising an amino acid sequence of formula (I): VH2-CH1 b -Linker-VH1-CL a ; b) a second polypeptide, the second polypeptide comprising an amino acid sequence of formula (II): VL1-CH1 a ; c) a third polypeptide comprising an amino acid sequence of formula (III): VH2-CH1 b ; d) a fourth polypeptide and a fifth polypeptide, each of which comprises an amino acid sequence of formula (IV): VL2-CL b .

[0284] In certain embodiments of the polypeptide complexes provided herein, CH1 a Comprising the amino acid sequence of SEQ ID NO: 100, CL a Comprising the amino acid sequence of SEQ ID NO: 101, CH1 b Comprising the amino acid sequence of SEQ ID NO:99, CL b Comprising the amino acid sequence of SEQ ID NO:102.

[0285] 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.

[0286] 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 certain embodiments, the first Fc and the second Fc comprise the amino acid sequences of SEQ ID NO: 69 and 84. In certain embodiments of these embodiments, the fusion polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide comprise the amino acid sequences of SEQ ID NO: 46, 45, 47, and 48, respectively. This polypeptide complex is also referred to as anti-CD20 xCD3 FORMAT NEW6.

[0287] FORMAT NEW 8

[0288] In certain embodiments, the polypeptide complex provided herein comprises: a) a fusion polypeptide comprising the amino acid sequence of formula (I): VH2-CH1b-linker-VL1-CH1a; b) a second polypeptide comprising the amino acid sequence of formula (II): VH1-CLa; c) a third polypeptide comprising the amino acid sequence of formula (III): VH2-CH1b; d) a fourth polypeptide and a fifth polypeptide, each of the fourth polypeptide and the fifth polypeptide comprising the amino acid sequence of formula (IV): VL2-CLb.

[0289] In certain embodiments of the polypeptide complexes provided herein, CH1 a comprising the amino acid sequence of SEQ ID NO: 105, CLa comprising 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.

[0290] 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.

[0291] 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 certain embodiments, the first Fc and the second Fc comprise the amino acid sequences of SEQ ID NO: 69 and 84. In certain embodiments of these embodiments, the fusion polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide comprise the amino acid sequences of SEQ ID NO: 62, 61, 63, and 64, respectively. This polypeptide complex is also referred to as anti-CD20 xCD3 FORMAT NEW8.

[0292] VI. Polynucleotides and Recombinant Methods

[0293] The disclosure provides a kind of nucleic acid, it comprises the nucleotide sequence of the fusion polypeptide or polypeptide complex that encodes this paper.As used herein, term " nucleic acid " or " nucleotide sequence " refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and its polymer in single-strand or double-stranded form.Unless otherwise indicated, otherwise specific polynucleotide sequence also implicitly covers its conservative modified variant (for example, degenerate codon substitution), allele, ortholog, SNP and complementary sequence and the sequence clearly indicated. Specifically, 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 Acids 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)).

[0294] The polynucleotides encoding the polypeptide complexes disclosed herein can be produced using methods known in the art. In certain embodiments, the sequence of the polynucleotide can be obtained based on the amino acid sequence of the polypeptide complex, and the nucleic acid can be produced using synthetic methods. Alternatively, the polynucleotides provided herein can also be obtained from another available nucleic acid, which encodes a polypeptide having a sequence homologous to the polypeptide in the polypeptide complex disclosed herein. DNA manipulation processes can then be applied to manipulate the sequence of the parent antibody encoding nucleic acid, such as introducing mutations, insertions, deletions, etc., to obtain nucleic acids encoding the polypeptide complex disclosed herein.

[0295] The nucleotide sequence encoding the fusion polypeptide or polypeptide complex can be inserted into one or more vectors using recombinant techniques known in the art for further cloning (amplification of DNA) or expression. Many vectors are available. Vector compositions typically 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.

[0296] The disclosure provides a vector comprising a nucleic acid provided herein. In certain embodiments, the nucleic acid encoding antibody provided herein, wherein at least one promoter (e.g., SV40, CMV, EF-1α) is operably connected to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0297] The vector containing the nucleotide sequence encoding the fusion polypeptide or polypeptide complex can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vector herein are the above-mentioned prokaryotes, yeast cells or higher eukaryotic cells. Suitable prokaryotes for this purpose include true bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia (e.g., Escherichia coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) (e.g., Salmonella typhimurium (Salmonella typhimurium), Serratia (e.g., Serratia marcescans) and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0298] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptide complexes. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species and strains are commonly used and suitable for use herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0299] Suitable host cells for expressing the glycosylated polypeptide complexes provided herein are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. A variety of baculovirus strains and variants and corresponding permissive insect host cells have been identified, and the permissive insect host cells are derived from hosts such as: Spodopterafrugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori. A variety of viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses herein according to the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.

[0300] However, most attention has been paid to vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (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 CCL 10); 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 CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical tumor cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CCL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); murine breast tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293, and derivatives thereof.

[0301] The host cell is transformed with the above-mentioned expression or cloning vector for antibody production, and the host cell is cultivated in a conventional nutrient medium, and the conventional nutrient medium is modified to be suitable for inducing promoters, selecting transformants or amplifying the gene of the desired sequence of encoding. In another embodiment, the antibody can be produced by homologous recombination as known in the art. In some embodiments, the host cell can produce the fusion polypeptide or polypeptide complex provided by this paper.

[0302] The disclosure also provides a method for expressing the fusion polypeptide or polypeptide complex provided herein, the method comprising culturing the host cell provided herein under the condition of expressing the carrier of the disclosure. The host cell for producing the antibody provided herein can be cultured in a variety of culture media. Commercially available culture 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, any culture medium described in the following literature can be used as a culture medium for the host cells: Ham et al., Methods in Enzymology 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Patent Reexamination Procedure 30,985. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drugs), trace elements (defined as inorganic compounds whose final concentrations are usually in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art.

[0303] When using recombinant technology, fusion polypeptide or polypeptide complex can be produced in intracellular, periplasmic space, or directly secreted into culture medium. If fusion polypeptide or polypeptide complex is produced intracellularly, then as a first step, the microparticle fragments of host cell or dissolved fragments can be removed, for example, by centrifugation or ultrafiltration. Carter et al., "Bio / Technology (Bio / Technology)" 10:163-167 (1992) describes the procedure for separating antibodies secreted into the periplasmic space of Escherichia coli. In short, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. In the case where fusion polypeptide or polypeptide complex is secreted into culture medium, usually firstly, the supernatant from such expression system is concentrated using commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0304] Fusion polypeptides or polypeptide complexes produced by cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.

[0305] In certain embodiments, protein A fixed on a solid phase is used for immunoaffinity purification of fusion polypeptides or polypeptide complexes. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the polypeptide complex. Protein A can be used for purifying antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., Journal of Immunological Methods (J.Immunol.Meth.) 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., Journal of the European Molecular Biology Association (EMBO J.) 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Compared with the flow rate and processing time that can be achieved with agarose, mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene can achieve faster flow rates and shorter processing times. In the case where the polypeptide complex comprises a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) can be used for purification. Other techniques for protein purification are also available, depending on the antibody to be recovered, 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 (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0306] Following any preliminary purification steps, the mixture comprising the fusion polypeptide or polypeptide complex of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5-4.5, preferably at low salt concentrations (e.g., about 0-0.25 M salt).

[0307] VII. Pharmaceutical Compositions

[0308] The present invention further provides a pharmaceutical composition comprising the polypeptide complex described herein and a pharmaceutically acceptable carrier.

[0309] As used herein, the term "pharmaceutically acceptable" means that the specified carrier, vehicle, diluent, excipient, salt and / or medium is generally chemically and / or physiologically compatible with the other ingredients, such as the active ingredients comprising the formulation (i.e., polypeptide complex or heterodimeric antibody or antigen-binding fragment thereof), and is physiologically compatible with the subject to whom the pharmaceutical composition is to be administered.

[0310] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation that is biologically acceptable and non-toxic to a subject, in addition to the active ingredient. In the context of the present disclosure, pharmaceutically acceptable carriers for the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending agents / distributing agents, sequestrants or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0311] In this article, suitable "component" can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, coloring agents, emulsifiers or stabilizers, such as sugar and cyclodextrin. Suitable "antioxidants" can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole (butylated hydroxanisol), butylated hydroxytoluene (butylated hydroxytoluene) and / or propyl gallate. As disclosed herein, one or more antioxidants (such as methionine) are included in the pharmaceutical composition provided herein to reduce the oxidation of polypeptide complexes or heterodimeric antibodies or their antigen-binding fragments. This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving the stability of the protein and extending the shelf life to the greatest extent. Therefore, in certain embodiments, a pharmaceutical composition is provided, which, in addition to the active ingredient (ie, the polypeptide complex or heterodimeric antibody or antigen-binding fragment thereof disclosed herein), further comprises one or more antioxidants such as methionine.

[0312] Pharmaceutically acceptable carriers can 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, antimicrobial agents at 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, emulsifiers such as polysorbate 80 (TWEEN-80), sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. The antimicrobial agent used as a carrier can be added to the pharmaceutical composition in the multidose container, and the antimicrobial agent includes phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or medicaments such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0313] Pharmaceutically acceptable "diluents" may include saline and aqueous buffer solutions.

[0314] Pharmaceutically acceptable "adjuvants" can include preservatives, wetting agents, emulsifiers and dispersants. Through the above-mentioned sterilization procedures and by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, etc.), it is possible to ensure the presence of microorganisms to prevent. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in these compositions. In addition, the extended absorption of injectable drug forms can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0315] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0316] In an embodiment, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solutions, suspensions, emulsions or solid forms suitable for producing liquid solutions, suspensions or emulsions. Injectable preparations can include sterile and / or pyrogen-free solutions prepared for injection, sterile dry soluble products prepared for combination with solvents before use, such as lyophilized powders, including subcutaneous injection tablets, sterile suspensions prepared for injection, sterile dry insoluble products prepared for combination with vehicles before use, and sterile and / or pyrogen-free emulsions. The solution can be aqueous or non-aqueous.

[0317] In certain embodiments, the unit dose parenteral formulation is packaged in an ampoule, a vial, or a syringe with a needle. As is known and practiced in the art, all preparations for parenteral administration should be sterile and pyrogen-free.

[0318] In certain embodiments, the sterile lyophilized powder is prepared by dissolving the polypeptide complex as disclosed herein in a suitable solvent. The solvent may contain an excipient that improves the stability of the powder or the reconstituted solution prepared from the powder or other pharmacological components. Excipients that can be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. In one embodiment, the solvent may include a buffer of about neutral pH, such as citrate, sodium phosphate or potassium phosphate or other such buffers known to those skilled in the art. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art to provide a desirable formulation. In one embodiment, the resulting solution is dispensed into vials for freeze-drying. Each vial may contain a single dose or multiple doses of a polypeptide complex, a polypeptide complex. It is acceptable for the vial to be overfilled beyond the smaller amount required for a single dose or a set of doses (e.g., about 10%) to facilitate accurate sample extraction and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0319] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. In one embodiment, sterile and / or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the selected therapy to be administered and can be determined empirically.

[0320] In certain embodiments, a composition is further provided, comprising a pharmaceutically acceptable carrier, diluent or adjuvant and an active ingredient. The active ingredient may be the polypeptide complex disclosed herein or a conjugate of the polypeptide complex disclosed herein.

[0321] VIII. Conjugates

[0322] The polypeptide complexes as provided herein can be used in unconjugated form or in conjugated form.

[0323] In conjugated form, the polypeptide complex is conjugated to one or more desired conjugate moieties (ie, heterologous moieties) to achieve certain functions, such as facilitating target detection or for imaging or therapy.

[0324] Herein, the present disclosure provides a conjugate comprising a polypeptide complex provided herein and a conjugate moiety (e.g., a payload) conjugated thereto. The payload may be any one of the group consisting of: a radioactive label, a fluorescent label, an enzyme-substrate label, an affinity purification tag, a tracer molecule, an anticancer drug, and a cytotoxic molecule.

[0325] Various conjugates can be linked to the polypeptide complexes provided herein by covalent binding, affinity binding, embedding, coordination binding, complexation, association, admixture or addition, etc. (see, e.g., "Conjugate Vaccines", in Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)).

[0326] In certain embodiments, the polypeptide complexes provided herein can be engineered to include specific sites outside of the epitope binding moiety that can be specifically used to bind to one or more conjugates. For example, such sites can include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent attachment to the conjugate.

[0327] In certain embodiments, the N-terminus and / or C-terminus of the polypeptide complex provided herein can also be used to provide a reactive group for conjugation. For example, the N-terminus can be conjugated to one moiety (e.g., polyethylene glycol (PEG), etc.), and the C-terminus can be conjugated to another moiety (e.g., biotin, etc.).

[0328] In certain embodiments, a polypeptide complex provided herein can be linked directly to a conjugate, or indirectly to a conjugate, for example, through another conjugate or through a linker.

[0329] For example, polypeptide complexes provided herein having reactive residues (e.g., cysteine) can be linked to thiol-reactive reagents, wherein the reactive group is, for example, maleimide, iodoacetamide, pyridyl disulfide, or other thiol-reactive conjugation ligands (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. Bioconjugate Chem. 3:3; Techniques (1996) San Diego Academic Press, pp. 40-55, 643-671).

[0330] For another example, the polypeptide complex provided herein can be conjugated to biotin and then indirectly conjugated to a second conjugate, which is conjugated to avidin. For another example, the polypeptide complex can be connected to a linker, which is further connected to the conjugate. Examples of linkers include bifunctional coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl diimidoadipate HCl), active esters (such as disuccinimidyl 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 2,6-diisocyanatotoluene) 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 bonds.

[0331] In certain embodiments, the conjugate moiety comprises an agent for detection or separation, such as a clearance modulator, 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 microtubule binding agent or other anti-cancer drug.

[0332] 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 can include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate 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 Bihe 32 P, other lanthanides, luminescent labels), chromophore moieties, digoxigenin, biotin / avidin, DNA molecules or gold for detection.

[0333] In some embodiments, the conjugate part can be a pharmacokinetic modification part, such as PEG that helps increase the half-life of the antibody. Other suitable polymers include copolymers such as carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol / propylene glycol, etc. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers connected to the antibody can vary, and if more than one polymer is connected, the polymer can be the same or different molecules. In some embodiments, the conjugate can be a purification part, such as magnetic beads.

[0334] In certain embodiments, the conjugate moiety may be a cytotoxic moiety. A "cytotoxic moiety" may be any agent that is harmful to cells or that can damage or kill cells. Examples of cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, and dactinomycin. D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C (mitomycin C), C) and dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).In some embodiments, the conjugate moiety comprises an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, carnation protein, Phytolaca americana protein, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and crescent toxin.

[0335] Methods for conjugating conjugate moieties to proteins such as antibodies, immunoglobulins, or fragments thereof are found, for example, in U.S. Pat. No. 5,208,020; U.S. Pat. No. 6,4411,163; WO 2005037992; WO 2005081711; and WO2006 / 034488, which are incorporated herein by reference in their entireties.

[0336] In certain embodiments, the polypeptide complexes provided herein are used as the basis for conjugates.

[0337] IX. Composition

[0338] In another aspect, the present invention provides a composition comprising the polypeptide complex or conjugate described herein, and a pharmaceutically acceptable carrier.

[0339] X. Medical Use

[0340] In another aspect, the present invention provides a method for treating a disease condition in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of a polypeptide complex of the present invention, a pharmaceutical composition described herein, a conjugate described herein, or a composition described herein.

[0341] As used herein, the term "subject" or "individual" or "animal" or "patient" refers to a human or non-human animal, including a mammal or primate, for whom diagnosis, prognosis, improvement, prevention and / or treatment of a disease or condition is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports or play animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, etc. In certain embodiments, the subject is a human.

[0342] As used herein, "treatment" of a condition can include alleviating the condition, slowing the rate of onset or development of the condition, delaying the development of symptoms associated with the condition, alleviating or ending symptoms associated with the condition, causing complete or partial regression of the condition, or some combination thereof.

[0343] As used herein, the terms "disorder," "disease," "condition," and the like refer to a condition affecting a subject that would still benefit from treatment with a polypeptide complex.

[0344] As used herein, the term "therapeutically effective amount" of a therapeutic agent refers to an amount of a therapeutic agent that can produce a sufficient therapeutic effect on a subject when taken in an appropriate manner. It should be understood that, as with other therapeutic drugs, the therapeutically effective amount of a polypeptide complex as provided above will be affected by various factors known in the art, such as the subject's weight, age, past medical history, current medication, health status and the possibility of cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease development. A person of ordinary skill in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage as indicated by these and other circumstances or requirements.

[0345] In certain embodiments, the polypeptide complexes as provided herein can be administered in a therapeutically effective amount of about 0.01 mg / kg to about 100 mg / kg. The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single dose can be administered, or several divided doses can be administered over time.

[0346] The polypeptide complexes described above and the methods disclosed herein can be used to treat a variety of diseases. In humans and other primates, diseases that can be treated by the polypeptide complexes described above and the methods disclosed herein may include the following:

[0347] (1) Cancer and other hyperproliferative disorders, including benign or malignant tumors, leukemias, and lymphoid malignancies. Examples include, depending on the cell type affected by the cancer or hyperproliferative disorder, neurons, glia, astrocytes, hypothalamus, glands, macrophages, epithelial, endothelial, and stromal malignancies. Examples include, depending on the organ / location affected by the cancer or hyperproliferative disorder, head cancer, neck cancer, eye cancer, oral cancer, laryngeal cancer, esophageal cancer, breast cancer, skin cancer, bone cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, stomach cancer, spleen cancer, kidney cancer, skeletal muscle cancer, subcutaneous tissue cancer, metastatic melanoma, endometrial cancer, prostate cancer, breast cancer, ovarian cancer, testicular cancer, thyroid cancer, blood cancer, lymph node cancer, kidney cancer, liver cancer, pancreatic cancer, brain cancer, or central nervous system cancer;

[0348] (2) Autoimmune and / or inflammatory disorders, including alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, disease), adrenal autoimmune diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Sjogren's syndrome, psoriasis, atherosclerosis, diabetic and other retinopathy, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangioma, thyroid hyperplasia (including Grave's disease), corneal and other tissue transplants and chronic inflammation, sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic 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 disease), bullous pemphigoid, cardiomyopathy, sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease disease), mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, 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, hypertensive arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as 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 caused by chronic viral or bacterial infection;.

[0349] (3) Infectious diseases and parasitic diseases, such as those caused by viruses (e.g., HBV, HCV, HIV, RSV, hMPV, PIV, coronavirus or influenza virus, etc.), fungi (e.g., Naegleria, Aspergillus, Blastomyces, Histoplasma, Candida or Tinea, etc.), eukaryotic microorganisms (Giardia, Toxoplasma, etc.), oplasma, Plasmodium, Trypanosoma and Entamoeba, etc.) and bacteria (Staphylococcus, Streptococcus, Pseudomonas, Clostridium, Borrelia, Vibro and Neiserria, etc.);

[0350] (4) Other diseases or conditions, including those not covered by any of (1)-(3) above, such as cardiovascular disease, neurological disease, neuropsychiatric symptoms, injury or coagulation disorder, etc.

[0351] In some embodiments, the disease is selected from the group consisting of cancer, inflammatory diseases, infectious or parasitic diseases, cardiovascular diseases, eye diseases, central nervous system (CNS) diseases, injuries, metabolic diseases, autoimmune diseases, or coagulation disorders. In some embodiments, the CNS disease is a neuropathy, a neuropsychiatric condition, neuroblastoma, glioblastoma, or Alzheimer's disease.

[0352] The polypeptide complexes disclosed herein can be administered by any route known in the art, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal or topical) routes.

[0353] In some embodiments, the polypeptide complexes disclosed herein can be administered alone or in combination with one or more additional therapeutic means or agents. For example, the polypeptide complexes disclosed herein can be administered in combination with another therapeutic agent, such as a chemotherapeutic agent or an anticancer drug.

[0354] In certain of these embodiments, the polypeptide complex as disclosed herein administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the polypeptide complex and the additional therapeutic agent may be administered as part of the same pharmaceutical composition. However, a polypeptide complex administered "in combination" with another therapeutic agent need not be administered simultaneously with the agent or in the same composition as the agent. When the phrase "in combination" is used herein, a polypeptide complex administered before or after another agent is considered to be administered "in combination" with the agent, even if the polypeptide complex and the second agent are administered by different routes. Where possible, the additional therapeutic agent administered in combination with the polypeptide complex disclosed herein is administered according to the schedule listed in the product information sheet of the additional therapeutic agent or according to the Physician's Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457 57th Edition (November 2002)) or a regimen well known in the art.

[0355] XI. Antigen Detection Methods

[0356] On the other hand, the present disclosure provides a method 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, such as a tumor-associated antigen. In certain embodiments, the polypeptide complex disclosed herein is used in a method for diagnosing a subject with a disease (e.g., cancer), the method comprising: determining the presence or amount of a disease-associated (e.g., tumor-associated) antigen in a sample obtained from a subject by contacting the sample with a polypeptide complex of the present disclosure and detecting the presence of an antigen-bound polypeptide complex.

[0357] Any sample suspected of containing tumor-associated antigens can be used, such as biological fluids, such as blood samples, plasma samples, urine samples, etc., and biopsies of diseased tissue (eg, tumor tissue).

[0358] The presence or level of a disease-associated (e.g., tumor-associated) antigen in a sample can be determined based on the presence or level of a complex of a disease-associated (e.g., tumor-associated) antigen bound by a polypeptide complex disclosed herein or an antigen-binding fragment thereof. Any suitable method can be used for such detection, for example, by immunoassay, such as immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (e.g., Western blot), flow cytometry (e.g., 15FACSTM), enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), and radioimmunoassay (RIA).

[0359] For a review of immunology and immunoassay procedures, see Basic and Clinical Immunology (Stites and Terr, eds., 7th ed. 1991). In addition, immunoassays can be performed in any of several configurations, which are extensively reviewed in Enzyme Immunoassay (Maggio, ed. 1980); and Harlow and Lane, supra. For a review of general immunoassays, see Methods in Cell Biology: Antibodies in Cell Biology, Vol. 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites and Terr, eds., 7th ed. 1991).

[0360] In certain embodiments, the polypeptide complexes disclosed herein are detectably labeled with a conjugated payload, or are unlabeled but reactive with a detectably labeled secondary molecule (eg, a detectably labeled secondary antibody).

[0361] In certain embodiments, the polypeptide complex disclosed herein can be immobilized on a solid substrate. Immobilization can be performed by covalent attachment 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 selection of detectably labeled solid phase materials and methods can be determined based on the desired assay format performance characteristics.

[0362] The level of antigen can be determined, for example, by normalization relative to a control value or a standard curve. The control value can be predetermined or determined simultaneously.

[0363] The assays and methods provided herein for measuring levels of antigens can be adapted or optimized for use in automated and semi-automated systems or point-of-care assay systems.

[0364] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials and methods described below fall within the scope of the invention in whole or in part. These specific compositions, materials and methods are not intended to limit the present invention, but are only used to illustrate specific embodiments falling within the scope of the present invention. Those skilled in the art can develop equivalent compositions, materials and methods without using inventive capabilities and without departing from the scope of the present invention. It should be understood that many changes can be made in the procedures described herein while still remaining within the limits of the present invention. It is the intention of the inventor of the present invention that such changes are all included within the scope of the present invention.

[0365] Example:

[0366] Example 1

[0367] 1.1 Antibody Construction

[0368] Using the new 2:1 format disclosed in the present invention (i.e., designated as FORMAT NEW hereinafter and in Figure 5A ) and the prior art 2:1 format known in the art (i.e., designated hereinafter as FORMAT EX and Figure 4A To demonstrate the advantages of FORMAT NEW over FORMAT EX, eight pairs of bispecific antibodies were constructed and expressed, which were identical in other respects except for the use of FORMAT EX or FORMAT NEW, thereby allowing a head-to-head comparison of the two formats.

[0369] Both FORMAT NEW and FORMAT EX are IgG-like bispecific antibodies that are mainly 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 ( Figure 4A and Figure 5A ). The first antibody heavy chain is named B1 (including VH1 and CH1a), and the first antibody light chain is named A1 (including VL1 and CLa), which are monovalent in the bispecific antibody. The second antibody heavy chain is named B2 (including VH2 and CH1b), and the second antibody light chain is named A2 (including VL2+CLb), which are divalent in the bispecific antibody. Use of linker (GGGGS) in bispecific antibodies 2 to connect different functional domains.

[0370] Two monoclonal antibodies targeting CD20 and CD3, respectively, were selected to construct the bispecific antibodies used in the examples. In the illustrative bispecific antibodies constructed, the anti-CD20 antibody was made divalent (i.e., corresponding to Figure 4A and Figure 5A A2 and B2 in ), and anti-CD3 is monovalent (i.e., corresponding to Figure 4A and Figure 5A A1 and B1 in (A1 and B1 in (B1). The anti-CD20 antibody used in this example is 2F2, and has been previously reported in US20040167319. The anti-CD3 antibody used in this example is SP34, and has been previously reported in Journal of the European Molecular Biology Association, Vol. 4, No. 2, pp. 337-344, 1985.

[0371] In order to reduce mismatching, mutations or crossover configurations were introduced into the Fab regions of the first antibody and the second antibody, respectively, and the details are shown in Table 1 below.

[0372] Table 1. Mutations or configurations introduced into bispecific antibodies

[0373]

[0374]

[0375] Mutations were also introduced into the CH3 region to promote heterodimerization of the first Fc region and the second Fc region. Specifically, one of the CH3 regions introduced T366S, L368A and Y407V, while the other introduced T366W mutations. The mutations in the CH3 region in the following examples provide a knob-in-hole design in a bispecific antibody.

[0376] Based on the bispecific antibody structure indicated above (Table 1), by using the novel 2:1 format of the present invention: FORMAT NEW (eg Figure 4A ) and by using the conventional 2:1 format: FORMAT EX (as Figure 5A ), 8 pairs of bispecific antibodies were constructed and expressed. These 8 pairs of bispecific antibodies (i.e., a total of 16 bispecific antibodies) were recombinantly expressed and named FORMAT NEW1 to FORMAT NEW8 and FORMAT EX1 to FORMAT EX8, respectively. For each antibody, the full-length amino acid sequence of each polypeptide chain is shown below, wherein the functional domains are marked with different underlines as shown in the annotations below the sequence.

[0377] FORMAT EX1

[0378] a) FORMAT EX1 Chain 2 (AH): CD20HC+CD3HC (knob) SEQ ID No.: 1

[0379]

[0380]

[0381] Annotation: “_”: VH2 (SEQ ID NO: 65); CH1b (SEQ ID NO: 66); VH1 (SEQ ID NO: 67); CH1a (SEQ ID NO: 68); Fc (SEQ ID NO:69)

[0382] b)FORMAT EX1 chain 1(AL): CD3 LC SEQ ID No.:2

[0383]

[0384] Annotation: “_”: VL1 (SEQ ID NO: 70); CLa (SEQ ID NO:71)

[0385] c) FORMAT EX1 chain 3 (BH): CD20 HC (hole) SEQ ID No.: 3

[0386]

[0387] Annotation: “_”: VH2 (SEQ ID NO: 65); CH1b (SEQ ID NO: 66); Fc (SEQ ID NO: 84)

[0388] d) FORMAT EX1 chain 4 (BL): CD20 LC SEQ ID No.: 4

[0389]

[0390] Annotation: “_”: VL2 (SEQ ID NO: 72); CLb (SEQ ID NO:73)

[0391] FORMAT NEW1

[0392] a) FORMAT NEW1 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID No.: 5

[0393]

[0394]

[0395] Annotation: “_”: VH1 (SEQ ID NO: 67); CH1 a (SEQ ID NO:68); Fc (SEQ ID NO:69)

[0396] b) FORMAT NEW1 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID No.: 6

[0397]

[0398] Annotation: “_”: VH2 (SEQ ID NO: 65); CH1 b (SEQ ID NO:66); VL1 (SEQ ID NO: 70); CL a (SEQ ID NO:71)

[0399] c) FORMAT NEW1 Chain 3 (B2): Anti-CD20 heavy chain (hole) SEQ ID No.: 7

[0400]

[0401] Annotation: “_”: VH2 (SEQ ID NO: 65); CH1 b (SEQ ID NO:66); Fc (SEQ ID NO: 84)

[0402] d) FORMAT NEW1 Chain 4 (A2): Anti-CD20 light chain SEQ ID No.: 8

[0403]

[0404] Note: “_”: VL2 (SEQ ID NO: 72); CL b (SEQ ID NO:73)

[0405] FORMAT EX2

[0406] a) FORMAT EX2 chain 2 (AH): CD20HC+CD3HC (knob) SEQ ID No.: 9

[0407]

[0408] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); VH1 (SEQ IDNO:76); "_": CH1 a (SEQ ID NO:77); Fc (SEQ ID NO:69)

[0409] b) FORMAT EX2 chain 1 (AL): CD3 LC SEQ ID No.: 10

[0410]

[0411] Annotation: “_”: VL1 (SEQ ID NO: 78); CL a (SEQ ID NO:79)

[0412] c) FORMAT EX2 chain 3 (BH): CD20 HC (hole) SEQ ID No.: 11

[0413]

[0414]

[0415] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); Fc (SEQ ID NO: 84)

[0416] d) FORMAT EX2 chain 4 (BL): CD20 LC SEQ ID No.: 12

[0417]

[0418] Note: “_”: VL2 (SEQ ID NO: 80); CL b (SEQ ID NO:81)

[0419] FORMAT NEW2

[0420] a) FORMAT NEW2 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID No.: 13

[0421]

[0422] Annotation: “_”: VH1 (SEQ ID NO: 76); CH1 a (SEQ ID NO:77); Fc (SEQ ID NO:69)

[0423] b) FORMAT NEW2 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID No.: 14

[0424]

[0425]

[0426] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); VL1 (SEQ ID NO: 78); CL a (SEQ ID NO:79)

[0427] c) FORMAT NEW2 Chain 3 (B2): Anti-CD20 heavy chain (hole) SEQ ID No.: 15

[0428]

[0429] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); Fc (SEQ ID NO: 84)

[0430] d) FORMAT NEW2 Chain 4 (A2): Anti-CD20 light chain SEQ ID No.: 16

[0431]

[0432] Note: “_”: VL2 (SEQ ID NO: 80); CL b (SEQ ID NO:81)

[0433] FORMAT EX3

[0434] a) FORMAT EX3 chain 2 (AH): CD20HC+CD3HC (knob) SEQ ID No.: 17

[0435]

[0436]

[0437] Annotation: “_”: VH2 (SEQ ID NO: 86); CH1 b (SEQ ID NO:87); VH1 (SEQ ID NO: 85); CH1 a (SEQ ID NO:88); Fc (SEQ ID NO:69)

[0438] b)FORMAT EX3 chain 1(AL): CD3LC SEQ ID No.:18

[0439]

[0440] Annotation: “_”: VL1 (SEQ ID NO: 89);

[0441] c) FORMAT EX3 chain 3 (BH): CD20 HC (hole) SEQ ID No.: 19

[0442]

[0443] Annotation: “_”: VH2 (SEQ ID NO: 86); CH1 b (SEQ ID NO:87); Fc (SEQ ID NO: 84)

[0444] d)FORMAT EX3 chain 4(BL): CD20LC SEQ ID No.:20

[0445]

[0446]

[0447] Note: “_”: VL2 (SEQ ID NO: 91); CL b (SEQ ID NO:92)

[0448] FORMAT NEW3

[0449] a) FORMAT NEW3 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID No.: 21

[0450]

[0451] Annotation: “_”: VH1 (SEQ ID NO: 85); CH1 a (SEQ ID NO:88); Fc (SEQ ID NO:69)

[0452] b) FORMAT NEW3 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID No.: 22

[0453]

[0454] Annotation: “_”: VH2 (SEQ ID NO: 86); CH1 b (SEQ ID NO:87); VL1 (SEQ ID NO: 89);

[0455] c) FORMAT NEW3 Chain 3 (B2): Anti-CD20 heavy chain (hole) SEQ ID No.: 23

[0456]

[0457]

[0458] Annotation: “_”: VH2 (SEQ ID NO: 86); CH1 b (SEQ ID NO:87); Fc (SEQ ID NO: 84)

[0459] d) FORMAT NEW3 Chain 4 (A2): Anti-CD20 light chain SEQ ID No.: 24

[0460]

[0461] Note: “_”: VL2 (SEQ ID NO: 91); CL b (SEQ ID NO:92)

[0462] FORMAT EX4

[0463] a) FORMAT EX4 chain 2 (AH): CD20HC+CD3HC (knob) SEQ ID No.: 25

[0464]

[0465] Annotation: “_”: VH2 (SEQ ID NO: 82); CH1 b (SEQ ID NO:75); VH1 (SEQ ID NO: 93); CH1 a (SEQ ID NO:83); Fc (SEQ ID NO:69)

[0466] b) FORMAT EX4 chain 1 (AL): CD3 LC SEQ ID No.: 26

[0467]

[0468] Note: “_”: VL1 (SEQ ID NO: 94);

[0469] c) FORMAT EX4 chain 3 (BH): CD20HC (hole) SEQ ID No.: 27

[0470]

[0471] Annotation: “_”: VH2 (SEQ ID NO: 82); CH1 b (SEQ ID NO:75); Fc (SEQ ID NO: 84)

[0472] d) FORMAT EX4 chain 4 (BL): CD20LC SEQ ID No.: 28

[0473]

[0474] Note: “_”: VL2 (SEQ ID NO: 96); CL b (SEQ ID NO:81)

[0475] FORMAT NEW4

[0476] a) FORMAT NEW4 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID No.: 29

[0477]

[0478]

[0479] Annotation: “_”: VH1 (SEQ ID NO: 93); CH1 a (SEQ ID NO:83); Fc (SEQ ID NO:69)

[0480] b) FORMAT NEW4 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID No.: 30

[0481]

[0482] Annotation: “_”: VH2 (SEQ ID NO: 82); CH1 b (SEQ ID NO:75); VL1 (SEQ ID NO: 94); CL a (SEQ ID NO:95)

[0483] c) FORMAT NEW4 Chain 3 (B2): Anti-CD20 heavy chain (mortise) SEQ ID No.: 31

[0484]

[0485] Annotation: “_”: VH2 (SEQ ID NO: 82); CH1 b (SEQ ID NO:75); Fc (SEQ ID NO: 84)

[0486] d) FORMAT NEW4 Chain 4 (A2): Anti-CD20 light chain SEQ ID No.: 32

[0487]

[0488]

[0489] Note: “_”: VL2 (SEQ ID NO: 96); CL b (SEQ ID NO:81)

[0490] FORMAT EX5

[0491] a) FORMAT EX5 chain 2 (AH): CD20HC+CD3HC (knob) SEQ ID No.: 33

[0492]

[0493] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); VH1 (SEQ ID NO: 76); TCRα (SEQ ID NO:97); Fc (SEQ ID NO:69)

[0494] b) FORMAT EX5 chain 1 (AL): CD3LC SEQ ID No.: 34

[0495]

[0496] Annotation: “_”: VL1 (SEQ ID NO: 78);

[0497] c) FORMAT EX5 chain 3 (BH): CD20 HC (hole) SEQ ID No.: 35

[0498]

[0499]

[0500] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); Fc (SEQ ID NO: 84)

[0501] d) FORMAT EX5 chain 4 (BL): CD20LC SEQ ID No.: 36

[0502]

[0503] Note: “_”: VL2 (SEQ ID NO: 80); CL b (SEQ ID NO:81)

[0504] FORMAT NEW5

[0505] a) FORMAT NEW5 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID No.: 37

[0506]

[0507] Annotation: “_”: VH1 (SEQ ID NO: 76); TCRα (SEQ ID NO:97); Fc (SEQ ID NO:69)

[0508] b) FORMAT NEW5 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID No.: 38

[0509]

[0510]

[0511] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); VL1 (SEQ ID NO: 78); TCRβ (SEQ ID NO:98)

[0512] c) FORMAT NEW5 chain 3 (B2): anti-CD20 heavy chain (hole) SEQ ID No.: 39

[0513]

[0514] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); Fc (SEQ ID NO: 84)

[0515] d) FORMAT NEW5 Chain 4 (A2): Anti-CD20 light chain SEQ ID No.: 40

[0516]

[0517] Note: “_”: VL2 (SEQ ID NO: 80); CL b (SEQ ID NO:81)

[0518] FORMAT EX6

[0519] a) FORMAT EX6 chain 2 (AH): CD20HC+CD3HC (knob) SEQ ID No.: 41

[0520]

[0521]

[0522] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:99); VL1 (SEQ ID NO: 78); CH1 a (SEQ ID NO: 100); Fc (SEQ ID NO:69)

[0523] b) FORMAT EX6 chain 1 (AL): CD3LC SEQ ID No.: 42

[0524]

[0525] Annotation: “_”: VH1 (SEQ ID NO: 76);

[0526] c)FORMAT EX6 chain 3 (BH): CD20HC (mortar) SEQ ID No.: 43

[0527]

[0528] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:99); Fc (SEQ ID NO: 84)

[0529] d) FORMAT EX6 chain 4 (BL): CD20LC SEQ ID No.: 44

[0530]

[0531]

[0532] Note: “_”: VL2 (SEQ ID NO: 80); CL b (SEQ ID NO: 102)

[0533] FORMAT NEW6

[0534] a) FORMAT NEW6 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID No.: 45

[0535]

[0536] Annotation: “_”: VL1 (SEQ ID NO: 78); CH1 a (SEQ ID NO: 100); Fc (SEQ ID NO: 69)

[0537] b) FORMAT NEW6 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID No.: 46

[0538]

[0539] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:99); VH1 (SEQ ID NO: 76);

[0540] c) FORMAT NEW6 chain 3 (B2): anti-CD20 heavy chain (hole) SEQ ID No.: 47

[0541]

[0542]

[0543] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:99); Fc (SEQ ID NO: 84)

[0544] d) FORMAT NEW6 Chain 4 (A2): Anti-CD20 light chain SEQ ID No.: 48

[0545]

[0546] Note: “_”: VL2 (SEQ ID NO: 80); CL b (SEQ ID NO: 102)

[0547] FORMAT EX7

[0548] a) FORMAT EX7 chain 2 (AH): CD20HC+CD3HC (knob) SEQ ID No.: 49

[0549]

[0550] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); VH1 (SEQ ID NO: 76); CH1 a (SEQ ID NO: 103); Fc (SEQ ID NO:69)

[0551] b) FORMAT EX7 chain 1 (AL): CD3LC SEQ ID No.: 50

[0552]

[0553] Annotation: “_”: VL1 (SEQ ID NO: 78); CL a (SEQ ID NO: 104)

[0554] c) FORMAT EX7 chain 3 (BH): CD20 HC (hole) SEQ ID No.: 51

[0555]

[0556] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); Fc (SEQ ID NO: 84)

[0557] d) FORMAT EX7 chain 4 (BL): CD20 LC SEQ ID No.: 52

[0558]

[0559] Note: “_”: VL2 (SEQ ID NO: 80); CL b (SEQ ID NO:81)

[0560] FORMAT NEW7

[0561] a) FORMAT NEW7 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID No.: 53

[0562]

[0563]

[0564] Annotation: “_”: VH1 (SEQ ID NO: 76); CH1 a (SEQ ID NO: 103); Fc (SEQ ID NO: 69)

[0565] b) FORMAT NEW7 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID No.: 54

[0566]

[0567] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); VL1 (SEQ ID NO: 78); CL a (SEQ ID NO: 104)

[0568] c) FORMAT NEW7 Chain 3 (B2): Anti-CD20 heavy chain (hole) SEQ ID No.: 55

[0569]

[0570] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); Fc (SEQ ID NO: 84)

[0571] d) FORMAT NEW7 Chain 4 (A2): Anti-CD20 light chain SEQ ID No.: 56

[0572]

[0573]

[0574] Note: “_”: VL2 (SEQ ID NO: 80); CL b (SEQ ID NO:81)

[0575] FORMAT EX8

[0576] a) FORMAT EX8 Chain 2 (AH): CD20HC+CD3VHLC (knob) SEQ ID No.: 57

[0577]

[0578] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); VH1 (SEQ ID NO: 76); CL a (SEQ ID NO: 106); Fc (SEQ ID NO:69)

[0579] b) FORMAT EX8 chain 1 (AL): CD3LC SEQ ID No.: 58

[0580]

[0581] Annotation: “_”: VL1 (SEQ ID NO: 78); CH1 a (SEQ ID NO: 105)

[0582] c)FORMAT EX8 chain 3 (BH): CD20HC (mortar) SEQ ID No.: 59

[0583]

[0584]

[0585] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); Fc (SEQ ID NO: 84)

[0586] d) FORMAT EX8 chain 4 (BL): CD20LC SEQ ID No.: 60

[0587]

[0588] Note: “_”: VL2 (SEQ ID NO: 80); CL b (SEQ ID NO:81)

[0589] FORMAT NEW8

[0590] a) FORMAT NEW8 Chain 2 (B1): Anti-CD3 heavy chain (knob) SEQ ID No.: 61

[0591]

[0592] Annotation: “_”: VH1 (SEQ ID NO: 76); CL a (SEQ ID NO: 106); Fc (SEQ ID NO:69)

[0593] b) FORMAT NEW8 Chain 1 (B2-A1): anti-CD20 heavy chain + anti-CD3 light chain SEQ ID No.: 62

[0594]

[0595]

[0596] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); VL1 (SEQ ID NO: 78); CH1 a (SEQ ID NO: 105)

[0597] c) FORMAT NEW8 Chain 3 (B2): Anti-CD20 heavy chain (hole) SEQ ID No.: 63

[0598]

[0599] Annotation: “_”: VH2 (SEQ ID NO: 74); CH1 b (SEQ ID NO:75); Fc (SEQ ID NO: 84)

[0600] d) FORMAT NEW8 Chain 4 (A2): Anti-CD20 light chain SEQ ID No.: 64

[0601]

[0602] Note: “_”: VL2 (SEQ ID NO: 80); CL b (SEQ ID NO:81)

[0603] 1.2 Gene synthesis, expression and purification of bispecific antibodies

[0604] Plasmids were constructed to encode each polypeptide chain of each bispecific antibody described above. Plasmids encoding each bispecific antibody were transfected into ExpiCHO-S cells to allow expression of the polypeptide chains, which were then assembled into the corresponding bispecific antibodies. The expressed bispecific antibodies were collected and purified with protein A. The specific experimental procedures are described below.

[0605] Before synthesis, the gene sequence encoding the polypeptide chain was codon optimized using OptimumGene. The gene sequence was constructed in the pcDNA3.4 vector and purified by PureLink TM Hipure prepared the confirmed recombinant vector DNA for transfection.

[0606] Use ExpiCHO Expression Medium to grow in CO 2 The cells were cultured in a shaker and when the density of ExpiCHO-S cells reached 7 × 10 6 -10×10 6 When the viability is >95%, 25 ml of cell suspension is treated with ExpiFectamine containing about 25 ul of DNA heavy chain and light chain. TM Transfection of CHO / DNA complexes.

[0607] On the 8th to 9th day after transfection, the culture was harvested, and the cell suspension was collected, centrifuged, and further purified using chromatography. Chromatographic 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: 0-100% B linear gradient elution. After separation, the eluate was collected in a volume of 1 ml / tube, and 0.1 ml of neutralization buffer C, pH about 6-7, was added to each 1 ml fraction.

[0608] 1.3 Expression results of each bispecific antibody

[0609] SEC-HPLC (see Figures 7A to 7P ) and SDS-PAGE (see Fig. 6A and 6B ) Analysis and characterization of the purified antibodies.

[0610] 1). SDS-PAGE:The sample is mixed with 4X sample buffer (15μl+5μl) in an Eppendorf tube, and heated at 100°C for 5-10 minutes, centrifuged, and the supernatant is collected simultaneously. Then, 5μg of the treated sample is pipetted, and carefully added to each well of the gel in sequence, and the marker is added to one well in the well. The electrode chamber is placed on the electrophoresis tank, the power is turned on, the positive and negative electrodes are aligned, and the voltage is set to 150V. After running for 5 minutes, the voltage is adjusted to 200V. Run electrophoresis again, continue for about 35 minutes, and then turn off the power to stop. Use Bio-rad gel imager to scan unstained SDS-PAGE gel and take photos, and use Image Lab5.2.1 to analyze photos to calculate protein purity.

[0611] 2). Capillary electrophoresis sodium dodecyl sulfate (CE-SDS): The sample was diluted to 2.5 mg / ml with ultrapure water, and 55 μL of sample buffer was added, followed by 2 μL of 10 kDa IS and 5 μL of 250 mM IAM, and mixed thoroughly. Next, the sample was heated at 70 ± 2 °C in a metal bath for 10 ± 2 minutes, then cooled to room temperature in a water bath, and transferred to the attached sample tube, labeled, and prepared for sampling.

[0612] 3). Size Exclusion-High Performance Liquid Chromatography (SEC-HPLC) :

[0613] The samples were centrifuged at 10,000 rpm for 5 minutes at 15°C. The supernatant was obtained and analyzed. The relevant SEC chromatography parameters are listed below:

[0614] Mobile phase A: 200 mM PBS pH 6.9;

[0615] Mobile phase B: ultrapure water;

[0616] Flow rate: 0.7 ml / min;

[0617] Wavelength: 280nm;

[0618] Column temperature: 25°C;

[0619] Sample analysis time: 25 minutes;

[0620] Injection size: 20 μg.

[0621] 1.4 Analysis results

[0622] Conventional 2:1 format (FORMAT EX)

[0623] According to the SEC-HPLC results ( Fig. 7A , 7C, 7E, 7G, 7I, 7K, 7M, and 7O), except for the FORMAT EX5 construct ( Fig.7I ), all samples seemed to show a clear single main peak of 200 kDa, indicating that almost all samples were of high purity. However, this result was inconsistent with the SDS-PAGE results (see Fig. 6A and 6B ), the latter showing multiple protein bands in the sample, indicating that the sample contained low-molecular impurities and therefore still needed to be purified.

[0624] The reason why the SEC-HPLC results were inconsistent with the SDS-PAGE results was that the molecular weight of most of the A1-B2 and A2-B1 mismatch products in the samples of FORMAT EX antibodies (FORMAT EX1 to FORMAT EX8) was about 200 kDa, which was the same as or very close to the molecular weight of the expected product, and therefore the SEC-HPLC method could not identify these mismatch products present in the samples. In contrast, such mismatch products could be shown by the SDS-PAGE method because Figure 5B The A1-B2 and A2-B1 mismatch products in the are not covalently bound by disulfide bonds and will dissociate during the SDS-PAGE process. In the present study, the SDS-PAGE results showed that there were 175kDa or even 150kDa products near the 200kDa band, which can be seen in FORMAT EX1, FORMAT EX2, FORMAT EX3, FORMAT EX4, FORMAT EX5, FORMAT EX7 and FORMAT EX8, but not in FORMAT EX6. These mismatch products are not seen in the SEC-HPLC results, and because their physicochemical properties are similar to those of the corresponding homologous pairing target products, they are difficult to remove through conventional purification processes.

[0625] New 2:1 format (FORMAT NEW)

[0626] As shown in the SDS-PAGE results (see Fig. 6A and 6B ), the impurities far from the target band on SDS-PAGE were mainly 150 kDa. These results are more consistent with the SEC-HPLC results (see Figure 7B , 7D , 7F, 7H, 7J, 7L, 7N and 7P), that is, the main peak of 200KDa on the SEC-HPLC spectrum is a single target product, while A1-B2 and A2-B1 mismatch products (see Figure 4B) are more easily separated by SEC-HPLC because of their large difference in molecular weight from the target product, and the final product is also more convenient to purify due to the significant difference in physicochemical properties.

[0627] Example 2

[0628] The anti-CD20 x CD3 bispecific molecule FORMAT New7 was constructed and purified using conventional methods, demonstrating the convenience of the present invention for purification.

[0629] 2.1 Antibody Construction:

[0630] Chain 1 (AL): B2-Linker-A1 (MW: about 50 kDa)

[0631] Chain 2 (AH): B1-1st Fc (MW: about 50 kDa)

[0632] Chain 3 (BH): B2-2Fc (MW: about 50 kDa)

[0633] Chain 4 (BL): A2 (MW: about 25 kDa)

[0634] The following mutations were introduced into CH1 of B1: V173C, S183K, C220S.

[0635] The following mutations were introduced into the CL of A1: Q160C, S176D, C214S.

[0636] 2.2 Expression of anti-CD20 x CD3 FORMAT New7 bispecific antibody:

[0637] Anti-CD20 x CD3 FORMAT New7 is a bispecific antibody composed of 2 pairs of different light and heavy chains. Host cells were co-transfected with CHO-K1 cells using two stable transfer vectors. After transfection, cells were plated at a density of 2×10e4 cells / well and incubated in CO 2Incubator, culture under the condition of selection pressure. After about 3 weeks of culture, supernatant is taken out from cell culture to test the combination with CD20 and CD3 respectively. Supernatant showing high binding activity to CD20 and CD3 is identified, and corresponding cell clones are expanded into 24-well cell culture plates. After 4 to 6 days, the binding activity of supernatant to CD20 and CD3 is tested. Cell clones with higher binding activity to CD20 and CD3 are expanded, and finally the cell line of stable transfection is selected and expanded. Stable cell line is passaged and cultured in shaking flask for 3 days, the binding activity to CD20 and CD3 is tested, and fed-batch evaluation and cryopreservation are carried out based on cell growth and expression. Expression and quality (SEC, non-reduced CE-SDS, CEX, SDS-PAGE) of test fed-batch samples are used to evaluate the growth, metabolism, productivity and product quality of stable cell pools, and stable cell pools are finally selected to carry out subsequent monoclonal cell line screening.

[0638] The above cell lines were plated into 96-well plates using a single cell sorter, with 1 cell per well, and cultured in CO 2 The cells were cultured in an incubator and photographed by scanning the plates with Solentim Cell Metric. Monoclonal cells were selected for fed-batch evaluation and cell cryopreservation. Expression and quality analysis (SEC, nrCE-SDS, iCIEF, N-glycans, molecular weight) was performed to evaluate the growth, metabolism, productivity and product quality of stable cell pools, and 6 monoclonal cell lines were selected for RCB library construction.

[0639] The selected monoclonal cell lines were revived, expanded, plated and cultured in shake flasks for 14 days. The supernatant was collected for purification and analysis.

[0640] 2.2.1 Analytical methods:

[0641] 1). SDS-PAGE :Supernatant samples are mixed with 4X sample buffer (15 μl+5 μl) in Eppendorf tubes, and heated at 100 ℃ for 5-10 minutes, centrifuged, and supernatant is collected simultaneously. Then, 5 μg of treated samples are pipetted, and carefully added to each well of the gel in sequence, and markers are added to one well in the well. The electrode chamber is placed on the electrophoresis tank, the power is turned on, the positive and negative electrodes are aligned, and the voltage is set to 150V. After running for 5 minutes, the voltage is adjusted to 200V. Run electrophoresis again, continue for about 35 minutes, and then turn off the power to stop. Use Bio-rad gel imager to scan unstained SDS-PAGE gel and take photos, and use ImageLab 5.2.1 to analyze photos to calculate protein purity.

[0642] 2). CE-SDS : The sample was diluted to 2.5 mg / ml with ultrapure water, and 55 μL of sample buffer was added, followed by 2 μL of 10 kDa IS and 5 μL of 250 mM IAM, and mixed thoroughly. Next, the sample was heated at 70 ± 2 °C for 10 ± 2 minutes, then cooled to room temperature, and transferred to the included sample tube, labeled, and prepared for sampling.

[0643] 3). SEC-HPLC : The samples were centrifuged at 10000 rpm for 5 minutes at 15°C. The supernatant was collected and analyzed. The relevant SEC chromatography parameters are listed below:

[0644] Mobile phase A: 200 mM PBS pH 6.9;

[0645] Mobile phase B: ultrapure water;

[0646] Flow rate: 0.7 ml / min;

[0647] Wavelength: 280nm;

[0648] Column temperature: room temperature;

[0649] Sample analysis time: 20 minutes;

[0650] Injection size: 20 μg.

[0651] 2.2.2 Analysis results

[0652] Using SDS-PAGE analysis, the purity of the expression product of the anti-CD20 x CD3 FORMAT New7 bispecific antibody was about 84.1% ( Figure 8 ).

[0653] Using CE-SDS analysis, the purity of the expression product of the anti-CD20 x CD3 FORMAT New7 bispecific antibody was about 82.91% (see Fig. 9 Peak 10 in the figure).

[0654] Using SEC-HPLC analysis, the purity of the expressed anti-CD20 x CD3 FORMAT New7 bispecific antibody was about 90.23% (see Fig.10 Peak 4 in the figure).

[0655] In addition to the target protein, there are also some low molecular weight (LMW) fragments in the expression product (see Figure 8 , 9 and 10) and high molecular weight aggregates (HMW, see Fig.10). The related products can be more clearly distinguished by SDS-PAGE, CE-SDS and SEC-HPLC. Figure 8-10 Shown in.

[0656] 2.3 Antibody purification

[0657] 2.3.1 Experimental methods

[0658] 1). Affinity chromatography (AC) : Supernatant samples obtained from cell culture containing the anti-CD20 x CD3 FORMAT New7 bispecific antibody were eluted by affinity chromatography at pH 4.0, and the elution peaks were then analyzed.

[0659] Chromatographic 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: 0-100% B linear gradient elution. After separation, the eluate was collected in a volume of 1 ml / tube, and 0.1 ml of neutralization buffer C, pH about 6-7, was added to each 1 ml fraction.

[0660] 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 sample CEX loading. Column: Capto S Impact was applied with the following buffer:

[0661] EQ / Wash / Elution Buffer A: 50 mM NaAc-HAc, pH 6.0

[0662] Elution buffer B: 50 mM NaAc-HAc + 1 M NaCl, pH 6.0

[0663] Linear gradient elution was performed: Gradient: 0→50% B, 60CV.

[0664] 2.3.2 Purification results:

[0665] 1). Affinity chromatography (AC) :

[0666] Purification curve Fig.11 As shown in Fig.11 As shown, the target product was successfully eluted from the affinity chromatography column by the eluent buffer, and a single peak was detected using the UV signal (see Fig.11 ), indicating that the purified product has high purity.

[0667] The purity of the eluate was analyzed by SEC-HPLC, and the result showed that the purity was 92.55% (see Fig.12 3 of the peak), which indicates that AC reduces the LMW content.

[0668] The purity of the eluate was also analyzed by SDS-PAGE, and the results showed that the purity was 85.7% (see Fig.13 ), which indicates that there is still LMW after purification in this step ( Fig.13 ).

[0669] Compared to the results observed with the unpurified sample in Example 1, the SEC-HPLC and SDS-PAGE results clearly reflected the improvement of purification by the AC method, which also revealed that the impurity was LMW and thus provided direction for the subsequent purification process.

[0670] 2). Cation exchange chromatography (CEX) :

[0671] In order to further remove LMW from the AC-purified samples, the eluate from the AC process was collected and further purified using CEX. The elution curve after CEX purification is Fig.14 A main single peak was detected by UV signal (see the peak from 100 ml of eluent to 120 ml of eluent), which indicated that the target product was successfully eluted by the eluent buffer through linear gradient elution.

[0672] Collect three eluate samples (i.e., C01-C03, see Fig.14 The purity was analyzed by SDS-PAGE and SEC, respectively.

[0673] SDS-PAGE showed that the purity of C01, C02 and C03 were 94.6%, 95.0% and 91.0%, respectively. Fig.15 SEC-HPLC showed that the purity of C01, C02 and C03 were 93.89%, 99.74% and 97.73% respectively (see Table 2 below). Fig.16 Shown in.

[0674] Table 2. Purity analysis of fractions eluted from CEX by SEC-HPLC

[0675] sample monomer% polymer% Segment % CEX Loading 96.06 3.94 / C01 93.89 0.11 / C02 99.74 0.26 / C03 97.73 2.27 /

[0676] It is noteworthy that after purification by CEX, the purity of the C02 fraction was over 99% as determined by SEC-HPLC ( Fig.16 ), and more than 95% ( Fig.15), which shows that the purity of the CEX loaded sample was significantly improved compared with that before CEX purification, removing most of the LMW and HMW in the AC purified product.

[0677] SEC-HPLC and SDS-PAGE clearly reflected the improved purity by CEX, which also showed that the purified final product had a high degree of homogeneity.

[0678] The above results show 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 purification process as simple as two steps can provide a high-purity sample with a purity of more than 95%, which shows that the FORMAT NEW antibody is significantly superior to the FORMAT EX antibody in antibody purification, and also shows that the FORMAT NEW antibody structure of the present invention is easy to use.

[0679] Although the examples used anti-CD20 x CD3 bispecific antibodies to demonstrate the convenience of purification and high purity, the inventors also tested other bispecific antibodies constructed using the novel bispecific antibody format provided in the present disclosure, and obtained similar results in terms of the convenience of purification and high purity. Therefore, it should be understood that the novel format provided in the present disclosure is applicable to various types of targets and can provide the convenience of purification and high purity for any bispecific antibody.

Claims

1. A fusion polypeptide comprising, from C-terminus to N-terminus: a) a first target binding fragment A1; b) a polypeptide linker; c) a second target binding fragment B2; in: The length of the polypeptide linker is short enough to minimize potential intramolecular interactions between A1 and B2. The A1 is capable of pairing with the first pairing fragment B1 to form a first target binding domain; The B2 is capable of pairing with the second pairing fragment A2 to form a second target binding domain; and The A1 is configured to exhibit less binding to the B2 relative to the B1, and the B2 is configured to exhibit less binding to the A1 relative to the A2.

2. A polypeptide complex comprising: a) The fusion polypeptide according to claim 1, b) a second polypeptide comprising the first paired fragment B1; c) a third polypeptide comprising said second target binding fragment B2; and d) a fourth polypeptide and a fifth polypeptide each comprising said second paired fragment A2; in: The A1 in the fusion polypeptide pairs with the B1 in the 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.

3. The fusion polypeptide according to claim 1 or the polypeptide complex according to claim 2, wherein at least one of the pair of B1 and A1 and the pair of B2 and A2 contains at least one conformation that can hinder mismatching between B1 and A2 and / or between B2 and A1.

4. The fusion polypeptide according to claim 1 or 3 or the polypeptide complex according to claim 2 or 3, wherein: A1 comprises a first antibody variable region VA1 selected from VH1 or VL1, The B1 comprises a first paired antibody variable region VB1 capable of pairing with VA1 to form the first target binding domain, wherein the VB1 is selected from VH1 or VL1, and The B2 comprises a second antibody variable region VB2 selected from VH2 or VL2, and The A2 comprises a second pairing antibody variable region VA2 that can pair with VB2 to form the second target binding domain, wherein the VA2 is selected from VH2 or VL2. 5 . The fusion polypeptide according to claim 4 or the polypeptide complex according to claim 4 , wherein the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VH2, and the VA2 comprises VL2.

6. The fusion polypeptide according to claim 4 or the polypeptide complex according to claim 4, wherein: a) the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VL2, and the VA2 comprises VH2; or b) the VB1 comprises VL1, the VA1 comprises VH1, the VB2 comprises VH2, and the VA2 comprises VL2.

7. The fusion polypeptide according to any one of claims 1 and 3 to 6 or the polypeptide complex according to any one of claims 2 to 6, wherein the A1 further comprises a first scaffold region SR operably linked to the VA1 a , and the B2 further comprises a second scaffold region SR operably connected to the VB2 b , where the SR a and the SR b is configured to hinder the SR a With the SR b The pairing between.

8. The fusion polypeptide according to claim 7 or the polypeptide complex according to claim 7, wherein the B1 further comprises a first paired scaffold region PSR a , the first paired scaffold region PSR a is operably connected to the VB1 and is capable of communicating with the SR a Combination, and wherein said A2 further comprises a second paired scaffold region PSR b , the second paired scaffold region PSR b is operably connected to the VA2 and is capable of communicating with the SR b In combination, optionally, the SR a and the PSR a Paired via the first disulfide bond, and / or the SR b and the PSR b Paired via a second disulfide bond.

9. The fusion polypeptide according to claim 7 or 8 or the polypeptide complex according to claim 7 or 8, wherein SR a / PSR a Yes or SR b / PSR b The pair is selected from the group consisting of: a) a heavy chain constant region 1 (CH1) and a light chain constant region (CL) pair; b) T cell receptor (TCR) constant region α (Cα) and TCR constant region β (Cβ) pair; c) TCR constant region γ (Cγ) and TCR constant region δ (Cδ) pair; d) the ligand binding domain of the receptor and the ligand pair; e) a PRD (proline-rich domain) and an SH3 domain pair; and f) Occultin and titin pair.

10. The fusion polypeptide according to any one of claims 7 to 9 or the polypeptide complex according to any one of claims 7 to 9, wherein the SR a and PSR a Different from the SR b and PSR b right.

11. The fusion polypeptide according to any one of claims 7 to 10 or the polypeptide complex according to any one of claims 7 to 10, wherein: a) The SR a and PSR a pairs are CH1 and CL pairs, and the SR b and PSR b The following are true: i) Cα and Cβ pairs, ii) Cγ and Cδ pairs, iii) the ligand binding domain of the receptor and the ligand pair; iv) a PRD (proline-rich domain) and an SH3 domain pair, or v) shielding protein and titin pair, or b) The SR b and PSR b pairs are CH1 and CL pairs, and the SR a and PSR a The following are true: i) Cα and Cβ pairs, ii) Cγ and Cδ pairs, iii) the ligand binding domain of the receptor and the ligand pair; iv) a PRD (proline-rich domain) and an SH3 domain pair, or v) Occultin and titin pair.

12. The fusion polypeptide according to any one of claims 7 to 9 or the polypeptide complex according to any one of claims 7 to 9, wherein the SR a / PSR a To and the SR b / PSR b is the same for both, and the SR a / PSR a To and / or the SR b / PSR b The pair is configured to block SR a / PSR b Between or SR b / PSR a The mismatch between.

13. The fusion polypeptide according to claim 12 or the polypeptide complex according to claim 12, wherein the SR a / PSR a The pair comprises a CH1 domain CH1a and a CL domain CLa, and the SR b / PSR b The pair comprises a CH1 domain CH1b and a CL domain CLb.

14. The fusion polypeptide according to claim 13 or the polypeptide complex according to claim 13, wherein the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VH2, and the VA2 comprises VL2, and wherein: a) The PSR a is the CL domain CLa, the SR a is the CH1 domain CH1a, the SR b is the CH1 domain CH1b, the PSR b is the CL domain CLb; or b) The PSR a is the CH1 domain CH1a, the SR a is the CL domain CLa, the SR b is the CL domain CLb, the PSR b It is the CH1 domain CH1b.

15. The fusion polypeptide according to claim 13 or the polypeptide complex according to claim 13, wherein the PSR a is the CH1 domain CH1a, the SR a is the CL domain CLa, the SR b is the CH1 domain CH1b, the PSR b is CL domain CLb, and wherein: a) the VB1 comprises VH1, the VA1 comprises VL1, the VB2 comprises VL2, and the VA2 comprises VH2; or b) the VB1 comprises VL1, the VA1 comprises VH1, the VB2 comprises VH2, and the VA2 comprises VL2.

16. The fusion polypeptide according to claim 13 or the polypeptide complex according to claim 13, wherein: The VB1 includes VH1, the VA1 includes VL1, the VB2 includes VH2, and the VA2 includes VL2, the PSR a is the CH1 domain CH1a, the SR a is the CL domain CLa, the SR b is the CH1 domain CH1b, the PSR b is CL domain CLb, and wherein: a) the fusion polypeptide comprises the 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, d) the fourth polypeptide and the fifth polypeptide each comprise an amino acid sequence of formula (IV): VL2-CLb; The CH1b / CLb pair and / or the CH1a / CLa pair are configured to hinder mismatching between CH1b and CLa and / or between CH1a and CLb.

17. The fusion polypeptide according to claim 16 or the polypeptide complex according to claim 16, wherein at least one of the CH1b / CLb pair and the CH1a / CLa pair has one or more of the following properties: 1) having at least one non-native disulfide bond that hinders mispairing between CH1b and CLa and / or between CH1a and CLb; 2) has one or more introduced amino acid mutations that form at least one or more introduced charged amino acid residues that hinder 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 hinders mispairing between CH1b and CLa or between CH1a and CLb.

18. The fusion polypeptide according to claim 17 or the polypeptide complex according to claim 17, wherein the first CH1 / CL pair and the second CH1 / CL pair are selected from CH1b / CLb and CH1a / CLa, and wherein the first CH1 / CL pair is associated through a non-natural first disulfide bond and optionally lacks a natural disulfide bond or has a destroyed natural disulfide bond.

19. The fusion polypeptide according to claim 18 or the polypeptide complex according to claim 18, wherein the second CH1 / CL pair is associated through a second disulfide bond, the second disulfide bond is formed at a position different from the first disulfide bond, and optionally the second disulfide bond is a native disulfide bond.

20. The fusion polypeptide according to claim 18 or 19 or the polypeptide complex according to claim 18 or 19, wherein the first disulfide bond is formed by two cysteine ​​residues introduced at a set of heavy chain-light chain EU positions, the set of heavy chain-light chain EU positions being 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) EU position 128 of the heavy chain to EU position 118 of the light chain.

21. The fusion polypeptide according to any one of claims 18 to 20 or the polypeptide complex according to any one of claims 18 to 20, wherein the native disulfide bond is located between EU position 220 of the heavy chain and EU position 214 of the light chain.

22. A fusion polypeptide according to any one of claims 18 to 21 or a polypeptide complex according to any one of claims 18 to 21, wherein the first CH1 / CL pair comprises CH1 and CL, wherein the CH1 comprises a substitution of a cysteine ​​residue at EU position 126 and a substitution of a non-cysteine ​​residue at EU position 220; and the CL comprises 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 according to any one of claims 17 to 22 or the polypeptide complex according to 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 wherein the first CH1 / CL pair contains at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of an oppositely charged residue for a charged residue, so that the first CH1 / CL pair contains a first pair of oppositely charged residues that are conducive to the pairing of the first CH1 / CL pair.

24. The fusion polypeptide according to claim 23 or the polypeptide complex according to claim 23, wherein the second CH1 / CL pair contains at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of an oppositely charged residue for a charged residue, so that the second CH1 / CL pair contains a second pair of oppositely charged residues that is conducive to the pairing of the second CH1 / CL pair, and optionally, the first pair of oppositely charged residues and the second pair of oppositely charged residues hinder the pairing between CH1a and CLb or hinder the pairing between CH1b and CLa.

25. The fusion polypeptide according to claim 24 or the polypeptide complex according to 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 charged residues or both have negatively charged residues, and / or CH1b and CLa both have positively charged residues or both have negatively charged residues.

26. The fusion polypeptide according to any one of claims 23 to 25 or the polypeptide complex according to any one of claims 23 to 25, wherein the first pair of oppositely charged residues and / or the second pair of oppositely charged residues are introduced at a set of heavy chain-light chain EU positions, the heavy chain-light chain EU positions being 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.

27. A fusion polypeptide according to any one of claims 22 to 26 or a polypeptide complex according to any one of claims 22 to 26, wherein the pairs of oppositely charged residues comprise 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).

28. The fusion polypeptide according to any one of claims 17 to 27 or the polypeptide complex according to 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 wherein the first CH1 / CL pair comprises one or more introduced amino acid mutations that form an orthogonal CH1-CL interface.

29. The fusion polypeptide of claim 28 or the polypeptide complex of claim 28, wherein the orthogonal CH1-CL interface is introduced at a set of heavy chain-light chain EU positions, the set of heavy chain-light chain EU positions comprising heavy chain EU positions H168A, F170G, light chain EU positions L135Y, S176W.

30. The fusion polypeptide according to any one of claims 17 to 29 or the polypeptide complex according to any one of claims 17 to 29, wherein 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, the one or more introduced amino acid mutations being selected from the group consisting of: a) Substitutions at EU positions A141I, F170S, S181M, S183A and V185A in the heavy chain and substitutions at EU positions F116A, A235V, S174A, S176F and T178V in the light chain.

31. A fusion polypeptide according to any one of claims 16 to 30 or a polypeptide complex according to 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 wherein the first VH / VL pair has at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of an oppositely charged residue for a charged residue, such that the first VH / VL contains a third pair of oppositely charged residues that are conducive to the pairing of the first VH / VL pair.

32. A fusion polypeptide according to claim 31 or a polypeptide complex according to claim 31, wherein the second VH / VL pair has at least one substitution of a charged residue for a non-charged residue, and / or at least one substitution of an oppositely charged residue for a charged residue, so that the second VH / VL contains a fourth pair of oppositely charged residues that is conducive to the pairing of the second VH / VL pair, and optionally, the third pair of oppositely charged residues and the fourth pair of oppositely charged residues hinder the pairing between VH1 and VL2 or hinder the pairing between VH2 and VL1.

33. The fusion polypeptide according to claim 32 or the polypeptide complex according to claim 32, wherein the third pair of oppositely charged residues and the fourth pair of oppositely charged residues are configured so that both VH1 and VL2 have positively charged residues or both have negatively charged residues, and / or both VH2 and VL1 have positively charged residues or both have negatively charged residues.

34. The fusion polypeptide according to any one of claims 31 to 33 or the polypeptide complex according to any one of claims 31 to 33, wherein the third pair of oppositely charged residues and / or the fourth pair of oppositely charged residues are introduced at a set of heavy chain-light chain EU positions, the heavy chain-light chain EU positions being 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.

35. The fusion polypeptide according to any one of claims 31 to 34 or the polypeptide complex according to any one of claims 31 to 34, wherein the oppositely charged residue pairs comprise positively charged amino acid residues and negatively charged amino acid residues, wherein the positively charged amino acid residues are selected from the group consisting of lysine (K), histidine (H) and arginine (R), and / or the negatively charged amino acid residues are selected from the group consisting of aspartic acid (D) and glutamic acid (E).

36. A polypeptide complex according to any of the preceding claims, wherein the second polypeptide and the third polypeptide further comprise a first operably linked dimerization domain and a second operably linked dimerization domain, respectively, the first dimerization domain and the second dimerization domain associate to form a dimer; optionally, the first dimerization domain comprises a first Fc region, and / or the second dimerization domain comprises a second Fc region. The polypeptide complex according to claim 36 , wherein the first Fc region and / or the second Fc region is derived from IgG1, IgG2, IgG3 or IgG4.

38. The polypeptide complex of claim 36, wherein the first Fc region and the second Fc region have different amino acid sequences and have at least one configuration that promotes heterodimerization of the first Fc region and the second Fc region.

39. The polypeptide complex of claim 38, wherein 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 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) the first Fc mutation comprises L351D or L368E, and the second Fc mutation comprises L351K or T366K, The numbering is based on the EU index.

40. The fusion polypeptide of any preceding claim or the polypeptide complex of any preceding claim, wherein the first target binding domain and the second target binding domain bind to different targets.

41. The fusion polypeptide of any preceding claim or the polypeptide complex of any preceding claim, wherein at least one of the first target binding domain and the second target binding domain is chimeric, humanized or fully human.

42. A fusion polypeptide according to any of the preceding claims or a polypeptide complex according to any 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, optionally, the disease-associated antigen is a tumor-associated antigen, an antigen associated with an autoimmune disease or an 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 disease.

43. A fusion polypeptide according to any of the preceding claims or a polypeptide complex according to any 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.

44. A nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide according to any one of the preceding claims or the polypeptide complex according to any one of the preceding claims.

45. A vector comprising the nucleic acid according to claim 44.

46. ​​A host cell comprising the nucleic acid of claim 44 or the vector of claim 45.

47. A pharmaceutical composition comprising the polypeptide complex according to any one of claims 1 to 43 and a pharmaceutically acceptable carrier.

48. A conjugate comprising a polypeptide complex according to any one of claims 1 to 43 and a payload conjugated to the polypeptide complex, 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 tracer molecule, an anti-cancer drug and a cytotoxic molecule.

49. A composition comprising the polypeptide complex according to any one of claims 1 to 43, or the conjugate according to claim 48, and a pharmaceutically acceptable carrier.

50. A method of treating or preventing a disease, condition or symptom, the method comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide complex of any one of claims 1 to 43, the pharmaceutical composition of claim 47, the conjugate of claim 48 or the composition of claim 49.

51. The method of claim 50, wherein the disease is selected from the group consisting of cancer, inflammatory disease, infectious or parasitic disease, cardiovascular disease, eye disease, central nervous system (CNS) disease, injury, metabolic disease, autoimmune disease, or coagulation disorder.

52. A method for detecting the presence or level of an antigen, the method comprising: contacting a sample suspected of containing the antigen with the polypeptide complex according to any one of claims 1 to 43; and determining the formation of a complex between the antigen and the polypeptide complex.

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