Modified antibodies and uses thereof

Abstract

The present disclosure provides modified antibodies, methods of making and uses thereof. In particular, the modified antibody is a bispecific antibody or a multispecific antibody. The heavy and light chains of the modified antibodies provided in the present disclosure pair with high assembly accuracy and stability.

Description

Technical Field

[0001] The present disclosure generally relates to modified antibodies and uses thereof. Background Art

[0002] At present, monoclonal antibodies have been used as specific and effective biological drugs, which can show excellent specificity for a single target antigen. However, for some cases, a single antibody with specificity for two (or more) different target antigens, usually referred to as a bispecific antibody (or multispecific antibody), is preferred. Bispecific or multispecific antibodies can simultaneously recognize two or more different antigens, neutralize different pathogenic mediators, recruit different types of effector cells and regulate signaling pathways, which makes them superior to monospecific antibodies in many aspects. In such cases, the development of bispecific or multispecific antibodies as therapeutic agents for human diseases has important clinical significance, and bispecific antibodies have become a widely used form in diagnostic and therapeutic applications in recent years.

[0003] However, the generation of bispecific or multispecific antibodies is still challenging. In the case of bispecific antibodies, a heavy chain that has specificity to a certain antigen must be paired with a light chain that has specificity to the same antigen. If the heavy chain is paired with a light chain that has specificity to other different antigens, the antigen-specificity of expectation may be destroyed or reduced. Because it is still impossible to control the correct pairing between the light chain and the corresponding heavy chain with natural IgG structure well, the effort of introducing other antibody specificity into a single antibody often causes the generation of wrongly assembled antibodies.

[0004] There remains a need to generate new modified antibodies with more than one specificity. Summary of the invention

[0005] On the one hand, the present disclosure provides an antibody or an antigen-binding fragment thereof, comprising a first arm and a second arm, the first arm being formed by a first heavy chain and a first light chain and an interchain disulfide bond formed therebetween, the second arm being formed by a second heavy chain and a second light chain and an interchain disulfide bond formed therebetween, wherein at least one non-cysteine ​​residue on the first heavy chain is substituted with cysteine, wherein the non-cysteine ​​residue is located at a position selected from the group consisting of: amino acid position 126, 128, 129, 136, 141, 168, 170, 173, 175 or 187 of the first heavy chain; and at least one non-cysteine ​​residue on the first light chain is substituted with cysteine, wherein the non-cysteine ​​residue is located at a position selected from the group consisting of: amino acid position 114, 116, 118, 124, 135, 137, 138, 160, 162 or 164 of the first light chain.

[0006] In some embodiments, the at least one non-cysteine ​​residue on the first heavy chain that is substituted with cysteine ​​is selected from the group consisting of: amino acid residues F126, L128, A129, S136, A141, H168, F170, V173, Q175 or T187 of the first heavy chain; and the at least one non-cysteine ​​residue on the first light chain that is substituted with cysteine ​​is selected from the group consisting of: amino acid residues S114, F116, F118, Q124, L135, N137, N138, Q160, S162 or T164 of the first light chain.

[0007] In some embodiments, the first light chain and the first heavy chain comprise at least one non-cysteine ​​substitution pair of a native cysteine ​​on the first light chain and a native cysteine ​​on the first heavy chain and at least one cysteine ​​substitution pair selected from the group consisting of: (i) S114C on the light chain and S136C on the heavy chain; (ii) F116C on the light chain and S136C on the heavy chain; (iii) F118C on the light chain and L128C on the heavy chain; (iv) F118C on the light chain and A129C on the heavy chain; (v) F118C on the light chain and A141C on the heavy chain; (vi) Q124C on the light chain and F126C on the heavy chain; (vii) L135C on the light chain and F170C on the heavy chain; (viii) F118C on the light chain and A129C on the heavy chain; (iii) N137C on the light chain and F170C on the heavy chain; (ix) N138C on the light chain and H168C on the heavy chain; (x) Q160C on the light chain and V173C on the heavy chain; (xi) Q160C on the light chain and Q175C on the heavy chain; (xii) S162C on the light chain and F170C on the heavy chain; (xiii) S162C on the light chain and T187C on the heavy chain; and (xiv) T164C on the light chain and F170C on the heavy chain; wherein the at least one cysteine ​​substitution pair forms at least one interchain disulfide bond between the first heavy chain and the first light chain; wherein the native cysteines on the first heavy chain and the first light chain are independently substituted by any one of non-cysteine ​​residues.

[0008] In some embodiments, the native cysteine ​​on the first heavy chain and the first light chain is independently replaced by any one of serine, alanine, glycine or valine. In some embodiments, the native cysteine ​​to be replaced is amino acid residue C214 in the light chain of the antibody and amino acid residue C220 in the heavy chain. In some embodiments, the first light chain includes mutation C214S, and the first heavy chain includes mutation C220S.

[0009] In some embodiments, the first heavy chain and the first light chain comprise one cysteine ​​substitution pair. In some embodiments, the first heavy chain and the first light chain comprise two cysteine ​​substitution pairs.

[0010] In some embodiments, the two cysteine ​​substitution pairs are selected from the group consisting of: (i) S114C / Q160C on the light chain and S136C / V173C on the heavy chain; (ii) F118C / Q124C on the light chain and L128C / F126C on the heavy chain; (iii) F118C / Q124C on the light chain and A129C / F126C on the heavy chain; (iv) F118C / Q124C on the light chain and A141C / F126C on the heavy chain; (v) F116C / Q124C on the light chain and S136C / F126C on the heavy chain; (vi) Q124C / T164C on the light chain and F126C / F170C on the heavy chain; (vii) (x) F118C / S162C on the light chain and L128C / F170C on the heavy chain; (xi) F118C / S162C on the light chain and A141C / F170C on the heavy chain; (xii) Q124C / S162C on the light chain and F126C / F170C on the heavy chain; (xiii) N137C / S162C on the light chain and F170C / T187C on the heavy chain.

[0011] In some embodiments, the first light chain comprises the mutations Q124C / T164C / C214S, and the first heavy chain comprises the mutations F126C / F170C / C220S. In some embodiments, the first light chain comprises the mutations Q124C / S162C / C214S, and the first heavy chain comprises the mutations F126C / F170C / C220S.

[0012] In some embodiments, the disulfide bond between the second heavy chain and the second light chain is formed between a native cysteine ​​on the second light chain and a native cysteine ​​on the second heavy chain; wherein the position of the native cysteine ​​on the second light chain corresponds to position C214 on the human κ chain, and the position of the native cysteine ​​on the second heavy chain corresponds to position C220 on human IgG1.

[0013] In some embodiments, the second light chain and the second heavy chain comprise at least one non-cysteine ​​substitution pair, wherein the native cysteine ​​on the light chain is substituted with serine and the native cysteine ​​on the heavy chain is substituted with serine, and at least one cysteine ​​substitution pair selected from the group consisting of: (i) S114C on the light chain and S136C on the heavy chain; (ii) F116C on the light chain and S136C on the heavy chain; (iii) F118C on the light chain and L128C on the heavy chain; (iv) F118C on the light chain and A129C on the heavy chain; (v) F118C on the light chain and A141C on the heavy chain; (vi) Q124C on the light chain and F126C on the heavy chain; (vii) L135C on the light chain and L128C on the heavy chain; (viii) N137C on the light chain and F170C on the heavy chain; (ix) N138C on the light chain and H168C on the heavy chain; (x) Q160C on the light chain and V173C on the heavy chain; (xi) Q160C on the light chain and Q175C on the heavy chain; (xii) S162C on the light chain and F170C on the heavy chain; (xiii) S162C on the light chain and T187C on the heavy chain; and (xiv) T164C on the light chain and F170C on the heavy chain; wherein the at least one cysteine ​​substitution pair forms at least one interchain disulfide bond between the second light chain and the second heavy chain, and the cysteine ​​substitution pair on the first arm is different from the cysteine ​​substitution pair on the second arm. A non-cysteine ​​substitution pair refers to a pair of native cysteines (one on the light chain and one on the heavy chain) that have been mutated to a non-cysteine ​​amino acid. A cysteine ​​substitution pair refers to a pair of native non-cysteine ​​amino acids (one on the light chain and one on the heavy chain) that have been mutated to cysteine.

[0014] In some embodiments, the substituted native cysteine ​​on the second light chain is located at position C214 on the human kappa chain, and the substituted native cysteine ​​on the second heavy chain is located at position C220 on the human IgG1.

[0015] In some embodiments, the second heavy chain and the second light chain comprise two cysteine ​​substitution pairs. In some embodiments, the two cysteine ​​substitution pairs are selected from the group consisting of: (i) S114C / Q160C on the light chain and S136C / V173C on the heavy chain; (ii) F118C / Q124C on the light chain and L128C / F126C on the heavy chain; (iii) F118C / Q124C on the light chain and A129C / F126C on the heavy chain. C; (iv) F118C / Q124C on the light chain and A141C / F126C on the heavy chain; (v) F116C / Q124C on the light chain and S136C / F126C on the heavy chain; (vi) Q124C / T164C on the light chain and F126C / F170C on the heavy chain; (vii) Q124C / N138C on the light chain and A141C / F126C on the heavy chain. (viii) Q160C / L135C on the light chain and Q175C / F170C on the heavy chain; (ix) S114C / S162C on the light chain and S136C / F170C on the heavy chain; (x) F118C / S162C on the light chain and L128C / F170C on the heavy chain; (xi) F118C / S162C on the light chain and L128C / F170C on the heavy chain; C / S162C and A141C / F170C on the heavy chain; (xii) Q124C / S162C on the light chain and F126C / F170C on the heavy chain; and (xiii) N137C / S162C on the light chain and F170C / T187C on the heavy chain; wherein the cysteine ​​substitution pair on the first arm is different from the cysteine ​​substitution pair on the second arm.

[0016] In some embodiments, the first heavy chain constant region and / or the second heavy chain constant region comprises human IgG1, IgG2, IgG3, or IgG4; and the first light chain constant region and / or the second light chain constant region comprises human κ light chain or human λ light chain.

[0017] In some embodiments, the first heavy chain and the second heavy chain form a heterodimer; and the Fc region of the first heavy chain constant region and / or the Fc region of the second heavy chain constant region comprises one or more modifications that promote heterodimerization.

[0018] In some embodiments, the Fc region of the first heavy chain interacts with the Fc region of the second heavy chain through a knob / hole structure. In some embodiments, the Fc region of the first heavy chain comprises a knob mutation and the Fc region of the second heavy chain comprises a hole mutation; or the Fc region of the first heavy chain comprises a hole mutation and the Fc region of the second heavy chain comprises a knob mutation. In some embodiments, the knob mutation comprises T366W and the hole mutation comprises T366S / L368A / Y407V.

[0019] In some embodiments, the modification that promotes heterodimerization comprises the introduction of a cysteine ​​residue capable of forming a disulfide bond.

[0020] In some embodiments, the Fc region of the first heavy chain constant region and the Fc region of the second heavy chain constant region each include a modification in the CH3 region; wherein the modifications in both CH3 regions are selected from the following:

[0021] Modification in one CH3 region Modification in another CH3 region T366S / L368A / Y407V T366W S354C / T366W Y349C / T366S / L368A / Y407V T366Y Y407T T366W Y407A T394W F405A T366Y / F405A T394W / Y407T T366W / F405W T394S / Y407A F405W T394S D399C K392C T366W / D399C T366S / L368A / K392C / Y407V T366W / K392C T366S / L368A / D399C / Y407V S354C / T366W Y349C / T366S / L368A / Y407V Y349C / T366W S354C / T366S / L368A / Y407V E356C / T366W Y349C / T366S / L368A / Y407V Y349C / T366W E356C / T366S / L368A / Y407V E357C / T366W Y349C / T366S / L368A / Y407V Y349C / T366W E357C / T366S / L368A / Y407V

[0022] In some embodiments, the Fc region of the first heavy chain constant region and the Fc region of the second heavy chain constant region each include a modification in the CH3 region; wherein the modifications in both CH3 regions are selected from the following:

[0023] Modification in one CH3 region Modification in another CH3 region K370E / D399K / K439D D356K / E357K / K409D K409D D399K K409E D399K K409E D399R K409D D399R D339K E356K E356K / D399K K392D / K409D E356K / D399K K409D / K439D E357K / D399K K370D / K409D D399K / E357K / E356K K370D / K392D / K409D E357K / D399K K392D / K409D K392D / K409D D399K K360D / K409D D399K

[0024] In some embodiments, the first arm and the second arm can specifically bind to an antigen selected from the group consisting of: SIRPα, CLDN18.2, Siglec15, HER2, EGFR, CD19, CD20, CD39, CD47, PD1, PDL1, CD3, NKG2D, NKG2A, Nkp46, CD137, OX40, CD40, LILRB1, LILRB2, LILRB4, GPC3, TROP2, CD112, TIGIT, FAP, VEGFA, DLL4, ANG-2, wherein the first arm and the second arm can specifically bind to different antigens.

[0025] In some embodiments, the Fc region of the first heavy chain constant region and / or the Fc region of the second heavy chain constant region further comprises a modification that improves the stability of the antibody or the antigen-binding fragment.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody or a multispecific antibody.

[0027] In some embodiments, the antibody or the antigen-binding fragment is linked to one or more conjugate moieties.

[0028] In some embodiments, the conjugate moiety comprises a second antibody fragment. In some embodiments, the antibody or antigen-binding fragment thereof comprises a Fab fragment linked to the C-terminus of the Fc region of the second antibody fragment.

[0029] In some embodiments, the conjugate moiety includes an agent for detection or separation, such as a clearance modifier, a luminescent label, a fluorescent label, an enzyme substrate label, or a purification moiety. In some embodiments, the conjugate moiety includes a therapeutic agent or drug.

[0030] In one aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof disclosed herein.

[0031] In one aspect, the present disclosure provides a vector comprising an isolated polynucleotide disclosed herein.

[0032] In one aspect, the present disclosure provides a host cell comprising a vector disclosed herein.

[0033] In one aspect, the present disclosure provides a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof or a polynucleotide disclosed herein; and (ii) one or more pharmaceutically acceptable carriers.

[0034] In one aspect, the present disclosure provides a method of expressing an antibody or antigen-binding fragment thereof disclosed herein, the method comprising culturing a host cell disclosed herein under conditions suitable for expression of a vector contained therein.

[0035] In one aspect, the present disclosure provides a method for treating, preventing or alleviating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or an antigen-binding fragment thereof, or a polynucleotide encoding the antibody or an antigen-binding fragment thereof, or a vector, or a host cell, or a pharmaceutical composition disclosed herein.

[0036] In some embodiments, the subject is a human. In some embodiments, the administering is oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular.

[0037] In one aspect, the present disclosure provides use of an antibody or an antigen-binding fragment thereof, or a polynucleotide encoding the antibody or an antigen-binding fragment thereof, or a vector, or a host cell, or a pharmaceutical composition disclosed herein in the preparation of a medicament for treating, preventing or alleviating a disease in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A schematic diagram of screening assay 1 is shown.

[0039] Figure 2 A schematic diagram of screening assay 2 is shown.

[0040] Figures 3A to 3D The structures of expression vectors used in screening assays 1 and 2 are shown. Figure 3A The structures of expression vectors for light chains with and without shifted disulfide bond mutations are shown. Figure 3B The structures of expression vectors for heavy chains with and without shifted disulfide bond mutations are shown. Figure 3C The structure of the expression vector for the wild-type light chain with the VHH domain fused at the N-terminus is shown. Figure 3D The structure of the expression vector of naked Fc with knob or hole residue substitution is shown.

[0041] Figure 4A and 4B Shown are the results of the relative abundance of engineered asymmetric Fab-Fc and VHH-Fab-Fc variants characterized by SEC-HPLC. Figure 4A Asymmetric Fab-Fc variants with different residue substitution pairs of CH1-CL interchain disulfide bond shifts, as tested by Screening Assay 1, are shown. Figure 4B An asymmetric VHH-Fab-Fc molecule with native CH1-CL interchain disulfide bonds, as detected by screening assay 2, is shown.

[0042] Figure 5 Shown is the schematic structure of a symmetric bivalent monospecific antibody with shifted interchain disulfide bonds in the two Fab arms.

[0043] Fig. 6A and 6B The yields of engineered symmetric antibody variants with different residue substitution pairs having CH1-CL interchain disulfide bond shifts are shown ( Fig. 6A ) and monomer percentage evaluation ( Figure 6B ) results.

[0044] Figures 7A to 7D Shown are the results of the binding activity assessment of all symmetrical bivalent anti-CLDN18.2 antibody variants to MC38 cells overexpressing CLDN18.2 by FACS assay.

[0045] Figure 8 Shown are the results of thermal stability analysis of symmetrical bivalent antibody variants with different residue substitution pairs of CH1-CL interchain disulfide bond offset using a thermal shift assay.

[0046] Fig. 9A schematic diagram of the Case 1 study is shown.

[0047] Fig.10 Shown are the results of assembly accuracy and thermal stability of the antibodies tested in the Case 1 study, as measured by LC-MS assay and differential scanning fluorimetry, respectively.

[0048] Fig.11 A schematic diagram of the Case 2 study is shown.

[0049] Fig.12 Shown are the results of assembly accuracy and thermal stability of the antibodies tested in the Case 2 study, as measured by LC-MS assay and differential scanning fluorimetry, respectively.

[0050] Fig.13A and 13B It is shown that in case 1 study ( Fig.13A ) and Case 2 Study ( Fig. 13B ) SDS-PAGE results of the antibodies tested in ).

[0051] Fig.14 The SDS-PAGE and SEC results of the tested antibody ACF_361 are shown.

[0052] Fig.15 LC-MS results for the test antibody ACF_361 are shown.

[0053] Fig.16A The results of the binding affinity of antibodies to MC38 / CD47 / CLDN18.2 cells obtained by FACS are shown. Fig. 16B The results of antibody binding affinity to CHO / SIRPαV1 cells obtained by FACS are shown. Fig. 16C The results of the phagocytic activity of the antibodies are shown.

[0054] Fig.17 The SDS-PAGE and SEC results of the tested antibody ACF_389 are shown.

[0055] Fig.18 and 19 Shown are the results of Mono S chromatography purification of the test antibody ACF_389.

[0056] Fig. 20 LC-MS results for the test antibody ACF_389 are shown.

[0057] Fig.21A The results of the binding affinity of antibodies to Raji / PDL1 cells obtained by FACS are shown. Fig.21B The results of antibody binding affinity to CHO / SIRPαV1 cells obtained by FACS are shown.

[0058] Fig. 22 and 23 The results of Mono S chromatography purification of the test antibody ESB07.451 are shown.

[0059] Fig.24 LC-MS results for the test antibody ESB07.451 are shown. DETAILED DESCRIPTION

[0060] The following description of the present disclosure is only intended to illustrate various embodiments of the present disclosure. In this way, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that such equivalent embodiments will be included herein. All documents cited in this article, including disclosures, patents and patent applications are incorporated herein by reference in their entirety. The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0061] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. For example, "an antibody" means one antibody or more than one antibody.

[0062] In addition, where the terms "including," "include," "having," "has," "with," or variations thereof are used in a particular specification and / or claim, such terms are intended to be inclusive in a manner similar to the term "comprising." Throughout this disclosure, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of the stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" means that the listed elements are required or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or facilitate the activities or behaviors specified for the listed elements in this disclosure. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present, depending on whether or not they affect the activity or function of the listed elements.

[0063] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" may mean within 1 or more than 1 standard deviation. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5% and still more preferably up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude of a value, preferably within 5-fold and more preferably within 2-fold. When describing a particular value in the present application and claims, unless otherwise indicated, it should be assumed that the term "about" means within an acceptable error range for the particular value.

[0064] References throughout this disclosure to "one embodiment," "an embodiment," "a particular embodiment," "some embodiments," or "certain embodiments," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the above phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner.

[0065] I. Antibody modification

[0066] In some aspects, the present disclosure provides modified antibodies with increased assembly fidelity and stability of the polypeptide chains of the antibody.

[0067] In some aspects, the modified antibodies are designed to reduce mispairing of heavy and light chains in bispecific or multispecific antibodies with two or more different antigen binding sites.

[0068] In some aspects, the modified antibodies have amino acid residue mutations wherein non-cysteine ​​residues are mutated to cysteine ​​residues to form new disulfide bonds between the heavy and light chains of the antibody.

[0069] In some aspects, the modified antibodies have amino acid residue mutations, wherein cysteine ​​residues are mutated to non-cysteine ​​residues to disrupt the formation of disulfide bonds between cysteine ​​residues.

[0070] 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. In addition, it should be noted that a horizontal line at the beginning or end of an amino acid residue sequence represents a peptide bond to another one or more amino acid residue sequences or a covalent bond to a carboxyl or hydroxyl end group. However, the absence of a dash should not be construed as meaning that there are no such peptide bonds or covalent bonds to the carboxyl or hydroxyl terminal groups, since dashes are typically omitted when expressing amino acid sequences.

[0071] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, bispecific antibody or monospecific antibody that binds to one or more specific antigens. A natural complete antibody includes two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ and μ, each heavy chain consisting of a variable region (VH) and a first constant region, a second constant region, a third constant region and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are classified as λ or κ, and each light chain consists of a variable region (VL) and a constant region. The antibody is "Y" shaped, wherein the stem of the Y consists of the second constant region and the third constant region of two heavy chains bound together by disulfide bonds. Each arm of the Y includes the variable region and the first constant region of a single heavy chain bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the two chains usually include three highly variable loops, called complementarity determining regions (CDRs) (the light chain CDRs include LCDR1, LCDR2 and LCDR3, and the heavy chain CDRs include HCDR1, HCDR2 and HCDR3).The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the Kabat, IMGT, Chothia or Al-Lazikani rules (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., Sequences of Proteins of Immunological Interest. 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)). The three CDRs are separated by side segments called framework regions (FRs) (light chain FRs include LFR1, LFR2, LFR3 and LFR4, and heavy chain FRs include HFR1, HFR2, HFR3 and HFR4), which are more highly conserved than CDRs and form a scaffold to support highly variable loops. The constant regions of heavy and light chains are not related to antigen binding, but exhibit a variety of effector functions. Antibodies can be divided into several categories based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, which are characterized by the presence of α, δ, ε, γ and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0072] As used herein, the term "variable domain" refers to an antibody variable region or fragment thereof including one or more CDRs. Although a variable domain may include a complete variable region (such as a HCVR or LCVR), it may also include less than a complete variable region but still retain the ability to bind to an antigen or form an antigen binding site.

[0073] As used herein, the term "antigen binding fragment" refers to an antibody fragment formed by a portion of an antibody including one or more CDRs, or any other antibody fragment that binds to an antigen but does not include a complete native antibody structure. Examples of antigen binding fragments include, but are not limited to, variable domains, variable regions, bifunctional antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and divalent domain antibodies. Antigen binding fragments can bind to the same antigen bound by a parent antibody. In certain embodiments, an antigen binding fragment may include one or more CDRs from a specific human antibody that is transplanted to a framework region from one or more different human antibodies. Further and detailed forms of antigen binding fragments (also referred to as antigen binding portions) are described in Spiess et al., 2015 Molecular Immunology, 67(2), pp. 95-106 (2015) and Brinkman et al., mAbs, 9(2), pp. 182-212 (2017), which are incorporated herein by reference in their entirety.

[0074] "Fab" with respect to antibodies refers to the portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by disulfide bonds.

[0075] "Fab'" refers to the Fab fragment including a portion of the hinge region.

[0076] "F(ab')2" refers to a dimer of Fab'.

[0077] In the case of antibodies (e.g., IgG, IgA or IgD isotypes), "Fc" refers to the portion of the antibody consisting of the second constant domain and the third constant domain of the first heavy chain combined with the second constant domain and the third constant domain of the second heavy chain via a disulfide bond. The Fc of IgM and IgE isotype antibodies further includes a fourth constant domain. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

[0078] In the context of antibodies, the "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region includes approximately 25 amino acid residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently.

[0079] As used herein, "CH2 domain" refers to the portion of a heavy chain molecule that includes and extends from about amino acid 244 to amino acid 360 of, for example, an IgG antibody using the conventional numbering scheme (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340, EU numbering system; see Kabat, E. et al., US Department of Health and Human Services, (1983)).

[0080] The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and includes approximately 108 amino acids. Certain immunoglobulin classes, such as IgM, further include a CH4 region.

[0081] In the case of antibodies, "Fv" refers to the smallest fragment of an antibody that carries a complete antigen binding site. The Fv fragment consists of the variable region of a single light chain combined with the variable region of a single heavy chain. Many Fv designs have been provided, including dsFv, in which the association between the two domains is enhanced by an introduced disulfide bond; and a peptide linker can be used to combine the two domains together as a single polypeptide to form scFv. Fv constructs containing variable domains of heavy or light immunoglobulin chains associated with the variable domains and constant domains of the corresponding immunoglobulin heavy or light chains have also been produced. Fv has also been multimerized to form bifunctional antibodies and trifunctional antibodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0082] A "camelized single domain antibody", "heavy chain antibody" or "HCAb" is a HHeavy chain antibodies are antibodies that contain heavy chain domains but not light chains (Riechmann L. and Muyldermans S., J Immunol Methods Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Pat. No. 6,005,079). Heavy chain antibodies were originally derived from the Camelidae (camels, dromedaries and llamas). Despite the absence of light chains, camelized antibodies have a realistic antigen binding repertoire (Hamers-Casterman C. et al., Nature June 3; 363(6428): 446-8 (1993); Nguyen VK. et al., Immunogenetics. April; 54(1): 39-47 (2002); Nguyen VK. et al., Immunology. May; 109(1): 93-101 (2003)). The variable domain of the heavy chain antibody (VHH domain) represents the smallest known antigen binding unit produced by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November; 21(13): 3490-8. Epub 2007 June 15 (2007)).

[0083] "Nanobody" refers to an antibody fragment consisting of the VHH domain from a heavy chain antibody and the two constant domains CH2 and CH3.

[0084] "Diabodies" or "dAbs" include small antibody fragments with two antigen-binding sites, wherein the fragments include a V-binding site in the same polypeptide chain. L Domain-linked V H Domain (V H -V L or V L -V H) (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15;90(14):6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen binding sites. The antigen binding sites can target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds bifunctional antibody" is a bifunctional antibody that targets two different antigens (or epitopes).

[0085] 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", "trivalent", "tetravalent" and "hexavalent" refer to the presence of two binding sites, three 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.

[0086] As used herein, a "bispecific" antibody refers to 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.

[0087] As used herein, a "multispecific" antibody refers to an artificial antibody having fragments derived from more than two different monoclonal antibodies and capable of binding to more than two different epitopes. The more than two epitopes may be present on the same antigen, or they may be present on more than two different antigens.

[0088] 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 include 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.

[0089] As used herein, the term "humanized" means that the antibody or antigen-binding fragment includes CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans when applicable.

[0090] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (ie, antibody) or fragment thereof and an antigen.

[0091] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen. Specific binding can be characterized by binding affinity, such as measured by K. D The value represents the ratio of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium (k off / k on ). D It can be determined by using any conventional method known in the art, including but not limited to surface plasmon resonance, microthermophoresis, HPLC-MS and flow cytometry (such as FACS). -6 M (e.g. ≤5×10 -7 M, ≤2×10 -7 M, ≤10 -7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≤10 -8 M, ≤5×10 -9 M, ≤4×10 -9 M, ≤3×10 -9 M, ≤2×10 -9 M or ≤10 -9 M) K D The value can indicate specific binding between the antibody or antigen-binding fragment thereof and the corresponding antigen.

[0092] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitope within the antigen. An epitope may be linear or conformational (i.e., comprising spaced-apart amino acid residues).

[0093] "Percentage (%) of sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percentage (%) of sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence to which it is compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. For example, publicly available tools such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., Journal of Mol. Biol., 215:403-410 (1990); Stephen F et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin et al., Methods in Enzymology, 267:383-402 (1997); MA et al. Bioinformatics (Bioinformatics) (Cambridge, England), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software are used to determine the comparison of amino acid (or nucleic acid) sequence identity percentage. Those skilled in the art can use the default parameters provided by the tool or can appropriately customize the parameters according to the needs of the comparison, for example, by selecting a suitable algorithm.

[0094] As used herein, the terms "mutation", "mutated" with respect to amino acid residues refer to a substitution, insertion or deletion of one or more amino acid residues.

[0095] As used herein, the term "substitution" or "substituted" with reference to an amino acid sequence refers to the replacement of a native amino acid residue with a different amino acid residue.

[0096] The term "conservative substitution" with respect to an amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As known in the art, conservative substitutions generally do not cause significant changes in protein conformational structure, and therefore the biological activity of the protein can be retained.

[0097] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians and reptiles. Except where noted, the terms "patient" or "subject" are used interchangeably herein.

[0098] The antibody or its antigen-binding fragment can include one or more modifications that introduce or remove glycosylation sites. Glycosylation sites are amino acid residues with side chains to which carbohydrate moieties (e.g., oligosaccharide structures) can be attached. Glycosylation of antibodies is typically N-connected or O-connected. N-connection refers to the side chain to which the carbohydrate moiety is connected to an asparagine residue (e.g., an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine), wherein X is any amino acid except proline. O-connected glycosylation refers to the connection of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly to serine or threonine.

[0099] Disulfide bond formation and cleavage

[0100] In some embodiments, the antibodies provided herein include a first arm and a second arm, wherein the first arm is formed by a first heavy chain and a first light chain and at least one disulfide bond formed therebetween, and the second arm is formed by a second heavy chain and a second light chain and at least one disulfide bond formed therebetween.

[0101] In some embodiments, the connection between the first heavy chain and the first light chain forming the first arm comprises an artificially introduced disulfide bond, and the connection between the second heavy chain and the second light chain forming the second arm comprises a naturally occurring disulfide bond. In some embodiments, the connection between the first heavy chain and the first light chain forming the first arm comprises an artificially introduced disulfide bond at a first set of positions, and the connection between the second heavy chain and the second light chain forming the second arm comprises an artificially introduced disulfide bond at a second set of positions different from the first set of positions.

[0102] In some aspects, the modified antibody has an amino acid residue mutation, wherein a pair of non-cysteine ​​residues (which can be any amino acid other than cysteine) respectively located on the heavy chain and light chain of the antibody mutates to a cysteine ​​residue to form a new disulfide bond therebetween. In some embodiments, according to EU numbering, the non-cysteine ​​residues on the heavy chain are located at amino acid positions 126, 128, 129, 136, 141, 168, 170, 173, 175 or 187, and according to EU numbering, the non-cysteine ​​residues on the light chain are located at amino acid positions 114, 116, 118, 124, 135, 137, 138, 160, 162 or 164. In some embodiments, according to EU numbering, the non-cysteine ​​residues on the heavy chain are amino acid residues of F126, L128, A129, S136, A141, H168, F170, V173, Q175 or T187. In certain embodiments, the non-cysteine ​​residue on the light chain is an amino acid residue of S114, F116, F118, Q124, L135, N137, N138, Q160, S162, or T164 according to EU numbering.

[0103] In certain embodiments, the modified antibody comprises one or more pairs of mutations from non-cysteine ​​residues to cysteine ​​residues at the following positions:

[0104] Table 1. Amino acid residues to be mutated from non-cysteine ​​residues to cysteine ​​residues.

[0105]

[0106] In certain embodiments, the modified antibody comprises one or more of the following mutation pairs from a non-cysteine ​​residue to a cysteine ​​residue:

[0107] Table 2. Amino acid residues to be mutated from non-cysteine ​​residues to cysteine ​​residues.

[0108]

[0109] In some aspects, the modified antibody has amino acid residue mutations, wherein the cysteine ​​residues are mutated to non-cysteine ​​residues to destroy the formation of disulfide bonds between the cysteine ​​residues. In certain embodiments, the cysteine ​​residue pair that is mutated to non-cysteine ​​residues is amino acid residue C214 in the light chain of the antibody and amino acid residue C220 in the heavy chain.

[0110] In certain embodiments, the modified antibody has one or more pairs of amino acid residue mutations at positions selected from Table 1 or Table 2, and the pair of cysteine ​​residues mutated to non-cysteine ​​residues is amino acid residue C214 in the light chain and amino acid residue C220 in the heavy chain of the antibody.

[0111] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure include a first arm formed by a first heavy chain and a first light chain and two new disulfide bonds formed therebetween, wherein the two disulfide bonds are formed by a mutation pair from non-cysteine ​​to cysteine ​​selected from the mutation pairs listed in Table 1 or Table 2.

[0112] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a first arm formed by a first heavy chain and a first light chain and a disulfide bond formed therebetween, and a second arm formed by a second heavy chain and a second light chain and a disulfide bond formed therebetween, wherein the first light chain and the first heavy chain comprise at least one pair of mutations from a non-cysteine ​​residue to a cysteine ​​residue selected from the group consisting of:

[0113] (i) S114C on the light chain and S136C on the heavy chain;

[0114] (ii) F116C on the light chain and S136C on the heavy chain;

[0115] (iii) F118C on the light chain and L128C on the heavy chain;

[0116] (iv) F118C on the light chain and A129C on the heavy chain;

[0117] (v) F118C on the light chain and A141C on the heavy chain;

[0118] (vi) Q124C on the light chain and F126C on the heavy chain;

[0119] (vii) L135C on the light chain and F170C on the heavy chain;

[0120] (viii) N137C on the light chain and F170C on the heavy chain;

[0121] (ix) N138C on the light chain and H168C on the heavy chain;

[0122] (x) Q160C on the light chain and V173C on the heavy chain;

[0123] (xi) Q160C on the light chain and Q175C on the heavy chain;

[0124] (xii) S162C on the light chain and F170C on the heavy chain;

[0125] (xiii) S162C on the light chain and T187C on the heavy chain; and

[0126] (xiv) T164C on the light chain and F170C on the heavy chain;

[0127] wherein at least one pair of mutations from a non-cysteine ​​residue to a cysteine ​​residue forms at least one disulfide bond between the first heavy chain and the first light chain, and the native cysteine ​​on the first light chain and the native cysteine ​​on the first heavy chain are mutated to a non-cysteine ​​amino acid. In certain embodiments, the non-cysteine ​​residue to which the native cysteine ​​is mutated is independently selected from S, A or G.

[0128] Unless otherwise indicated, all amino acid residues and positions on the heavy and light chains of the antibodies described in the present disclosure are numbered according to the EU numbering system. As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant regions, such as Edelman, GM et al., Proc. Natl. Acad. Sci. USA, 63, 78-85 (1969) and Kabat et al., Protein Sequences with Immunological Significance, U.S. Department of Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0129] In some embodiments, the naturally occurring disulfide bond formed between the native cysteine ​​on the second light chain CL region and the native cysteine ​​on the second heavy chain CH1 region is destroyed by substitution of non-cysteine ​​on the two cysteines. In some embodiments, the native cysteine ​​is independently substituted by any non-cysteine ​​residue, as long as there is no interaction between the artificially introduced new residue pairs. In some embodiments, the native cysteine ​​is independently substituted by serine (S), alanine (A) or glycine (G). In some embodiments, the native cysteine ​​is substituted by serine (S).

[0130] In some embodiments, the artificially introduced disulfide bonds are formed by a combination of mutants selected from the group consisting of the mutations listed in Table 3.

[0131] Table 3. Mutation sites on CH1 and CL that form an artificially introduced disulfide bond between CH1 and CL.

[0132] Mutant sites in CL Mutant sites in CH1 S114C / C214S S136C / C220S F116C / C214S S136C / C220S F118C / C214S L128C / C220S F118C / C214S A129C / C220S F118C / C214S A141C / C220S Q124C / C214S F126C / C220S Q160C / C214S Q175C / C220S Q160C / C214S V173C / C220S L135C / C214S F170C / C220S N137C / C214S H170C / C220S N138C / C214S H168C / C220S S162C / C214S F170C / C220S S162C / C214S T187C / C220S T164C / C214S F170C / C220S S114C / Q160C / C214S S136C / V173C / C220S F118C / Q124C / C214S L128C / F126C / C220S F118C / Q124C / C214S A129C / F126C / C220S F118C / Q124C / C214S A141C / F126C / C220S F116C / Q124C / C214S S136C / F126C / C220S Q124C / T164C / C214S F126C / F170C / C220S Q124C / N138C / C214S F126C / H168C / C220S Q160C / L135C / C214S Q175C / F170C / C220S S114C / S162C / C214S S136C / F170C / C220S F118C / S162C / C214S L128C / F170C / C220S F118C / S162C / C214S A141C / F170C / C220S Q124C / S162C / C214S F126C / F170C / C220S N137C / S162C / C214S F170C / T187C / C220S

[0133] In some embodiments, the connection between the first heavy chain and the first light chain forming the first arm comprises two pairs of artificially introduced disulfide bonds formed by a combination of mutants selected from the group consisting of:

[0134] (i) S114C / Q160C on the light chain and S136C / V173C on the heavy chain;

[0135] (ii) F118C / Q124C on the light chain and L128C / F126C on the heavy chain;

[0136] (iii) F118C / Q124C on the light chain and A129C / F126C on the heavy chain;

[0137] (iv) F118C / Q124C on the light chain and A141C / F126C on the heavy chain;

[0138] (v) F116C / Q124C on the light chain and S136C / F126C on the heavy chain;

[0139] (vi) Q124C / T164C on the light chain and F126C / F170C on the heavy chain;

[0140] (vii) Q124C / N138C on the light chain and F126C / H168C on the heavy chain;

[0141] (viii) Q160C / L135C on the light chain and Q175C / F170C on the heavy chain;

[0142] (ix) S114C / S162C on the light chain and S136C / F170C on the heavy chain;

[0143] (x) F118C / S162C on the light chain and L128C / F170C on the heavy chain;

[0144] (xi) F118C / S162C on the light chain and A141C / F170C on the heavy chain;

[0145] (xii) Q124C / S162C on the light chain and F126C / F170C on the heavy chain; and

[0146] (xiii) N137C / S162C on the light chain and F170C / T187C on the heavy chain.

[0147] In some embodiments, the connection between the first heavy chain and the first light chain forming the first arm comprises two pairs of artificially introduced disulfide bonds formed by a combination of mutants selected from the group consisting of:

[0148] (i) F118C / Q124C on the light chain and L128C / F126C on the heavy chain;

[0149] (ii) Q124C / T164C on the light chain and F126C / F170C on the heavy chain;

[0150] (iii) F118C / S162C on the light chain and L128C / F170C on the heavy chain;

[0151] (iv) F118C / S162C on the light chain and A141C / F170C on the heavy chain; and

[0152] (v) Q124C / S162C on the light chain and F126C / F170C on the heavy chain.

[0153] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein include a heavy chain CH1 region that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline heavy chain CH1 region. In some embodiments, the human germline heavy chain CH1 region has the amino acid sequence set forth in SEQ ID NO: 5 provided herein.

[0154] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein include a light chain CL region that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline light chain CL region. In some embodiments, the human germline light chain CL region has the amino acid sequence set forth in SEQ ID NO: 4 provided herein.

[0155] Formation of bispecific antibodies

[0156] By repositioning the disulfide bonds formed by cysteine ​​pairs from the CH1 region and the CL region, the present disclosure provides modified antibodies whose heavy and light chains are paired with high assembly accuracy and stability.

[0157] As used herein, a "bispecific antibody" is an antibody that has binding specificities for at least two separate antigens or different epitopes within the same antigen.

[0158] In certain embodiments, the bispecific antibody comprises a first binding site for one epitope and a second binding site for another epitope. In certain embodiments, the two antigen targets are selected from the group consisting of: SIRPα, CLDN18.2, Siglec15, HER2, EGFR, CD19, CD20, CD39, CD47, PD1, PDL1, CD3, NKG2D, NKG2A, Nkp46, CD137, OX40, CD40, LILRB1, LILRB2, LILRB4, GPC3, TROP2, CD112, TIGIT, FAP, VEGFA, DLL4, ANG-2. In certain embodiments, one of the antigen binding arms of the bispecific antibody can target SIRPα, and the other antigen binding arm of the bispecific antibody can target Claudin 18.2.

[0159] In certain embodiments, the binding affinity of a bispecific antibody to two independent antigens (or epitopes on the same antigen) is approximately the same. In certain embodiments, the binding affinity of a bispecific antibody to two independent antigens (or epitopes on the same antigen) is different. In some embodiments, the affinity of a bispecific antibody to two independent antigens (or epitopes on the same antigen) can differ by 1, 2, 3, 4 or more times.

[0160] Mutations in the Fc region

[0161] The antibodies or antigen-binding fragments thereof disclosed herein also encompass Fc variants that may include one or more amino acid residue modifications or substitutions at the Fc region and / or hinge region.

[0162] In some embodiments, heterodimer pairing is achieved by engineering the Fc regions of the two heavy chains so that they exclusively form heterodimers. In some embodiments, mutations are introduced into the CH3 region of the Fc region.

[0163] In certain embodiments, the antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions in the interaction interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include introducing a protrusion into a first Fc polypeptide and introducing a cavity into a second Fc polypeptide, 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. Methods for producing antibodies with these modifications are known in the art, as described in U.S. Patent No. 5,731,168.

[0164] In some embodiments, a "knob" is produced by replacing one or more small amino acid side chains from the active interface of the first antibody molecule with a larger side chain (e.g., tyrosine or tryptophan). A compensatory "hole" of the same or similar size as the large side chain is produced on the active interface of the second antibody molecule by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). CH3 modifications for enhancing heterodimerization include, for example, Y407V / T366S / L368A on one heavy chain and T366W on the other heavy chain; S354C / T366W on one heavy chain and Y349C / Y407V / T366S / L368A on the other heavy chain. Table 4 provides such modifications that produce a protrusion on one chain and a cavity on the other chain.

[0165] Table 4. Mutations that form the knob and hole structures of Fc heterodimerization.

[0166] Modification in one CH3 region Modification in another CH3 region T366S / L368A / Y407V T366W S354C / T366W Y349C / T366S / L368A / Y407V T366Y Y407T T366W Y407A T394W F405A T366Y / F405A T394W / Y407T T366W / F405W T394S / Y407A F405W T394S D399C K392C T366W / D399C T3 66S / L368A / K392C / Y407V T366W / K392C T366S / L368A / D399C / Y407V S354C / T366W Y349C / T366S / L368A / Y407V Y349C / T366W S354C / T366S / L368A / Y407V E356C / T366W Y349C / T366S / L368A / Y407V Y349C / T366W E356C / T366S / L368A / Y407V E357C / T366W Y349C / T366S / L368A / Y407V Y349C / T366W E357C / T366S / L368A / Y407V

[0167] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein includes a first CH3 region and a second CH3 region, wherein the first CH3 region or the second CH3 region includes an amino acid sequence different from the wild-type IgG amino acid sequence, so 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 the corresponding positions in the CH3 region. Alternatively, the first CH3 region or the second CH3 region includes a van amino acid sequence different from the wild-type IgG amino acid sequence, so 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 the corresponding positions in the CH3 region. In some embodiments, the modifications in the two CH3 regions are selected from the groups listed in Table 5.

[0168] Table 5. Mutations that alter the charge polarity of Fc heterodimerization.

[0169] Modification in one CH3 region Modification in another CH3 region K370E / D399K / K439D D356K / E357K / K409D K409D D399K K409E D399K K409E D399R K409D D399R D339K E356K E356K / D399K K392D / K409D E356K / D399K K409D / K439D E357K / D399K K370D / K409D E356K / E357K / D399K K370D / K392D / K409D E357K / D399K K392D / K409D K392D / K409D D399K K360D / K409D D399K

[0170] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment thereof may be derived from human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the light chain constant region of the antibody or antigen-binding fragment thereof may be derived from human κ chain or human λ chain.

[0171] II. Conjugate

[0172] In some embodiments, the antibody or antigen-binding fragment thereof further comprises one or more conjugate moieties.In certain embodiments, the antibody or antigen-binding fragment thereof provided herein is used as the basis for a conjugate.

[0173] The conjugate part can be connected to an antibody or its Fab. The conjugate part is a part that can be connected to an antibody or its Fab. It is envisioned that a variety of conjugate parts can be connected to an antibody or its Fab provided herein (see, for example, "Conjugate Vaccines", "Contributions to Microbiology and Immunology", JM Cruse and RE Lewis, Jr. (eds.), Carcer Press, New York, (1989)). These conjugate parts can be connected to an antibody or its Fab by covalent bonding, affinity bonding, embedding, coordination bonding, compounding, association, blending or adding and other methods. In some embodiments, an antibody or its Fab can be connected to one or more conjugates by a linker.

[0174] III. Preparation method

[0175] The present disclosure provides polynucleotides encoding the antibodies or antigen-binding fragments thereof provided herein.

[0176] As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise indicated, a specific polynucleotide sequence also implicitly encompasses variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences thereof and sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acids Research 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0177] The nucleic acid or polynucleotide encoding the antibody or its antigen-binding fragment provided herein can be constructed using recombinant technology. For this reason, conventional procedures can be used (e.g., by using oligonucleotide probes that can bind to the genes of the heavy chain and light chain of the encoded antibody) to separate the DNA encoding the antigen-binding fragment (such as CDR or variable region) of the parent antibody and sequence it. As an example, obtain and operably connect the polynucleotide sequence encoding the variable domain (VH) and the polynucleotide sequence encoding CH1, CH2 and CH3 to allow transcription and expression in a host cell to produce a heavy chain polypeptide. Similarly, the polynucleotide sequence encoding VL is operably connected to the polynucleotide sequence encoding CL, thereby allowing the light chain to be expressed in a host cell.

[0178] Mutations can be introduced by various methods known in the art. In some embodiments, primers with designed mutations (eg, including point mutations, deletions of polynucleotide fragments, or insertions of polynucleotide fragments) can be used to introduce certain mutations using PCR techniques.

[0179] In some embodiments, polynucleotides with or without mutations can be obtained by synthetic methods. Chemical DNA synthesis methods are well known in the art. Generally, the method comprises the following steps. The 5' end of the first nucleotide is protected by dimethoxytrityl (DMT), and the linker of silica connects the OH end. The reactive groups of all nucleotides are chemically protected. Afterwards, the DMT is removed by washing, and the next nucleotide is activated and connected to the 3'-OH group. Using iodine, oxidize the 5' to 3' bond to produce a phosphotriester bond (one O of the phosphate group is methylated). The reaction continues until the desired chain length is reached. In this way, about 70-80 kinds of residual polymers can be prepared. The process also has an automated version, and conventional synthesis services are commercially available. The DNA encoding the antibody disclosed herein or its antigen-binding fragment is easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0180] The encoding polynucleotide sequence may further be operably linked to one or more regulatory sequences, optionally in an expression vector, so that expression or production of the heavy and light chains is feasible and under appropriate control.

[0181] The present disclosure provides a vector including a polynucleotide encoding the present invention. As used herein, the term "vector" refers to a medium into which a polynucleotide encoding a protein can be operably inserted so as to cause the expression of the protein. Typically, a construct also includes a suitable regulatory sequence. For example, a polynucleotide molecule may include a regulatory sequence located in the 5'-flanking region of a nucleotide sequence encoding a guide RNA and / or a nucleotide sequence encoding a site-directed modified polypeptide, and the regulatory sequence is operably connected to the coding sequence in a manner that the desired transcript / gene can be expressed in a host cell. The vector can be used to transform, transduce or transfect a host cell so that the genetic elements it carries are expressed in the host cell. Examples of vectors include plasmids, phagemids, clay mids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1, phages such as lambda phages or M13 phages, and animal viruses. The categories of animal viruses used as vectors include retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). The vector may include a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may include a replication origin. The vector may also include materials that assist its entry into cells, including but not limited to viral particles, liposomes, or protein coatings.

[0182] Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), λ phage and M13 phage, 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.

[0183] Vectors comprising polynucleotide sequences encoding antibodies or antigen-binding fragments thereof can be introduced into host cells for cloning or gene expression. As used herein, the term "host cell" refers to a cell into which exogenous polynucleotides and / or vectors have been introduced.

[0184] Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast cells or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include true bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example, Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium. typhimurium), Serratia, for example, Serratia marcescans and Shigella; and Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa and Streptomyces.

[0185] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among 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, e.g., 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.

[0186] Suitable host cells for expressing the glycosylated antibodies or antigen-binding fragments thereof 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 Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori. A variety of virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain variant of Bombyx mori NPV, and such viruses can all be used as viruses herein according to the disclosure, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.

[0187] In certain embodiments, the host cell is a vertebrate cell. 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 CRL 1651); 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 (BRL3A, ATCC CRL 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.

[0188] Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in a conventional nutrient medium modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0189] The disclosure also provides a method for expressing the antibody or its Fab 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 or its Fab provided herein can be cultivated in various 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 of the media described in 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. Pat. App. Rev. 30,985 can be used as culture medium for host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM Drugs), trace elements (defined as inorganic compounds with final concentrations generally 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.

[0190] When using recombinant technology, antibodies can be produced in intracellular, periplasmic space, or directly secreted into culture medium. If antibodies are produced intracellularly, then as a first step, the particulate fragments of the fragments of host cells or dissolution can be removed, for example, by centrifugation or ultrafiltration. Carter et al., " Bio / Technology (Bio / Technology) " 10:163-167 (1992) describes a procedure for separating antibodies secreted into the periplasmic space of Escherichia coli. In short, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) through about 30 minutes. Cell debris can be removed by centrifugation. When antibodies are secreted into culture medium, usually first commercially available protein concentration filters (for example, Amicon or Millipore Pellicon ultrafiltration units) are used to concentrate the supernatant from such expression systems. Protease inhibitors such as PMSF can be included in any step in the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0191] In certain embodiments, the present disclosure provides a method for producing an antibody or antigen-binding fragment thereof provided herein, the method comprising: a) introducing into a host cell: a first polynucleotide encoding a first heavy chain, a second polynucleotide encoding a first light chain, a third polynucleotide encoding a second heavy chain, and a fourth polynucleotide encoding a second light chain, wherein the first light chain and the first heavy chain comprise a non-cysteine ​​substitution pair of C214X located on the first light chain and C220X located on the first heavy chain and at least one cysteine ​​substitution pair; b) allowing the host cell to express the polypeptide complex. In certain embodiments, the at least one cysteine ​​substitution pair is selected from the group consisting of: (i) S114C on the light chain and S136C on the heavy chain; (ii) F116C on the light chain and S136C on the heavy chain; (iii) F118C on the light chain and L128C on the heavy chain; (iv) F118C on the light chain and A129C on the heavy chain; (v) F118C on the light chain and A141C on the heavy chain; (vi) Q124C on the light chain and F126C on the heavy chain; (viii) F118C on the light chain and A129C on the heavy chain; (iviv) F118C on the light chain and A141C on the heavy chain; (viv) F118C on the light chain and A129C on the heavy chain; (vii) Q124C on the light chain and F126C on the heavy chain; (viii) F118C on the light chain and A129C on the heavy chain; (viviv) F118C on the light chain and A141C on the heavy chain; (viii) F118C on the light chain and A129C on the heavy chain; (viv ... (ii) L135C on the light chain and F170C on the heavy chain; (viii) N137C on the light chain and F170C on the heavy chain; (ix) N138C on the light chain and H168C on the heavy chain; (x) T164C on the light chain and F170C on the heavy chain; (xi) Q160C on the light chain and V173C on the heavy chain; (xii) Q160C on the light chain and Q175C on the heavy chain; and (xiii) S162C on the light chain and F170C on the heavy chain.

[0192] In certain embodiments, the method further comprises isolating the antibody or antigen-binding fragment thereof.The term "isolated" is intended to mean that the compound of interest has been separated or purified from components that accompany it in nature or during preparation and is provided in an enriched form.

[0193] Antibodies or antigen-binding fragments thereof produced by the cells can be separated using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography.

[0194] In certain embodiments, protein A fixed on a solid phase is used for immunoaffinity purification of antibodies and antigen-binding fragments thereof. Whether protein A is suitable as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., Journal of Immunological Methods 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, a mechanically stable matrix (such as controlled pore glass or poly (styrene divinyl) benzene) can achieve faster flow rates and shorter processing times. When the antibody comprises a CH3 domain, Bakerbond ABX TM Resins (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 ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, SEPHAROSE TM Chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0195] Following any preliminary purification steps, the mixture comprising the antibody or antigen-binding fragment thereof of interest and contaminants can be subjected to hydrophobic interaction chromatography or ion exchange chromatography using a gradient elution of salt concentrations.

[0196] In certain embodiments, the antibody or its Fab provided herein can be easily purified with high yield using conventional methods. One of the advantages of the antibody or its Fab is that the mispairing between the heavy chain variable domain sequence and the light chain variable domain sequence is significantly reduced. This reduces the generation of unwanted by-products and makes it possible to obtain a high-purity product with high yield using a relatively simple purification process.

[0197] IV. Pharmaceutical compositions and administration

[0198] The present disclosure further provides pharmaceutical compositions comprising antibodies or antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.

[0199] The present disclosure further provides a pharmaceutical composition comprising a polynucleotide encoding an antibody or its antigen-binding fragment and one or more pharmaceutically acceptable carriers. The antibodies provided herein can also be produced in vivo by delivering polynucleotides encoding the antibodies or their antigen-binding fragments provided herein (e.g., in vitro transcribed mRNA or expression vectors). Methods for polynucleotide delivery of in vivo antibody expression are known in the art, see, for example, Rybakova, Y. et al., Molecular Therapy, Vol. 27 (8), pp. 1415-1423 (2019); Deal, CE et al., Vaccines, 2021, 9, 108.

[0200] The present disclosure further provides a pharmaceutical composition comprising an expression vector comprising a polynucleotide encoding an antibody or an antigen-binding fragment thereof and one or more pharmaceutically acceptable carriers.

[0201] In some embodiments, the expression vector comprises a viral vector or a non-viral vector. Examples of viral vectors include, but are not limited to, adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, and adenoviral vectors. Examples of non-viral vectors include, but are not limited to, naked DNA, plasmids, exosomes, mRNA, etc. In some embodiments, the expression vector is suitable for human gene therapy. Suitable vectors for gene therapy include, for example, adeno-associated virus (AAV) or adenoviral vectors. In some embodiments, the expression vector comprises a DNA vector or an RNA vector. In some embodiments, the pharmaceutically acceptable carrier is a polymer excipient, such as, but not limited to, microspheres, microcapsules, polymer micelles, and dendrimers. The polynucleotides and / or polynucleotide vectors disclosed herein can be encapsulated, adhered or coated on a polymer-based component by methods known in the art (see, e.g., W. Heiser, Nonviral gene transfer technologies, published by Humana Press, 2004; U.S. Pat. No. 6,025,337; Advanced Drug Delivery Reviews, 57(15):2177-2202 (2005)).

[0202] Pharmaceutically acceptable carriers for the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquids, gels or solid phase carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / partitioning agents, sequestering agents or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0203] Suitable components can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers, such as sugars and cyclodextrins. 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 and / or propyl gallate. As disclosed herein, including one or more antioxidants such as methionine in the composition including the antibody or its Fab and conjugate provided herein reduces the oxidation of the antibody or its Fab. This oxidation reduction can prevent or reduce the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Therefore, in certain embodiments, a pharmaceutical composition is provided, which comprises one or more antibodies or its Fab as disclosed herein and one or more antioxidants such as methionine. Further provided are methods for preventing oxidation, extending shelf life, and / or improving therapeutic efficacy of the antibodies or antigen-binding fragments provided herein by mixing the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0204] To further illustrate, pharmaceutically acceptable carriers may include, for example: aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; 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, methylparaben and propylparaben, 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.

[0205] 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.

[0206] In some embodiments, pharmaceutical composition is formulated into injectable composition.Injectable pharmaceutical composition can be prepared in any conventional form, such as liquid solution, suspension, emulsion or solid form suitable for producing liquid solution, suspension or emulsion.Injection preparation can include sterile and / or pyrogen-free solution prepared for injection, sterile dry soluble product (such as lyophilized powder, including subcutaneous injection tablets) prepared for injection before use with solvent combination, sterile suspension prepared for injection, sterile dry insoluble product prepared for use with vehicle combination before use and sterile and / or pyrogen-free emulsion.Solution can be aqueous or non-aqueous.

[0207] 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.

[0208] In certain embodiments, a sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment thereof as disclosed herein in a suitable solvent. The solvent may include an excipient that can improve the stability of the powder or a reconstituted solution prepared from the powder or other pharmacological components. Excipients that can be used include, but are not limited to, water, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may include a buffer such as citrate, sodium phosphate or potassium phosphate, or other such buffers known to those skilled in the art, and in one embodiment, the buffer is about neutral pH. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art to provide the desired 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 an antibody or antigen-binding fragment thereof or a composition thereof. It is acceptable to overfill the vial with an amount slightly higher than that required for each dose or multiple doses (e.g., about 10%) to facilitate accurate sampling and accurate dosing. The freeze-dried powder may be stored under appropriate conditions (e.g., at about 4°C to room temperature).

[0209] 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.

[0210] The pharmaceutical compositions described herein comprising a pharmaceutically acceptable carrier (such as an addition salt or hydrate thereof) can be delivered to a patient by a variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal and parenteral administration, including intramuscular, subcutaneous and intravenous injection. Preferably, the pharmaceutical compositions of the invention comprising an antibody or antibody fragment as a targeting moiety are administered parenterally, more preferably intravenously.

[0211] As used herein, the terms "administering" and "administration" are intended to encompass all means of direct and indirect delivery of a pharmaceutical composition to its intended site of action.

[0212] The pharmaceutical compositions described herein, or pharmaceutically acceptable salts and / or hydrates thereof, may be administered alone, and / or in combination with other therapeutic agents. Of course, the choice of therapeutic agents that may be co-administered with the pharmaceutical compositions of the present disclosure will depend in part on the condition being treated.

[0213] For example, when administered to a patient suffering from a disease state caused by an organism dependent on an autoinducer, the pharmaceutical compositions of the present disclosure can be administered in the form of a mixture containing agents for treating pain, infection, and other symptoms and side effects typically associated with the disease. Such agents include, for example, analgesics, antibiotics, and the like.

[0214] When administered to a subject undergoing cancer treatment, the pharmaceutical composition can be administered in the form of a mixture containing an anticancer agent and / or an adjuvant synergist. The pharmaceutical composition can also be administered in the form of a mixture containing agents for treating the side effects of radiotherapy (such as antiemetics, radioprotectants, etc.).

[0215] V. Reagent test kit

[0216] In another aspect, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof provided herein.

[0217] In certain embodiments, the disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent. In certain embodiments, the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anticancer drugs, radiotherapy, immunotherapeutic agents, anti-angiogenic agents, targeted therapy, cell therapy, gene therapy, hormone therapy, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

[0218] As will be apparent to those skilled in the art, such kits may further include one or more of the various conventional pharmaceutical kit components, such as a container with one or more pharmaceutically acceptable carriers, additional containers, etc., if desired. Instructions (as an insert or as a label) indicating the amounts of the components to be administered, administration instructions, and / or instructions for mixing the components may also be included in the kit. The kit may also include one or more vials, test tubes, flasks, bottles, or syringes.

[0219] Other formats of kits will be apparent to those skilled in the art and are within the scope of the present disclosure.

[0220] VI. Medical Uses

[0221] Therapeutic Uses

[0222] In another aspect, the present disclosure provides a method for treating a disease or condition in a subject in need of such treatment, the method comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0223] The disease or condition to be treated is related to the antigen targeted by the antibody or its antigen-binding fragment. In some embodiments, the targeted antigen is selected from the group consisting of SIRPα, CLDN18.2, Siglec15, HER2, EGFR, CD19, CD20, CD39, CD47, PD1, PDL1, CD3, NKG2D, NKG2A, Nkp46, CD137, OX40, CD40, etc.

[0224] In some embodiments, the use of the pharmaceutical compositions disclosed herein includes the preparation of a medicament for treating a disease (such as cancer) in a subject (such as a human). "Cancer" herein refers to a pathological condition in humans characterized by unregulated cell proliferation.

[0225] In some embodiments, the disease is a SIRPα-related disease, disorder or condition characterized by expression or overexpression of SIRPα and / or SIRPα signature genes. In certain embodiments, SIRPα-related diseases, disorders or conditions include, but are not limited to, cancer, solid tumors, chronic infections, inflammatory diseases, multiple sclerosis, autoimmune diseases, nervous system diseases, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant dysfunction or arthritis.

[0226] In some embodiments, the disease is a Claudin 18.2-associated disease, disorder or condition characterized by expression or overexpression of Claudin 18.2. Non-limiting examples of Claudin 18.2-associated diseases include cancer. In some embodiments, the cancer is an epithelial cell-derived cancer. In some embodiments, the cancer is anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma (non-Hodgkin lymphoma), In some embodiments, the cancer is gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer or its metastasis.

[0227] The term "treating" or "treatment" as used herein refers to both therapeutic treatment and prophylactic or preventative treatment, wherein the objective is to reduce or prevent a target pathological condition or symptom. In certain embodiments, "treatment" or "treating" includes (1) inhibiting a disease in a subject experiencing or displaying the pathology or symptoms of a disease; (2) ameliorating a disease in a subject experiencing or displaying the pathology or symptoms of a disease; and / or (3) achieving any measurable reduction in a disease in a subject or patient experiencing or displaying the pathology or symptoms of a disease.

[0228] The term "therapeutically effective amount" means in this article that the active ingredient (i.e., antibody or antigen binding fragment thereof) of the pharmaceutical composition provided herein is effective in "treating" the amount of the condition of a subject or mammal. Pharmaceutical compositions suitable for use in the present disclosure include compositions in which the active ingredient is in a therapeutically effective amount, i.e., in an amount that effectively achieves its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. The determination of an effective amount is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosure herein.

[0229] In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells, reduce tumor size, inhibit cancer cell infiltration into peripheral organs, inhibit tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer.

[0230] In some embodiments, the antibody or its antigen binding fragment provided herein can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibody or its antigen binding fragment disclosed herein can be administered in combination with a second therapeutic agent, such as a chemotherapeutic agent, an anticancer drug, radiotherapy, an immunotherapeutic agent, an anti-angiogenic agent, targeted therapy, cell therapy, gene therapy, hormone therapy, antiviral agent, antibiotic, analgesic, antioxidant, metal chelator or cytokine.

[0231] In certain of these embodiments, an antibody or antigen-binding fragment thereof provided herein administered in combination with one or more additional therapeutic agents can be administered simultaneously with one or more additional therapeutic agents, and in certain of these embodiments, the antibody or antigen-binding fragment thereof and the additional therapeutic agent can be administered as part of the same pharmaceutical composition.

[0232] Diagnostic Uses

[0233] In another aspect, the present disclosure provides a method for diagnosing a disease, disorder or condition in a subject, the method comprising: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; b) determining the presence or amount of an antigen targeted by the antibody or antigen-binding fragment thereof in the sample; and c) correlating the presence or amount of the relevant antigen with the presence or status of the disease, disorder or condition in the subject.

[0234] The terms "diagnosis", "diagnose" or "diagnosing" refer to the identification of a pathological state, disease or condition, such as the identification of a specific antigen-associated disease, or to the identification of a subject with a specific antigen-associated disease who may benefit from a particular treatment regimen. In some embodiments, diagnosis includes identifying an abnormal amount or activity of a specific antigen. In some embodiments, diagnosis refers to identifying a cancer or autoimmune disease in a subject.

[0235] As used herein, the term "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. 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.

[0236] In another aspect, the present disclosure provides a kit, comprising an antibody or antigen-binding fragment thereof provided herein, the antibody or antigen-binding fragment thereof optionally conjugated to a detectable portion, the kit being useful for detecting a disease, disorder or condition. The kit may further include instructions for use.

[0237] 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 present disclosure 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 abilities 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 disclosure. It is the intention of the inventor of the present invention that such changes are all included within the scope of the present invention.

[0238] All amino acid position numbering in the following examples is according to the EU numbering system.

[0239] Example:

[0240] Example 1: Construction and analysis of asymmetric monovalent antibodies with shifted CH1-CL interchain disulfide bonds

[0241] Multiple potential sites for new disulfide bond formation on the antibody were identified. Potential sites were selected from amino acid residues in the CL region of the light chain and in the CH1 region of the heavy chain. A pair of non-cysteine ​​residues (one in the CL region and the other in the CH1 region) were both replaced with cysteine ​​to form a new disulfide bond therebetween. One or two pairs of such residues that could potentially form a new interchain disulfide bond were selected for each design and mutated to cysteine.

[0242] In addition, the conserved native disulfide bond between Cys220 in CH1 and Cys214 in CL was disrupted by replacing both residues with serine. For the initial screening, an assay system that can be easily read by SEC-HPLC was designed ( Figure 1 and Figure 2 ).

[0243] Specifically, the system consists of 4 polypeptide chains. Chain 1 is a full-length HC with a cysteine ​​mutation in the CH1 region (from a non-cysteine ​​residue to a cysteine ​​residue) (chain 1a is used for screening assay 1, Figure 1 ) or without cysteine ​​mutation (chain 1b for screening assay 2, Figure 2), and each chain 1 has a knob or hole mutation in the Fc region. Chain 2 is a full-length LC with a compatible cysteine ​​mutation in the CL region. Chain 3 is a wild-type LC with a single domain antibody (VHH) fused at the N-terminus. Chain 4 is a null Fc with a hole or knob mutation compatible with the knob or hole mutation in the full-length HC (i.e., chain 1a or chain 1b). In the case of this system, the major product (which is a monovalent heterodimer) consists of two possible molecular species that differ in size and can be easily distinguished and easily quantified in the SEC-HPLC profile ( Figure 1 and Figure 2 ). In both screening assays, heterodimers formed between mutant and wild-type peptide chains were considered mismatched heterodimers, whereas heterodimers between designed mutant chains or between wild-type chains were considered correctly matched heterodimers. The relative abundance (RA) of different heterodimers is a direct measure of the pairing preferences of different CL and CH1 chains.

[0244] The sequences of the different polypeptide chains of the two assay systems are listed in Table 6 below. The VH (SEQ ID NO: 3) and VL (SEQ ID NO: 2) sequences are derived from humanized anti-CLDN18.2 antibodies. The heavy chain constant region, including the CH1 region (SEQ ID NO: 5) and the Fc region (SEQ ID NO: 6), is derived from human IgG1. The light chain constant region CL (SEQ ID NO: 7) is derived from human kappa light chain. The VHH domain (SEQ ID NO: 1) is connected at the N-terminus of the VL region by a linker (SEQ ID NO: 9). All DNA fragments were chemically synthesized. Each of the four polypeptide chains was cloned into the pcDNA3.4 vector ( Figures 3A to 3D ). The four plasmids were mixed and co-transfected into ExpiCHO-S cells (Life Technologies). The expressed protein variants were then produced in ExpiCHO-S cells using a 24-well plate production system. The culture supernatant was collected by centrifugation 7 days after transfection and incubated with protein A magnetic beads (GenScript). The bound antibodies were eluted with 0.05M citric acid buffer (pH 3.4) and immediately neutralized with Tris buffer (pH 9.0). The purified antibodies were evaluated by SDS-PAGE and SEC-HPLC (Agilent).

[0245] Table 6. Amino acid sequences of polypeptide chains used in screening assays 1 and 2

[0246]

[0247]

[0248] One or two non-cysteine ​​residues at various positions (including positions 114, 116, 118, 121, 124, 135, 137, 138, 160, 164) in the CL region of chain 2 are mutated to cysteine, and one or two non-cysteine ​​residues at various positions (including positions 126, 128, 129, 136, 141, 168, 170, 173, 175, 187) in the CH1 region of chain 1 are mutated to cysteine. Multiple mutation pairs between chain 1 and chain 2 are formed using the above mutation sites, and the multiple mutation pairs are used in screening assay 1.

[0249] In screening assay 1, wild-type and mutant CL compete for mutant CH 1. Correct pairing is formed between chain 2 (with mutant CL) and chain 1a (with mutant CH 1), while mismatch is formed between chain 3 (with wild-type CL) and chain 1a (with mutant CH 1).

[0250] In screening assay 1, many combinations of mutations of VL-CL (with mutations in the CL region) and VH-CH1-Fc (with mutations in the CH1 region and knob / hole mutations in the Fc region) were tested. Table 7 and Figure 4A RA values ​​for some mutant pairs that produce high RA values ​​are shown. ACF_003 was used as a benchmark in this screening assay 1. ACF_003 (comprising a mutation combination of S121C on the light chain and F126C on the heavy chain) has corresponding mutations of V12 in the CL and CH1 regions as disclosed in U.S. Pat. No. 9,527,927.

[0251] Table 7 Combinations of mutations tested in screening assay 1 showing high RA values

[0252]

[0253]

[0254]

[0255] In screening assay 2, mutant and wild-type CL competed for wild-type CH1. The RA values ​​of the mutant pairs used in screening assay 2 were calculated by the same equation as in screening assay 1, except that the correct pairing was formed only between chain 3 (with wild-type CL) and chain 1b (with wild-type CH1), and the mismatch was formed between chain 2 (with mutant CL) and chain 1b (with wild-type CH1).

[0256] Mutation pairs that produce high RA values ​​are listed in Table 8. ACF_245 was used as a benchmark in this screening assay 2. ACF_245 (including the S121C mutation on the light chain and no mutation on the heavy chain) has mutations in the CL region corresponding to ACF_003, except that there are no mutations on the heavy chain. Table 8 and Figure 4B The RA of some mutant pairs that yielded high RA values ​​are shown.

[0257] Table 8 Combinations of mutations tested in screening assay 2 showing high RA values.

[0258]

[0259]

[0260] Example 2: Construction and analysis of symmetrical bivalent monospecific antibodies with offset CH1-CL disulfide bonds

[0261] To further evaluate the expression level and stability of the selected mutants in the reductionist system, some mutant pairs selected from the experiments in Example 1 were converted into symmetrical bivalent monospecific antibodies ( Figure 5 , Table 9) for yield and purity testing.

[0262] All mutations were generated based on the parent antibody hu28. The amino acid sequences of the VL region and VH region of hu28 are shown in SEQ ID NOs: 2 and 3, respectively. The amino acid sequence of the CL region of hu28 is shown in SED ID NO: 4. The amino acid sequence of the CH1 region of hu28 is shown in SED ID NO: 5. The amino acid sequence of the Fc region of hu28 is shown in SED ID NO: 6.

[0263] Table 9. Combinations of mutations in bivalent monospecific antibodies tested.

[0264]

[0265]

[0266] All DNA fragments were synthesized and cloned into the pcDNA3.4 vector. The LC and HC vectors containing the selected residue substitution pairs were co-transfected into ExpiCHO-S cells using a 24-well plate production system and purified using protein A beads. The purity of the purified antibodies was first assessed by SDS-PAGE and HPLC-SEC. Fig. 6A and 6B The yield and purity of each antibody are shown in . ACF-hu28_001 containing the residue substitution pair reported by MedImmune in US Pat. No. 9,527,927 was used as a reference for comparison.

[0267] like Fig. 6A and 6B The results in 4. ACF-hu28_032, ACF-hu28_014, ACF-hu28_016, ACF-hu28_012, ACF-hu28_011, ACF-hu28_004, ACF-hu28_015, ACF -hu28_020, ACF-hu28_035, ACF-hu28_022, ACF-hu28_018, ACF-hu28_036, ACF-hu28_010, ACF-hu28_030, ACF-hu28_039, ACF-hu28_019, ACF-hu28_024, ACF-hu28_028, ACF-hu28_023, ACF-hu28_005, and ACF-hu28_003 have comparable or higher yields and monomer percentages than the reference monoclonal antibody ACF hu28_001 (S121C / C214S, F126C / C220S).

[0268] Example 3. Antigen binding measurements and thermal stability comparisons of bivalent monospecific antibodies

[0269] 3.1 Evaluation of binding activity by FACS assay

[0270] Using MC38 cells overexpressing CLDN18.2, all selected bivalent variants were compared for binding to CLDN18.2 by FACS ( Figures 7A to 7D The buffer of all proteins was exchanged into PBS (pH 7.2) and diluted into different concentrations (100 nM, 20 nM, 4 nM, 0.8 nM) with FACS buffer (BD). Hu28 mAb was used as a positive control.

[0271] No significant differences in antigen binding activity were observed between the variants and the positive control mAb.

[0272] 3.2 Thermal stability analysis of mutant mAbs

[0273] The thermal stability of the purified variants was assessed using a thermal shift assay ( Figure 8). The buffer of the antibody variants was first exchanged into PBS (pH 7.2) before mixing with freshly diluted protein thermal shift dye (Thermo Fisher Scientific). The mixture was then transferred to a 384-well plate and loaded onto a QuantStudio Real-Time PCR System (Thermo Fisher Scientific) for Tm measurement. The temperature was scanned from 25°C to 99°C with a ramp rate of 1.6°C / sec and a hold time of 2 minutes. The obtained melting curves were analyzed with protein thermal shift software. The data showed that all variants had comparable thermal stability to the wild-type monoclonal antibody.

[0274] Example 4. Production and LC-MS analysis of asymmetric bispecific antibodies

[0275] To quantitatively assess the efficiency of cognate CH1 and CL chain pairing in a bispecific context, two series of bispecific antibodies were generated and analyzed by LC-MS.

[0276] In both series, one arm of the bispecific antibody was derived from an anti-CLDN 18.2 antibody (hu28) and the other arm was derived from an anti-SIRPα antibody (hu25). The knob mutation was introduced into the CH3 region of the hu28 arm, and the hole mutation was introduced into the CH3 region of the hu25 arm. In addition, a mutation that abolished protein A binding was introduced into the hu25 arm to simplify the system.

[0277] The amino acid sequences of the VH and VL regions of hu25 are shown in SEQ ID NOs: 10 and 11, respectively (as shown in Table 10). The amino acid sequences of the VH and VL regions of hu28 are shown in SEQ ID NOs: 2 and 3, respectively. The amino acid sequence of the non-mutated CL region of hu25 or hu28 is shown in SEQ ID NO: 4. The amino acid sequence of the non-mutated CH1 region of hu25 or hu28 is shown in SEQ ID NO: 5. The amino acid sequence of the Fc of hu25 or hu28 (with a "hole" mutation) is shown in SEQ ID NO: 7. The amino acid sequence of the Fc of hu25 or hu28 (with a "knob" mutation) is shown in SEQ ID NO: 8.

[0278] Table 10. Amino acid sequence of hu25.

[0279]

[0280] In case 1 study, different pairs of mutations that shifted disulfide bond formation were introduced into the CH1 and CL regions of the hu28 arm, and the wild-type sequence was retained in the hu25 arm ( Fig. 9 and 10 , Table 11).

[0281] In case 2 study, the same set of mutation pairs was introduced into the hu25 arm, and the wild-type sequence was retained in the hu28 arm ( Fig.11 and 12 , Table 11).

[0282] Table 11. Different construct designs in Case 1 and Case 2

[0283]

[0284] All mutation combinations constructed for testing are shown in Table 12.

[0285] Table 12. Combinations of mutations in Case 1 and Case 2 studies used for testing.

[0286]

[0287]

[0288] To generate bispecific antibodies, plasmids of hu28 HC and LC were mixed with plasmids of hu25 HC and LC before co-transfection. Variants were produced in ExpiCHO-S cells (Life Technologies) using a 24-well plate production system. The culture supernatant was collected by centrifugation 7 days after transfection and incubated with protein A magnetic beads (GenScript). The bound antibodies were eluted with 0.05 M citrate buffer (pH 3.4) and immediately neutralized with Tris buffer (pH 9.0). The purified antibodies were first analyzed by SDS-PAGE ( Fig.13A and 13B ).

[0289] In order to quantitatively evaluate the efficiency of homologous CH1 and CL chain pairing, the purified antibodies were analyzed by LC-MS under non-reducing conditions. Due to the denaturing nature of the sample processing conditions in the LC-MS analysis, molecules containing mismatched HC-LC (CH1 mutated HC paired with wild-type LC, or HC without CH1 modification paired with CL mutated LC) will be dissociated and only the LC peak will be shown in the LC-MS spectrum. Therefore, in theory, all molecules containing heterodimeric HCs will be detected. Heterodimeric HCs with two correctly paired LCs are the desired bispecific intact antibodies. Heterodimeric HCs with mismatched LCs are detected by mismatched and dissociated LC peaks in the LC-MS spectrum. By evaluating the intensity of the MS signal of the LC peak only derived from the CH1-CL mismatch, the homologous CH1 and CL chain pairing efficiencies of different mutation pairs can be ranked ( Fig. 9 and 11 ).

[0290] The thermal stability of the antibodies was assessed by differential scanning fluorimetry.

[0291] In the case 1 study, hu025 LCs of ACF_329, 333, 337, 338, and 339 exhibited lower signals, and hu028 LCs exhibited equivalent signals compared to the benchmark ACF_326 (designed by MedImmune). Fig.10 ). These data indicate that the mutation pairs introduced in ACF_329, 333, 337, 338 and 339 provide higher efficiency of pairing of cognate CH1 and CL chains in this case.

[0292] In the case 2 study, ACF_356, 359, 361, 364, 365, 366, 367, 368 showed signals of hu025 LC and hu028 LC comparable to the benchmark ACF_355 (designed by MedImmune) ( Fig.12 ). These data indicate that the mutation pairs introduced in ACF_356, 359, 361, 364, 365, 366, 367 and 368 provide high homologous CH1 and CL chain pairing efficiency.

[0293] Notably, the mutation pairs listed below provided high cognate CH1 and CL chain pairing efficiency in both cases: (i) F118C / Q124C on the light chain and L128C / F126C on the heavy chain; (ii) Q124C / T164C on the light chain and F126C / F170C on the heavy chain; (iii) F118C / S162C on the light chain and L128C / F170C on the heavy chain; (iv) F118C / S162C on the light chain and A141C / F170C on the heavy chain; and (v) Q124C / S162C on the light chain and F126C / F170C on the heavy chain.

[0294] Example 5. Further generation and analysis of asymmetric bispecific antibodies

[0295] 5.1 Protein expression and purification

[0296] 5.1.1 Constructs and amino acid sequences of the tested antibodies

[0297] Three bispecific antibodies, Antibody 1, Antibody 2, and Antibody 3, were expressed and purified.

[0298] In the antibody 1 study, the mutation pair of Q124C / T164C / C214S located on the light chain and F126C / F170C / C220S located on the heavy chain was introduced into the hu25 arm, and the wild-type sequence was retained in the hu28 arm. Antibody 1 has the same structure and amino acid sequence as antibody ACF_361 in Example 4. Therefore, it is indicated as ACF_361 or ACF361 hereinafter.

[0299] In the antibody 2 study, the mutation pair of Q124C / T164C / C214S located on the light chain and F126C / F170C / C220S located on the heavy chain was introduced into the hu25 arm, and the wild-type sequence was retained in the hu29 (source sequence: Atezolizumab, an anti-PDL1 antibody) arm. Antibody 2 is indicated as ACF_389 or ACF389 hereinafter.

[0300] The amino acid sequences of the VH region and VL region of hu25 are shown in SEQ ID NOs: 10 and 11, respectively (Table 10). The amino acid sequences of the VH region and VL region of hu29 are shown in SEQ ID NOs: 12 and 13, respectively (Table 11). The amino acid sequence of the non-mutated CL region of hu25 or hu29 is shown in SEQ ID NO: 4. The amino acid sequence of the non-mutated CH1 region of hu25 or hu29 is shown in SEQ ID NO: 5. The amino acid sequence of the Fc of hu25 or hu29 (with a "hole" mutation) is shown in SEQ ID NO: 7. The amino acid sequence of the Fc of hu25 or hu29 (with a "knob" mutation) is shown in SEQ ID NO: 8.

[0301] Table 11. Amino acid sequences of the heavy chain variable region and light chain variable region of hu29

[0302]

[0303] In the Antibody 3 study, the mutation pair of Q124C / S162C / C214S located on the light chain and F126C / F170C / C220S located on the heavy chain was introduced into the H95 arm, and the wild-type sequence was retained in the H43 arm. Antibody 3 is hereinafter indicated as ESB07.451.

[0304] The amino acid sequences of the VH region and VL region of H95 are shown in SEQ ID NOs: 14 and 15, respectively. The amino acid sequences of the VH region and VL region of H43 are shown in SEQ ID NOs: 16 and 17, respectively. The amino acid sequence of the non-mutated CL region of H95 or H43 is shown in SEQ ID NO: 19. The amino acid sequence of the non-mutated CH1 region of H95 or H43 is shown in SEQ ID NO: 18. The amino acid sequence of the Fc of H43 (with a "hole" mutation) is shown in SEQ ID NO: 21. The amino acid sequence of the Fc of H95 (with a "knob" mutation) is shown in SEQ ID NO: 20.

[0305] Table 12. Amino acid sequences of H95 and H43

[0306]

[0307] 5.1.2 Expression and purification of test antibodies

[0308] To generate bispecific antibodies, plasmids of hu28 HC and LC were mixed with plasmids of hu25 HC and LC, plasmids of hu29 HC and LC were mixed with plasmids of hu25 HC and LC, and plasmids of H95 HC and LC were mixed with plasmids of H43 HC and LC before co-transfection.

[0309] Variants were produced in ExpiCHO-S cells (Life Technologies). Seven days after transfection, culture supernatants were collected by centrifugation and incubated with alkali-resistant protein A medium (AT protein ADiamond, Bestchrom). The bound antibodies were eluted with 0.05M citric acid buffer (pH 3.4) and immediately neutralized with Tris buffer (pH 9.0). The purified antibodies were first analyzed by SDS-PAGE and SEC. Fig.14 , Fig.17 and Fig. 22 Results for ACF_361, ACF_389 and ESB07.451 are shown respectively.

[0310] For ACF_389 and ESB07.451, in order to obtain purer samples, Mono S chromatography (5 / 50 GL, Cytiva) was used. The antibodies were loaded and washed with buffer A (10 mM sodium acetate, pH 5.2), and then eluted with a buffer B gradient (10 mM sodium acetate, pH 5.2, 250 mM NaCl). Fig.18 and 19 The results for ACF_389 are shown in . Fig. 22 and 23 The results for ESB07.451 are shown in .

[0311] To quantitatively assess the efficiency of cognate CH1 and CL chain pairing, purified antibodies were analyzed by LC-MS under non-reducing conditions. Fig.15 , Fig. 20 and Fig.24 Results for ACF_361, ACF_389 and ESB07.451 are shown, respectively. According to the LC-MS results, the peak near 145 KD is the homologous CH1 / CL pairing, which indicates that the three antibodies are correctly assembled.

[0312] 5.2 Binding affinity determination

[0313] 5.2.1 Binding affinity to cells overexpressing CLDN18.2 and SIRPα

[0314] The dual binding properties of the bispecific antibody ACF_361 to cells were analyzed by FACS.

[0315] 1×10 5 MC38 cells expressing CLDN18.2 were incubated with 100 nM to 0.05 nM of each purified antibody for 1 hour, and then incubated with the secondary antibody Alexa Flour 647 anti-human IgG (H+L).

[0316] In this assay, ES028-h26, ES028-h28, AE016-201, and ES028-005-08-26H1L2-201 were used as positive controls, and hIgG1 (an isotype control that does not specifically bind to CLDN18.2 or SIRPα, the same below) was used as a negative control. Fig.16A The information of each antibody tested is shown in . Both ES028-h26 and ES028-h28 are monospecific IgG antibodies targeting CLDN18.2. AE016-201 is a bispecific antibody targeting CLDN18.2 and SIRPα (wherein the scFv is linked to the IgG). ES028-005-08-26H1L2-201 is a bispecific antibody targeting CLDN18.2 and SIRPα (wherein the scFv is linked to the IgG).

[0317] Fig.16A The binding curves of the antibodies to MC38-CLDN18.2 cells are shown in FIG.

[0318] 1×10 5 CHO / SIRPαV1 cells were incubated with 100 Nm to 0.05 Nm of each purified antibody for 1 hour, and then incubated with the secondary antibody Alexa Flour 647 anti-human IgG (H+L).

[0319] In this assay, ES001-025.201-IgG1LALA, ES028-005-08-26H1L2-201, AE016-201 were used as positive controls and the hIgG1 isotype was used as a negative control. Fig. 16B The information of each antibody tested is shown in . ES001-025.201-IgG1LALA is a monospecific IgG antibody targeting SIRPα, with a LALA mutation on the Fc region of the antibody. ES028-005-08-26H1L2-201 and AE016-201 are both bispecific antibodies targeting CLDN18.2 and SIRPα (with scFv linked to IgG).

[0320] Fig. 16BThe binding curve of the antibody to CHO / SIRPαV1 cells is shown in FIG.

[0321] 5.2.2 Binding affinity to cells overexpressing PDL1 and SIRPα

[0322] The dual binding properties of the bispecific antibody ACF_389 to cells were analyzed by FACS. 5 PDL1-expressing Raji cells were incubated with 100 nM to 0.05 nM of each purified antibody for 1 hour, and then incubated with the secondary antibody Alexa Flour 647 anti-human IgG (H+L).

[0323] In this assay, atezolizumab was used as a positive control, and ES004-025.201-IgG4, hIgG1, and hIgG4 (isotype controls that do not specifically bind to CLDN18.2 or SIRPα, the same below) were used as negative controls. Atezolizumab is a known anti-PDL1 antibody, detailed information of which can be found in, for example, US9873740B2. ES004-025.201-IgG4 is a monospecific antibody targeting SIRPα.

[0324] Fig.21A The binding curves of antibodies to Raji / PDL1 cells are shown in FIG.

[0325] 1×10 5 CHO / SIRP V1 cells were incubated with 100 nM to 0.05 nM of each purified antibody for 1 hour, and then incubated with the secondary antibody Alexa Flour 647 anti-human IgG (H+L).

[0326] In this assay, ES004-025.201-IgG4 was used as a positive control, and atezolizumab, hIgG1, and hIgG4 were used as negative controls. The information of the antibodies is described above.

[0327] Fig.21B The binding curve of the antibody to CHO / SIRPαV1 cells is shown in FIG.

[0328] 5.3 Phagocytic activity of bispecific antibodies

[0329] The phagocytic activity of ACF_361 was tested in this assay.

[0330] To obtain a single-cell suspension, microphages were digested with cell digestion solution at 37°C for 10 min, and then cell culture medium was added to stop the digestion. After centrifugation at 400 g for 5 min, macrophages were centrifuged at 3 × 10 4 / well were re-seeded in 96-well plates and recovered overnight at 37°C. Mouse BMDM cells were stained with CD11b and F4 / 80 to assess quality and purity. The next day, 25 μl of diluted Abs with concentrations ranging from 0.05 nM to 10 nM and 50 μl of 6 × 10 4 Violet CSFE labeled Raji-CLDN18.2 cells / well and incubated at 37°C for 3 hours. Wash the cells with DPBS and digest with cell digestion solution at 37°C for 10 minutes, and then wash the digested cells with FACS buffer. Incubate the cells with anti-mouse F4 / 80APC at 4°C for 30 minutes. Wash and suspend the stained cells with FACS buffer. Determine the percentage of phagocytosis by FACS.

[0331] In this assay, ES028-005-08-26H1L2-201 and AE016-201 were used as positive controls, and hIgG1+hIgG4, anti-SIRPα monospecific antibodies, two anti-CLDN18.2 antibodies (anti-CLDN18.2.h26 and anti-CLDN18.2.h28) and combinations were used as negative controls. Both ES028-005-08-26H1L2-201 and AE016-201 are bispecific antibodies targeting CLDN18.2 and SIRPα (wherein scFv is linked to IgG). Equimolar anti-CLDN18.2 hu26 (hIgG1) and anti-SIRPα hu25.060 (hIgG4) were added as a combined treatment group (ie, combination). Equimolar hIgG1 and hIgG4 were added as isotype control groups (ie, hIgG1+hIgG4).

[0332] Fig. 16C The summary data is shown in .

[0333] 5.4 Hydrophobicity evaluation

[0334] The retention time in HIC-HPLC determination is a factor for evaluating the hydrophobicity of the antibody. 15 μl of 0.25 mg / ml antibody was injected into the MacPac-10 HIC column in the Agilent 1260 system, and the signal at 280 nm / 214 nm was detected. As shown in Table 13, the retention times of ACF_361, ACF_389 and ESB07.451 in MacPac-10 HIC were 8.413 minutes, 13.583 minutes and 9.9 minutes, respectively, indicating that the hydrophobicity of the three bispecific antibodies was low.

[0335] Table 13. Retention time of antibodies in HIC-HPLC assay

[0336] Sample name RT-HIC-UPLC (minutes) ACF_361 8.413 ACF_389 13.583 ESB07.451 9.9

[0337] 5.5 Stability Test

[0338] 5.5.1 Thermal stability evaluation

[0339] The melting temperature (Tm) value of the bispecific antibody in PBS buffer was detected to predict its thermal stability. In short, the bispecific antibody was dissolved in 10mM PBS buffer (pH 7.2-7.4). The Tm value was then detected by differential scanning fluorimetry (DSF) using QuantStudio 7Flex real-time PCR system. As shown in Table 14, the Tm values ​​of ACF_361, ACF_389 and ESB07.451 indicate that the bispecific antibody has good thermal stability.

[0340] Table 14. Tm values ​​of antibodies

[0341] Sample name Tm1-DSF(℃) Tm2-DSF(℃) ACF_361 68.7 80.7 ACF_389 68.7 84.8 ESB07.451 69.2 74.3

[0342] 5.5.2 Low pH stability assessment

[0343] Good stability under low pH conditions is important for antibodies because the purification process usually involves exposure to acidic solution conditions and commonly used low pH virus inactivation. Low pH values ​​usually produce soluble, insoluble aggregates and accelerate fragmentation. Therefore, in order to assess the stability of proteins under low pH conditions, the bispecific antibodies in PBS buffer were titrated to pH 3.0-3.5 with acetic acid. The stability of the antibody was then assessed by monitoring the total soluble protein (recovery) and purity changes before and after being exposed to low pH solutions for 2 hours.

[0344] The results are shown in Table 15, where T0 indicates the data of the original sample, and Low pH 2 hours indicates the data at low pH after 2 hours. It can be seen that exposure to a low pH solution for 2 hours causes almost no change in overall purity, indicating that all three antibodies ACF_361, ACF_389, and ESB07.451 have good stability under low pH conditions.

[0345] Table 15. Low pH stability of antibodies

[0346]

[0347] 5.5.3 Stability under freeze-thaw stress

[0348] Freeze-thaw stability is often explored to determine the sensitivity of antibodies to temperature cycles to which the product is often exposed. For example, drug substances are usually frozen to enable long-term storage. As part of the freeze-drying process, drug products may be exposed to freezing temperatures. The main degradation pathway of freeze-thaw is aggregates, including precipitates, particles, and soluble particles. Therefore, the freeze-thaw stability of bispecific antibodies is evaluated by monitoring the purity changes before and after repeated freeze-thaw treatments. In short, the antibodies in 20mM PBS (pH 6.0-6.2) were frozen to -80°C for more than 12 hours and then thawed at room temperature. After 5 repeated freeze-thaw cycles, the concentration and purity of the stress-treated samples were determined.

[0349] The results are shown in Table 16, where T0 indicates the data of the original sample and FT5 indicates the data after five freeze-thaw cycles. It can be seen that ACF_361, ACF_389 and ESB07.451 did not show obvious aggregation and fragmentation increase after multiple freeze-thaw cycles, indicating that all three antibodies have good freeze-thaw stability.

[0350] Table 16: Antibody stability under freeze-thaw stress

[0351]

[0352] 5.5.4 Stability under thermal stress

[0353] Thermal stress at temperatures exceeding normal storage conditions may accelerate degradation, thereby increasing the detectability of potential degradation pathways to provide information about long-term degradation under expected storage conditions. Thermal stress testing of bispecific antibodies in PBS was performed at 25°C and / or 40°C for 4 weeks. SEC purity was tested to monitor insoluble or soluble aggregates.

[0354] The results are shown in Table 17, where T0 indicates data of the original sample, 25C-2W indicates data for 2W below 25°C, and other naming conventions are similar. It can be seen that after incubation at 25°C for 4 weeks, the purity of ACF_361 and ACF_389 in PBS did not change much, and after incubation at 25°C and 40°C for 4 weeks, the purity of ESB07.451 in PBS did not change much.

[0355] Table 17. Antibody stability under thermal stress

[0356]

[0357]

Claims

1. An antibody or an antigen-binding fragment thereof, comprising a first arm and a second arm, wherein the first arm is formed by a first heavy chain and a first light chain and an interchain disulfide bond formed therebetween, and the second arm is formed by a second heavy chain and a second light chain and an interchain disulfide bond formed therebetween, wherein at least one non-cysteine ​​residue on the first heavy chain is substituted with cysteine, wherein the non-cysteine ​​residue is located at a position selected from the group consisting of: amino acid position 126, 128, 129, 136, 141, 168, 170, 173, 175 or 187 of the first heavy chain; and At least one non-cysteine ​​residue on the first light chain is substituted with cysteine, wherein the non-cysteine ​​residue is located at a position selected from the group consisting of: amino acid position 114, 116, 118, 124, 135, 137, 138, 160, 162 or 164 of the first light chain.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The at least one non-cysteine ​​residue on the first heavy chain that is substituted with cysteine ​​is selected from the group consisting of amino acid residues F126, L128, A129, S136, A141, H168, F170, V173, Q175, or T187 of the first heavy chain; and The at least one non-cysteine ​​residue on the first light chain that is substituted with cysteine ​​is selected from the group consisting of amino acid residues S114, F116, F118, Q124, L135, N137, N138, Q160, S162 or T164 of the first light chain.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the first light chain and the first heavy chain comprise at least one non-cysteine ​​substitution pair for a native cysteine ​​on the first light chain and a native cysteine ​​on the first heavy chain and at least one cysteine ​​substitution pair selected from the group consisting of: (i) S114C on the light chain and S136C on the heavy chain; (ii) F116C on the light chain and S136C on the heavy chain; (iii) F118C on the light chain and L128C on the heavy chain; (iv) F118C on the light chain and A129C on the heavy chain; (v) F118C on the light chain and A141C on the heavy chain; (vi) Q124C on the light chain and F126C on the heavy chain; (vii) L135C on the light chain and F170C on the heavy chain; (viii) N137C on the light chain and F170C on the heavy chain; (ix) N138C on the light chain and H168C on the heavy chain; (x) Q160C on the light chain and V173C on the heavy chain; (xi) Q160C on the light chain and Q175C on the heavy chain; (xii) S162C on the light chain and F170C on the heavy chain; (xiii) S162C on the light chain and T187C on the heavy chain; and (xiv) T164C on the light chain and F170C on the heavy chain; wherein the at least one cysteine ​​substitution pair forms at least one interchain disulfide bond between the first heavy chain and the first light chain; wherein the native cysteine ​​on the first heavy chain and the first light chain are independently substituted by any one of non-cysteine ​​residues. 4 . The antibody or antigen-binding fragment thereof according to claim 3 , wherein the native cysteine ​​on the first heavy chain and the first light chain is independently substituted with any one of serine, alanine, glycine or valine.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the first heavy chain and the first light chain comprise a cysteine ​​substitution pair.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the first heavy chain and the first light chain comprise two cysteine ​​substitution pairs.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the two cysteine ​​substitution pairs are selected from the group consisting of: (i) S114C / Q160C on the light chain and S136C / V173C on the heavy chain; (ii) F118C / Q124C on the light chain and L128C / F126C on the heavy chain; (iii) F118C / Q124C on the light chain and A129C / F126C on the heavy chain; (iv) F118C / Q124C on the light chain and A141C / F126C on the heavy chain; (v) F116C / Q124C on the light chain and S136C / F126C on the heavy chain; (vi) Q124C / T164C on the light chain and F126C / F170C on the heavy chain; (vii) Q124C / N138C on the light chain and F126C / H168C on the heavy chain; (viii) Q160C / L135C on the light chain and Q175C / F170C on the heavy chain; (ix) S114C / S162C on the light chain and S136C / F170C on the heavy chain; (x) F118C / S162C on the light chain and L128C / F170C on the heavy chain; (xi) F118C / S162C on the light chain and A141C / F170C on the heavy chain; (xii) Q124C / S162C on the light chain and F126C / F170C on the heavy chain; (xiii) N137C / S162C on the light chain and F170C / T187C on the heavy chain; (xiv) Q124C / T164C / C214S on the light chain and F126C / F170C / C220S on the heavy chain; and (xv) Q124C / S162C / C214S on the light chain and F126C / F170C / C220S on the heavy chain.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein the interchain disulfide bond between the second heavy chain and the second light chain is formed between a native cysteine ​​on the second light chain and a native cysteine ​​on the second heavy chain; The position of the native cysteine ​​on the second light chain corresponds to position C214 on the human κ chain, and the position of the native cysteine ​​on the second heavy chain corresponds to position C220 on human IgG1.

9. The antibody or antigen-binding fragment thereof of claim 1, wherein the second light chain and the second heavy chain comprise at least one non-cysteine ​​substitution pair, wherein a native cysteine ​​on the light chain is substituted with a serine and a native cysteine ​​on the heavy chain is substituted with a serine, and at least one cysteine ​​substitution pair selected from the group consisting of: (i) S114C on the light chain and S136C on the heavy chain; (ii) F116C on the light chain and S136C on the heavy chain; (iii) F118C on the light chain and L128C on the heavy chain; (iv) F118C on the light chain and A129C on the heavy chain; (v) F118C on the light chain and A141C on the heavy chain; (vi) Q124C on the light chain and F126C on the heavy chain; (vii) L135C on the light chain and F170C on the heavy chain; (viii) N137C on the light chain and F170C on the heavy chain; (ix) N138C on the light chain and H168C on the heavy chain; (x) Q160C on the light chain and V173C on the heavy chain; (xi) Q160C on the light chain and Q175C on the heavy chain; (xii) S162C on the light chain and F170C on the heavy chain; (xiii) S162C on the light chain and T187C on the heavy chain; and (xiv) T164C on the light chain and F170C on the heavy chain; wherein the at least one cysteine ​​substitution pair forms at least one interchain disulfide bond between the second light chain and the second heavy chain, and the cysteine ​​substitution pair on the first arm is different from the cysteine ​​substitution pair on the second arm.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein the native cysteine ​​substituted on the second light chain is located at position C214 on the human κ chain, and the native cysteine ​​substituted on the second heavy chain is located at position C220 on the human IgG1.

11. The antibody or antigen-binding fragment thereof of claim 1, wherein the second heavy chain and the second light chain comprise two cysteine ​​substitution pairs.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein the two cysteine ​​substitution pairs are selected from the group consisting of: (i) S114C / Q160C on the light chain and S136C / V173C on the heavy chain; (ii) F118C / Q124C on the light chain and L128C / F126C on the heavy chain; (iii) F118C / Q124C on the light chain and A129C / F126C on the heavy chain; (iv) F118C / Q124C on the light chain and A141C / F126C on the heavy chain; (v) F116C / Q124C on the light chain and S136C / F126C on the heavy chain; (vi) Q124C / T164C on the light chain and F126C / F170C on the heavy chain; (vii) Q124C / N138C on the light chain and F126C / H168C on the heavy chain; (viii) Q160C / L135C on the light chain and Q175C / F170C on the heavy chain; (ix) S114C / S162C on the light chain and S136C / F170C on the heavy chain; (x) F118C / S162C on the light chain and L128C / F170C on the heavy chain; (xi) F118C / S162C on the light chain and A141C / F170C on the heavy chain; (xii) Q124C / S162C on the light chain and F126C / F170C on the heavy chain; and (xiii) N137C / S162C on the light chain and F170C / T187C on the heavy chain; wherein the cysteine ​​substitution pair on the first arm is different from the cysteine ​​substitution pair on the second arm.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein The first heavy chain constant region and / or the second heavy chain constant region comprises human IgG1, IgG2, IgG3 or IgG4; and The first light chain constant region and / or the second light chain constant region comprises a human kappa light chain or a human lambda light chain.

14. The antibody or antigen-binding fragment thereof of claim 1, wherein the first heavy chain and the second heavy chain form a heterodimer; and the Fc region of the first heavy chain constant region and / or the Fc region of the second heavy chain constant region include one or more modifications that promote heterodimerization.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the Fc region of the first heavy chain interacts with the Fc region of the second heavy chain via a Knob / Hole structure.

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein the Fc region of the first heavy chain comprises a knob mutation, and the Fc region of the second heavy chain comprises a hole mutation; or the Fc region of the first heavy chain comprises a hole mutation, and the Fc region of the second heavy chain comprises a knob mutation.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the knob mutation comprises T366W, and the hole mutation comprises T366S / L368A / Y407V.

18. The antibody or antigen-binding fragment thereof according to claim 14, wherein the modification promoting heterodimerization comprises introducing a cysteine ​​residue capable of forming an interchain disulfide bond.

19. The antibody or antigen-binding fragment thereof according to claim 14, wherein the Fc region of the first heavy chain constant region and the Fc region of the second heavy chain constant region each comprise a modification in the CH3 region; wherein the modifications in the two CH3 regions are selected from the following: 。 20. The antibody or antigen-binding fragment thereof according to claim 14, wherein the Fc region of the first heavy chain constant region and the Fc region of the second heavy chain constant region each comprise a modification in the CH3 region; wherein the modifications in the two CH3 regions are selected from the following: 。 21. The antibody or antigen-binding fragment thereof according to claim 1, wherein the first arm and the second arm are capable of specifically binding to an antigen selected from the group consisting of: SIRPα, CLDN18.2, Siglec15, HER2, EGFR, CD19, CD20, CD39, CD47, PD1, PDL1, CD3, NKG2D, NKG2A, Nkp46, CD137, OX40, CD40, LILRB1, LILRB2, LILRB4, GPC3, TROP2, CD112, TIGIT, FAP, VEGFA, DLL4, ANG-2, wherein the first arm and the second arm can specifically bind to different antigens.

22. The antibody or antigen-binding fragment thereof according to claim 14, wherein the Fc region of the first heavy chain constant region and / or the Fc region of the second heavy chain constant region further comprises a modification that improves the stability of the antibody or the antigen-binding fragment.

23. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a humanized antibody or a chimeric antibody.

24. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a bispecific antibody or a multispecific antibody.

25. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, linked to one or more conjugate moieties.

26. The antibody or antigen-binding fragment thereof of claim 25, wherein the conjugate moiety comprises a second antibody fragment.

27. The antibody or antigen-binding fragment thereof according to claim 26, wherein the antibody or antigen-binding fragment thereof comprises a Fab fragment linked to the C-terminus of the Fc region of the second antibody fragment.

28. The antibody or antigen-binding fragment thereof of claim 25, wherein the conjugate moiety comprises an agent for detection or separation, such as a clearance modifier, a luminescent label, a fluorescent label, an enzyme substrate label, or a purification moiety.

29. The antibody or antigen-binding fragment thereof of claim 25, wherein the conjugate moiety comprises a therapeutic agent or drug.

30. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of the preceding claims.

31. A vector comprising the isolated polynucleotide according to claim 30.

32. A host cell comprising the vector according to claim 31.

33. A pharmaceutical composition comprising: (i) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 29 or the polynucleotide according to claim 30; and (ii) one or more pharmaceutically acceptable carriers.

34. A method of expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, the method comprising culturing the host cell according to claim 32 under conditions suitable for expressing the vector contained in the host cell.

35. A method for treating, preventing or alleviating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, or a polynucleotide encoding the antibody or antigen-binding fragment thereof according to claim 30, or a vector according to claim 31, or a host cell according to claim 32, or a pharmaceutical composition according to claim 33.

36. The method of claim 35, wherein the subject is a human.

37. The method of claim 35 or 36, wherein the administration is performed orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.

38. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, or a polynucleotide encoding the antibody or antigen-binding fragment thereof according to claim 30, or a vector according to claim 31, or a host cell according to claim 32, or a pharmaceutical composition according to claim 33 in the preparation of a medicament for treating, preventing or alleviating a disease in a subject.

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