Modified antibodies and uses thereof

Abstract

Provided is a modified antibody, its preparation method and its use. In particular, the modified antibody is a bispecific or multispecific antibody. The heavy and light chains of the modified antibody are paired with high assembly accuracy and stability.

Description

[Technical field]

[0001]

[0001] This disclosure relates generally to modified antibodies and uses thereof. [Background technology]

[0002]

[0002] Currently, monoclonal antibodies are used as specific and effective biological drugs, and can show exquisite specificity for a single target antigen. However, in some situations, a single antibody with specificity for two (or more) different target antigens, often called a bispecific antibody (or multispecific antibody), is preferred. Bispecific or multispecific antibodies are superior to monospecific antibodies in many aspects because they can simultaneously recognize two or more different antigens, neutralize different pathogenic mediators, recruit different types of effector cells, and modulate signal pathways. In this context, the development of bispecific and multispecific antibodies as therapeutic agents for human diseases is of great clinical significance, and bispecific antibodies have been widely used in diagnostic and therapeutic applications in recent years.

[0003] However, the production of bispecific and polyspecific antibodies has been difficult. In bispecific antibodies, a heavy chain specific for one antigen must be paired with a light chain specific for the same antigen. Pairing a heavy chain with a light chain specific for an antigen different from that of the heavy chain can destroy or reduce the intended antigen specificity. Because the correct pairing of a light chain with its corresponding heavy chain with the native IgG structure cannot yet be controlled, attempts to introduce additional antibody specificities into a single antibody often result in the production of misassembled species. Summary of the Invention [Problem to be solved by the invention]

[0004]

[0004] There remains a need for the generation of novel modified antibodies with multiple specificities. [Means for solving the problem]

[0005]

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

[0006]

[0006] In some embodiments, at least one non-cysteine ​​residue on the first heavy chain that is replaced with a 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 at least one non-cysteine ​​residue on the first light chain that is replaced with a 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 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 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 non-cysteine ​​substitution pair of a native cysteine ​​on the first light chain and a native cysteine ​​on the first heavy chain. (x) N138C in the light chain and H168C in the heavy chain; (x) Q160C in the light chain and V173C in the heavy chain; (xi) Q160C in the light chain and Q175C in the heavy chain; (xii) S162C in the light chain and F170C in 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; the at least one cysteine ​​substitution pair forms at least one interchain disulfide bond between the first heavy chain and the first light chain; and the native cysteines on the first heavy chain and the first light chain are independently replaced with any one of the non-cysteine ​​residues.

[0008] In some embodiments, the native cysteines on the first heavy chain and the first light chain are independently replaced with one of serine, alanine, glycine, or valine. In some embodiments, the replaced native cysteines are amino acid residue C214 of the light chain and amino acid residue C220 of the heavy chain of the antibody. In some embodiments, the first light chain comprises the mutation C214S and the first heavy chain comprises the 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: (i) S114C / Q160C on the light chain and S136C / V173C on the heavy chain; (ii) F118C / Q124C of the light chain and L128C / F126C of the heavy chain; (iii) F118C / Q124C of the light chain and A129C / F126C of the heavy chain; (iv) F118C / Q124C of the light chain and A141C / F126C of the heavy chain; (v) F116C / Q124C of the light chain and S136C / F126C of the heavy chain; (vi) Q124C / T164C of the light chain and F126C / F170C of the heavy chain; (vii) Q124C (viii) Q160C / L135C in the light chain and Q175C / F170C in the heavy chain; (ix) S114C / S162C in the light chain and S136C / F170C in the heavy chain; (x) F118C / S162C in the light chain and L128C / F170C in the heavy chain; (xi) F118C / S162C in the light chain and A141C / F170C in 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.

[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]

[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; the position of the native cysteine ​​on the second light chain corresponds to position C214 on a human κ chain and the position of the native cysteine ​​on the second heavy chain corresponds to position C220 on a human IgG1.

[0013] In some embodiments, the second light chain and the second heavy chain contain at least one non-cysteine ​​substitution pair of a native cysteine ​​substituted for serine on the light chain and a native cysteine ​​substituted for serine on the heavy chain, and (i) S114C on the light chain and S136C on the heavy chain; (ii) F116C in the light chain and S136C in the heavy chain; (iii) F118C in the light chain and L128C in the heavy chain; (iv) F118C in the light chain and A129C in the heavy chain; (v) F118C in the light chain and A141C in the heavy chain; (vi) Q124C in the light chain and F126C in the heavy chain; (vii) L135C in the light chain and F170C in the heavy chain; (viii) N137C in the light chain and N137C in the heavy chain. and F170C in the heavy chain; (ix) N138C in the light chain and H168C in the heavy chain; (x) Q160C in the light chain and V173C in the heavy chain; (xi) Q160C in the light chain and Q175C in the heavy chain; (xii) S162C in the light chain and F170C in 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 in the light chain and one in the heavy chain) that are mutated together to a non-cysteine ​​amino acid. A cysteine ​​substitution pair refers to a pair of native non-cysteine ​​amino acids (one in the light chain and one in the heavy chain) that are mutated together to a cysteine.

[0014]

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

[0015] In some embodiments, the second heavy chain and the second light chain comprise two pairs of cysteine ​​substitutions. In some embodiments, the two cysteine ​​substitution pairs are: (i) S114C / Q160C on the light chain and S136C / V173C on the heavy chain; (ii) F118C / Q124C in the light chain and L128C / F126C in the heavy chain; (iii) F118C / Q124C in the light chain and A129C / F126C in the heavy chain; (iv) F118C / Q124C in the light chain and A141C / F126C in the heavy chain; (v) F116C / Q124C in the light chain and S136C / F126C in the heavy chain; (vi) Q124C / T164C in the light chain and F126C / F170C in the heavy chain; (vii) Q124C / N138C in the light chain and F126C / H168C in the heavy chain; (viii) Q160C / L135C in the light chain and Q175C / F170C in the heavy chain; (ix) S114C / S162C in the light chain and S136C / F170C in the heavy chain; (x) F118C / S162C in the light chain and L128C / F170C in the heavy chain; (xi) F118C / S162C in the light chain and A141C / F170C in 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;

[0016]

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

[0017]

[0017] In some embodiments, the first heavy chain and the second heavy chain form a heterodimer; the Fc region of the constant region of the first heavy chain and / or the Fc region of the constant region of the second heavy chain contain 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 via 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]

[0019] In some embodiments, the modification that promotes heterodimerization includes 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 comprise a modification in the CH3 region; the modifications in the two CH3 regions are selected from the following:

[0020] [Table 1]

[0021] In some embodiments, the Fc region of the constant region of the first heavy chain and the Fc region of the constant region of the second heavy chain each comprise a modification in the CH3 region; the modifications in the two CH3 regions are selected from the following:

[0022] [Table 2]

[0023]

[0022] 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, and ANG-2, and the first arm and the second arm specifically bind to different antigens.

[0024]

[0023] In some embodiments, the Fc region of the constant region of the first heavy chain and / or the Fc region of the constant region of the second heavy chain further comprises a modification that improves the stability of the antibody or antigen-binding fragment.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof is a humanized or chimeric antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific or multispecific antibody.

[0026]

[0025] In some embodiments, the antibody or antigen-binding fragment thereof is linked to one or more conjugate moieties. 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.

[0027] In some embodiments, the conjugate moiety comprises an agent for detection or isolation, 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 comprises a therapeutic agent or drug.

[0028]

[0028] In one respect, the disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof disclosed herein.

[0029] In one respect, the disclosure provides a vector comprising an isolated polynucleotide disclosed herein.

[0029]

[0030] In one respect, the disclosure provides a host cell comprising the vector disclosed herein.

[0031] In one respect, the disclosure provides a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof, or a polynucleotide disclosed herein, and (ii) one or more pharma- ceutically acceptable carriers.

[0030]

[0032] In one respect, the 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 the vector contained therein.

[0031]

[0033] In certain respects, the disclosure provides a method of treating, preventing, or ameliorating a disease in a subject, the method comprising administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or a polynucleotide encoding the antibody or antigen-binding fragment thereof, or a vector, or a host cell, or a pharmaceutical composition disclosed herein.

[0032]

[0034] In some embodiments, the subject is a human. In some embodiments, administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

[0035] In certain respects, the disclosure provides for the use of an antibody or antigen-binding fragment thereof, or a polynucleotide encoding the antibody or antigen-binding fragment thereof, or a vector, or a host cell, or a pharmaceutical composition disclosed herein in the manufacture of a medicament for treating, preventing, or ameliorating a disease in a subject. [Brief description of the drawings]

[0033] [Figure 1]

[0036] FIG. 1 is a schematic diagram of screening assay 1. [Diagram 2]

[0037] FIG. 2 is a schematic diagram of screening assay 2. [Diagram 3]

[0038] Figures 3A to 3D show the structures of expression vectors used in screening assays 1 and 2. Figure 3A shows the structure of the expression vector for the light chain with or without a mutation for a shifted disulfide bond. Figure 3B shows the structure of the expression vector for the heavy chain with or without a mutation for a shifted disulfide bond. Figure 3C shows the structure of the expression vector for the wild-type light chain with a VHH domain fused to the N-terminus. Figure 3D shows the structure of the expression vector for the naked Fc with a substituted knob or hole residue. [Figure 4-1]

[0039] Figures 4A and 4B show the results of the relative abundance of artificial asymmetric Fab-Fc and VHH-Fab-Fc variants characterized by SEC-HPLC. Figure 4A shows asymmetric Fab-Fc variants with different residue substitution pairs for CH1-CL interchain disulfide bond shift detected by screening assay 1. Figure 4B shows asymmetric VHH-Fab-Fc molecules with native CH1-CL interchain disulfide bond detected by screening assay 2. [Figure 4-2] Same as above. [Diagram 5]

[0040] FIG. 5 shows the schematic structure of a symmetric bivalent monospecific antibody in which the interchain disulfide bonds of both Fab arms have been transferred. [Figure 6]

[0041] 6A and 6B show the results of the evaluation of the yield (FIG. 6A) and monomer ratio (FIG. 6B) of artificial symmetric antibody variants with different residue substitution pairs for CH1-CL interchain disulfide bond shift. [Figure 7-1]

[0042] 7A to 7D show the results of evaluating the binding activity of symmetric bivalent anti-CLDN18.2 antibody variants to CLDN18.2-overexpressing MC38 cells by FACS assay. [Figure 7-2] Same as above. [Figure 8]

[0043] FIG. 8 shows the results of heat resistance analysis of symmetric bivalent antibody variants with different residue substitution pairs for CH1-CL interchain disulfide bond shift using a Thermo Shift assay. [Figure 9]

[0044] FIG. 9 is a schematic diagram of the Case 1 study. [Figure 10]

[0045] FIG. 10 shows the results of assembly accuracy and thermostability of the antibodies tested in the Case 1 study, as measured by LC-MS assay and differential scanning fluorimetry, respectively. [Figure 11]

[0046] Figure 11 is a schematic diagram of the Case 2 study. [Figure 12]

[0047] FIG. 12 shows the results of assembly accuracy and thermostability of the antibodies tested in the Case 2 study, as measured by LC-MS assay and differential scanning fluorimetry, respectively. [Figure 13]

[0048] Figures 13A and 13B show the SDS-PAGE results of the antibodies tested in the case 1 study (Figure 13A) and case 2 study (Figure 13B). [Figure 14]

[0049] FIG. 14 shows the results of SDS-PAGE and SEC of the test antibody ACF_361. [Figure 15]

[0050] FIG. 15 shows the LC-MS results of the tested antibody, ACF_361. [Figure 16-1]

[0051] Figure 16A shows the results of antibody binding affinity to MC38 / CD47 / CLDN18.2 cells by FACS. Figure 16B shows the results of antibody binding affinity to CHO / SIRPαV1 cells by FACS. Figure 16C shows the results of antibody phagocytic activity. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 17]

[0052] FIG. 17 shows the results of SDS-PAGE and SEC of the tested antibody ACF_389. [Figure 18]

[0053] FIG. 18 shows the results of Mono S chromatography purification of the test antibody ACF_389. [Figure 19] FIG. 19 shows the results of Mono S chromatography purification of the test antibody ACF_389. [Figure 20]

[0054] FIG. 20 shows the results of LC-MS of the test antibody ACF_389. [Figure 21-1]

[0055] Figure 21A shows the results of antibody binding affinity to Raji / PDL1 cells by FACS, and Figure 21B shows the results of antibody binding affinity to CHO / SIRPαV1 cells by FACS. [Figure 21-2] Same as above. [Figure 22]

[0056] FIG. 22 shows the results of Mono S chromatography purification of the tested antibody ESB07.451. [Diagram 23] FIG. 23 shows the results of Mono S chromatography purification of the tested antibody ESB07.451. [Figure 24]

[0057] FIG. 24 shows the LC-MS results of the test antibody ESB07.451. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034]

[0058] The following description of the disclosure is intended to merely illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed are not to be interpreted as limitations on the scope of the disclosure. It is clear to those skilled in the art that various equivalents, changes and modifications can be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are included herein. All references cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety. The terms used herein are only for describing specific embodiments and are not intended to limit the present invention.

[0035]

[0059] 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. By way of example, "an antibody" means one antibody or more than one antibody.

[0036]

[0060] Additionally, to the extent that "containing", "containing", "having", "having", "with", or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising". Throughout this disclosure, unless otherwise required by context, the words "comprising", "including" and "comprising" are understood to mean the inclusion of a recited step or element or group of steps or elements, but not the exclusion of other steps or elements or group of steps or elements. "Consisting of" means containing and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and other elements must not be present. "Consisting essentially of" means that the recited elements following the phrase are contained, and only other elements that do not interfere with or contribute to the activity or action identified in the disclosure of the recited elements are contained. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0037]

[0061] 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 error range depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in the present specification and claims, the term "about" should be assumed to mean within an acceptable error range for the particular value, unless otherwise indicated.

[0038]

[0062] Throughout this disclosure, references to "one embodiment," "one embodiment," "particular embodiment," "some embodiments," or "an embodiment," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with an embodiment are included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. I. Antibody modification

[0063] In some aspects, the present disclosure provides modified antibodies, in which the polypeptide chains of the antibodies have greater assembly precision and stability.

[0039]

[0064] In some embodiments, the modified antibodies are designed to reduce mispairing of heavy and light chains in bispecific or multispecific antibodies having two or more different antigen binding sites.

[0040]

[0065] In some embodiments, the modified antibodies have amino acid residue mutations in which non-cysteine ​​residues are mutated to cysteine ​​residues to form new disulfide bonds between the heavy and light chains of the antibody.

[0041]

[0066] In some embodiments, the modified antibodies have amino acid residue mutations in which cysteine ​​residues are mutated to non-cysteine ​​residues to disrupt disulfide bond formation between the cysteine ​​residues.

[0042]

[0067] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to a linear series of amino acid residues linked together by peptide bonds, including proteins, polypeptides, oligopeptides, peptides, and fragments thereof. Proteins may be composed of naturally occurring and / or synthetic (e.g., modified or non-naturally occurring) amino acids. Thus, as used herein, "amino acid" or "peptide residue" refers to both naturally occurring and synthetic amino acids. The terms "polypeptide", "peptide" and "protein" include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, fusions with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, such as fusion proteins with fluorescent proteins, β-galactosidase, luciferase, etc. as fusion partners; and the like. It should further be noted that a dash at the beginning or end of an amino acid residue sequence indicates either a peptide bond to a further sequence comprising one or more amino acid residues, or a covalent bond to the carboxyl or hydroxyl terminal group, but since it is conventional to omit such things in the representation of amino acid sequences, the absence of a dash should not be taken to imply the absence of such a peptide bond or a covalent bond to the carboxyl or hydroxyl terminal group.

[0043]

[0068] The term "antibody" as used herein includes all immunoglobulins, monoclonal antibodies, polyclonal antibodies, multivalent antibodies, bivalent antibodies, monovalent antibodies, multispecific antibodies, bispecific antibodies or monospecific antibodies that bind to one or more specific antigens. Naturally occurring intact antibodies are composed of two heavy chains (H) and two light chains (L). Mammalian heavy chains are classified as α, δ, ε, γ, μ, each heavy chain composed of a variable region (VH) and a first, second, third and optionally fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are classified as λ or κ, each light chain composed of a variable region (VL) and a constant region. Antibodies are "Y" shaped, with the stem of the Y composed of the second and third constant regions of two heavy chains linked via disulfide bonds. Each arm of the Y contains the variable region and first constant region of a single heavy chain bound to the variable 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 both chains contain three highly variable loops, commonly called complementarity determining regions (CDRs) (the CDRs of the light chain contain LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain contain HCDR1, HCDR2, and HCDR3).The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be determined by the methods described in Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83 (1989); Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of The three CDRs may be defined or identified by the convention of "framework regions" (Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77(2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells(second edition), chapter 26, 481-514, (2015)). The three CDRs are sandwiched between flanking stretches known as framework regions (FRs) (light chain FRs contain LFR1, LFR2, LFR3, LFR4, heavy chain FRs contain HFR1, HFR2, HFR3, HFR4), which are more highly conserved than the CDRs and form a scaffold supporting the highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified into classes based on the amino acid sequence of the constant region of the heavy chain.The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the 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), and IgA2 (α2 heavy chain).

[0044]

[0069] The term "variable domain" as used herein with respect to an antibody refers to an antibody variable region or fragment thereof comprising one or more CDRs. A variable domain may comprise a complete variable region (such as HCVR or LCVR), but may not comprise a complete variable region that retains the ability to bind antigen or form an antigen-binding site.

[0045]

[0070] The term "antigen-binding fragment" as used herein refers to an antibody fragment comprising one or more CDRs and formed from a portion of an antibody or other antibody fragment that binds to an antigen but does not constitute a complete native antibody structure. Examples of antigen-binding fragments include, but are not limited to, variable domains, variable regions, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bispecific antibodies (ds bispecific antibodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen as the parent antibody binds. In certain embodiments, antigen-binding fragments may comprise one or more CDRs grafted from a particular human antibody onto framework regions from one or more different human antibodies. Many of the antigen-binding fragments (also called antigen-binding sites) and their detailed configurations 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 entireties.

[0046]

[0071] "Fab," in reference to an antibody, refers to the portion of an antibody that consists of a single light chain (both variable and constant region) to which the variable region and first constant region of a single heavy chain are attached by disulfide bonds.

[0047]

[0072] "Fab'" refers to a Fab fragment that contains part of the hinge region.

[0073] "F(ab')2" refers to a Fab' dimer.

[0074] "Fc" with respect to an antibody (e.g., IgG, IgA, or IgD isotype) refers to the portion of the antibody that consists of the second and third constant domains of a first heavy chain and the second and third constant domains of a second heavy chain bound thereto via a disulfide bond. Fc with respect to antibodies of IgM and IgE isotypes further comprises a fourth constant domain. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), etc., but does not function in antigen binding.

[0048]

[0075] The term "hinge region" of an antibody includes the portion of the heavy chain molecule that connects the CH1 and CH2 domains. This hinge region contains approximately 25 amino acid residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently.

[0049]

[0076] As used herein, a "CH2 domain" includes the portion of a heavy chain molecule extending from amino acid 244 to amino acid 360 of an IgG antibody, for example, using the conventional numbering scheme (amino acids 244-360, Kabat numbering system; and amino acids 231-340, EU numbering system; see Kabat, E, et al, USDepartment of Health and Human Services, (1983)).

[0050]

[0077] The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 amino acids. Certain immunoglobulin classes, such as IgM, further contain a CH4 region.

[0051]

[0078] "Fv" in reference to an antibody refers to the smallest fragment of an antibody that contains an intact antigen-binding site. The Fv fragment consists of the variable region of a single heavy chain bound to the variable region of a single light chain. Many Fv designs are available, including dsFv, in which the bond between the two domains is strengthened by the introduction of a disulfide bond; and scFv, which is formed by joining the two domains as a single polypeptide using a peptide linker. Fv constructs have also been made that contain variable domains of heavy or light immunoglobulin chains, in which the variable domains are associated with the variable and constant domains of the corresponding immunoglobulin heavy or light chains. Fvs have also been multimerized to form bispecific and trispecific antibodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0052]

[0079] "Camelized single domain antibodies", "heavy chain antibodies", or "HCAbs" are antibodies that consist of two V HIt refers to antibodies that contain heavy chains and do not contain 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. Patent No. 6,005,079). Heavy chain antibodies are originally derived from camelids (camels, dromedaries, and llamas). Although they lack light chains, camelized antibodies have a genuine antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al. Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al. Immunology. May; 109(1): 93-101 (2003)). The variable domains of heavy chain antibodies (VHH domains) are known to be the smallest antigen-binding units generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)).

[0053]

[0080] "Nanobody" refers to an antibody fragment that consists of a VHH domain derived from a heavy chain antibody and two constant domains, CH2 and CH3.

[0081] A "bispecific antibody" or "dAb" comprises a small antibody fragment with two antigen-binding sites, the fragment being located at the V L V linked to domain H Domain Included (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 the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, thereby forming two antigen-binding sites. The antigen-binding sites can target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds bispecific antibody" is a bispecific antibody that targets two different antigens (or epitopes).

[0054]

[0082] As used herein, the term "valent" refers to the presence of a particular number of antigen-binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen-binding fragment that has only a single antigen-binding site, and the term "multivalent" refers to an antibody or antigen-binding fragment that has multiple antigen-binding sites. Thus, the terms "bivalent", "trivalent", "tetravalent", and "hexavalent" refer to the presence of two, three, four, and six binding sites, respectively, in an antigen-binding molecule. In some embodiments, an antibody or antigen-binding fragment thereof is bivalent.

[0055]

[0083] 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, which may be present on the same antigen or on two different antigens.

[0056]

[0084] As used herein, a "multispecific" antibody refers to an artificial antibody that has fragments derived from two or more different monoclonal antibodies and is capable of binding to two or more different epitopes, which may be present on the same antigen or on two or more different antigens.

[0057]

[0085] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of a heavy and / or light chain from one species and the remaining portion of the heavy and / or light chain from a different species. Illustratively, a chimeric antibody may contain a constant region from a human and a variable region 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.

[0058]

[0086] As used herein, the term "humanized" means that an antibody or antigen-binding fragment contains CDRs derived from a non-human animal, FR regions derived from a human, and, where appropriate, a constant region derived from a human.

[0059]

[0087] As used herein, the term "affinity" refers to the strength of non-covalent interactions between an immunoglobulin molecule (i.e., an antibody) or fragment thereof and an antigen.

[0088] As used herein, the term "specifically bound" or "specifically binds" refers to a non-random binding reaction between two molecules, such as, for example, between an antibody and an antigen. Specific binding can be, for example, D value, i.e., 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 The binding affinity can be characterized by the binding affinity, which is expressed as K D K can be determined using any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). D The value of is ≦10 -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 -9M, ≤2×10 -9 M, or ≤ 10 -9 M) indicates specific binding of the antibody or antigen-binding fragment thereof to the corresponding antigen.

[0060]

[0089] As used herein, the term "epitope" refers to the 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 epitope or closely related epitopes within the antigen. Epitopes may be linear or conformational (i.e., containing amino acid residues spaced apart).

[0061]

[0090] "Percent 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 maximize the number of identical amino acids (or nucleic acids). In other words, the percent sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) that are identical to the reference sequence being compared by the total number of amino acid residues (or bases) in either the candidate or reference sequence, whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be accomplished, for example, using BLASTN, BLASTp (available on the US National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the European Bioinformatics Institute website, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One skilled in the art may use the default parameters provided by the tool or may customize the parameters as appropriate for the alignment, e.g., by selecting an appropriate algorithm.

[0062]

[0091] As used herein, the terms "mutation" and "mutated" in reference to amino acid residues refer to the substitution, insertion or deletion of one or more amino acid residues.

[0092] As used herein, the term "substitution" or "substituted" in reference to an amino acid sequence refers to the replacement of a native amino acid residue with a different amino acid residue.

[0063]

[0093] The term "conservative substitution" in relation to amino acid sequence refers to the substitution of an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions are 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 is known in the art, conservative substitutions usually do not cause significant changes in the conformation of a protein, and therefore can retain the biological activity of the protein.

[0064]

[0094] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, goats, dogs, cows, chickens, amphibians, and reptiles. Except as otherwise noted, the terms "patient" and "subject" are used interchangeably herein.

[0065]

[0095] An antibody or antigen-binding fragment thereof may contain one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of antibodies is usually either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of a sugar, either N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine.

[0066]

[0096] Disulfide bond formation and breakdown

[0097] In some embodiments, the antibodies provided herein comprise a first arm and a second arm, the first arm being formed by a first heavy chain and a first light chain having at least one disulfide bond therebetween, and the second arm being formed by a second heavy chain and a second light chain having at least one disulfide bond therebetween.

[0067]

[0098] In some embodiments, the connection between the first heavy chain and the first light chain forming the first arm contains an artificially introduced disulfide bond, whereas the connection between the second heavy chain and the second light chain forming the second arm contains a naturally occurring disulfide bond. In some embodiments, the connection between the first heavy chain and the first light chain forming the first arm contains an artificially introduced disulfide bond at a first set of positions, whereas the connection between the second heavy chain and the second light chain forming the second arm contains an artificially introduced disulfide bond at a second set of positions that is different from the first set of positions.

[0068]

[0099] In some embodiments, the modified antibody has amino acid residue mutations in which a pair of non-cysteine ​​residues (which may be any amino acid other than cysteine) located in the heavy and light chains of the antibody, respectively, are mutated to cysteine ​​residues to form new disulfide bonds between them. In certain embodiments, 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 according to EU numbering, and 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 according to EU numbering. In certain embodiments, the non-cysteine ​​residue on the heavy chain is an amino acid residue at F126, L128, A129, S136, A141, H168, F170, V173, Q175, or T187 according to EU numbering. In certain embodiments, the non-cysteine ​​residue on the light chain is an amino acid residue at S114, F116, F118, Q124, L135, N137, N138, Q160, S162, or T164 according to EU numbering.

[0069]

[0100] In certain embodiments, the modified antibody contains one or more pairs of mutations of non-cysteine ​​residues to cysteine ​​residues at the following positions:

[0101]

[0070] [Table 3]

[0071]

[0102] In certain embodiments, the modified antibody contains one or more of the following pairs of non-cysteine ​​to cysteine ​​residue mutations:

[0103]

[0072] [Table 4]

[0073]

[0104] In some embodiments, the modified antibody has amino acid residue mutations in which cysteine ​​residues are mutated to non-cysteine ​​residues to disrupt disulfide bond formation between the cysteine ​​residues. In certain embodiments, the pair of cysteine ​​residues mutated to non-cysteine ​​residues is amino acid residue C214 of the light chain and amino acid residue C220 of the heavy chain of the antibody.

[0074]

[0105] 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 one pair of cysteine ​​residues mutated to non-cysteine ​​residues is amino acid residue C214 of the light chain and amino acid residue C220 of the heavy chain of the antibody.

[0075]

[0106] In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises a first arm formed by a first heavy chain and a first light chain with two new disulfide bonds formed therebetween, the two disulfide bonds being formed by a pair of non-cysteine ​​to cysteine ​​mutations selected from the mutation pairs listed in Table 1 or Table 2.

[0076]

[0107] In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises a first arm formed by a first heavy chain and a first light chain with a disulfide bond formed therebetween, and a second arm formed by a second heavy chain and a second light chain with a disulfide bond formed therebetween, wherein the first light chain and the first heavy chain are (i) S114C on the light chain and S136C on the heavy chain; (ii) F116C in the light chain and S136C in the heavy chain; (iii) F118C in the light chain and L128C in the heavy chain; (iv) F118C in the light chain and A129C in the heavy chain; (v) F118C in the light chain and A141C in the heavy chain; (vi) Q124C in the light chain and F126C in the heavy chain; (vii) L135C in the light chain and F170C in the heavy chain; (viii) N137C in the light chain and F170C in the heavy chain; (ix) N138C in the light chain and H168C in the heavy chain; (x) Q160C in the light chain and V173C in the heavy chain; (xi) Q160C in the light chain and Q175C in the heavy chain; (xii) S162C in the light chain and F170C in 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 containing at least one pair of mutations from a non-cysteine ​​residue to a cysteine ​​residue selected from the group consisting of: At least one pair of mutations of non-cysteine ​​residues to cysteine ​​residues forms at least one disulfide bond between the first heavy chain and the first light chain, and a native cysteine ​​on the first light chain and a 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.

[0077]

[0108] The numbering of the amino acid residues and positions on the heavy and light chains of the antibody described in this disclosure is all according to the EU numbering system unless otherwise specified.As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant region, which is described in Edelman, GM et al., Proc.Natl.Acad.USA, 63, 78-85 (1969) and Kabat et al, Sequences of Proteins of Immunological Interest, USDept.Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0078]

[0109] 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 disrupted by substituting both cysteines with non-cysteines. In some embodiments, the native cysteines are independently substituted with any one of the non-cysteine ​​residues, so long as the artificially introduced new pair of residues has no interaction between them. In some embodiments, the native cysteines are independently substituted with serine (S), alanine (A), or glycine (G). In some embodiments, both native cysteines are substituted with serine (S).

[0079]

[0110] In some embodiments, the artificially introduced disulfide bond is formed by a combination of mutations selected from the group consisting of the mutations listed in Table 3.

[0111]

[0080] [Table 5]

[0081]

[0112] In some embodiments, the connection between the first heavy chain and the first light chain forming the first arm is (i) S114C / Q160C on the light chain and S136C / V173C on the heavy chain; (ii) F118C / Q124C in the light chain and L128C / F126C in the heavy chain; (iii) F118C / Q124C in the light chain and A129C / F126C in the heavy chain; (iv) F118C / Q124C in the light chain and A141C / F126C in the heavy chain; (v) F116C / Q124C in the light chain and S136C / F126C in the heavy chain; (vi) Q124C / T164C in the light chain and F126C / F170C in the heavy chain; (vii) Q124C / N138C in the light chain and F126C / H168C in the heavy chain; (viii) Q160C / L135C in the light chain and Q175C / F170C in the heavy chain; (ix) S114C / S162C in the light chain and S136C / F170C in the heavy chain; (x) F118C / S162C in the light chain and L128C / F170C in the heavy chain; (xi) F118C / S162C in the light chain and A141C / F170C in 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 The present invention comprises two artificially introduced disulfide bonds formed by a combination of mutations selected from the group consisting of:

[0082]

[0113] In some embodiments, the connection between the first heavy chain and the first light chain forming the first arm is (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 The present invention comprises two artificially introduced disulfide bonds formed by a combination of mutations selected from the group consisting of:

[0083]

[0114] In some embodiments, an antibody or antigen-binding fragment thereof disclosed herein comprises 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.

[0084]

[0115] In some embodiments, an antibody or antigen-binding fragment thereof disclosed herein comprises 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.

[0085]

[0116] Formation of bispecific antibodies

[0117] By rearranging the disulfide bonds formed by pairs of cysteines from the CH1 and CL regions, the present disclosure provides modified antibodies in which the heavy and light chains pair with high assembly precision and stability.

[0086]

[0118] 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.

[0119] In certain embodiments, the bispecific antibody contains 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 may target SIRPα and the other of the antigen binding arms of the bispecific antibody may target claudin-18.2.

[0087]

[0120] 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) may differ by 1-fold, 2-fold, 3-fold, 4-fold or more.

[0088]

[0121] Fc Region Mutations

[0122] The antibodies or antigen-binding fragments thereof disclosed herein also encompass Fc variants, which may comprise modification or substitution of one or more amino acid residues in the Fc region and / or hinge region.

[0089]

[0123] In some embodiments, heterodimeric pairing is achieved by engineering the Fc regions of the two heavy chains to form heterodimers exclusively, hi some embodiments, mutations are introduced into the CH3 region of the Fc region.

[0090]

[0124] In certain embodiments, the antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions in the interface of the Fc region, which amino acid substitutions favor and / or promote heterodimerization. These modifications include the introduction of a protuberance into the first Fc polypeptide and a cavity into the second Fc polypeptide, which may be located within the cavity, thereby promoting the interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for producing antibodies with such modifications are known in the art and are described, for example, in U.S. Patent No. 5,731,168.

[0091]

[0125] In some embodiments, the "knobs" are generated by replacing one or more small amino acid side chains from the interface of the first antibody molecule with larger side chains (e.g., tyrosine or tryptophan). Compensatory "holes" of identical or similar size to the large side chains are created in the interface of the second antibody molecule by replacing the large amino acid side chains with smaller side chains (e.g., alanine or threonine). CH3 modifications that promote heterodimerization include, for example, Y407V / T366S / L368A in one heavy chain and T366W in the other heavy chain; S354C / T366W in one heavy chain and Y349C / Y407V / T366S / L368A in the other heavy chain. Such modifications that result in a protuberance in one chain and a cavity in the other chain are shown in Table 4.

[0092]

[0126]

[0093] [Table 6]

[0094]

[0127] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein comprises a first CH3 region and a second CH3 region, and the first CH3 region or the second CH3 region comprises an amino acid sequence different from the wild-type IgG amino acid sequence, for example, one or more positively charged amino acids (e.g., lysine, histidine and arginine) in the wild-type human IgG amino acid sequence are replaced with one or more negatively charged amino acids (e.g., aspartic acid and glutamic acid) at the corresponding positions in the CH3 region. Alternatively, the first CH3 region or the second CH3 region comprises a van amino acid sequence different from the wild-type IgG amino acid sequence, for example, one or more negatively charged amino acids in the wild-type human IgG amino acid sequence are replaced with one or more positively charged amino acids at the corresponding positions in the CH3 region. In some embodiments, the modifications in the two CH3 regions are selected from the group listed in Table 5.

[0095]

[0128]

[0096] [Table 7]

[0097]

[0129] In some embodiments, the constant region of the heavy chain of the antibody or antigen-binding fragment thereof may be derived from human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the constant region of the light chain of the antibody or antigen-binding fragment thereof may be derived from a human κ chain or a human λ chain. II. Conjugates

[0130] 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.

[0098]

[0131] The conjugate moiety can be attached to an antibody or its antigen-binding fragment. A conjugate moiety is a site that can bind to an antibody or its antigen-binding fragment. It is contemplated that various conjugate moieties can be linked to the antibody or its antigen-binding fragment 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 moieties can be linked to the antibody or its antigen-binding fragment by methods such as covalent binding, affinity binding, insertion, coordinate binding, complexing, association, mixing, or addition. In some embodiments, the antibody or its antigen-binding fragment can be linked to one or more conjugates via a linker. III. Method of Preparation

[0132] The present disclosure provides polynucleotides encoding the antibodies or antigen-binding fragments thereof provided herein.

[0099]

[0133] As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and polymers thereof in either single-stranded or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the 3-position of one or more selected (or all) codons is replaced with mixed bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0100]

[0134] Nucleic acids or polynucleotides encoding the antibodies or antigen-binding fragments thereof provided herein can be constructed using recombinant techniques. For this purpose, DNA encoding the antigen-binding fragments (such as CDRs or variable regions) of the parent antibody can be isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). As an example, a polynucleotide sequence encoding the variable domain (VH) and a polynucleotide sequence encoding CH1, CH2 and CH3 are obtained and operably linked to allow transcription and expression in a host cell to produce a heavy chain polypeptide. Similarly, a polynucleotide sequence encoding VL is operably linked to a polynucleotide sequence encoding CL to allow expression of the light chain in a host cell.

[0101]

[0135] Mutations can be introduced by various methods known in the art. In some embodiments, primers with designed mutations (e.g., containing point mutations, deletions of polynucleotide segments, or insertions of polynucleotide segments) may be used to introduce specific mutations using PCR techniques.

[0102]

[0136] In some embodiments, polynucleotides with or without mutations can be obtained by synthetic methods. Methods of chemical DNA synthesis are well known in the art. Typically, the method includes the following steps: The 5' end of the first nucleotide is protected by dimethoxytrityl (DMT), while a linker to silica is attached to the OH end. The reactive groups of all nucleotides are chemically protected. The DMT is then removed by washing, and the next nucleotide is activated and coupled to the 3'-OH group. The 5'-3' bond is oxidized using iodine to generate a phosphotriester bond (one of the O's of the phosphate group is methylated). The reaction is continued until the desired chain length is reached. Polymers of about 70-80 residues can be made in this way. There are also automated versions of this process, and routine synthesis services are commercially available. DNA encoding the antibodies or antigen-binding fragments thereof disclosed herein can be easily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0103]

[0137] The encoding polynucleotide sequence may further be operably linked to one or more regulatory sequences, optionally in an expression vector, so that the expression or production of the heavy and light chains is under feasible and appropriate control.

[0104]

[0138] The present disclosure provides a vector comprising the encoding polynucleotide provided herein. As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to express the protein. Typically, the construct also contains appropriate regulatory sequences. For example, the polynucleotide molecule may contain a regulatory sequence located in the 5'-flanking region of the nucleotide sequence encoding the guide RNA and / or the nucleotide sequence encoding the site-directed modifying polypeptide, and operably linked to the coding sequence in a manner that allows the desired transcript / gene to be expressed in the host cell. The vector may be used to transform, transduce, or transfect a host cell to cause expression of the genetic element carried in the host cell. Vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, animal viruses, and the like. Categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papoviruses (e.g., SV40). Vectors may contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, vectors may contain origins of replication. Vectors may contain substances that aid in their entry into cells, including, but not limited to, viral particles, liposomes, protein coatings, and the like.

[0105]

[0139] Examples of vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papoviruses (e.g., SV40), lambda phage, and M13 phage, and the 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, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM, pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

[0106]

[0140] A vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof can be introduced into a host cell for cloning or gene expression. As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0107]

[0141] Suitable host cells for cloning or expressing DNA in the vector herein are the above-mentioned prokaryote, yeast, or higher eukaryote cells.Prokaryotes suitable for this purpose include eubacteria, such as gram-negative or gram-positive organisms, such as Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, Enterobacteriaceae, such as Shigella, and Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0108]

[0142] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi and yeast, are suitable hosts for cloning and expressing antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, Kluyveromyces hosts, such as, for example, Schizosaccharomyces pombe; 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 spp. (EP 402,226); Pichia pastoris (EP 183,070); Candida spp.; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces spp., such as Schwanniomyces occidentalis; and members of the genera Neurospora, Penicillium, Tolypocladium, Aspergillus, Many other genera, species and strains are commonly available and useful herein, such as filamentous fungi such as the Aspergillus genus, such as Aspergillus nidulans and Aspergillus niger.

[0109]

[0143] The host cell suitable for expressing the glycosylated antibody or its antigen fragment provided herein is derived from a multicellular organism. Examples of invertebrate cells include plant cells and insect cells. A large number of baculovirus strains and variants and corresponding permissive insect host cells have been identified, such as Spodoptera frugiperda (cutworm moth), Aedes aegypti (Aedes aegypti), Aedes albopictus (Aedes albopictus), Drosophila melanogaster (Drosophila melanogaster), Bombyx mori, etc. Various virus strains for transfection are publicly available, including, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as the virus herein according to the present disclosure, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be utilized as hosts.

[0110]

[0144] In certain embodiments, the host cell is a vertebrate cell. Propagation of vertebrate cells in culture (tissue culture) is a routine procedure. Examples of useful mammalian host cell lines are the SV40 transformed monkey kidney CV1 line (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 carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC5 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 their derivatives.

[0111]

[0145] For antibody production, host cells are transformed with the above-described expression or cloning vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0112]

[0146] The present disclosure also provides a method of expressing an antibody or antigen-binding fragment thereof provided herein, comprising culturing a host cell provided herein under conditions in which a vector of the present disclosure is expressed. The host cells used to produce the antibody or antigen-binding fragment thereof provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Reissue Patent Application No. 30,985 can be used as culture media for host cells. These media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug Gentamicin®), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), glucose or an equivalent energy source. Other necessary nutritional supplements may be contained 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.

[0113]

[0147] When using recombinant techniques, antibodies may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, the first step is to remove particulate debris, such as host cells and lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cellular debris can be removed by centrifugation. If the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

[0114]

[0148] In certain embodiments, the disclosure provides a method of producing an antibody or antigen-binding fragment thereof provided herein, comprising: a) introducing 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 into a host cell, wherein the first light chain and the first heavy chain comprise a non-cysteine ​​substitution pair of C214X on the first light chain and C220X on the first heavy chain, and at least one cysteine ​​substitution pair; and b) allowing the host cell to express the polypeptide complex. In certain embodiments, at least one cysteine ​​substitution pair is: (i) S114C on the light chain and S136C on the heavy chain; (ii) F116C in the light chain and S136C in the heavy chain; (iii) F118C in the light chain and L128C in the heavy chain; (iv) F118C in the light chain and A129C in the heavy chain; (v) F118C in the light chain and A141C in the heavy chain; (vi) Q124C in the light chain and F126C in the heavy chain; (vii) L128C in the light chain and L128C in the heavy chain; (viii) N137C in the light chain and F170C in the heavy chain; (ix) N138C in the light chain and H168C in the heavy chain; (x) T164C in the light chain and F170C in the heavy chain; (xi) Q160C in the light chain and V173C in 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.

[0115]

[0149] 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 which accompany it in nature or during manufacture and is provided in an enriched form.

[0116]

[0150] The antibody or antigen-binding fragment thereof prepared from the cells can be isolated using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, affinity chromatography, and the like.

[0117]

[0151] In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of Protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX® resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE®, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), isoelectric focusing, SDS-PAGE, ammonium sulfate precipitation, etc., are also available depending on the antibody to be recovered.

[0118]

[0152] After any preliminary purification steps, the mixture containing the antibody of interest or antigen-binding fragment thereof and contaminants may be subjected to hydrophobic interaction or ion exchange chromatography using a salt gradient elution.

[0119]

[0153] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein can be easily purified with high yield using conventional methods.One advantage of the antibody or antigen-binding fragment thereof is that the mismatch between the heavy and light chain variable domain sequences is significantly reduced.This reduces the production of unwanted by-products, and allows for a relatively simple purification step to obtain a high yield of highly pure product. IV. Pharmaceutical Compositions and Administration

[0154] The present disclosure further provides pharmaceutical compositions comprising the antibody or antigen-binding fragment thereof and one or more pharma- ceutically acceptable carriers.

[0120]

[0155] The present disclosure further provides a pharmaceutical composition comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof and one or more pharma- ceutically acceptable carriers. The antibodies provided herein may be produced in vivo by delivering a polynucleotide encoding an antibody or antigen-binding fragment thereof, such as by in vitro transcribed mRNA, or an expression vector. Methods of polynucleotide delivery for 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.

[0121]

[0156] The disclosure further provides a pharmaceutical composition comprising an expression vector comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof, and one or more pharma- ceutically acceptable carriers.

[0122]

[0157] In certain embodiments, the expression vector comprises a viral vector or a non-viral vector. Viral vectors include, but are not limited to, adeno-associated virus (AAV) vectors, lentivirus vectors, retrovirus vectors, and adenovirus vectors. Non-viral vectors include, but are not limited to, naked DNA, plasmids, exosomes, mRNA, and the like. In certain embodiments, the expression vector is suitable for gene therapy in humans. Vectors suitable for gene therapy include, but are not limited to, adeno-associated virus (AAV) or adenovirus vectors. In certain embodiments, the expression vector comprises a DNA vector or an RNA vector. In certain embodiments, the pharmaceutically acceptable carrier is a polymeric excipient, such as, but not limited to, a microsphere, a microcapsule, a polymeric micelle, and a dendrimer. The polynucleotides or polynucleotide vectors of the present disclosure may be encapsulated, attached, or coated with polymer-based moieties by methods known in the art (see, e.g., W. Heiser, Nonviral gene transfer techniques, Published by Humana Press, 2004; U.S. Patent No. 6,025,337; Advanced Drug Delivery Reviews, 57(15):2177-2202 (2005)).

[0123]

[0158] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include pharma- ceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other ingredients known in the art, or various combinations thereof.

[0124]

[0159] Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers and stabilizers such as sugars and cyclodextrins. Suitable antioxidants include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in a composition comprising an antibody or antigen-binding fragment thereof and a conjugate provided herein reduces oxidation of the antibody or antigen-binding fragment thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving the stability of the antibody and maximizing shelf life. Thus, in certain embodiments, pharmaceutical compositions are provided that include one or more antibodies or antigen-binding fragments thereof disclosed herein and one or more antioxidants, such as methionine. Additionally, methods are provided to prevent oxidation, extend shelf life, and / or improve efficacy of the antibodies or antigen-binding fragments provided herein by combining the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0125]

[0160] By way of further example, pharma- ceutically acceptable carriers may contain, for example, aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactated Ringer's Injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents in bacteriostatic or fungistatic concentrations; isotonicity agents such as sodium chloride or glucose; buffers such as phosphate buffer or citrate buffer; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending or dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifying agents such as polysorbate 80 (TWEEN®-80); Antibacterial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers containing phenol or cresol, mercuria, benzyl alcohol, chlorobutanol, p-hydroxybenzoic acid methyl ester and propyl ester, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients include, for example, water, saline, glucose, glycerol, or ethanol. Suitable non-toxic auxiliary substances include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0126]

[0161] The pharmaceutical compositions may be in the form of liquids, suspensions, emulsions, pills, capsules, tablets, sustained release formulations, or powders. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0127]

[0162] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition.The injectable pharmaceutical composition may be prepared in any conventional form, such as, for example, a liquid, a suspension, an emulsion, or a solid form suitable for forming a liquid, a suspension, or an emulsion.The injectable preparation may include a sterile and / or non-pyrogenic solution ready for injection, a sterile dry soluble preparation, such as a freeze-dried powder, including a hypodermic tablet, ready to be combined with a solvent immediately before use, a sterile suspension ready for injection, a sterile dry insoluble preparation ready to be mixed with a vehicle immediately before use, and a sterile and / or non-pyrogenic emulsion.The solution may be aqueous or non-aqueous.

[0128]

[0163] In certain embodiments, unit dose parenteral preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration should be sterile and nonpyrogenic, as known and practiced in the art.

[0129]

[0164] In certain embodiments, the sterile lyophilized powder is prepared by dissolving the antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain excipients that improve stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents. The solvent may contain a buffer such as citrate, sodium or potassium phosphate, or other buffers known to those skilled in the art, in one embodiment at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In one embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial may contain a single dose or multiple doses of the antibody or antigen-binding fragment thereof or composition thereof. To facilitate accurate sampling and accurate dosing, it is acceptable to fill the vial with a small amount (e.g., about 10%) in excess of the amount needed for a single dose or set of doses. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0130]

[0165] The lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration.In one embodiment, for reconstitution, sterile water and / or nonpyrogenic water or other carrier is added to the lyophilized powder as appropriate liquid.The exact amount depends on the selected therapeutic method to be administered and can be empirically determined.

[0131]

[0166] The pharmaceutical compositions described herein contain pharmaceutical acceptable carriers, such as their addition salts or hydrates, and can be delivered to patients using a wide variety of administration routes or modes.Suitable administration routes include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal administration, and parenteral administration, including intramuscular injection, subcutaneous injection, and intravenous injection.Preferably, the pharmaceutical compositions of the present invention that contain antibody or antibody fragment as a targeting moiety are administered parenterally, more preferably intravenously.

[0132]

[0167] As used herein, the terms "administer" and "administration" are intended to encompass all means for delivering a pharmaceutical composition directly and indirectly to its intended site of action.

[0133]

[0168] The pharmaceutical compositions described herein, or pharma- ceutically acceptable salts and / or hydrates thereof, can be administered alone and / or in combination with other therapeutic agents. Of course, the choice of therapeutic agent that may be co-administered with the pharmaceutical compositions of the present disclosure will depend, in part, on the condition being treated.

[0134]

[0169] 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 as a cocktail containing drugs used to treat pain, infection, and other symptoms and side effects commonly associated with the disease, such as, for example, painkillers, antibiotics, etc.

[0135]

[0170] When administered to a subject undergoing cancer treatment, the pharmaceutical composition may be administered as a cocktail containing anti-cancer drugs and / or boosting agents. The pharmaceutical composition may also be administered as a cocktail containing agents that treat the side effects of radiation therapy, such as antiemetics, radiation protectants, etc. V.Kit

[0171] In another aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein.

[0136]

[0172] In certain embodiments, the present 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 a chemotherapeutic agent, an anticancer drug, a radiation therapy agent, an immunotherapy agent, an antiangiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic agent, an antioxidant, a metal chelator, and a cytokine.

[0137]

[0173] Such kits can optionally further contain one or more of the various components of a conventional pharmaceutical kit, such as, for example, containers housing one or more pharma- ceutically acceptable carriers, additional containers, etc. Instructions indicating the amounts of components to be administered, directions for administration, and / or directions for mixing the components, can be included in the kit as an insert or label. The kit can also contain one or more vials, test tubes, flasks, bottles, or syringes.

[0138]

[0174] Other formats of the kit will be apparent to those of skill in the art and are within the scope of this disclosure. VI. Medical Use

[0175] therapeutic use

[0176] In another aspect, the present disclosure provides a method for treating a subject in need of treatment for a disease or condition, 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 pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0139]

[0177] The disease or condition to be treated is related to the antigen targeted by the antibody or antigen-binding fragment thereof. 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, and others.

[0140]

[0178] In some embodiments, uses of the pharmaceutical compositions disclosed herein include the manufacture of a medicament for treating a disease, such as cancer, in a subject, such as a human. As used herein, "cancer" refers to a pathological condition in humans characterized by unregulated cell proliferation.

[0141]

[0179] In some embodiments, the disease is a SIRPα-associated disease, disorder, or condition characterized by expression or overexpression of SIRPα and / or the SIRPα signature gene. In certain embodiments, the SIRPα-associated disease, disorder, or condition includes, but is not limited to, cancer, solid tumors, chronic infections, inflammatory diseases, multiple sclerosis, autoimmune diseases, neurological diseases, brain injury, neurological injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant failure, or arthritis.

[0142]

[0180] 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 a cancer derived from epithelial cells. In some embodiments, the cancer is anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, gastric cancer, lung cancer, bronchial cancer, bone cancer, liver cancer 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, urethral cancer, bladder cancer, head and neck cancer, spinal cancer, brain tumor, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rect ... The cancer is selected from the group consisting of cancers of the esophagus, gastrointestinal tract, skin, prostate, pituitary, vaginal, thyroid, pharyngeal, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T-cell or B-cell lymphoma, GI organ interstitialoma, soft tissue tumor, hepatocellular carcinoma, or adenocarcinoma, or metastasis thereof. In some embodiments, the cancer is gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, or metastasis thereof.

[0143]

[0181] As used herein, the term "treat" or "treatment" refers to therapeutic and prophylactic or preventative treatment, the purpose of which is to alleviate or prevent a targeted pathological disorder or condition. In certain embodiments, "treatment" or "treating" includes (1) inhibiting a disease in a subject experiencing or exhibiting a disease symptom or symptom, (2) ameliorating a disease in a subject experiencing or exhibiting a disease symptom or symptom, and / or (3) affecting any measurable reduction in a disease in a subject or patient experiencing or exhibiting a disease symptom or symptom.

[0144]

[0182] As used herein, the term "therapeutically effective amount" refers to an amount of the active ingredient (i.e., an antibody or antigen-binding fragment thereof) in the pharmaceutical compositions provided herein effective to "treat" a disorder in a subject or mammal. Pharmaceutical compositions suitable for use in the present disclosure include compositions in which the active ingredient contains a therapeutically effective amount, i.e., an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. Determination of the effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein.

[0145]

[0183] In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells, reduce tumor size, inhibit the infiltration of cancer cells 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.

[0146]

[0184] In some embodiments, the antibody or antigen-binding fragment thereof provided herein can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibody or antigen-binding fragment thereof disclosed herein can be administered in combination with a second therapeutic agent, such as a chemotherapeutic agent, an anticancer drug, a radiotherapy agent, an immunotherapy agent, an antiangiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic agent, an antioxidant, a metal chelator, or a cytokine.

[0147]

[0185] In certain of these embodiments, the antibodies or antigen-binding fragments thereof provided herein, when administered in combination with one or more additional therapeutic agents, may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the antibodies or antigen-binding fragments thereof and the additional therapeutic agents may be administered as part of the same pharmaceutical composition.

[0148]

[0186] Diagnostic Uses

[0187] In another aspect, the disclosure provides a method of diagnosing a disease, disorder, or condition in a subject comprising the steps of: 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 in the sample that is targeted by the antibody or antigen-binding fragment thereof; and c) correlating the presence or amount of the relevant antigen with the presence or condition of the disease, disorder, or condition in the subject.

[0149]

[0188] The terms "diagnosis," "diagnose," or "diagnosing" refer to the identification of a pathological state, disease, or condition, such as a disease associated with a particular antigen, or to the identification of a subject having a disease associated with a particular antigen and who may benefit from a particular treatment regimen. In some embodiments, diagnosis includes the identification of an abnormal amount or activity of a particular antigen. In some embodiments, diagnosis refers to the identification of cancer or an autoimmune disease in a subject.

[0150]

[0189] As used herein, the term "sample" refers to a biological composition obtained or derived from a subject of interest, which contains cells and / or other molecular entities that are to be characterized and / or identified, for example, 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, which are obtained by methods known to those of skill in the art.

[0151]

[0190] In another aspect, the disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein, optionally conjugated to a detectable moiety, useful for detecting a disease, disorder, or condition. The kit may further comprise instructions for use.

[0152]

[0191] The following examples are provided to better illustrate the claimed invention and are not to be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below are 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 merely to illustrate specific embodiments that fall within the scope of the present invention. Those skilled in the art can develop equivalent compositions, materials, and methods without exerting inventive ability and without departing from the scope of the present invention. It will be understood that many variations can be made in the procedures described herein while remaining within the scope of the present disclosure. It is the intention of the inventors that such variations are included within the scope of the present invention.

[0153]

[0192] In the following examples, all amino acid positions are numbered according to the EU numbering system. EXAMPLES

[0154] Example 1 Construction and analysis of asymmetric monovalent antibodies with a transferred CH1-CL interchain disulfide bond

[0193] Multiple sites on the antibody where new disulfide bonds could potentially form were identified. Candidate sites were selected from amino acid residues in the CL region of the light chain and the CH1 region of the heavy chain. A pair of non-cysteine ​​residues, one located in the CL region and the other in the CH1 region, were both substituted with cysteines to allow the formation of a new disulfide bond between them. One or two pairs of residues with the potential to form new interchain disulfide bonds were selected for each design and mutated to cysteines.

[0155]

[0194] In addition, the conserved native disulfide bond between Cys220 of CH1 and Cys214 of CL was disrupted by substituting both residues with serine.For initial screening, an assay system was designed that allowed for easy readout by SEC-HPLC (Figures 1 and 2).

[0156]

[0195] Specifically, the system consists of four polypeptide chains. Chain 1 is a full-length HC with (chain 1a used in screening assay 1, Figure 1) or without (chain 1b used in screening assay 2, Figure 2) a cysteine ​​mutation (non-cysteine ​​to cysteine ​​residue) in the CH1 region, 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 fused to a single domain antibody (VHH) at its N-terminus. Chain 4 is an empty 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 this system, the main product is a monovalent heterodimer consisting of two possible, differently sized species that are easily distinguished in the SEC-HPLC profile and can be easily quantified (Figures 1 and 2). In both screening assays, heterodimers formed between mutant and wild-type peptide chains were considered as mismatched heterodimers, whereas heterodimers between the designed mutant or wild-type chains were considered as correctly matched heterodimers. Thus, the relative abundance (RA) of different heterodimers was defined as:

[0157]

number

[0158] It is a direct measure of the pairing preference of different CL and CH1 chains.

[0196] The sequences of the different polypeptide chains of the two assay systems are shown in Table 6 below. The VH (SEQ ID NO: 3) and VL (SEQ ID NO: 2) sequences were derived from humanized anti-CLDN18.2 antibody. The heavy chain constant region, containing the CH1 region (SEQ ID NO: 5) and the Fc region (SEQ ID NO: 6), was derived from human IgG1. The light chain constant region CL (SEQ ID NO: 7) was derived from human kappa light chain. The VHH domain (SEQ ID NO: 1) was linked to the N-terminus of the VL region via a linker (SEQ ID NO: 9). All DNA fragments were synthesized by chemical process. Each of the four polypeptide chains was cloned into pcDNA3.4 vector (Figure 3A to 3D). The four plasmids were mixed and transfected together into ExpiCHO-S cells (Life Technologies). The expressed protein variants were then produced in ExpiCHO-S cells using a 24-well plate production system. Seven days after transfection, the culture supernatant was collected by centrifugation and incubated with Protein A magnetic beads (GenScript). Bound antibodies were eluted with 0.05 M citrate buffer (pH 3.4) and immediately neutralized with Tris buffer (pH 9.0). Purified antibodies were evaluated by SDS-PAGE and SEC-HPLC (Agilent).

[0159]

[0197]

[0160] [Table 8]

[0161]

[0198] 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 were 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 were mutated to cysteine. Multiple mutation pairs were generated between chain 1 and chain 2 using the above mutation sites and used in screening assay 1.

[0162]

[0199] In screening assay 1, wild-type and mutant CL were competed against mutant CH1. A correct match was formed between strand 2 (with mutant CL) and strand 1a (with mutant CH1), and a mismatch was formed between strand 3 (with wild-type CL) and strand 1a (with mutant CH1).

[0163]

[0200] In screening assay 1, multiple mutation combinations 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 show the RA values ​​of some mutant pairs that showed high RA values. In this screening assay 1, ACF_003 was used as a reference. ACF_003 (containing a combination of mutations S121C on the light chain and F126C on the heavy chain) has corresponding mutations in the CL and CH1 regions, similar to V12 disclosed in U.S. Patent No. 9,527,927.

[0164]

[0201]

[0165] [Table 9-1]

[0166] [Table 9-2]

[0167]

[0202] In screening assay 2, mutant CL and wild-type CL were competed against wild-type CH1. The RA values ​​of the mutant pairs used in screening assay 2 were calculated by the same formula as in screening assay 1, except that correct pairing was formed only between strand 3 (with wild-type CL) and strand 1b (with wild-type CH1), and mispairing was formed between strand 2 (with mutant CL) and strand 1b (with wild-type CH1).

[0168]

[0203] The mutant pairs that showed high RA values ​​are shown in Table 8. ACF_245 is used as the standard for this screening assay 2. ACF_245 (containing the S121C mutation on the light chain and no mutation on the heavy chain) has a mutation corresponding to the CL region similar to ACF_003, except that it has no mutation on the heavy chain. Table 8 and Figure 4B show the RA of some mutant pairs that showed high RA values.

[0169]

[0204]

[0170] [Table 10]

[0171] Example 2 Construction and analysis of a symmetric bivalent monospecific antibody with a shifted CH1-CL disulfide bond

[0205] To further evaluate the expression levels and stability of selected mutants in a reductive system, several selected mutant pairs from the experiment in Example 1 were converted into symmetric bivalent monospecific antibodies (Figure 5, Table 9) for yield and purity testing.

[0172]

[0206] All mutations were made based on the parent antibody hu28. The amino acid sequences of the VL and VH regions of hu28 were as set forth in SEQ ID NO:2 and 3, respectively. The amino acid sequence of the CL region of hu28 was as set forth in SEQ ID NO:4. The amino acid sequence of the CH1 region of hu28 was as set forth in SEQ ID NO:5. The amino acid sequence of the Fc region of hu28 was as set forth in SEQ ID NO:6.

[0173]

[0207]

[0174] [Table 11]

[0175]

[0208] All DNA fragments were synthesized and cloned into 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 by protein A beads. Purified antibodies were first evaluated for purity by SDS-PAGE and HPLC-SEC. The yield and purity of each antibody are shown in Figure 6A and 6B, respectively. ACF-hu28_001, which contains the residue substitution pairs reported by MedImmune in U.S. Patent No. 9,527,927, was used as a reference for comparison.

[0176]

[0209] As shown in the results in Figures 6A and 6B, ACF-hu28_025, ACF-hu28_038, ACF-hu28_029, ACF-hu28_037, ACF-hu28_002, ACF-hu28_031, ACF-hu28_034, ACF-hu28_032, ACF-hu28_014, ACF-hu28_016, ACF-hu28_012, ACF-hu28_011, ACF-hu28_004, ACF-hu28_0 15, 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, ACF-hu28_003 have comparable or higher production yields and percentage monomer compared to the reference monoclonal antibody ACF hu28_001 (S121C / C214S, F126C / C220S).

[0177] Example 3 Antigen binding assay and heat resistance comparison of bivalent monospecific antibodies

[0210] 3.1 Evaluation of binding activity by FACS assay

[0211] All selected bivalent variants were compared for binding to CLDN18.2 by FACS using MC38 cells overexpressing CLDN18.2 (Figures 7A-7D). All proteins were buffer exchanged into PBS (pH 7.2) and diluted to different concentrations (100 nM, 20 nM, 4 nM, 0.8 nM) in FACS buffer (BD). Hu28 mAb was used as a positive control.

[0178]

[0212] No significant difference in antigen-binding activity was observed between the variants and the positive control mAb.

[0213] 3.2 Analysis of heat resistance of mutant mAbs

[0214] The thermostability of the purified variants was assessed using a thermal shift assay (Figure 8). The antibody variants were first buffer exchanged into PBS (pH 7.2) and then mixed 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 / s and a hold time of 2 min. The resulting melting curves were analyzed with Protein Thermal Shift Software. The data suggested that all variants had thermostability comparable to that of the wild-type monoclonal antibody.

[0179] Example 4 Generation of asymmetric bispecific antibodies and their analysis by LC-MS

[0215] To quantitatively evaluate the efficiency of pairing between CH1 and CL chains in bispecific antibodies, two series of bispecific antibodies were generated and analyzed by LC-MS.

[0180]

[0216] In both series, one arm of the bispecific antibody was derived from an anti-CLDN18.2 antibody (hu28) and the other arm was derived from an anti-SIRPα antibody (hu25). Knob mutations were introduced into the CH3 region of the hu28 arm, and hole mutations were introduced into the CH3 region of the hu25 arm. In addition, mutations that disrupt protein A binding were introduced into the hu25 arm to simplify the system.

[0181]

[0217] The amino acid sequences of the VH and VL regions of hu25 were as set forth 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 were as set forth in SEQ ID NOs: 2 and 3, respectively. The amino acid sequence of the non-mutated CL region of hu25 or hu28 was as set forth in SEQ ID NO: 4. The amino acid sequence of the non-mutated CH1 region of hu25 or hu28 was as set forth in SEQ ID NO: 5. The amino acid sequence of the Fc (with "hole" mutations) of hu25 or hu28 was as set forth in SEQ ID NO: 7. The amino acid sequence of the Fc (with "knob" mutations) of hu25 or hu28 was as set forth in SEQ ID NO: 8.

[0182]

[0218]

[0183] [Table 12]

[0184]

[0219] In case 1 studies, different pairs of mutations to form shifted disulfide bonds were introduced into the CH1 and CL regions of the hu28 arms, while the wild-type sequence was retained in the hu25 arms (Figures 9 and 10, Table 11).

[0185]

[0220] In the case 2 study, the same set of paired mutations was introduced into the arms of hu25, while the wild-type sequence was retained in the arms of hu28 (Figures 11 and 12, Table 11).

[0221]

[0186] [Table 13]

[0187]

[0222] All mutation combinations constructed for testing are shown in Table 12.

[0223]

[0188] [Table 14]

[0189]

[0224] To produce bispecific antibodies, hu28 HC and LC plasmids were mixed with hu25 HC and LC plasmids before co-transfection. The variants were produced in ExpiCHO-S cells (Life Technologies) using a 24-well plate production system. Seven days after transfection, culture supernatants were collected by centrifugation and incubated with Protein A magnetic beads (GenScript). 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 (Figures 13A and 13B).

[0190]

[0225] To quantitatively evaluate the pairing efficiency of the cognate CH1 and CL chains, purified antibodies were analyzed by LC-MS under non-reducing conditions. Due to the denaturing nature of the sample processing conditions in LC-MS analysis, molecules containing mispaired HC-LCs (CH1 mutated HC and wild-type LC pairs, or CH1 unmodified HC and CL mutated LC pairs) dissociate and show an LC-only peak in the LC-MS profile. Thus, in theory, all molecules containing heterodimeric HCs will be detected. Heterodimeric HCs with two correctly paired LCs were desired for bispecific complete antibodies. Heterodimeric HCs with mispaired LCs are detected by the mispaired and dissociated LC peak in the LC-MS profile. By evaluating the intensity of the MS signal of the LC-only peak derived from the CH1-CL mispairing, the pairing efficiency of different mutant pairs of cognate CH1 and CL chains can be ranked (Figures 9 and 11).

[0191]

[0226] The heat resistance of the antibody was assessed by differential scanning fluorimetry.

[0227] In the case 1 study, ACF_329, 333, 337, 338 and 339 had lower LC signals for hu025 and equivalent LC signals for hu028 than the benchmark ACF_326 (designed by MedImmune) (Figure 10). These data indicate that in this case, the mutation pairs introduced into ACF_329, 333, 337, 338 and 339 led to high efficiency of pairing with the cognate CH1 and CL chains.

[0192]

[0228] In the case 2 study, ACF_356, 359, 361, 364, 365, 366, 367, and 368 showed comparable hu025 LC and hu028 LC signals to the reference ACF_355 (designed by MedImmune) (Figure 12). These data indicate that the mutation pairs introduced into ACF_356, 359, 361, 364, 365, 366, 367, and 368 resulted in high efficiency of pairing with the cognate CH1 and CL chains.

[0193]

[0229] Of note, the following mutation pairs resulted in high efficiency of pairing of the cognate CH1 with the CL chain in each case: (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, (v) Q124C / S162C on the light chain and F126C / F170C on the heavy chain.

[0194] Example 5 Further generation and analysis of asymmetric bispecific antibodies

[0230] 5.1 Protein expression and purification

[0231] 5.1.1 Constructs and amino acid sequences of tested antibodies

[0232] Three bispecific antibodies, Antibody 1, Antibody 2, and Antibody 3, were expressed and purified.

[0195]

[0233] In the study of antibody 1, the mutation pairs Q124C / T164C / C214S on the light chain and F126C / F170C / C220S on the heavy chain were introduced into the arms of hu25, and the wild-type sequences were retained in the arms of hu28. Antibody 1 has the same structure and amino acid sequence as antibody ACF_361 in Example 4. Therefore, it is hereinafter referred to as ACF_361 or ACF361.

[0196]

[0234] In the study of antibody 2, the mutation pairs Q124C / T164C / C214S on the light chain and F126C / F170C / C220S on the heavy chain were introduced into the arms of hu25, and the wild-type sequence was retained in the arms of hu29 (source sequence: atezolizumab, an anti-PDL1 antibody). Hereafter, antibody 2 is referred to as ACF_389 or ACF389.

[0197]

[0235] The amino acid sequences of the VH and VL regions of hu25 are set forth in SEQ ID NOs: 10 and 11, respectively (Table 10). The amino acid sequences of the VH and VL regions of hu29 are set forth in SEQ ID NOs: 12 and 13, respectively (Table 11). The amino acid sequences of the non-mutated CL region of hu25 or hu29 are set forth in SEQ ID NO: 4. The amino acid sequence of the non-mutated CH1 region of hu25 or hu29 is set forth in SEQ ID NO: 5. The amino acid sequence of the Fc of hu25 or hu29 (with "hole" mutations) is set forth in SEQ ID NO: 7. The amino acid sequence of the Fc of hu25 or hu29 (with "knob" mutations) is set forth in SEQ ID NO: 8.

[0198]

[0236]

[0199] [Table 15]

[0200]

[0237] In the study of antibody 3, the mutation pairs Q124C / S162C / C214S on the light chain and F126C / F170C / C220S on the heavy chain were introduced into the H95 arm, and the wild-type sequence was retained in the H43 arm. Hereinafter, antibody 3 is referred to as ESB07.451.

[0201]

[0238] The amino acid sequences of the VH and VL regions of H95 are set forth in SEQ ID NO: 14 and 15, respectively. The amino acid sequences of the VH and VL regions of H43 are set forth in SEQ ID NO: 16 and 17, respectively. The amino acid sequences of the non-mutated CL regions of H95 or H43 are set forth in SEQ ID NO: 19. The amino acid sequences of the non-mutated CH1 regions of H95 or H43 are set forth in SEQ ID NO: 18. The amino acid sequence of the Fc of H43 (with the "hole" mutation) is set forth in SEQ ID NO: 21. The amino acid sequence of the Fc of H95 (with the "knob" mutation) is set forth in SEQ ID NO: 20.

[0202]

[0239]

[0203] [Table 16]

[0204]

[0240] 5.1.2 Expression and purification of test antibodies

[0241] To produce bispecific antibodies, hu28 HC and LC plasmids were mixed with hu25 HC and LC plasmids, hu29 HC and LC plasmids were mixed with hu25 HC and LC plasmids, and H95 HC and LC plasmids were mixed with H43 HC and LC plasmids prior to cotransfection.

[0205]

[0242] The variants were produced in ExpiCHO-S cells (Life Technologies). Seven days after transfection, the culture supernatants were collected by centrifugation and incubated with alkaline-resistant Protein A medium (AT Protein A Diamond, Bestchrom). 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 and SEC. The results for ACF_361, ACF_389, and ESB07.451 are shown in Figure 14, Figure 17, and Figure 22, respectively.

[0206]

[0243] For ACF_389 and ESB07.451, Mono S chromatography (5 / 50GL, Cytiva) was used to obtain samples with higher purity. The antibody was loaded and washed with buffer A (10 mM sodium acetate, pH 5.2), and then eluted with a gradient of buffer B (10 mM sodium acetate, pH 5.2, 250 mM NaCl). The results for ACF_389 are shown in Figures 18 and 19. The results for ESB07.451 are shown in Figures 22 and 23.

[0207]

[0244] To quantitatively evaluate the pairing efficiency of the cognate CH1 and CL chains, the purified antibodies were analyzed by LC-MS under non-reducing conditions. The results for ACF_361, ACF_389, and ESB07.451 are shown in Figures 15, 20, and 24, respectively. The LC-MS results showed that the peak at around 145 KD was the cognate CH1 / CL pairing, indicating that the three antibodies were correctly assembled.

[0208]

[0245] 5.2 Binding affinity assays

[0246] 5.2.1 Binding affinity of CLDN18.2 to cells overexpressing SIRPα

[0247] The dual binding properties of the bispecific antibody ACF_361 to cells were analyzed by FACS.

[0209]

[0248] 1 x 10 per well 5CLDN18.2-expressing MC38 cells were incubated with 100 nM to 0.05 nM of each purified antibody for 1 hour, followed by incubation with secondary antibody Alexa Flour 647 anti-human IgG (H+L).

[0210]

[0249] 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, as below) was used as a negative control. Information on each antibody tested is shown in Figure 16A. ES028-h26 and ES028-h28 were both monospecific IgG antibodies targeting CLDN18.2. AE016-201 was a bispecific antibody (whose scFv is bound to IgG) targeting CLDN18.2 and SIRPα. ES028-005-08-26H1L2-201 was a bispecific antibody (whose scFv is bound to IgG) targeting CLDN18.2 and SIRPα.

[0211]

[0250] The binding curves of the antibodies to MC38-CLDN18.2 cells are shown in Figure 16A.

[0251] 1 x 10 per well 5 CHO / SIRPαV1 cells were incubated with each purified antibody at 100 to 0.05 Nm for 1 hour, followed by incubation with the secondary antibody Alexa Flour 647 anti-human IgG (H+L).

[0212]

[0252] In this assay, ES001-025.201-IgG1LALA, ES028-005-08-26H1L2-201, AE016-201 were used as positive controls, and hIgG1 isotype was used as negative control. Information of each tested antibody is shown in Figure 16B. ES001-025.201-IgG1LALA was a monospecific IgG antibody targeting SIRPα with LALA mutation in the Fc region of the antibody. ES028-005-08-26H1L2-201 and AE016-201 were bispecific antibodies (whose scFvs are bound to IgG) targeting CLDN18.2 and SIRPα.

[0213]

[0253] The binding curves of the antibodies to CHO / SIRPαV1 cells are shown in FIG. 16B.

[0254] 5.2.2 Binding affinity to PDL1 and SIRPα overexpressing cells

[0255] The dual binding properties of the bispecific antibody ACF_389 to cells were analyzed by FACS. 1 × 10 per well 5 PDL1-expressing Raji cells were incubated with 100 nM to 0.05 nM of each purified antibody for 1 hour, followed by incubation with the secondary antibody Alexa Flour 647 anti-human IgG (H+L).

[0214]

[0256] 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, hereinafter the same) were used as negative controls. Atezolizumab is a known anti-PDL1 antibody, the details of which are described, for example, in US Patent No. 9873740 (B2). ES004-025.201-IgG4 was a monospecific antibody that targets SIRPα.

[0215]

[0257] The binding curves of the antibodies to Raji / PDL1 cells are shown in FIG. 21A.

[0258] 1 x 10 per well 5CHO / SIRP V1 cells were incubated with 100 nM to 0.05 nM of each purified antibody for 1 hour, followed by incubation with the secondary antibody Alexa Flour 647 anti-human IgG (H+L).

[0216]

[0259] In this assay, ES004-025.201-IgG4 was used as a positive control, and atezolizumab, hIgG1 and hIgG4 were used as negative controls. Antibody information is as above.

[0217]

[0260] The binding curves of the antibodies to CHO / SIRPαV1 cells are shown in FIG. 21B.

[0261] 5.3 Phagocytic activity of bispecific antibodies

[0262] The phagocytic activity of ACF_361 was tested in this assay.

[0218]

[0263] To obtain a single cell suspension, the macrophage cells were digested with Accutase at 37°C for 10 min, and then cell culture medium was added to stop the digestion. After centrifugation at 400g for 5 min, the macrophage cells were plated in a 96-well plate at 3 × 10 4 Mouse BMDM cells were stained with CD11b and F4 / 80 to assess quality and purity. The following day, 25ul of antibody was added at concentrations ranging from 0.05nM to 10nM, and 6x10 4 50ul of Violet CSFE-labeled Raji-CLDN18.2 cells per well were added and incubated at 37℃ for 3 hours. The cells were washed with DPBS and digested with Accutase at 37℃ for 10 minutes, after which the digested cells were washed with FACS buffer. The cells were incubated with anti-mouse F4 / 80 APC at 4℃ for 30 minutes. The stained cells were washed and suspended in FACS buffer. The phagocytosis rate was measured by FACS.

[0219]

[0264] In this assay, ES028-005-08-26H1L2-201 and AE016-201 were used as positive controls, and hIgG1+hIgG4, anti-SIRPα monospecific antibody, two anti-CLDN18.2 antibodies (anti-CLDN18.2.h26 and anti-CLDN18.2.h28) and their combination were used as negative controls. Both ES028-005-08-26H1L2-201 and AE016-201 are bispecific antibodies (whose scFvs are linked to IgG) targeting CLDN18.2 and SIRPα. Anti-CLDN18.2 hu26 (hIgG1) and anti-SIRPα hu25.060 (hIgG4) were added in equimolar amounts to represent the combination treatment group (i.e., combination). Equimolar amounts of hIgG1 and hIgG4 were added to represent the isotype control group (i.e., hIgG1+hIgG4).

[0220]

[0265] Summarized data is shown in FIG. 16C.

[0266] 5.4 Hydrophobicity evaluation

[0267] The retention time in HIC-HPLC assay is a factor for evaluating the hydrophobicity of antibodies. 15ul of 0.25mg / ml antibody was injected into the MacPac-10 HIC column of an Agilent 1260 system, and the signals at 280nm / 214nm were detected. As shown in Table 13, the retention times of ACF_361, ACF_389, and ESB07.451 in MacPac-10HIC were 8.413min, 13.583min, and 9.9min, respectively, indicating that the three bispecific antibodies have low hydrophobicity.

[0221]

[0268]

[0222] [Table 17]

[0223]

[0269] 5.5 Stability testing

[0270] 5.5.1 Heat resistance evaluation

[0271] The melting temperature (Tm) values ​​of the bispecific antibodies in PBS buffer were detected to predict their heat resistance. Briefly, the bispecific antibodies were decomposed in 10 mM PBS buffer (pH 7.2-7.4). Then, the Tm values ​​were detected by differential scanning fluorometry (DSF) using a QuantStudio7 Flex Real time PCR system. As shown in Table 14, the Tm values ​​of ACF_361, ACF_389 and ESB07.451 indicate that the bispecific antibodies have good heat resistance.

[0224]

[0272]

[0225] [Table 18]

[0226]

[0273] 5.5.2 Low pH stability evaluation

[0274] High stability under low pH conditions is important for antibodies because purification steps often require exposure to acidic solution conditions, and the commonly used low pH viral inactivation. Low pH typically induces soluble and insoluble aggregation and promotes fragmentation. To evaluate protein stability under low pH conditions, bispecific antibodies in PBS buffer were titrated to pH 3.0-3.5 with acetic acid. Antibody stability was then evaluated by monitoring the change in total soluble protein (recovery) and purity before and after 2 h exposure to low pH solutions.

[0227]

[0275] The results are shown in Table 15, where TO indicates the data for the original sample and Low pH 2h indicates the data at low pH after 2 hours. As a result of exposure to low pH solution for 2 hours, the total purity was almost unchanged, indicating that all three antibodies ACF_361, ACF_389 and ESB07.451 have good stability under low pH conditions.

[0228]

[0276]

[0229] [Table 19]

[0230]

[0277] 5.5.3 Stability under freeze-thaw stress

[0278] Freeze-thaw stability is often studied to examine the sensitivity of antibodies to temperature cycles to which products are frequently exposed. For example, drug substances are often frozen to enable long-term storage. Pharmaceuticals may be exposed to freezing temperatures during the process of lyophilization. The main degradation pathways of freeze-thawing are precipitates, particles, and aggregates containing soluble particles. Therefore, the freeze-thaw stability of a bispecific antibody was evaluated by monitoring the change in purity before and after repeated freeze-thaw treatments. Briefly, the antibody in 20 mM PBS (pH 6.0-6.2) was frozen at -80 °C for more than 12 h and thawed at room temperature. After five freeze-thaw cycles, the concentration and purity of the stressed samples were measured.

[0231]

[0279] The results are shown in Table 16, where TO indicates the data for the original sample and FT5 indicates the data after five freeze-thaw cycles. No obvious increase in aggregates or fragments was observed for ACF_361, ACF_389, or ESB07.451 even after multiple freeze-thaw cycles, indicating that all three antibodies have good freeze-thaw stability.

[0232]

[0280]

[0233] [Table 20]

[0234]

[0281] 5.5.4 Stability under heat stress

[0282] Heat stress at temperatures above normal storage conditions can accelerate degradation, thus increasing the detectability of potential degradation pathways and providing information on long-term degradation under intended storage conditions. Heat stress testing of bispecific antibodies in PBS was performed at 25°C and / or 40°C for 4 weeks. SEC purity was detected to monitor for insoluble or soluble aggregates.

[0235]

[0283] The results are shown in Table 17, where TO indicates the data of the original sample, 25C-2W indicates the data of 2 weeks at 25°C, and other naming conventions are similar. It can be seen that the purity of ACF_361 and ACF_389 in PBS hardly changes after 4 weeks of incubation at 25°C, and the purity of ESB07.451 in PBS hardly changes after 4 weeks of incubation at 25°C and 40°C.

[0236]

[0284]

[0237] [Table 21]

Claims

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

2. at least one non-cysteine ​​residue on the first heavy chain that is replaced with a 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; 2. The antibody or antigen-binding fragment thereof of claim 1, wherein at least one non-cysteine ​​residue on the first light chain that is replaced with a 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 first light chain and the first heavy chain have 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 (i) S114C on the light chain and S136C on the heavy chain; (ii) F116C in the light chain and S136C in the heavy chain; (iii) F118C in the light chain and L128C in the heavy chain; (iv) F118C in the light chain and A129C in the heavy chain; (v) F118C in the light chain and A141C in the heavy chain; (vi) Q124C in the light chain and F126C in the heavy chain; (vii) L135C in the light chain and F170C in the heavy chain; (viii) N137C in the light chain and F170C in the heavy chain; (ix) N138C in the light chain and H168C in the heavy chain; (x) Q160C in the light chain and V173C in the heavy chain; (xi) Q160C of the light chain and Q175C of the heavy chain; (xii) S162C in the light chain and F170C in 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 and at least one cysteine ​​substitution pair selected from the group consisting of: at least one pair of cysteine ​​substitutions forms at least one interchain disulfide bond between the first heavy chain and the first light chain; 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the native cysteines on the first heavy chain and the first light chain are independently replaced with any one of non-cysteine ​​residues.

4. 4. The antibody or antigen-binding fragment thereof of claim 3, wherein the native cysteines on the first heavy chain and the first light chain are independently replaced 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 one 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. Two cysteine ​​substitution pairs: (i) S114C / Q160C on the light chain and S136C / V173C on the heavy chain; (ii) F118C / Q124C in the light chain and L128C / F126C in the heavy chain; (iii) F118C / Q124C in the light chain and A129C / F126C in the heavy chain; (iv) F118C / Q124C in the light chain and A141C / F126C in the heavy chain; (v) F116C / Q124C in the light chain and S136C / F126C in the heavy chain; (vi) Q124C / T164C in the light chain and F126C / F170C in the heavy chain; (vii) Q124C / N138C in the light chain and F126C / H168C in the heavy chain; (viii) Q160C / L135C in the light chain and Q175C / F170C in the heavy chain; (ix) S114C / S162C in the light chain and S136C / F170C in the heavy chain; (x) F118C / S162C in the light chain and L128C / F170C in the heavy chain; (xi) F118C / S162C in the light chain and A141C / F170C in the heavy chain; (xii) Q124C / S162C in the light chain and F126C / F170C in the heavy chain; (xiii) N137C / S162C in the light chain and F170C / T187C in 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 The antibody or antigen-binding fragment thereof of claim 6, selected from the group consisting of:

8. an 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 antibody or antigen-binding fragment thereof of claim 1, wherein the position of a native cysteine ​​on the second light chain corresponds to position C214 on a human κ chain and the position of a native cysteine ​​on the second heavy chain corresponds to position C220 on a human IgG1.

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

10. The antibody or antigen-binding fragment thereof of claim 1, wherein the substituted native cysteine ​​on the second light chain is at position C214 on a human κ chain and the substituted native cysteine ​​on the second heavy chain is at position C220 on a 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. Two cysteine ​​substitution pairs: (i) S114C / Q160C on the light chain and S136C / V173C on the heavy chain; (ii) F118C / Q124C in the light chain and L128C / F126C in the heavy chain; (iii) F118C / Q124C in the light chain and A129C / F126C in the heavy chain; (iv) F118C / Q124C in the light chain and A141C / F126C in the heavy chain; (v) F116C / Q124C in the light chain and S136C / F126C in the heavy chain; (vi) Q124C / T164C in the light chain and F126C / F170C in the heavy chain; (vii) Q124C / N138C in the light chain and F126C / H168C in the heavy chain; (viii) Q160C / L135C in the light chain and Q175C / F170C in the heavy chain; (ix) S114C / S162C in the light chain and S136C / F170C in the heavy chain; (x) F118C / S162C in the light chain and L128C / F170C in the heavy chain; (xi) F118C / S162C in the light chain and A141C / F170C in 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 selected from the group consisting of: The antibody or antigen-binding fragment thereof of claim 11, wherein the pair of cysteine ​​substitutions on the first arm is different from the pair of cysteine ​​substitutions on the second arm.

13. the first heavy chain constant region and / or the second heavy chain constant region comprises human IgG1, IgG2, IgG3, or IgG4; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the constant region of the first light chain and / or the constant region of the second light chain comprises a human κ light chain or a human λ 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 constant region of the first heavy chain and / or the Fc region of the constant region of the second heavy chain contain 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 of 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. 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 of claim 14, wherein the modification that promotes heterodimerization comprises the introduction of a cysteine ​​residue capable of forming an interchain disulfide bond.

19. The first heavy chain constant region Fc region and the second heavy chain constant region Fc region each comprise a modification in the CH3 region; the modifications in the two CH3 regions are as follows: 【Table 1】 The antibody or antigen-binding fragment thereof of claim 14, selected from the group consisting of:

20. The first heavy chain constant region Fc region and the second heavy chain constant region Fc region each comprise a modification in the CH3 region; the modifications in the two CH3 regions are as follows: 【Table 2】 The antibody or antigen-binding fragment thereof of claim 14, selected from the group consisting of:

21. The antibody or antigen-binding fragment thereof according to claim 1, wherein 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, and ANG-2, and the first arm and the second arm specifically bind to different antigens.

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

23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, which is a humanized or chimeric antibody.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, which is a bispecific or multispecific antibody.

25. 25. The antibody or antigen-binding fragment thereof of any one of claims 1 to 24, linked to one or more conjugate moieties.

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

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

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

29. 26. 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 of any one of claims 1 to 29.

31. A vector comprising the isolated polynucleotide of claim 30.

32. A host cell comprising the vector of claim 31.

33. (i) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, or a polynucleotide according to claim 30; (ii) one or more pharma- ceutically acceptable carriers; 23. A pharmaceutical composition comprising:

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

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

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

37. 37. The method of claim 35 or 36, wherein administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

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

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