Bispecific antibodies with selectively matched interchain cysteine ​​and their use

Abstract

Engineered bispecific antibodies are described in which the interchain disulfide bond of one arm is shifted and the second arm maintains the native interchain disulfide bond. Anti-CD47 / FRα bispecific antibodies and antigen-binding fragments thereof are also described. Nucleic acids encoding these antibodies, compositions comprising these antibodies, and methods of producing these antibodies and using them to treat or prevent diseases, such as cancer and / or related complications, are also described.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims priority to U.S. Provisional Application No. 62 / 948,953, filed December 17, 2019; U.S. Provisional Application No. 62 / 952,747, filed December 23, 2019; U.S. Provisional Application No. 62 / 988,144, filed March 11, 2020; U.S. Provisional Application No. 63 / 007,996, filed April 10, 2020; U.S. Provisional Application No. 62 / 704,973, filed June 5, 2020; and U.S. Provisional Application No. 62 / 706,511, filed August 21, 2020. The entire disclosure of each is incorporated herein by reference in its entirety.

[0002] [Field of the Invention] The present invention relates to engineered bispecific antibodies in which the inter - chain disulfide bonds of one arm are shifted and the second arm maintains native inter - chain disulfide bonds. These bispecific antibodies are excellent in stability and productivity and can be used for therapeutic purposes. The present invention relates to bispecific antibodies, nucleic acids encoding the antibodies and expression vectors, recombinant cells containing the vectors, and compositions containing the antibodies. Methods for producing the antibodies and methods for using the antibodies for treating diseases including cancer and / or related complications are also provided.

[0003] [Reference to Electronically Submitted Sequence Listing] This application includes a sequence listing submitted electronically via EFS - Web as an ASCII format sequence listing with the file name "065799.30WO1 Sequence Listing", creation date November 24, 2020, and size 36 kb. The sequence listing submitted via EFS - Web is part of this specification and is incorporated herein by reference in its entirety.

Background Art

[0004] Antibodies (immunoglobulins) are naturally occurring proteins that play a crucial role in the immune system's function in defending the body from foreign substances such as bacteria and viruses. Naturally occurring antibodies exist as Y-shaped proteins consisting of two arms, each containing an identical heavy chain (HC) and an identical light chain (LC). The heavy chain contains one variable region (VH) and three constant regions (CH1, CH2, and HC3, respectively), arranged in the order VH, CH1, CH2, and C3 from the N-terminus to the C-terminus. The light chain contains one variable region (VL) and one constant region (CL), arranged in the order VL and CL from the N-terminus to the C-terminus. The association of the heavy and light chains in each arm, involving VH, CH1, VL, and CL, is usually called "pairing." VH and VL physically interact with each other to form the binding domain of the antibody against the antigen, so that the Y-shaped antibody is bivalent and monospecific (a typical characteristic of monoclonal antibodies) because it has two identical binding domains (one on each arm) for the same antigen.

[0005] Furthermore, as part of the antibody structure, CH1 and CL physically interact with each other, including physical contact and interchain disulfide bonds (also called "disulfide bridges") formed by the two natural free cysteine ​​thiol groups on CH1 and CL, respectively. Interchain disulfide bonds help stabilize the overall structure formed by the heavy and light chains on each arm. In addition, intrachain disulfide bonds are also formed as part of the natural antibody structure. The C-terminuses of the heavy chains (CH2 and CH3) form a robust structure that is important for the divalent nature of natural antibodies.

[0006] Monoclonal antibodies (mAbs) have been an excellent platform for protein-based therapeutics due to their high antigen-binding affinity, long in vivo half-life, naturally occurring stable structure, ability to activate the immune system against drug targets, and many other advantages. However, mAbs cannot fulfill the purpose when a therapeutic strategy requires targeting two distinct antigens with a single antibody (e.g., two tumor-specific antigens on the same cancer cell). In such cases, bispecific antibodies are created that target two different antigens on the same cell (one arm binds to the primary antigen, and the other arm binds to the secondary antigen). While monovalent with respect to each antigen, binding to both antigens on the same cell compensates for the loss of avidity due to the bivalent deletion for each antigen. Bispecific antibodies offer higher selectivity compared to mAbs because they bind more readily to cells expressing both antigens than to cells expressing only one antigen. This is particularly important in mitigating safety concerns when normal cells or tissues express one of the two antigens. Bispecific antibodies can target two pathways simultaneously when they bind to two different cell surface antigens or soluble ligands / proteins, which is another advantage over mAbs.

[0007] When a bispecific antibody is produced from two different mAbs, the product will contain two distinct arms (each arm having a unique heavy chain and a unique light chain) derived from these two mAbs. Expressing a bispecific antibody in the producing cells during the manufacturing process requires the expression of four different proteins (two different heavy chains and two different light chains). The goal is to pair each HC with its corresponding LC on each arm of the bispecific antibody during production; however, pairing errors (LC from one arm pairing with HC from the other arm) often occur, resulting in undesirable products, which makes the production and isolation of the intended bispecific antibody product difficult. Several approaches have been employed to improve the manufacturing aspects of bispecific antibodies. One example is the identification of a common light chain through protein engineering. However, domain swapping and numerous mutations can significantly alter the native antibody structure, potentially increasing the risk of aggregation and / or reducing stability.

[0008] FRα expression is elevated in certain solid tumors, such as ovarian cancer, lung cancer, and breast cancer (Toffoli et al., Int J Cancer 1997;74:193-198 and Boogerd et al., Oncotarget 2016;7:17442-17454), but its expression is low in limited normal human tissue (Weitman et al., Cancer Res 1992;52:3396-3401). Consistent with this observation, Phase 1 clinical trials using small and large molecules targeting FRα have demonstrated good drug tolerability (Cheung et al., Oncotarget 2016;7:52553-52574). Therefore, FRα is an ideal target for cancer treatment. Furthermore, CD47, which mediates the "don't eat me" signal, is overexpressed in many tumors. A bispecific antibody (called an anti-CD47 / FRα bispecific antibody) with one arm binding to CD47 and the other arm binding to FRα can selectively target cells expressing both antigens. Because the bispecific antibody binds to both antigens on the same cell, it can achieve higher affinity compared to an antibody with only one arm due to its avidity. Compared to a bivalent anti-CD47 mAb, the bispecific antibody is expected to have weaker activity against cells expressing only CD47 (but not FRα) due to its lack of avidity, which may broaden its safety and / or tolerability. Anti-CD47 / FRα bispecific antibodies can selectively inhibit the CD47 / SIRPα interaction on cells expressing both CD47 and FRα, activating the innate immune system against cancer cells and other cells. Thus, anti-CD47 / FRα bispecific antibodies may be an effective treatment for ovarian cancer and other tumors that express both CD47 and FRα at significant levels on their cell surface. [Overview of the project]

[0009] In a general embodiment, the present invention relates to an isolated bispecific antibody or an antigen-binding fragment thereof, a. First heavy chain H1; b. Second heavy chain H2; c. First light chain L1; and d. Second light chain L2 Includes, H1 and L1 form a first arm containing a first antigen-binding domain that specifically binds to a first antigen, preferably a first antigen of human origin, and H2 and L2 form a second arm that includes a second antigen-binding domain that specifically binds to a second antigen, preferably a second antigen of human origin. (a) H1 comprises the CH1 region of human IgG1, IgG2, IgG3, or IgG4, and (b) L1 includes the CL region of the human κ light chain or human λ light chain, The CH1 and CL regions contain amino acid substitutions or native amino acids at the amino acid residues corresponding to the amino acid positions of SEQ ID NOs. 15, 21, 22, or 23 for CH1, and at the amino acid residues corresponding to the amino acid positions of SEQ ID NOs. 19 or 24 for CL. The amino acid substitutions or native amino acids in the CH1 and CL regions are: (1) K133C and C220X for CH1, and F209C and C214X for CL; (2) S131C and C220X of CH1, and P119C and C214X of CL; (3) K133C and C220X for CH1, and K207C and C214X for CL; (4) F170C and C220X for CH1, and S176C and C214X for CL; (5) P171C and C220X of CH1, and S162C and C214X of CL; (6) V173C and C220X for CH1, and Q160C and C214X for CL; (7) F170C and C131X of CH1, and S176C and C214X of CL; (8) P171C and C131X of CH1, and S162C and C214X of CL; (9) V173C and C131X of CH1, and Q160C and C214X of CL; (10) A129C and C220X of CH1, and S121C and C214X of CL; (11) K133C and C220X of CH1, and I117C and C214X of CL; (12) C131 of CH1, and P119C and C214X of CL; (13) A129C and C131X of CH1, and S121C and C214X of CL; (14) R133C and C131X of CH1, and K207C and C214X of CL; (15) R133C and C131X of CH1, and I117C and C214X of CL; (16) R133C and C131X of CH1, and L117C and C214X of CL; (17) K133C and C220X of CH1, and L117C and C214X of CL; (18) R133C and C131X of CH1, and F209C and C214X of CL; (19) R133C and C131X of CH1, and V209C and C214X of CL; or (20) CH1's K133C and C220X, and CL's V209C and C214X Selected from, Here, X is selected from S, A, or G. This relates to isolated bispecific antibodies or their antigen-binding fragments.

[0010] In another general embodiment, the present invention relates to an isolated bispecific antibody or an antigen-binding fragment thereof, a. First heavy chain, H1; b. The second heavy chain, H2; c. First light chain, L1; and d. Second light chain, L2 Includes, H1 and L1 form a first arm containing a first antigen-binding domain that specifically binds to a first antigen, preferably a first antigen of human origin, and H2 and L2 form a second arm that includes a second antigen-binding domain that specifically binds to a second antigen, preferably a second antigen of human origin. (a) H1 comprises the CH1 region and the heavy chain variable region (VH region) of human IgG1, IgG2, IgG3, or IgG4, and (b) L1 includes the CL region and the variable light chain region (VL region) of the human κ light chain or the human λ light chain, The CH1 region, VH region, CL region, and VL region contain amino acid substitutions at the amino acid residues corresponding to the amino acid positions of SEQ ID NO: 15, 21, 22, or 23 for CH1, SEQ ID NO: 13 for VH, SEQ ID NO: 19 or 24 for CL, and SEQ ID NO: 17 for VL, The amino acid substitutions in the CH1 region, VH region, CL region, and VL region are (1) C220X of CH1, G44C of VH, C214X of CL, and G101C of VL; or (2) C131X of CH1, G44C of VH, C214X of CL, and G101C of VL selected from, where X is selected from S, A, or G, relating to an isolated bispecific antibody or an antigen-binding fragment thereof.

[0011] In certain embodiments, the first antigen-binding domain is a CD47-binding domain. In certain embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 1, the CH1 region contains the amino acid sequence of SEQ ID NO: 2, the VL region contains the amino acid sequence of SEQ ID NO: 3, and the CL region contains the amino acid sequence of SEQ ID NO: 4.

[0012] In certain embodiments, the second arm containing H2 and L2 does not contain amino acid substitutions of the first arm containing H1 and L1. In certain embodiments, the two heavy chains H1 and H2 each contain a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the VH region has a different amino acid sequence. In certain embodiments, the two heavy chains H1 and H2 each contain a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the CH1 region has a different amino acid sequence. In certain embodiments, the two heavy chains H1 and H2 each contain a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the Fc region has a different amino acid sequence. In certain embodiments, the two light chains L1 and L2 each contain a VL region and a CL region, where the VL region has a different amino acid sequence. In certain embodiments, the two light chains L1 and L2 each contain a VL region and a CL region, where the CL region has a different amino acid sequence.

[0013] In certain embodiments, H1 and H2 form a heterodimer.

[0014] In certain embodiments, isolated humanized anti-CD47 / anti-FRα bispecific antibodies or their antigen-binding fragments can block the binding of signal-regulating protein α (SIRPα) to CD47 on cancer cells expressing both FRα and CD47.

[0015] In certain embodiments, isolated humanized anti-CD47 / anti-FRα bispecific antibodies or their antigen-binding fragments can induce macrophage-mediated phagocytosis in cancer cells expressing both FRα and CD47.

[0016] In certain embodiments, isolated humanized anti-CD47 / anti-FRα bispecific antibodies or their antigen-binding fragments can bind to cancer cells expressing both FRα and CD47, while binding to human erythrocytes (RBCs) is minimal to undetectable.

[0017] The present invention also provides isolated nucleic acids encoding isolated bispecific antibodies or antigen-binding fragments.

[0018] Furthermore, vectors comprising isolated nucleic acids encoding bispecific antibodies or antigen-binding fragments disclosed in the present invention are also provided.

[0019] The present invention also provides host cells comprising a vector containing an isolated nucleic acid encoding a bispecific antibody or antigen-binding fragment disclosed in the present invention.

[0020] In certain embodiments, a pharmaceutical composition is provided comprising an isolated bispecific antibody or an antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

[0021] Furthermore, the present invention provides a method for targeting FRα and CD47, both of which are expressed on the surface of cancer cells, in a target that requires such a method, and includes administering a pharmaceutical composition containing the isolated anti-CD-47 / anti-FRα bispecific antibody of the present invention or its antigen-binding fragment to the target.

[0022] Furthermore, a method is also provided for blocking the binding of SIRPα to CD47 on cancer cells expressing both FRα and CD47 in a target that requires such action, the method comprising administering a pharmaceutical composition containing the isolated anti-CD-47 / anti-FRα bispecific antibody of the present invention or its antigen-binding fragment to the target.

[0023] Furthermore, a method is also provided for inducing macrophage-mediated phagocytosis of cancer cells expressing both FRα and CD47 in a target that requires such action, the method comprising administering a pharmaceutical composition containing the isolated anti-CD-47 / anti-FRα bispecific antibody of the present invention or its antigen-binding fragment to the target.

[0024] Furthermore, the present invention provides a method for binding cancer cells expressing both FRα and CD47 to a target subject where such binding is minimal to undetectable, using an isolated anti-CD-47 / anti-FRα bispecific antibody or its antigen-binding fragment, the method comprising administering a pharmaceutical composition containing the isolated anti-CD-47 / anti-FRα bispecific antibody or its antigen-binding fragment to the target subject.

[0025] A method for treating cancer in a subject requiring it is also provided, comprising the step of administering the pharmaceutical composition of the present invention to the subject. The cancer may be any liquid cancer or solid tumor, and may be selected from, for example, lung cancer, gastric cancer, esophageal cancer, bile duct cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0026] The invention also provides a method for producing an isolated bispecific antibody or its antigen-binding fragment, comprising culturing cells containing nucleic acids encoding the antibody or its antigen-binding fragment under conditions for producing the antibody or its antigen-binding fragment, and recovering the antibody or its antigen-binding fragment from the cells or culture.

[0027] Furthermore, the present invention also provides a method for producing a pharmaceutical composition comprising an isolated bispecific antibody or an antigen-binding fragment thereof, the method comprising compounding the antibody or the antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0028] The above summary and the following detailed description of preferred embodiments of this application will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that this application is not limited to the embodiments shown in the drawings. [Brief explanation of the drawing]

[0029] [Figure 1A] Figures 1A and 1B show the structural diagrams of a bispecific antibody having a right arm for one antigen (e.g., CD47) and a left arm for a second antigen (e.g., FRα). Each arm has a different heavy chain VH region and light chain VL region. The heavy chain (HC) and light chain (LC) of the bispecific antibody are, for example, within the frameworks of κ and IgG1, respectively. To promote heterodimer formation, a "Knob in the hole (KiH)" mutation is introduced into the CH3 region of both HCs. Furthermore, cysteine ​​residues are introduced into each of the two CH3 regions to promote the formation of interchain disulfide bonds and stabilize the heterodimer. In Figure 1A, the native interchain disulfide bond between CH1 and CL (shown by the dashed line) is removed by converting the native cysteine ​​forming this disulfide bond to serine. Two native non-cysteine ​​residues on CH1 and CL are converted to cysteine, respectively, to form a new interchain disulfide bond between CH1 and CL. H1 and L1 are the heavy and light chains of the mAb1 arm, respectively, and H2 and L2 are the heavy and light chains of the mAb2 arm, respectively. [Figure 1B]Figures 1A and 1B are structural diagrams of a bispecific antibody having a right arm for one antigen (e.g., CD47) and a left arm for a second antigen (e.g., FRα). Each arm has a different heavy chain (VH region) and light chain (VL region). The heavy chain (HC) and light chain (LC) of the bispecific antibody are, for example, within the frameworks of κ and IgG1, respectively. To promote heterodimer formation, a "Knob in the hole (KiH)" mutation is introduced into the CH3 regions of both HCs. Furthermore, cysteine ​​residues are introduced into each of the two CH3 regions to promote the formation of interchain disulfide bonds and stabilize the heterodimer. In Figure 1B, the same method as in Figure 1A was used, but the newly formed interchain disulfide bond between the HC and LC of the right arm is located between the VH and VL regions. H1 and L1 are the heavy and light chains of the mAb1 arm, respectively, and H2 and L2 are the heavy and light chains of the mAb2 arm, respectively. [Figure 2A] Figures 2A to 2F show the sequences of various antibody components. Figures 2A to 2D show the VH (Figure 2A), CH1 (Figure 2B), VL (Figure 2C), and CL (Figure 2D) sequences of mAb1 (on the human IgG1 heavy chain and κ light chain) and mAb2 (on the human IgG1 heavy chain and κ light chain), respectively. VH of mAb1 (SEQ ID NO: 13); VH of mAb2 (SEQ ID NO: 14); CH1 of mAb1 (SEQ ID NO: 15); CH1 of mAb2 (SEQ ID NO: 16); VL of mAb1 (SEQ ID NO: 17); VL of mAb2 (SEQ ID NO: 18); CL of mAb1 (SEQ ID NO: 19); CL of mAb2 (SEQ ID NO: 20). The CDR region determined by the Kabat method is highlighted. Cysteine ​​residues involved in the formation of interchain disulfide bonds between the heavy and light chains of each arm are also highlighted. * indicates the site of an allelic variation of a known allele. [Figure 2B]Figures 2A to 2F show the sequences of various antibody components. Figures 2A to 2D show the VH (Figure 2A), CH1 (Figure 2B), VL (Figure 2C), and CL (Figure 2D) sequences of mAb1 (on the human IgG1 heavy chain and κ light chain) and mAb2 (on the human IgG1 heavy chain and κ light chain), respectively. VH of mAb1 (SEQ ID NO: 13); VH of mAb2 (SEQ ID NO: 14); CH1 of mAb1 (SEQ ID NO: 15); CH1 of mAb2 (SEQ ID NO: 16); VL of mAb1 (SEQ ID NO: 17); VL of mAb2 (SEQ ID NO: 18); CL of mAb1 (SEQ ID NO: 19); CL of mAb2 (SEQ ID NO: 20). The CDR region determined by the Kabat method is highlighted. Cysteine ​​residues involved in the formation of interchain disulfide bonds between the heavy and light chains of each arm are also highlighted. * indicates the site of an allelic variation of a known allele. [Figure 2C] Figures 2A to 2F show the sequences of various antibody components. Figures 2A to 2D show the VH (Figure 2A), CH1 (Figure 2B), VL (Figure 2C), and CL (Figure 2D) sequences of mAb1 (on the human IgG1 heavy chain and κ light chain) and mAb2 (on the human IgG1 heavy chain and κ light chain), respectively. VH of mAb1 (SEQ ID NO: 13); VH of mAb2 (SEQ ID NO: 14); CH1 of mAb1 (SEQ ID NO: 15); CH1 of mAb2 (SEQ ID NO: 16); VL of mAb1 (SEQ ID NO: 17); VL of mAb2 (SEQ ID NO: 18); CL of mAb1 (SEQ ID NO: 19); CL of mAb2 (SEQ ID NO: 20). The CDR region determined by the Kabat method is highlighted. Cysteine ​​residues involved in the formation of interchain disulfide bonds between the heavy and light chains of each arm are also highlighted. * indicates the site of an allelic variation of a known allele. [Figure 2D]Figures 2A to 2F show the sequences of various antibody components. Figures 2A to 2D show the VH (Figure 2A), CH1 (Figure 2B), VL (Figure 2C), and CL (Figure 2D) sequences of mAb1 (on the human IgG1 heavy chain and κ light chain) and mAb2 (on the human IgG1 heavy chain and κ light chain), respectively. VH of mAb1 (SEQ ID NO: 13); VH of mAb2 (SEQ ID NO: 14); CH1 of mAb1 (SEQ ID NO: 15); CH1 of mAb2 (SEQ ID NO: 16); VL of mAb1 (SEQ ID NO: 17); VL of mAb2 (SEQ ID NO: 18); CL of mAb1 (SEQ ID NO: 19); CL of mAb2 (SEQ ID NO: 20). The CDR region determined by the Kabat method is highlighted. Cysteine ​​residues involved in the formation of interchain disulfide bonds between the heavy and light chains of each arm are also highlighted. * indicates the site of an allelic variation of a known allele. [Figure 2E] Figures 2A to 2F show the sequences of various antibody components. The CDR region determined by the Kabat method is highlighted. Cysteine ​​residues involved in the formation of interchain disulfide bonds between the heavy and light chains of each arm are also highlighted. Figure 2E shows the alignment of the CH1 region of human IgG2 (SEQ ID NO: 21), IgG3 (SEQ ID NO: 22), and IgG4 (SEQ ID NO: 23). * indicates the site of known allelic variation. [Figure 2F] Figures 2A to 2F show the sequences of various antibody components. The CDR region determined by the Kabat method is highlighted. Cysteine ​​residues involved in the formation of interchain disulfide bonds between the heavy and light chains of each arm are also highlighted. Figure 2F shows the CL region of the human λ light chain (SEQ ID NO: 24). * indicates the site of a known allelelic variation. [Figure 3A]Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate the native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate the new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3A represents the 3D model of bsAb1. [Figure 3B] Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate the native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate the new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3B shows the 3D model of bsAb2. [Figure 3C] Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate the native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate the new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3C represents the 3D model of bsAb3. [Figure 3D]Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate the native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate the new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3D represents the 3D model of bsAb4. [Figure 3E] Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate the native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate the new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3E shows the 3D model of bsAb5. [Figure 3F] Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate the native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate the new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3F shows the 3D model of bsAb6. [Figure 3G]Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate the native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate the new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3G represents the 3D model of bsAb7. [Figure 3H] Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate the native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate the new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3H represents the 3D model of bsAb8. [Figure 3I] Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate the native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate the new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3I represents the 3D model of bsAb9. [Figure 3J]Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate the native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate the new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3J represents the 3D model of bsAb10. [Figure 3K] Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate the native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate the new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3K represents the 3D model of bsAb11. [Figure 3L] Figures 3A–3L show 3D models of the Fab region (including VH, CH1, VL, and CL) within the mAb1 arm to identify potential sites for cysteine ​​"knock-in" to form new interchain disulfide bonds between HC and LC. Dashed lines indicate native interchain disulfide bonds between HC and LC (the native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in these four bsAb models), and solid lines indicate new interchain disulfide bonds that may be formed by newly knocked-in cysteine. Figure 3L represents the 3D model of bsAb12. [Figure 4A]Figures 4A to 4H show the RP-HPLC profiles of mutant mAbs containing differently shifted interchain disulfide bonds under reducing and non-reducing conditions. [Figure 4B] Figures 4A to 4H show the RP-HPLC profiles of mutant mAbs containing differently shifted interchain disulfide bonds under reducing and non-reducing conditions. [Figure 4C] Figures 4A to 4H show the RP-HPLC profiles of mutant mAbs containing differently shifted interchain disulfide bonds under reducing and non-reducing conditions. [Figure 4D] Figures 4A to 4H show the RP-HPLC profiles of mutant mAbs containing differently shifted interchain disulfide bonds under reducing and non-reducing conditions. [Figure 4E] Figures 4A to 4H show the RP-HPLC profiles of mutant mAbs containing differently shifted interchain disulfide bonds under reducing and non-reducing conditions. [Figure 4F] Figures 4A to 4H show the RP-HPLC profiles of mutant mAbs containing differently shifted interchain disulfide bonds under reducing and non-reducing conditions. [Figure 4G] Figures 4A to 4H show the RP-HPLC profiles of mutant mAbs containing differently shifted interchain disulfide bonds under reducing and non-reducing conditions. [Figure 4H] Figures 4A to 4H show the RP-HPLC profiles of mutant mAbs containing differently shifted interchain disulfide bonds under reducing and non-reducing conditions. [Figure 4I] Figures 4I to 4J show the binding of mutant mAbs containing differently shifted interchain disulfide bonds to CD47 in ELISA assays. [Figure 4J] Figures 4I to 4J show the binding of mutant mAbs containing differently shifted interchain disulfide bonds to CD47 in ELISA assays. [Figure 5A]Figures 5A to 5G show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in a heat resistance test performed by incubating the samples at different temperatures for 5 minutes. Chromatograms for each temperature are shown. The table in each figure shows the %AUC (area under the curve) of the different peaks at each given temperature. Figure 5A shows mutant mAbs designed in M1. HMW is a high molecular weight species, MMW is a medium molecular weight species (mutant mAb), and LMW is a low molecular weight species. [Figure 5B] Figures 5A to 5G show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in a heat resistance test performed by incubating the samples at different temperatures for 5 minutes. Chromatograms for each temperature are shown. The table in each figure shows the %AUC (area under the curve) of different peaks at each given temperature. Figure 5B shows mutant mAbs designed for M2. HMW is a high molecular weight species, MMW is a medium molecular weight species (mutant mAb), and LMW is a low molecular weight species. [Figure 5C] Figures 5A to 5G show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in a heat resistance test performed by incubating the samples at different temperatures for 5 minutes. Chromatograms for each temperature are shown. The table in each figure shows the %AUC (area under the curve) of different peaks at each given temperature. Figure 5C shows mutant mAbs designed in Z1. HMW is a high molecular weight species, MMW is a medium molecular weight species (mutant mAb), and LMW is a low molecular weight species. [Figure 5D] Figures 5A to 5G show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in a heat resistance test performed by incubating the samples at different temperatures for 5 minutes. Chromatograms for each temperature are shown. The table in each figure shows the %AUC (area under the curve) of different peaks at each given temperature. Figure 5D shows mutant mAbs designed in Z2. HMW is a high molecular weight species, MMW is a medium molecular weight species (mutant mAb), and LMW is a low molecular weight species. [Figure 5E] Figures 5A to 5G show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in a heat resistance test performed by incubating the samples at different temperatures for 5 minutes. Chromatograms for each temperature are shown. The table in each figure shows the %AUC (area under the curve) of different peaks at each given temperature. Figure 5E shows mutant mAbs designed as bsAb10. HMW is a high molecular weight species, MMW is a medium molecular weight species (mutant mAb), and LMW is a low molecular weight species. [Figure 5F] Figures 5A to 5G show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in a heat resistance test performed by incubating the samples at different temperatures for 5 minutes. Chromatograms for each temperature are shown. The table in each figure shows the %AUC (area under the curve) of different peaks at each given temperature. Figure 5F shows mutant mAbs designed as bsAb11. HMW is a high molecular weight species, MMW is a medium molecular weight species (mutant mAb), and LMW is a low molecular weight species. [Figure 5G] Figures 5A to 5G show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in a heat resistance test performed by incubating the samples at different temperatures for 5 minutes. Chromatograms for each temperature are shown. The tables in each figure show the %AUC (area under the curve) of different peaks at each given temperature. Figure 5G shows mutant mAbs designed as bsAb12. HMW is a high molecular weight species, MMW is a medium molecular weight species (mutant mAb), and LMW is a low molecular weight species. [Figure 6A]Figures 6A to 6H show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in pH stability tests performed by incubating samples at room temperature and pH 3.0 for different time periods. Chromatograms are shown for each incubation time (0, 1, 3, 5, and 7 hours). The chromatogram for 0 hours shows the sample that was not incubated at pH 3.0. The table in each figure shows the %AUC (area under the curve) of different peaks at each given incubation time. Figure 6A shows mutant mAbs designed in M1. HMW is a high molecular weight species, MMW is a medium molecular weight species (mutant mAb), and LMW is a low molecular weight species. [Figure 6B] Figures 6A to 6H show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in pH stability tests performed by incubating samples at room temperature and pH 3.0 for different time periods. Chromatograms are shown for each incubation time (0, 1, 3, 5, and 7 hours). The chromatogram for 0 hours shows the sample that was not incubated at pH 3.0. The table in each figure shows the %AUC (area under the curve) of different peaks at each given incubation time. Figure 6B shows mutant mAbs designed for M2. HMW is a high molecular weight species, MMW is a medium molecular weight species (mutant mAb), and LMW is a low molecular weight species. [Figure 6C] Figures 6A to 6H show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in pH stability tests performed by incubating samples at room temperature and pH 3.0 for different time periods. Chromatograms are shown for each incubation time (0, 1, 3, 5, and 7 hours). The chromatogram for 0 hours shows the sample that was not incubated at pH 3.0. The table in each figure shows the %AUC (area under the curve) of different peaks at each given incubation time. Figure 6C shows mutant mAbs designed in Z1. HMW is a high molecular weight species, MMW is a medium molecular weight species (mutant mAb), and LMW is a low molecular weight species. [Figure 6D]Figures 6A to 6H show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in pH stability tests performed by incubating samples at room temperature and pH 3.0 for different time periods. Chromatograms are shown for each incubation time (0, 1, 3, 5, and 7 hours). The chromatogram for 0 hours shows the sample that was not incubated at pH 3.0. The table in each figure shows the %AUC (area under the curve) of different peaks at each given incubation time. Figure 6D shows mutant mAbs designed in Z2. HMW is a high molecular weight species, MMW is a medium molecular weight species (mutant mAb), and LMW is a low molecular weight species. [Figure 6E] Figures 6A to 6H show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in pH stability tests performed by incubating samples at room temperature and pH 3.0 for different time periods. Chromatograms are shown for each incubation time (0, 1, 3, 5, and 7 hours). The chromatogram for 0 hours shows the sample that was not incubated at pH 3.0. The table in each figure shows the %AUC (area under the curve) of different peaks at each given incubation time. Figure 6E shows mutant mAbs designed for bsAb5. HMW is the high molecular weight species, MMW is the medium molecular weight species (mutant mAb), and LMW is the low molecular weight species. [Figure 6F] Figures 6A to 6H show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in pH stability tests performed by incubating samples at room temperature and pH 3.0 for different time periods. Chromatograms are shown for each incubation time (0, 1, 3, 5, and 7 hours). The chromatogram for 0 hours shows the sample that was not incubated at pH 3.0. The table in each figure shows the %AUC (area under the curve) of different peaks at each given incubation time. Figure 6F shows mutant mAbs designed as bsAb10. HMW is the high molecular weight species, MMW is the medium molecular weight species (mutant mAb), and LMW is the low molecular weight species. [Figure 6G] Figures 6A to 6H show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in pH stability tests performed by incubating samples at room temperature and pH 3.0 for different time periods. Chromatograms are shown for each incubation time (0, 1, 3, 5, and 7 hours). The chromatogram for 0 hours shows the sample that was not incubated at pH 3.0. The table in each figure shows the %AUC (area under the curve) of different peaks at each given incubation time. Figure 6G shows mutant mAbs designed as bsAb11. HMW is the high molecular weight species, MMW is the medium molecular weight species (mutant mAb), and LMW is the low molecular weight species. [Figure 6H] Figures 6A to 6H show the SEC (size exclusion chromatography) profiles of mutant mAbs containing differently shifted interchain disulfide bonds in pH stability tests performed by incubating samples at room temperature and pH 3.0 for different time periods. Chromatograms are shown for each incubation time (0, 1, 3, 5, and 7 hours). The chromatogram for 0 hours shows the sample that was not incubated at pH 3.0. The table in each figure shows the %AUC (area under the curve) of different peaks at each given incubation time. Figure 6H shows mutant mAbs designed as bsAb12. HMW is the high molecular weight species, MMW is the medium molecular weight species (mutant mAb), and LMW is the low molecular weight species. [Figure 7A] Figures 7A to 7D show SDS-PAGE images of bispecific antibodies purified using protein A chromatography. [Figure 7B] Figures 7A to 7D show SDS-PAGE images of bispecific antibodies purified using protein A chromatography. [Figure 7C] Figures 7A to 7D show SDS-PAGE images of bispecific antibodies purified using protein A chromatography. In Figure 7C, SDS-PAGE was performed under non-reducing conditions. [Figure 7D]Figures 7A to 7D show SDS-PAGE images of bispecific antibodies purified using protein A chromatography. In Figure 7D, SDS-PAGE was performed under reducing conditions. [Figure 8A] Figures 8A to 8J show graphs illustrating the results of bridging ELISA assays in which purified bispecific antibodies for protein A bind to both antigens (i.e., CD47 and FRα). [Figure 8B] Figures 8A to 8J show graphs illustrating the results of bridging ELISA assays in which purified bispecific antibodies for protein A bind to both antigens (i.e., CD47 and FRα). [Figure 8C] Figures 8A to 8J show graphs illustrating the results of bridging ELISA assays in which purified bispecific antibodies for protein A bind to both antigens (i.e., CD47 and FRα). [Figure 8D] Figures 8A to 8J show graphs illustrating the results of bridging ELISA assays in which purified bispecific antibodies for protein A bind to both antigens (i.e., CD47 and FRα). [Figure 8E] Figures 8A to 8J show graphs illustrating the results of bridging ELISA assays in which purified bispecific antibodies for protein A bind to both antigens (i.e., CD47 and FRα). [Figure 8F] Figures 8A to 8J show graphs illustrating the results of bridging ELISA assays in which purified bispecific antibodies for protein A bind to both antigens (i.e., CD47 and FRα). [Figure 8G] Figures 8A to 8J show graphs illustrating the results of bridging ELISA assays in which purified bispecific antibodies for protein A bind to both antigens (i.e., CD47 and FRα). [Figure 8H] Figures 8A to 8J show graphs illustrating the results of bridging ELISA assays, in which purified bispecific antibodies for protein A bind to both antigens (i.e., CD47 and FRα). [Figure 8I]Figures 8A to 8J show graphs illustrating the results of bridging ELISA assays, in which purified bispecific antibodies for protein A bind to both antigens (i.e., CD47 and FRα). [Figure 8J] Figures 8A to 8J show graphs illustrating the results of bridging ELISA assays, in which purified bispecific antibodies for protein A bind to both antigens (i.e., CD47 and FRα). [Figure 9A] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by hydrophobic interaction chromatography (HIC) following protein A chromatography (Figures 9F to 9N). Figure 9A shows the SEC profile of bsAb1. [Figure 9B] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by hydrophobic interaction chromatography (HIC) following protein A chromatography (Figures 9F to 9N). Figure 9B shows the SEC profile of bsAb5. [Figure 9C] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by hydrophobic interaction chromatography (HIC) following protein A chromatography (Figures 9F to 9N). Figure 9C shows the SEC profile of bsAb6. [Figure 9D] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by hydrophobic interaction chromatography (HIC) following protein A chromatography (Figures 9F to 9N). Figure 9D shows the SEC profile of bsAb7. [Figure 9E]Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by hydrophobic interaction chromatography (HIC) following protein A chromatography (Figures 9F to 9N). Figure 9E shows the SEC profile of bsAb8. [Figure 9F] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by hydrophobic interaction chromatography (HIC) following protein A chromatography (Figures 9F to 9N). Figure 9F shows the SEC profile of bsAb5b(E / K). [Figure 9G] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by hydrophobic interaction chromatography (HIC) following protein A chromatography (Figures 9F to 9N). Figure 9G shows the SEC profile of bsAb10(E / K). [Figure 9H] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by hydrophobic interaction chromatography (HIC) following protein A chromatography (Figures 9F to 9N). Figure 9H shows the SEC profile of bsAb12(E / K). [Figure 9I] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by protein A chromatography followed by hydrophobic interaction chromatography (HIC) (Figures 9F to 9N). Figure 9I shows the SEC profile of bsAb5b(K / E). [Figure 9J]Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by protein A chromatography followed by hydrophobic interaction chromatography (HIC) (Figures 9F to 9N). Figure 9J shows the SEC profile of bsAb10(K / E). [Figure 9K] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by hydrophobic interaction chromatography (HIC) following protein A chromatography (Figures 9F to 9N). Figure 9K shows the SEC profile of bsAb12(K / E). [Figure 9L] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by hydrophobic interaction chromatography (HIC) following protein A chromatography (Figures 9F to 9N). Figure 9L shows the SEC profile of bsAb5b. [Figure 9M] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by protein A chromatography followed by hydrophobic interaction chromatography (HIC) (Figures 9F to 9N). Figure 9M shows the SEC profile of bsAb10. [Figure 9N] Figures 9A to 9N show the size exclusion chromatography (SEC) profiles of bispecific antibodies purified using protein A chromatography (Figures 9A to 9E), or bispecific antibodies purified by hydrophobic interaction chromatography (HIC) following protein A chromatography (Figures 9F to 9N). Figure 9N shows the SEC profile of bsAb12. [Figure 10A]Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10A shows the RP-HPLC profile of bsAb6. [Figure 10B] Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10B shows the RP-HPLC profile of bsAb7. [Figure 10C] Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10C shows the RP-HPLC profile of bsAb8. [Figure 10D] Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10D shows the RP-HPLC profile of bsAb5b(E / K). [Figure 10E] Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10E shows the RP-HPLC profile of bsAb10(E / K). [Figure 10F] Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10F shows the RP-HPLC profile of bsAb12(E / K). [Figure 10G] Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10G shows the RP-HPLC profile of bsAb5b(K / E). [Figure 10H] Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10H shows the RP-HPLC profile of bsAb10(K / E). [Figure 10I] Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10I shows the RP-HPLC profile of bsAb12(K / E). [Figure 10J]Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10J shows the RP-HPLC profile of bsAb5b. [Figure 10K] Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10K shows the RP-HPLC profile of bsAb10. [Figure 10L] Figures 10A to 10L show the RP-HPLC profiles of bispecific antibodies purified by HIC under non-reducing conditions. Figure 10L shows the RP-HPLC profile of bsAb12. [Figure 11A] Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11A shows the RP-HPLC profile of control antibody #1, purified by protein A chromatography using a combination of H1, H2, and L1 of bsAb7b. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11B]Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11B shows the RP-HPLC profile of control antibody #2, purified by protein A chromatography using a combination of H1, H2, and L2 of bsAb7b. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the HC and LC of the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11C] Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11C shows the RP-HPLC profile of bsAb6 purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11D] Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11D shows the RP-HPLC profile of bsAb7 purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11E] Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11E shows the RP-HPLC profile of bsAb8 purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11F] Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11F shows the RP-HPLC profile of bsAb5b(E / K) purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11G] Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11G shows the RP-HPLC profile of bsAb10(E / K) purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11H]Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11H shows the RP-HPLC profile of bsAb12(E / K) purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11I] Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11I shows the RP-HPLC profile of bsAb5b(K / E) purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11J] Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11J shows the RP-HPLC profile of bsAb10(K / E) purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11K]Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11K shows the RP-HPLC profile of bsAb12(K / E) purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11L] Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11L shows the RP-HPLC profile of bsAb5b purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the HC and LC of the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11M] Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11M shows the RP-HPLC profile of bsAb10 purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 11N]Figures 11A to 11N show the RP-HPLC profiles of bispecific antibodies purified by HIC under reducing conditions. Figure 11N shows the RP-HPLC profile of bsAb12 purified by HIC. H1 and L1 represent the HC and LC of the mAb1 (anti-CD47) arm, respectively, and H2 and L2 represent the HC and LC of the mAb2 (anti-FRα) arm (Figures 11A to 11E) or the mAb2b (anti-FRα) arm (Figures 11F to 11N), respectively. The predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain, and the area % represents the AUC ratio calculated using the AUC of all four peaks of each bispecific antibody in RP-HPLC under reducing conditions. [Figure 12A] Figures 12A to 12F show graphs illustrating the binding of HIC-purified bispecific antibodies to both antigens (i.e., CD47 and FRα) in bridging ELISA assays. Figure 12A shows bridging ELISA data for bsAb6, bsAb7, and bsAb8. [Figure 12B] Figures 12A to 12F show graphs illustrating the binding of HIC-purified bispecific antibodies to both antigens (i.e., CD47 and FRα) in bridging ELISA assays. Figure 12B shows bridging ELISA data for bsAb5b(E / K), bsAb10(E / K), and bsAb12(E / K). [Figure 12C] Figures 12A to 12F show graphs illustrating the binding of HIC-purified bispecific antibodies to both antigens (i.e., CD47 and FRα) in bridging ELISA assays. Figure 12C shows the bridging ELISA data for bsAb5b(K / E). [Figure 12D] Figures 12A to 12F show graphs illustrating the binding of HIC-purified bispecific antibodies to both antigens (i.e., CD47 and FRα) in bridging ELISA assays. Figure 12D shows the bridging ELISA data for bsAb10(K / E). [Figure 12E]Figures 12A to 12F show graphs illustrating the binding of HIC-purified bispecific antibodies to both antigens (i.e., CD47 and FRα) in bridging ELISA assays. Figure 12E shows bridging ELISA data for bsAb12(K / E). [Figure 12F] Figures 12A to 12F show graphs illustrating the binding of HIC-purified bispecific antibodies to both antigens (i.e., CD47 and FRα) in bridging ELISA assays. Figure 12F shows bridging ELISA data for bsAb5b, bsAb10, and bsAb12. [Figure 13A] Figures 13A to 13C show the binding of HIC-purified bispecific antibodies to SK-OV-3 cells, which are known to express both CD47 and FRα. [Figure 13B] Figures 13A to 13C show the binding of HIC-purified bispecific antibodies to SK-OV-3 cells, which are known to express both CD47 and FRα. [Figure 13C] Figures 13A to 13C show the binding of HIC-purified bispecific antibodies to SK-OV-3 cells, which are known to express both CD47 and FRα. [Figure 14A] Figures 14A to 14C show the mass spectrometry (MS) profiles of papain digestion samples of bispecific antibodies purified by HIC. Fab fragments derived from both arms (mAb1 arm and mAb2 arm) of each bispecific antibody were identified. Figure 14A shows the MS profile of papain digested bsAb6. [Figure 14B] Figures 14A–14C show the mass spectrometry (MS) profiles of papain digestion samples of bispecific antibodies purified by HIC. Fab fragments derived from both arms (mAb1 and mAb2 arms) of each bispecific antibody were identified. Figure 14B shows the MS profile of papain digested bsAb7. [Figure 14C]Figures 14A to 14C show the mass spectrometry (MS) profiles of papain digestion samples of bispecific antibodies purified by HIC. Fab fragments derived from both arms (mAb1 arm and mAb2 arm) of each bispecific antibody were identified. Figure 14C shows the MS profile of papain digested bsAb8. [Figure 15A] Figures 15A to 15F show the MS profiles of trypsin-digested samples of bispecific antibodies purified by HIC. Disulfide-crosslinked peptide fragments derived from the mAb1 arm of each bispecific antibody were identified. Cysteine, which forms the disulfide bond, is shown as a thick line. Figure 15A shows the MS profile of the disulfide-crosslinked peptide fragment derived from the mAb1 arm of trypsin-digested bsAb6. [Figure 15B] Figures 15A to 15F show the MS profiles of trypsin-digested samples of bispecific antibodies purified by HIC. Disulfide-crosslinked peptide fragments derived from the mAb1 arm of each bispecific antibody were identified. Cysteine ​​forming the disulfide bond is shown as a thick line. Figure 15B shows the MS profile of the disulfide-crosslinked peptide fragment derived from the mAb1 arm of trypsin-digested bsAb7. [Figure 15C] Figures 15A to 15F show the MS profiles of trypsin-digested samples of bispecific antibodies purified by HIC. Disulfide-crosslinked peptide fragments derived from the mAb1 arm of each bispecific antibody were identified. Cysteine ​​forming the disulfide bond is shown as a thick line. Figure 15C shows the MS profile of the disulfide-crosslinked peptide fragment derived from the mAb1 arm of trypsin-digested bsAb8. [Figure 15D] Figures 15A to 15F show the MS profiles of trypsin-digested samples of bispecific antibodies purified by HIC. Disulfide-crosslinked peptide fragments derived from the mAb1 arm of each bispecific antibody were identified. Cysteine ​​forming the disulfide bond is shown as a thick line. Figure 15D shows the MS profile of the disulfide-crosslinked peptide fragment derived from the mAb1 arm of trypsin-digested bsAb5b. [Figure 15E]Figures 15A to 15F show the MS profiles of trypsin-digested samples of bispecific antibodies purified by HIC. Disulfide-crosslinked peptide fragments derived from the mAb1 arm of each bispecific antibody were identified. Cysteine ​​forming the disulfide bond is shown as a thick line. Figure 15E shows the MS profile of the disulfide-crosslinked peptide fragment derived from the mAb1 arm of trypsin-digested bsAb10. [Figure 15F] Figures 15A to 15F show the MS profiles of trypsin-digested samples of bispecific antibodies purified by HIC. Disulfide-crosslinked peptide fragments derived from the mAb1 arm of each bispecific antibody were identified. Cysteine ​​forming the disulfide bond is shown as a thick line. Figure 15F shows the MS profile of the disulfide-crosslinked peptide fragment derived from the mAb1 arm of trypsin-digested bsAb12. [Figure 16A] Figures 16A to 16C show the MS profiles of IdeZ protease digestion samples of bispecific antibodies purified by HIC. (Fab')2 was identified from each bispecific antibody. Figure 16A shows the MS profile of bsAb6(Fab')2. [Figure 16B] Figures 16A to 16C show the MS profiles of IdeZ protease digestion samples of bispecific antibodies purified by HIC. (Fab')2 was identified from each bispecific antibody. Figure 16B shows the MS profile of bsAb7(Fab')2. [Figure 16C] Figures 16A to 16C show the MS profiles of IdeZ protease digestion samples of bispecific antibodies purified by HIC. (Fab')2 was identified from each bispecific antibody. Figure 16C shows the MS profile of bsAb8(Fab')2. [Figure 17A]Figures 17A to 17C show the inhibition of antibody binding to SK-OV-3 cells by F(ab')2 derived from anti-CD47 or anti-FRα parental mAbs in FACS assays. Figure 17A shows the case of bsAb5b. The anti-FRα parental mAb is the original mAb from which the anti-FRα arm was obtained for constructing the bispecific antibodies bsAb5b, bsAb10, and bsAb12. The anti-CD47 parental mAb is the original mAb from which the anti-CD47 arm was obtained for constructing the bispecific antibodies bsAb5b, bsAb10, and bsAb12. Ab represents antibody. The Ab concentrations used in the assay are shown below each graph. The inhibitory effect was evaluated using 5,000 nM F(ab')2. [Figure 17B] Figures 17A to 17C show the inhibition of antibody binding to SK-OV-3 cells by F(ab')2 derived from anti-CD47 or anti-FRα parental mAbs in the FACS assay. Figure 17B shows the case for bsAb10. The anti-FRα parental mAb is the original mAb from which the anti-FRα arm was obtained for constructing the bispecific antibodies bsAb5b, bsAb10, and bsAb12. The anti-CD47 parental mAb is the original mAb from which the anti-CD47 arm was obtained for constructing the bispecific antibodies bsAb5b, bsAb10, and bsAb12. Ab represents antibody. The Ab concentrations used in the assay are shown below each graph. The inhibitory effect was evaluated using 5,000 nM F(ab')2. [Figure 17C] Figures 17A to 17C show the inhibition of antibody binding to SK-OV-3 cells by F(ab')2 derived from anti-CD47 or anti-FRα parental mAbs in FACS assays. Figure 17C shows the case for bsAb12. The anti-FRα parental mAb is the original mAb from which the anti-FRα arm was obtained for constructing the bispecific antibodies bsAb5b, bsAb10, and bsAb12. The anti-CD47 parental mAb is the original mAb from which the anti-CD47 arm was obtained for constructing the bispecific antibodies bsAb5b, bsAb10, and bsAb12. Ab represents antibody. The Ab concentrations used in the assay are shown below each graph. The inhibitory effect was evaluated using 5,000 nM F(ab')2. [Figure 18A]Figures 18A and 18B show the sequential binding of two antigens (CD47 and FRα) to the bispecific antibody bsAb12 in Biacore. [Figure 18B] Figures 18A and 18B show the sequential binding of two antigens (CD47 and FRα) to the bispecific antibody bsAb12 in Biacore. [Figure 19A] Figures 19A to 19C show the MS profiles of Fab fragments obtained from papain digestion samples of bispecific antibodies purified with protein A. Figure 19A shows the profile of a WT bispecific antibody purified with protein A. A WT bispecific antibody is a bispecific antibody produced by co-expressing the mAb1 and mAb2b arms (both arms have natural interchain disulfide bonds) without introduced mutations. H1 and L1 are the heavy and light chains of the mAb1 arm, respectively, and H2 and L2 are the heavy and light chains of the mAb2 arm, respectively. H1 / L2 Fab and H2 / L1 Fab represent Fab fragments of heavy / light chain mismatch species. ND indicates "not detected". [Figure 19B] Figures 19A to 19C show the MS profiles of Fab fragments obtained from papain digestion samples of bispecific antibodies purified with protein A. Figure 19B shows the profile for bsAb10 purified with protein A. A WT bispecific antibody is a bispecific antibody produced by co-expressing the mAb1 and mAb2b arms (both arms have natural interchain disulfide bonds) without introduced mutations. H1 and L1 are the heavy and light chains of the mAb1 arm, respectively, and H2 and L2 are the heavy and light chains of the mAb2 arm, respectively. H1 / L2 Fab and H2 / L1 Fab represent Fab fragments of heavy / light chain mismatch species. ND indicates "not detected". [Figure 19C]Figures 19A to 19C show the MS profiles of Fab fragments obtained from papain digestion samples of bispecific antibodies purified with protein A. Figure 19C shows the profile for bsAb12 purified with protein A. A WT bispecific antibody is a bispecific antibody produced by co-expressing the mAb1 and mAb2b arms (both arms have native interchain disulfide bonds) without introduced mutations. H1 and L1 are the heavy and light chains of the mAb1 arm, respectively, and H2 and L2 are the heavy and light chains of the mAb2 arm, respectively. H1 / L2 Fab and H2 / L1 Fab represent Fab fragments of heavy / light chain mismatch species. ND indicates "not detected". [Figure 20] This is the MS profile of the Fab fragment obtained from papain digested bsAb12, which was purified by HIC following protein A purification. No mismatch species were detected. [Modes for carrying out the invention]

[0030] Various publications, documents, and patents are cited or referenced throughout the background art and this specification, and each of these references is incorporated herein by reference in its entirety. Discussions of documents, laws (acts), materials, devices, articles, etc., included herein are intended to provide background to the invention. Such discussions do not constitute an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which the present invention relates. Otherwise, any specific terms used herein have the meanings set forth herein.

[0032] As used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple subjects unless otherwise explicitly indicated in this context.

[0033] Unless otherwise stated, any numerical value, such as concentrations or concentration ranges, as described herein should be understood in all examples to be modified by the term “approximately.” Therefore, numerical values ​​typically include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Where used herein, the use of numerical ranges explicitly includes all possible subranges, including integers and fractions of their values, and all individual numerical values ​​within that range, unless the context otherwise clearly indicates otherwise.

[0034] Unless otherwise indicated, the term “at least” preceding a set of elements should be understood to refer to all elements within that set. Those skilled in the art will be able to recognize or confirm many equivalents of the specific embodiments of the invention described herein without using anything beyond conventional experimentation. Such equivalents are intended to be encompassed within the invention.

[0035] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or any other variation thereof, mean the inclusion of the integer or group of integers listed, but not the exclusion of any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that includes an enumeration of elements is not necessarily limited to those elements alone, and may include other elements not expressly enumerated or specific to such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated otherwise, “or” means comprehensive or not exclusive or. For example, condition A or B satisfies one of the following: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); and both A and B are true (or exist).

[0036] As used herein, the connecting term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within its meaning and therefore satisfies the requirements of the term "and / or" as used herein. The simultaneous application of one or more of these options is also understood to be included in this meaning and therefore satisfies the requirements of the term "and / or."

[0037] As used herein, the term "consists of," or variations thereof, such as "consist of" or "consisting of," as used throughout this specification and the claims, indicates the inclusion of any described integer or group of integers, but does not mean that any further integers or groups of integers are added to any particular method, structure, or composition.

[0038] As used herein, the term "consists essentially of," or variations thereof, such as "consist essentially of" or "consisting essentially of," as used throughout this specification and the claims, indicates the inclusion of any described integer or group of integers, and the inclusion of any selection of any described integer or group of integers that does not substantially alter the fundamental or novel properties of a particular method, structure, or composition. See MPEP §2111.03.

[0039] As used herein, “subject” means any animal, preferably a mammal, most preferably a human. The term “mammal” as used herein encompasses any mammal. Examples of mammals, but not limited to, include cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and more preferably humans.

[0040] The terms "right," "left," "down," and "up" indicate the direction in which they are referred to in the drawings.

[0041] When referring to the dimensions or features of preferred components of an invention, the terms used herein, such as “about,” “approximately,” “generally,” and “substantially,” should be understood as not excluding minor variations of those dimensions / features that are functionally the same or similar, as understood by those skilled in the art, rather than being strict boundaries or parameters. At the very least, such references, including numerical parameters, should include variations that do not change at least the least significant digit when using mathematical and industrial principles acceptable in the art (e.g., rounding errors, measurement errors or other systematic errors, manufacturing tolerances, etc.).

[0042] As used herein, throughout the specification and claims, the terms “different heavy chains” or “different light chains” refer to heavy chains or light chains having non-identical arrangements.

[0043] In the context of two or more nucleic acid or polypeptide sequences (e.g., bispecific antibodies, anti-FRα antibodies, anti-CD47 antibodies, anti-CD47 / anti-FRα bispecific antibodies, FRα polypeptides and encoding polynucleotides, and CD47 polypeptides and encoding polynucleotides), the term “identical” or “identity” percentage means two or more sequences or subsequences that are identical or have a specific percentage of identical amino acid residues or nucleotides when compared and aligned to the greatest match using one of the following sequence comparison algorithms or by visual inspection.

[0044] In sequence comparison, typically one sequence functions as the reference sequence against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are input into the computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentage for the test sequence compared to the reference sequence, based on the specified program parameters.

[0045] The optimal sequence alignment for comparison can be determined, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by running these algorithms on a computer (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI's GAP, BESTFIT, FASTA, and TFASTA), or by visual inspection (generally, Current Protocols in Molecular Biology, edited by FM Ausubel et al., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, This can be done through a joint venture of Inc. (see (1995 Addendum) (Ausubel)).

[0046] Suitable algorithms for determining sequence identity percentage and sequence similarity are the BLAST and BLAST2.0 algorithms, described in Altschul et al. (1990) J. Mol. Biol. 215: pp. 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: pp. 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in the database sequence, either match or satisfy a certain positive threshold score T. T is called the adjacent word score threshold (Altschul et al., cited above). These initial adjacent word hits act as seeds to initiate a search for longer HSPs that contain them. Next, this word hit is stretched bidirectionally along each sequence, as long as the cumulative alignment score can increase.

[0047] For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; always greater than 0) and N (penalty score for mismatched residues; always less than 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Word hit extension in each direction stops if the cumulative alignment score falls by amount X from its maximum achieved value; if the cumulative score becomes 0 or less due to the accumulation of one or more negative scoring residue alignments; or if it reaches the end of either sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and comparison of both strands as defaults. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:109 p. 15 (1989)) as default settings.

[0048] In addition to calculating the sequence identity percentage, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90: pp. 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that the match between two nucleotide or amino acid sequences occurs by chance. For example, if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0049] A further indicator that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is typically substantially identical to the second polypeptide when, for example, the two peptides differ only by conservative substitutions. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize with each other under stringent conditions.

[0050] As used herein, the term “polynucleotide” is synonymous with “nucleic acid molecule,” “nucleotide,” or “nucleic acid,” and refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotide” includes, but is not limited to, single-stranded DNA and double-stranded DNA, DNA which is a mixture of single-stranded and double-stranded regions, single-stranded RNA and double-stranded RNA, RNA which is a mixture of single-stranded and double-stranded regions, hybrid molecules containing single-stranded or more typically double-stranded DNA and RNA, or mixtures of single-stranded and double-stranded regions. Furthermore, “polynucleotide” refers to a triple-stranded region containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA whose backbone has been modified for stability or other reasons. “Modified” bases include, for example, tritylated bases and inosine and other abnormal bases. Various modifications can be made to DNA and RNA. Therefore, "polynucleotides" include chemically, enzymatically, or metabolically modified forms of polynucleotides commonly found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotides" also include relatively short nucleic acid chains (often called oligonucleotides).

[0051] As used herein, the term “vector” refers to a replicon in which another nucleic acid segment has been inserted by genetic engineering to result in the replication or expression of that segment.

[0052] As used herein, the term “host cell” refers to a cell containing the nucleic acid molecule of the present invention. A “host cell” is any type of cell, such as a primary cell, a cultured cell, or a cell obtained from a cell line. In one embodiment, a “host cell” is a cell transfected with the nucleic acid molecule of the present invention. In other embodiments, a “host cell” is a cell that is or may be a descendant of such a transfected cell. The descendants of a cell may be identical to the parent cell, or they may not be identical due to mutations occurring, for example, during the integration of the nucleic acid molecule into the host cell's genome or during passaging, or due to environmental influences.

[0053] As used herein, the term "expression" refers to the biosynthesis of a gene product. This term includes the transcription of a gene into RNA. It also includes the translation of RNA into one or more polypeptides, and further encompasses all spontaneously occurring post-transcriptional and post-translational modifications. Expressed bispecific antibodies may be present in the cytoplasm of host cells, enter the extracellular environment such as growth media in cell cultures, or be immobilized on the cell membrane.

[0054] As used herein, the terms “peptide,” “polypeptide,” or “protein” refer to molecules composed of amino acids that can be recognized as proteins by those skilled in the art. Conventional one- or three-letter codes are used to represent amino acid residues. The terms “peptide,” “polypeptide,” and “protein” are interchangeable herein to refer to polymers of any length consisting of amino acids. The polymers may be linear or branched, may contain modified amino acids, or may have non-amino acid intercalations. The terms also encompass amino acid polymers that are naturally occurring or modified by intervention (e.g., disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, etc.) or any other operation or modification (e.g., conjugation with a labeling component). Furthermore, this definition includes polypeptides containing, for example, one or more analogues of amino acids (e.g., non-natural amino acids) and other modifications known in the art.

[0055] Peptide sequences described herein are written according to convention, with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although amino acid isomers are known, the L-form of the amino acids is described unless otherwise specified.

[0056] [Bispecific antibody] This invention relates to isolated bispecific antibodies in which a native cysteine ​​residue in the interchain disulfide bond between the CH1 region of the heavy chain and the CL region of the light chain of one arm has been removed by converting this native cysteine ​​to a non-cysteine ​​residue (e.g., a native cysteine ​​residue to a serine residue). Two native non-cysteine ​​residues in the CH1 and CL regions, or in the VH and VL regions of the same arm, are converted to cysteine ​​simultaneously or sequentially so that a new interchain disulfide bond is formed between the heavy and light chains. These two native non-cysteine ​​residues (one each in the CH1 and CL regions, or one each in the VH and VL regions) were identified by structural modeling of their proximity and the possibility of forming an interchain disulfide bond when converted to cysteine. This amino acid substitution minimizes the perturbation it introduces to the overall structure of the antigen-binding domain. The overall effect of the present invention is that the innate interchain disulfide bond between the CH1 region and the CL region in one arm of the bispecific antibody is shifted to a different site by recombination through the introduction of cysteine, while the innate interchain disulfide bond in the second arm remains unchanged.

[0057] The present invention also relates to isolated bispecific antibodies in which one arm has the above-described shift in interchain disulfide bond and the other arm has a native interchain disulfide bond, forming a heterodimer, where one arm contains the shifted interchain disulfide bond and the second arm contains a native interchain disulfide bond. Bispecific antibodies can bind to two antigens. Bispecific antibodies formed with two different heavy chains (HC) and light chains (LC) are difficult to produce because they tend to mispair the two heavy chains and two light chains, resulting in the production of unwanted products that are difficult to remove during the manufacturing process. Even if the unwanted products resulting from mispairing can be removed during purification, the mispairing reduces the production efficiency of the intended bispecific antibody product. Other strategies have been attempted to reduce or eliminate mispairing of HC and LC, but many of them involve protein engineering, including domain swapping and mutagenesis, which increases the risk of aggregation and immunogenicity. By introducing a "shifted interchain disulfide bond" in one arm while maintaining the innate interchain disulfide bond in the second arm, and simultaneously introducing a "knob in the hole" and a cysteine ​​mutation that forms a disulfide bond in the Fc region, heterodimeric bispecific antibodies can be efficiently produced. The production of such bispecific antibodies (including, but not limited to, anti-CD47 / anti-FRα bispecific antibodies) can be achieved by co-expression of two heavy chains and two light chains.

[0058] antibody The present invention generally relates to isolated bispecific antibodies having a shifted interchain disulfide bond on one arm and maintaining a native interchain disulfide bond on the second arm. In particular, the present invention generally relates to anti-CD47 / anti-FRα bispecific antibodies, nucleic acids and expression vectors encoding the antibody, recombinant cells containing the vector, and compositions comprising the antibody. Methods for producing the antibody and methods for treating diseases (including cancer) using the antibody are also provided. The antibodies of the present invention possess one or more desirable functional properties (including, but not limited to, high binding affinity to FRα and CD47, high specificity to FRα and CD47, ability to induce effector-mediated tumor cell lysis, ability to stimulate complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cell-mediated cytotoxicity (ADCC) in cells expressing FRα and / or CD47, ability to mediate the recruitment of conjugate drugs, and ability to inhibit tumor growth when administered alone or in combination with other anticancer therapies in subjects and animal models).

[0059] As used herein, the term “antibody” is used in a broad sense and includes immunoglobulins or antibody molecules, which are monoclonal or polyclonal, and include human, humanized, complex, and chimeric antibodies and antibody fragments. Generally, an antibody is a protein or peptide chain that exhibits binding specificity to a particular antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) according to the amino acid sequence of their heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Therefore, the antibodies of the present invention may be of any of the five major classes or their corresponding subclasses. Preferably, the antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. The antibody light chains of vertebrate species can be assigned to one of two distinct types, namely kappa and lambda, based on the amino acid sequence of their constant domains. Therefore, the antibodies of the present invention may contain kappa or lambda light chain constant domains. According to certain embodiments, the antibody of the present invention comprises heavy chain and / or light chain constant regions derived from a rat or human antibody. In addition to the heavy chain and light chain constant domains, the antibody comprises a light chain variable region and an antigen-binding region composed of a heavy chain variable region, each of which comprises three domains (i.e., complementarity-determining regions 1-3; CDR1, CDR2, and CDR3). The domains of the light chain variable region are instead referred to as LCDR1, LCDR2, and LCDR3, and the domains of the heavy chain variable region are instead referred to as HCDR1, HCDR2, and HCDR3.

[0060] Several systems are used for numbering the amino acid residues of antibodies. The Kabat numbering system is a scheme based on the variable region of the antibody (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition (1991)). The EU numbering system is widely used for constant domains (including CH1, hinge, and part of Fc) (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition (1991)).

[0061] As used herein, the term “isolated antibody” refers to an antibody that substantially contains no other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to FRα substantially contains no antibodies that do not bind to FRα, an isolated antibody that specifically binds to CD47 substantially contains no antibodies that do not bind to CD47, and a bispecific antibody that specifically binds to both CD47 and FRα substantially contains no antibodies that do not bind to CD47 and FRα). Furthermore, an isolated antibody substantially contains no other cellular material and / or chemical substances.

[0062] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies constituting the population are identical except for any naturally occurring mutations that may be present in small amounts. The monoclonal antibodies of the present invention can be produced by hybridoma, phage display techniques, single lymphocyte gene cloning techniques, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas containing B cells obtained from transgenic non-human animals having a genome containing human heavy chain and light chain transgenes, such as transgenic mice or rats.

[0063] As used herein, the term “antigen-binding fragment” refers to an antibody fragment, such as a diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (dsdiabody), single-chain antibody molecule (scFv), single-domain antibody (sdab), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelized single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment can bind to the same antigen to which a parent antibody or parent antibody fragment binds. According to certain embodiments, an antigen-binding fragment includes a light chain variable region, a light chain constant region, and a heavy chain Fd segment. According to other certain embodiments, an antigen-binding fragment includes Fab and F(ab').

[0064] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art that includes a heavy-chain variable region and a light-chain variable region linked by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single-domain antibody" refers to a conventional single-domain antibody in the art that includes a heavy-chain variable region and a heavy-chain constant region, or includes only the heavy-chain variable region.

[0065] As used herein, the term “human antibody” means an antibody produced by a human or an antibody having an amino acid sequence corresponding to a human-produced antibody prepared using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide.

[0066] As used herein, the term “humanized antibody” refers to a non-human antibody that has been modified to increase sequence homology to that of a human antibody, such that the antibody’s antigen-binding properties are retained, but its antigenicity in the human body is reduced.

[0067] As used herein, the term “chimeric antibody” refers to an antibody whose immunoglobulin molecule’s amino acid sequence originates from two or more species. Often, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) that possesses the desired specificity, affinity, and capability, while the constant region corresponds to the sequence of an antibody derived from another mammalian species (usually human) to avoid inducing an immune response in that species.

[0068] As used herein, the term “multispecific antibody” refers to an antibody comprising multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence has binding specificity to a first epitope, and a second immunoglobulin variable domain sequence has binding specificity to a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or the same subunit of a polymer protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes do not overlap or substantially overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a polymer protein). In one embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a triplicate antibody molecule, or a quadruplicate antibody molecule.

[0069] As used herein, the term “bispecific antibody” refers to a multispecific antibody that binds to only two epitopes or two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or the same subunit of a polymer protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a polymer protein). In one embodiment, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a second epitope. In one embodiment, the bispecific antibody comprises a semi-antibody or fragment thereof having binding specificity to a first epitope, and a semi-antibody or fragment thereof having binding specificity to a second epitope. In another embodiment, the bispecific antibody comprises an scFv or fragment thereof having binding specificity to a first epitope, and an scFv or fragment thereof having binding specificity to a second epitope.

[0070] As used herein, the term "FRα" refers to folate receptor α (also known as folate receptor 1 (FOLR1) or folate-binding protein (FBP)), a glycosylphosphacidylinositol (GPI)-anchored membrane protein on the cell surface, which has high affinity for active forms of folate, 5-methyltetrahydrofolate (5-MTF), and their derivatives, and transports them into the cell (Salazar and Ratnam, Cancer Metastasis Rev 2007;26:141-52). FRα has been targeted in oncology because it is overexpressed in certain solid tumors such as ovarian cancer, lung cancer, and breast cancer (Toffoli et al., Int J Cancer 1997;74:pp. 193-198 and Boogerd et al., Oncotarget 2016;7:pp. 17442-17454), but its expression level is low in limited normal human tissues (Weitman et al., Cancer Res 1992;52:pp. 3396-3401). Consistent with this observation, Phase 1 clinical trials conducted to date using FRα-targeted small and large molecules have shown good drug tolerability (Cheung et al., Oncotarget 2016;7:pp. 52553-52574). Therefore, FRα is a tumor-associated / tumor-specific antigen, and anti-FRα monoclonal antibodies (mAbs) and bispecific antibodies hold potential for anticancer drug therapy. Furthermore, FRα can be used to specifically target therapeutic molecules to cancer cells. A representative example of the amino acid sequence of human FRα is GenBank accession number NP_057937.1.

[0071] As used herein, the term “CD47” refers to a multi-pass transmembrane receptor belonging to the immunoglobulin superfamily, which has been shown to be involved in multiple cellular processes, including cell migration, adhesion, and T cell function. Also known as integrin-related protein (IAP), ovarian cancer antigen (OA3), Rh-related antigen, and MER6, CD47 was originally identified as a tumor antigen on human ovarian cancer and later found to be expressed in a number of human tumor types, including both hematological and solid tumors. The interaction between CD47 and signal regulatory protein α (SIRPα) (an inhibitory protein expressed on macrophages) interferes with the phagocytosis of cells expressing CD47. CD47 is also expressed at low levels in virtually all non-malignant cells. The term “human CD47” refers to CD47 of human origin. The exemplary amino acid sequence of human CD47 is GenBabk accession number NP_001768.1.

[0072] When used herein, an antibody that "specifically binds to CD47 and / or FRα" is defined as 1 × 10⁶ antibodies. -7 M or less, preferably 1 × 10 -8 M or less, more convenient 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 × 10 -10 This refers to an antibody with a KD of M or less that binds to CD47 and / or FRα, preferably human CD47 and / or human FRα. The term "KD" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as molar concentration (M). The KD value for an antibody can be determined using methods in the art in view of this disclosure. For example, the KD of an antibody can be determined using surface plasmon resonance, for example, using a biosensor system, for example, the Biacore® system, or using biolayer interferometry, for example, the Octet RED96 system.

[0073] The lower the KD value of an antibody, the higher its affinity for binding to the target antigen.

[0074] According to another specific embodiment, the present invention relates to an isolated bispecific antibody or an antigen-binding fragment thereof, a. First heavy chain H1; b. Second heavy chain H2; c. First light chain L1; and d. Second light chain L2 Includes, H1 and L1 form a first arm containing a first antigen-binding domain that specifically binds to a first antigen, preferably a first antigen of human origin, and H2 and L2 form a second arm that includes a second antigen-binding domain that specifically binds to a second antigen, preferably a second antigen of human origin. (a) H1 comprises the CH1 region of human IgG1, IgG2, IgG3, or IgG4, and (b) L1 includes the CL region of the human κ light chain or human λ light chain, The CH1 and CL regions contain amino acid substitutions or native amino acids at the amino acid residues corresponding to the amino acid positions of SEQ ID NOs. 15, 21, 22, or 23 for CH1, and at the amino acid residues corresponding to the amino acid positions of SEQ ID NOs. 19 or 24 for CL. The amino acid substitutions or native amino acids in the CH1 and CL regions are: (1) K133C and C220X for CH1, and F209C and C214X for CL; (2) S131C and C220X of CH1, and P119C and C214X of CL; (3) K133C and C220X for CH1, and K207C and C214X for CL; (4) F170C and C220X for CH1, and S176C and C214X for CL; (5) P171C and C220X of CH1, and S162C and C214X of CL; (6) V173C and C220X for CH1, and Q160C and C214X for CL; (7) F170C and C131X of CH1, and S176C and C214X of CL; (8) P171C and C131X of CH1, and S162C and C214X of CL; (9) V173C and C131X of CH1, and Q160C and C214X of CL; (10) A129C and C220X of CH1, and S121C and C214X of CL; (11) K133C and C220X of CH1, and I117C and C214X of CL; (12) C131 of CH1, and P119C and C214X of CL; (13) A129C and C131X of CH1, and S121C and C214X of CL; (14) R133C and C131X of CH1, and K207C and C214X of CL; (15) R133C and C131X of CH1, and I117C and C214X of CL; (16) R133C and C131X of CH1, and L117C and C214X of CL; (17) K133C and C220X of CH1, and L117C and C214X of CL; (18) R133C and C131X of CH1, and F209C and C214X of CL; (19) R133C and C131X of CH1, and V209C and C214X of CL; or (20) CH1's K133C and C220X, and CL's V209C and C214X Selected from, Here, X is selected from S, A, or G. This relates to isolated bispecific antibodies or their antigen-binding fragments.

[0075] According to another specific embodiment, the present invention relates to an isolated bispecific antibody or an antigen-binding fragment thereof, a. First heavy chain, H1; b. The second heavy chain, H2; c. First light chain, L1; and d. Second light chain, L2 Includes, H1 and L1 form a first arm containing a first antigen-binding domain that specifically binds to a first antigen, preferably a first antigen of human origin, and H2 and L2 form a second arm that includes a second antigen-binding domain that specifically binds to a second antigen, preferably a second antigen of human origin. (a) H1 comprises the CH1 region and the heavy chain variable region (VH region) of human IgG1, IgG2, IgG3, or IgG4, and (b) L1 includes the CL region and the variable light chain region (VL region) of the human κ light chain or human λ light chain, The CH1 region, VH region, CL region, and VL region include amino acid substitutions at amino acid residues corresponding to the amino acid positions of SEQ ID NOs. 15, 21, 22, or 23 for CH1, SEQ ID NOs. 13 for VH, SEQ ID NOs. 19 or 24 for CL, and SEQ ID NOs. 17 for VL. The amino acid substitutions in the CH1 region, VH region, CL region, and VL region are as follows: (1) C220X for CH1, G44C for VH, C214X for CL, and G101C for VL; or (2) CH1 C131X, VH G44C, CL C214X, and VL G101C Selected from, Here, X is selected from S, A, or G. This relates to isolated bispecific antibodies or their antigen-binding fragments.

[0076] In another specific embodiment, the present invention provides a first antigen-binding domain which is a CD47-binding domain. In one particular embodiment, the VH region comprises the amino acid sequence of SEQ ID NO: 1, the CH1 region comprises the amino acid sequence of SEQ ID NO: 2, the VL region comprises the amino acid sequence of SEQ ID NO: 3, and the CL region comprises the amino acid sequence of SEQ ID NO: 4.

[0077] According to another specific embodiment, (a) The second arm, which includes H2 and L2, does not contain any amino acid substitutions of the first arm, which includes H1 and L1. (b) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the VH region has a different amino acid sequence; (c) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the CH1 region has a different amino acid sequence; (d) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the Fc region has a different amino acid sequence; (e) Each of the two light chains L1 and L2 includes a VL region and a CL region, where the VL region has a different amino acid sequence; and / or (f) Each of the two light chains, L1 and L2, contains a VL region and a CL region, where the CL region has a different amino acid sequence.

[0078] In another specific embodiment, H1 and H2 form a heterodimer.

[0079] In another specific embodiment, (a) the VH region of H1 and the VL region of L1 have the substitution mutations Q39E and Q38K, respectively, and the VH region of H2 and the VL region of L2 have the substitution mutations Q39K and Q38E, respectively; or (b) the VH region of H1 and the VL region of L1 have the substitution mutations Q39K and Q38E, respectively, and the VH region of H2 and the VL region of L2 have the substitution mutations Q39E and Q38K, respectively.

[0080] In other specific embodiments, the isolated bispecific antibody or its antigen-binding fragment is an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment. In one embodiment, the anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment can block the binding of signal-regulating protein α (SIRPα) to CD47 on cancer cells expressing both FRα and CD47. In one embodiment, the isolated humanized anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment can induce macrophage-mediated phagocytosis in cancer cells expressing both FRα and CD47. In one embodiment, the isolated humanized anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment can bind to cancer cells expressing both FRα and CD47, with binding to human erythrocytes (RBCs) ranging from minimal to undetectable.

[0081] In other specific embodiments, the first antigen-binding domain has the VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 17, and the second antigen-binding domain has the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 35, or the first antigen-binding domain has the VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 17, and the second antigen-binding domain has the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 18.

[0082] The full-length bispecific antibodies of the present invention can be created, for example, by introducing substitutions at the interface of the heavy chain CH3 in each antibody half to facilitate heterodimerization of two antibody half halves having distinct specificities, either in vitro in a cell-free environment or using co-expression, using Fab arm exchange (half-body exchange) between two monospecific bivalent antibodies. The Fab arm exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domain. It reduces the heavy chain disulfide bond in the hinge region of the parent monospecific antibody. As a result, the free cysteine ​​of one parent monospecific antibody forms a disulfide bond between the cysteine ​​residue and the heavy chain of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody detaches and reforms through dissociation-association. The CH3 domain of the Fab arm can be genetically engineered so that heterodimerization is preferred over homodimerization. The resulting product is a bispecific antibody having two Fab arms or halves, each binding to a separate epitope (i.e., an epitope on CD47 and an epitope on FRα).

[0083] As used herein, "homodimerization" refers to the interaction of two heavy chains having the same CH3 amino acid sequence. As used herein, "homodimer" refers to an antibody having two heavy chains with the same CH3 amino acid sequence.

[0084] As used herein, "heterodimerization" refers to the interaction of two heavy chains having different CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains with different CH3 amino acid sequences.

[0085] The "knob-in-hole" strategy (see, for example, International Patent Publication No. WO2006 / 028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect the interaction of the CH3 domain, thereby promoting the formation of heterodimers. Amino acids with small side chains (holes) are introduced into the heavy chain of an antibody that specifically binds to the first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds to the second antigen. After co-expression of the two antibodies, the heavy chains with "holes" and "knobs" preferentially interact, resulting in the formation of heterodimers. Examples of CH3 substitution pairs that form "bumps" and "indentations" are as follows (represented by the modification position in the first CH3 domain of the first heavy chain / modification position in the second CH3 domain of the second heavy chain): T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0086] Other strategies can be used, such as promoting heavy chain heterodimerization using electrostatic interactions by substituting a positively charged residue on one CH3 surface and a negatively charged residue on the second CH3 surface, as described in U.S. Patent Publication No. 2010 / 0015133, U.S. Patent Publication No. 2009 / 0182127, U.S. Patent Publication No. 2010 / 028637, or U.S. Patent Publication No. 2011 / 0123532. In other strategies, heterodimerization can be promoted by the following substitutions (represented as modification position in the first CH3 domain of the first heavy chain / modification position in the second CH3 domain of the second heavy chain): L351Y_F405AY407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y407A / T366A_K409F, or T350V_L351Y_F405A Y407V / T350V_T366L_K392L_T394W (described in U.S. Patent Publication No. 2012 / 0149876 or U.S. Patent Publication No. 2013 / 0195849)

[0087] In addition to the above method, the bispecific antibody of the present invention can be produced in vitro in a cell-free environment by introducing asymmetric mutations into the CH3 region of two monospecific homodimer antibodies under reducing conditions, according to the method described in International Patent Publication No. 2011 / 131746, thereby enabling isomerization of the disulfide bond and forming a bispecific homodimer antibody from two parent monospecific homodimer antibodies. In this method, the first monospecific bivalent antibody and the second monospecific bivalent antibody are genetically engineered to perform certain substitutions in the CH3 domain to promote heterodimer stability. These antibodies are incubated together under reducing conditions sufficient to cause isomerization of the disulfide bond at the cysteine ​​in the hinge region, thereby exchanging the Fab arms to produce a bispecific antibody. The incubation conditions may optionally be returned to non-reducing conditions. Examples of reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tri(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol. Preferably, the reducing agent is selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tri(2-carboxyethyl)phosphine. For example, the mixture can be incubated at pH 5-8 (e.g., pH 7.0 or pH 7.4) at at least 20°C for at least 90 minutes in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol.

[0088] In another general embodiment, the present invention relates to isolated nucleic acids encoding the bispecific antibody or its antigen-binding fragment. It will be recognized by those skilled in the art that the coding sequence of a protein can be altered (e.g., by substitution, deletion, insertion, etc.) without altering the amino acid sequence of the protein. Therefore, it will be understood by those skilled in the art that the nucleic acid sequence encoding the antibody or its antigen-binding fragment of the present invention can be altered without altering the amino acid sequence of the protein.

[0089] In another general embodiment, the present invention relates to a vector comprising an isolated nucleic acid encoding a bispecific antibody or its antigen-binding fragment. In view of this disclosure, any vector known to those skilled in the art, such as plasmids, cosmids, phage vectors, or viral vectors, may be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any elements for establishing the conventional function of an expression vector, such as a promoter, ribosome-binding element, terminator, enhancer, selection marker, and origin of replication. The promoter may be a constitutive, inductive, or repressive promoter. Many expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein for the production of antibodies or their antigen-binding fragments in cells. According to embodiments of the present invention, recombinant expression vectors can be generated using conventional cloning techniques or artificial gene synthesis. Such techniques are well known to those skilled in the art in relation to this disclosure.

[0090] In another general embodiment, the present invention relates to a host cell comprising a vector containing an isolated nucleic acid encoding the bispecific antibody or its antigen-binding fragment. In view of this disclosure, any host cell known to those skilled in the art can be used for recombinant expression of the antibody or its antigen-binding fragment. In some embodiments, the host cell is Escherichia coli (E. coli) TG1 or BL21 cell (e.g., for the expression of scFv or Fab antibody), CHO-DG44 or CHO-K1 cell, or HEK293 cell (e.g., for the expression of full-length IgG antibody). According to a particular embodiment, the recombinant expression vector is transformed into a host cell by a conventional method, e.g., chemical transfection, heat shock, or electroporation, and the recombinant expression vector is stably incorporated into the host cell genome so that the recombinant nucleic acid is effectively expressed.

[0091] In another general embodiment, the present invention relates to a method for producing the bispecific antibody or its antigen-binding fragment, comprising the steps of culturing cells containing nucleic acids encoding the bispecific antibody or its antigen-binding fragment under conditions for producing the bispecific antibody or its antigen-binding fragment, and recovering the bispecific antibody or its antigen-binding fragment from the cells or cell culture (e.g., from the supernatant). The expressed antibody or its antigen-binding fragment can be collected from cells and purified according to conventional techniques known in the art and described herein.

[0092] Pharmaceutical composition In another general embodiment, the present invention relates to a pharmaceutical composition comprising an isolated bispecific antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier. The term “pharmaceutical composition” as used herein means a product comprising the antibody of the present invention together with a pharmaceutically acceptable carrier. The antibodies of the present invention and compositions comprising them are also useful in the manufacture of pharmaceuticals for therapeutic applications as referred herein.

[0093] As used herein, the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, phase solvent, oil, lipid, vesicle-containing lipid, microsphere, liposome encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of a carrier, excipient, or diluent depend on the route of administration for a particular application. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the efficacy or biological activity of the composition according to the present invention. Depending on the particular embodiment, any pharmaceutically acceptable carrier suitable for use in antibody pharmaceutical compositions may be used in the present invention in view of this disclosure.

[0094] Formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, for example, as described in Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005), and any subsequent editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tonicity modifiers, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used in the formulation of the pharmaceutical compositions of the present invention.

[0095] In one embodiment of the present invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation containing water. The liquid formulation may include a solution, suspension, emulsion, microemulsion, gel, etc. The aqueous formulation typically contains at least 50% w / w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w / w water.

[0096] In one embodiment, the pharmaceutical composition can be formulated as an injectable agent that can be administered, for example, via an injection device (e.g., a syringe or infusion pump). The injection can be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, intravitreously, or intravenously.

[0097] In another embodiment, the pharmaceutical composition is a solid formulation, such as a freeze-dried or spray-dried composition, which may be used as is or may be used with the addition of a solvent and / or diluent by a physician or patient before use. Examples of solid dosage forms include tablets, such as compressed tablets and / or coated tablets, and capsules (such as hard or soft gelatin capsules). The pharmaceutical composition may also be in the form of, for example, sachets, sugar-coated tablets, powders, granules, lozenges, or powders for reconstitution.

[0098] The dosage form may be immediate-release (in which case it may contain a water-soluble or dispersible carrier), or it may be delayed-release, sustained-release, or modified-release (in which case it may contain a water-insoluble polymer that modulates the dissolution rate of the dosage form in the gastrointestinal tract or subcutaneously).

[0099] In other embodiments, the pharmaceutical composition may be delivered intranasally, intracheek, or sublingually.

[0100] The pH of the aqueous formulation may be between pH 3 and pH 10. In one embodiment of the present invention, the pH of the formulation is about 7.0 to about 9.5. In another embodiment of the present invention, the pH of the formulation is about 3.0 to about 7.0.

[0101] In another embodiment of the present invention, the pharmaceutical composition includes a buffering agent. Non-limiting examples of buffering agents include arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine, and tris(hydroxymethyl)-aminomethane, as well as mixtures thereof. The buffering agents may be present individually or in combination at concentrations ranging from about 0.01 mg / ml to about 50 mg / ml, for example, from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions containing each of these specific buffering agents constitute alternative embodiments of the present invention.

[0102] In another embodiment of the present invention, the pharmaceutical composition includes a preservative. Non-limiting examples of preservatives include benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorohexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidourea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thimerosal, and mixtures thereof. The preservatives may be present individually or in combination at concentrations ranging from about 0.01 mg / ml to about 50 mg / ml, for example, from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions containing each of these specific preservatives constitute alternative embodiments of the present invention.

[0103] In another embodiment of the present invention, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of isotonic agents include salts (e.g., sodium chloride), amino acids (e.g., glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), algitols (e.g., glycerol, 1,2-propanediol propylene glycol, 1,3-propanediol, and 1,3-butanediol), polyethylene glycol (e.g., PEG400), and mixtures thereof. Another example of an isotonic agent is a sugar. Non-limiting examples of sugars may be monosaccharides, disaccharides, or polysaccharides, or water-soluble glucans, such as fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose. Another example of an isotonic agent is a sugar alcohol, the term “sugar alcohol” is defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. A pharmaceutical composition comprises each of the isotonic agents listed in this paragraph, which constitute an alternative embodiment. The isotonic agents may be present individually or in combination at concentrations ranging from about 0.01 mg / ml to about 50 mg / ml, for example, from about 0.1 mg / ml to about 20 mg / ml. A pharmaceutical composition comprising each of these specific isotonic agents constitutes an alternative embodiment of the present invention.

[0104] In another embodiment of the present invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, salts of ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agents may be present individually or in combination at concentrations ranging from about 0.01 mg / ml to about 50 mg / ml, for example, from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific chelating agents constitute alternative embodiments of the present invention.

[0105] In another embodiment of the present invention, the pharmaceutical composition includes a stabilizer. Non-limiting examples of the stabilizer include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0106] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer, which is carboxy / hydroxycellulose and its derivatives (e.g., HPC, HPC-SL, HPC-L, and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (e.g., PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (e.g., sodium chloride), sulfur-containing substances (e.g., monothioglycerol), or thioglycolic acid. The stabilizers may be present individually or in combination at concentrations ranging from about 0.01 mg / ml to about 50 mg / ml, for example, from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific stabilizers constitute alternative embodiments of the present invention.

[0107] In further embodiments of the present invention, the pharmaceutical composition comprises one or more surfactants, preferably one surfactant, at least one surfactant, or two different surfactants. The term “surfactant” refers to any molecule or ion composed of a water-soluble (hydrophilic) part and a lipid-soluble (lipophilic) part. Surfactants can be selected from the group consisting of, for example, anionic surfactants, cationic surfactants, nonionic surfactants, and / or bipolar ionic surfactants. The surfactants may be present individually or collectively at a concentration of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific surfactants constitute alternative embodiments of the present invention.

[0108] In further embodiments of the present invention, the pharmaceutical composition comprises one or more protease inhibitors, such as EDTA and / or benzamidine hydrochloride (HCl). The protease inhibitors may be present individually or collectively at concentrations ranging from about 0.1 mg / ml to about 20 mg / mml. Pharmaceutical compositions comprising each of these specific protease inhibitors constitute alternative embodiments of the present invention.

[0109] In another general embodiment, the present invention relates to a method for producing a pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof, comprising the step of compounding the bispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0110] How to use In another general embodiment, the present invention relates to a method for targeting FRα and CD47, both expressed on the surface of cancer cells, in a subject requiring such method, comprising the step of administering an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment or a pharmaceutical composition of the present invention to a subject requiring such method. Binding of the bispecific antibody or antigen-binding fragment to FRα and / or CD47 may mediate complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cell-mediated cytotoxicity (ADCC), or other effects resulting in the death of target cancer cells. The bispecific antibody or its antigen-binding fragment may, for example, serve to recruit a conjugate drug to mediate the death of target cancer cells.

[0111] In another general embodiment, the present invention relates to a method for blocking the binding of SIRPα to CD47 on cancer cells expressing both FRα and CD47 in a subject requiring such blocking. The method comprises administering to a subject requiring such blocking an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment or a pharmaceutical composition of the present invention.

[0112] In another general embodiment, the present invention relates to a method for inducing macrophage-mediated phagocytosis of cancer cells expressing both FRα and CD47 in a subject requiring such induction. This method comprises administering to a subject requiring induction an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment or a pharmaceutical composition of the present invention.

[0113] In another general embodiment, the present invention relates to a method for conjugating a humanized anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment to cancer cells expressing both FRα and CD47, such that binding to human erythrocytes (RBCs) is minimal to undetectable, in subjects requiring such conjugation. This method comprises administering an isolated humanized anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment, or a pharmaceutical composition of the present invention, to subjects requiring such conjugation. The humanized anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment of the present invention has high selectivity for cancer cells and binding to human erythrocytes (RBCs) is minimal to undetectable.

[0114] The functional activity of bispecific antibodies and their antigen-binding fragments that bind to both FRα and CD47 can be characterized by methods known in the art and by methods described herein. Methods for characterizing bispecific antibodies and their antigen-binding fragments that bind to both FRα and CD47 include, but are not limited to, affinity and specificity assays including Biacore, ELISA, FACS, and OctetRed analysis. According to certain embodiments, methods for characterizing bispecific antibodies and their antigen-binding fragments that bind to both FRα and CD47 include those described below.

[0115] In another general embodiment, the present invention relates to a method for treating cancer in a subject requiring such treatment, comprising the step of administering an isolated humanized anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment or a pharmaceutical composition of the present invention to the subject. Cancer can be any liquid or solid tumor, but may be selected from, for example, lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0116] According to embodiments of the present invention, a pharmaceutical composition comprises a therapeutically effective amount of the anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment. As used herein, the term “therapeutically effective amount” refers to the amount of the active ingredient or component that elicits a desired biological or medical response in a subject. The therapeutically effective amount can be determined empirically and conventionally in relation to the purposes described.

[0117] When used herein in relation to an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment, therapeutically effective dose means the amount of the anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment that modulates the immune response in a subject requiring such action. Also, when used herein in relation to an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment, therapeutically effective dose means the amount of the anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment that results in the treatment of a disease, disorder, or condition, prevents or delays the progression of a disease, disorder, or condition, or reduces or completely alleviates the symptoms associated with a disease, disorder, or condition.

[0118] According to certain embodiments, the disease, disorder, or condition to be treated is cancer, preferably lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, and other solid tumors, as well as cancer selected from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors. According to other certain embodiments, the disease, disorder, or condition to be treated is inflammatory diseases, metabolic diseases, or any other disease in which bispecific antibodies can be used in therapy.

[0119] According to a particular embodiment, the therapeutically effective dose refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of the disease, disorder, or condition being treated, or the symptoms associated therewith; (ii) shortening the duration of the disease, disorder, or condition being treated, or the symptoms associated therewith; (iii) preventing the progression of the disease, disorder, or condition being treated, or the symptoms associated therewith; (iv) causing regression of the disease, disorder, or condition being treated, or the symptoms associated therewith; (v) preventing the progression or onset of the disease, disorder, or condition being treated, or the symptoms associated therewith. (vi) preventing recurrence of the disease, disorder or condition being treated, or symptoms associated therewith; (vii) reducing hospitalizations in subjects with the disease, disorder or condition being treated, or symptoms associated therewith; (viii) shortening the length of hospitalization in subjects with the disease, disorder or condition being treated, or symptoms associated therewith; (ix) increasing the survival rate of subjects with the disease, disorder or condition being treated, or symptoms associated therewith; (xi) inhibiting or reducing the disease, disorder or condition being treated, or symptoms associated therewith in subjects; and / or (xii) enhancing or improving the preventive or therapeutic effect of another treatment.

[0120] The therapeutically effective dose or effective dosage may vary depending on various factors, such as the disease, disorder, or condition being treated, the means of administration, the target site, the physiological state of the subject (including age, weight, and health), whether the subject is human or animal, other pharmaceuticals administered, and whether the treatment is prophylactic or therapeutic. The therapeutic dose is optimally dose-set to optimize safety and efficacy.

[0121] According to certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to the subject. For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.

[0122] As used herein, the terms “treat,” “treating,” and “treatment” are intended to mean improvement or enhancement of at least one measurable physical parameter related to cancer, which may or may not be recognizable in the subject. The terms “treat,” “treating,” and “treatment” may also mean causing regression of a disease, disorder, or condition, preventing its progression, or at least slowing its rate of progression. In certain embodiments, “treat,” “treating,” and “treatment” mean alleviating, preventing the progression or onset of, or shortening the duration of, one or more symptoms associated with a disease, disorder, or condition, such as a tumor, more preferably cancer. In certain embodiments, “treat,” “treating,” and “treatment” mean preventing the recurrence of a disease, disorder, or condition. In certain embodiments, “treat,” “treating,” and “treatment” refer to improving the survival rate of an object having a disease, disability, or condition. In certain embodiments, “treat,” “treating,” and “treatment” refer to the disappearance of a disease, disability, or condition in an object.

[0123] According to certain embodiments, compositions used in the treatment of cancer are also provided. In cancer therapy, the compositions may be used in combination with other treatments, including but not limited to chemotherapy, anti-TIM-3 mAb, anti-LAG-3 mAb, anti-CD73 mAb, anti-Apelin mAb, anti-CTLA-4 antibody, anti-EGFR mAb, anti-HER-2 mAb, anti-CD19 mAb, anti-CD20 mAb, anti-CD33 mAb, anti-TIP-1 mAb, anti-DLL3 mAb, anti-CLDN18.2 mAb, anti-PD-L1 antibody, anti-PD-1 antibody, PD-1 / PD-L1 therapy, other immuno-oncology drugs, anti-angiogenic drugs, radiotherapy, antibody-drug conjugates (ADCs), targeted therapies, or other anticancer drugs.

[0124] As used herein, the term “combined” refers to the use of two or more treatments in the context of administering two or more treatments to a subject. The use of the term “combined” does not limit the order in which the treatments are administered to the subject. For example, a first treatment (e.g., one of the compositions described herein) may be administered before administering a second treatment to a subject (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior), simultaneously with the administration, or after administering a second treatment (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later).

[0125] Embodiment The present invention also provides the following non-limiting embodiments.

[0126] Embodiment 1 is an isolated bispecific antibody or its antigen-binding fragment, a. First heavy chain H1; b. Second heavy chain H2; c. First light chain L1; and d. Second light chain L2 Includes, H1 and L1 form a first arm containing a first antigen-binding domain that specifically binds to a first antigen, preferably a first antigen of human origin, and H2 and L2 form a second arm that includes a second antigen-binding domain that specifically binds to a second antigen, preferably a second antigen of human origin. (a) H1 comprises the CH1 region of human IgG1, IgG2, IgG3, or IgG4, and (b) L1 includes the CL region of the human κ light chain or human λ light chain, The CH1 and CL regions contain amino acid substitutions or native amino acids at the amino acid residues corresponding to the amino acid positions of SEQ ID NOs. 15, 21, 22, or 23 for CH1, and at the amino acid residues corresponding to the amino acid positions of SEQ ID NOs. 19 or 24 for CL. The amino acid substitutions or native amino acids in the CH1 and CL regions are: (1) K133C and C220X for CH1, and F209C and C214X for CL; (2) S131C and C220X of CH1, and P119C and C214X of CL; (3) K133C and C220X for CH1, and K207C and C214X for CL; (4) F170C and C220X for CH1, and S176C and C214X for CL; (5) P171C and C220X of CH1, and S162C and C214X of CL; (6) V173C and C220X for CH1, and Q160C and C214X for CL; (7) F170C and C131X of CH1, and S176C and C214X of CL; (8) P171C and C131X of CH1, and S162C and C214X of CL; (9) V173C and C131X of CH1, and Q160C and C214X of CL; (10) A129C and C220X of CH1, and S121C and C214X of CL; (11) K133C and C220X of CH1, and I117C and C214X of CL; (12) C131 of CH1, and P119C and C214X of CL; (13) A129C and C131X of CH1, and S121C and C214X of CL; (14) R133C and C131X of CH1, and K207C and C214X of CL; (15) R133C and C131X of CH1, and I117C and C214X of CL; (16) R133C and C131X of CH1, and L117C and C214X of CL; (17) K133C and C220X of CH1, and L117C and C214X of CL; (18) R133C and C131X of CH1, and F209C and C214X of CL; (19) R133C and C131X of CH1, and V209C and C214X of CL; or (20) CH1's K133C and C220X, and CL's V209C and C214X Selected from, Here, X is selected from S, A, or G. This refers to an isolated bispecific antibody or its antigen-binding fragment.

[0127] Embodiment 2 is an isolated bispecific antibody or its antigen-binding fragment, a. First heavy chain, H1; b. The second heavy chain, H2; c. First light chain, L1; and d. Second light chain, L2 Includes, H1 and L1 form a first arm containing a first antigen-binding domain that specifically binds to a first antigen, preferably a first antigen of human origin, and H2 and L2 form a second arm that includes a second antigen-binding domain that specifically binds to a second antigen, preferably a second antigen of human origin. (a) H1 comprises the CH1 region and the heavy chain variable region (VH region) of human IgG1, IgG2, IgG3, or IgG4, and (b) L1 includes the CL region and the variable light chain region (VL region) of the human κ light chain or human λ light chain, The CH1 region, VH region, CL region, and VL region include amino acid substitutions at amino acid residues corresponding to the amino acid positions of SEQ ID NOs. 15, 21, 22, or 23 for CH1, SEQ ID NOs. 13 for VH, SEQ ID NOs. 19 or 24 for CL, and SEQ ID NOs. 17 for VL. The amino acid substitutions in the CH1 region, VH region, CL region, and VL region are as follows: (1) C220X for CH1, G44C for VH, C214X for CL, and G101C for VL; or (2) CH1 C131X, VH G44C, CL C214X, and VL G101C Selected from, Here, X is selected from S, A, or G. This refers to an isolated bispecific antibody or its antigen-binding fragment.

[0128] Embodiment 3 is an isolated bispecific antibody or antigen-binding fragment thereof according to Embodiment 1 or 2, wherein the first antigen-binding domain is a CD47-binding domain.

[0129] Embodiment 4 is an isolated bispecific antibody or antigen-binding fragment thereof as described in Embodiment 3, wherein the VH region contains the amino acid sequence of SEQ ID NO: 1, the CH1 region contains the amino acid sequence of SEQ ID NO: 2, the VL region contains the amino acid sequence of SEQ ID NO: 3, and the CL region contains the amino acid sequence of SEQ ID NO: 4.

[0130] Embodiment 5 is, (a) The second arm, which includes H2 and L2, does not contain any amino acid substitutions of the first arm, which includes H1 and L1. (b) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the VH region has a different amino acid sequence; (c) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the CH1 region has a different amino acid sequence; (d) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the Fc region has a different amino acid sequence; (e) Each of the two light chains L1 and L2 includes a VL region and a CL region, where the VL region has a different amino acid sequence; and / or (f) Each of the two light chains L1 and L2 contains a VL region and a CL region, where the CL region has a different amino acid sequence. This is an isolated bispecific antibody or its antigen-binding fragment described in any of Embodiments 1 to 4.

[0131] Embodiment 6 is an isolated bispecific antibody or its antigen-binding fragment as described in Embodiment 5, wherein H1 and H2 form a heterodimer.

[0132] Embodiment 7 is, (a) The VH region of H1 and the VL region of L1 have the substitution mutations Q39E and Q38K, respectively, and the VH region of H2 and the VL region of L2 have the substitution mutations Q39K and Q38E, respectively; or (b) The VH region of H1 and the VL region of L1 have the substitution mutations Q39K and Q38E, respectively, and the VH region of H2 and the VL region of L2 have the substitution mutations Q39E and Q38K, respectively. This is an isolated bispecific antibody or its antigen-binding fragment described in any of Embodiments 1 to 6.

[0133] Embodiment 8 is an isolated bispecific antibody or antigen-binding fragment according to any one of Embodiments 1 to 7, wherein the isolated bispecific antibody or antigen-binding fragment is an anti-CD47 / anti-FRα bispecific antibody or antigen-binding fragment, the first antigen-binding domain specifically binds to CD47, preferably human CD47, and the second antigen-binding domain specifically binds to folate receptor α (FRα), preferably human FRα.

[0134] Embodiment 9 is, (a) The first antigen-binding domain has the VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 17, and the second antigen-binding domain has the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 35; or (b) The first antigen-binding domain has the VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 17, and the second antigen-binding domain has the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 18. This is an isolated bispecific antibody or its antigen-binding fragment described in any of Embodiments 1 to 8.

[0135] Embodiment 10 describes an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment, Blocking the binding of signal regulatory protein α (SIRPα) to CD47 on cancer cells expressing both FRα and CD47, Inducing macrophage-mediated phagocytosis in cancer cells expressing both FRα and CD47, and / or Binding to human red blood cells (RBCs) is minimal to undetectable, while binding to cancer cells expressing both FRα and CD47. It is possible This is an isolated bispecific antibody or its antigen-binding fragment as described in Embodiment 8 or 9.

[0136] Embodiment 11 is an isolated nucleic acid encoding a bispecific antibody or antigen-binding fragment described in any of Embodiments 1 to 10.

[0137] Embodiment 12 is a vector containing the isolated nucleic acid described in Embodiment 11.

[0138] Embodiment 13 is a host cell containing the vector described in Embodiment 12.

[0139] Embodiment 14 is a pharmaceutical composition comprising an isolated bispecific antibody or its antigen-binding fragment described in any of Embodiments 1 to 10 and a pharmaceutically acceptable carrier.

[0140] Embodiment 15 is, A method for targeting FRα and CD47, both of which are expressed on the surface of cancer cells, in a target that requires this. A method for blocking the binding of SIRPα to CD47 on cancer cells expressing both FRα and CD47 in subjects requiring it, A method for inducing macrophage-mediated phagocytosis in cancer cells expressing both FRα and CD47 in subjects requiring it, A method for binding to cancer cells expressing both FRα and CD47, while binding to human red blood cells (RBCs) is minimal to undetectable in the target population, and / or A method of treating cancer in those who require it, The method involves administering a pharmaceutical composition comprising an isolated anti-CD-47 / anti-FRα bispecific antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier as described in any of Embodiments 8 to 10, Cancer is optionally selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors. It is a method.

[0141] Embodiment 16 is a method for producing a bispecific antibody or its antigen-binding fragment according to any one of Embodiments 1 to 10, comprising culturing cells containing nucleic acids encoding a bispecific antibody or its antigen-binding fragment under conditions for producing a bispecific antibody or its antigen-binding fragment, and recovering the bispecific antibody or its antigen-binding fragment from the cells or culture.

[0142] Embodiment 17 is a method for producing a pharmaceutical composition containing a bispecific antibody or an antigen-binding fragment thereof as described in any of Embodiments 1 to 10, the method comprising compounding the bispecific antibody or the antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0143] (Examples) Example 1 [Construction of a bispecific antibody with modified cysteine ​​moiety] Figures 1A and 1B illustrate a bispecific antibody having two different heavy chains and two different light chains as H1H2 heterodimers. This can be easily performed using common methods such as the "knob-in-hole" and "charged pairs" method. The native interchain disulfide bond in the H1L1 arm can be removed by converting two native cysteine ​​residues that form an interchain disulfide bond to non-cysteine ​​residues (dashed lines in Figures 1A and 1B), while two native non-cysteine ​​residues that are close to each other in the 3D structure of H1L1 can be converted to cysteine ​​residues to form a new interchain disulfide bond (solid lines in Figures 1A and 1B). The newly formed interchain disulfide bond may be between CH1 and CL of H1L1 (Figure 1A) or between VH and VL of H1L1 (Figure 1B). By shifting the interchain disulfide bond sites within H1L1, the two light chains can preferentially pair with their respective heavy chains. Furthermore, the asymmetric structure resulting from the shifting of interchain disulfide bonds on the mAb1 arm makes it easier to identify the physical properties and potential impurities of the bispecific antibody, facilitating the purification and production of the intended bispecific antibody. Several pairs of native non-cysteine ​​residues (also called "knock-in sites") capable of forming new interchain disulfide bonds within H1L1 have been identified. The following examples demonstrate this using bispecific antibodies of the VH and CH1 heavy chains and the VL and CL sequences of the κ light chains of human IgG1. This concept can also be applied to the construction of bispecific antibodies using the CH1 heavy chain and CL human λ light chain, whenever a maintained knock-in site is present. If the knock-in site happens to be a natural cysteine ​​(and not one involved in the formation of a natural interchain disulfide bond), it can always be used directly to form a new interchain disulfide bond.

[0144] Bispecific anti-CD47 / anti-FRα antibodies were constructed using a humanized anti-CD47 mAb (described in International Patent Publication No. WO2019 / 217145, referred to herein as mAb1) and two humanized anti-FRα mAbs (described in International Patent Publication No. WO2019 / 177854, referred herein as mAb2 and mAb2b, respectively). Figures 1A and 1B illustrate the structure of a desired bispecific antibody having mAb1 (anti-CD47) as the right arm and mAb2 (anti-FRα) as the left arm (in some bispecific antibodies, mAb2b is used as the left arm rather than mAb2). The VH and VL regions of the bispecific antibody were fused to the constant region (HC) and constant region (LC) of the human IgG1 heavy chain and κ light chain, respectively. mAb1 HC has the T366W (EU numbering) mutation, forming a "knob," while mAb2 HC has the T366S, L368A, and Y407V mutations, forming a "hole." The two heavy chains preferentially form bispecific antibodies with heterodimer HC (mAb1 HC / mAb2 HC) rather than homodimer HC (mAb1 HC / mAb1 HC or mAb2 HC / mAb2 HC). Furthermore, the heterodimer pairing of the heterodimer heavy chains was stabilized by introducing the S354C cysteine ​​mutation onto mAb1 HC and the Y349C cysteine ​​mutation onto mAb2 HC (Merchant et al., Nat. Biotechnol. 16(7):pp. 677-681 (1998)). By using a strategy to shift the interchain disulfide bonds between HC and LC on the mAb1 arm, we preferentially achieved the expression, purification, and / or production of the intended bispecific antibodies when two HCs and two LCs were simultaneously transfected into cells.To achieve this goal, two native cysteines on the HC and LC of the mAb1 arm that form interchain disulfide bonds were each converted to serine residues, and two native non-cysteines on the HC and LC of the mAb1 arm were, once cysteine was introduced at these sites (referred to herein as "knock-in cysteine" or "substitute cysteine"), each identified by structural modeling of their close proximity such that the cysteine residues potentially form new interchain disulfide bonds. The newly formed interchain disulfide bonds by pairing of substitute cysteines may be between the CH1 region and the CL region as shown in FIG. 1A, or may be between the VH region and the VL region as shown in FIG. 1B. The native interchain disulfide bond between the CH1 region and the CL region of the mAb1 arm is shown by a dashed line, and the newly formed interchain disulfide bond by the knock-in cysteine on the mAb1 arm is shown by a solid line. Since no mutations were introduced into the VH, VL, CH1 or CL regions of the mAb2 arm, the mAb2 arm has the same native interchain disulfide bonds.

[0145] The VH, CH1, VL, and CL sequences of mAb1 and mAb2, as well as the CH1 sequences of IgG2, IgG3, IgG4, and λCL, are listed in Figures 2A-2F and Table 1 (SEQ ID NOs. 13, 15, 17, and 19 for mAb1; SEQ ID NOs. 14, 16, 18, and 20 for mAb2; SEQ ID NOs. 21, 22, 23, and 24 for IgG2 CH1, IgG3 CH1, IgG4 CH1, and λCL, respectively). The VH, CH1, VL, and CL sequences of mAb 2b are listed in Table 1 (SEQ ID NOs. 33, 34, 35, and 36, respectively). The anti-CD47 antibody mAb1 was selected as the arm with the modified interchain disulfide bond. Homology models of many antibody structures previously elucidated in the public domain were constructed using Schrodinger BioLuminate® (Schrodinger; New York, NY). Using a cysteine ​​mutation tool with a beta-carbon cutoff distance of 7 Å, potential interchain disulfide crosslinks were identified. The identified pairs were further analyzed individually, and for each pair, a mutagenesis tool was used to identify non-cysteine-to-cysteine ​​mutations (which minimized perturbation to the overall structure, and while a single non-cysteine-to-cysteine ​​mutation reduced interchain binding affinity, paired cysteine ​​mutations did not significantly affect binding affinity). The most promising candidates were selected for expression and experimental validation. As shown in Figures 3A-3L, disulfide bonds potentially formed by knock-in cysteine ​​sites are shown by solid lines, while native interchain disulfide bonds are shown by dashed lines. The native interchain disulfide region was not included in the models of bsAb9, 10, 11, and 12, so there are no dashed lines in the models of these four bsAbs. Each of the mAb1 arms with different pairs of knock-in cysteines was paired with one native mAb2 arm to form bispecific antibodies (bsAbs). Amino acid substitutions on the mAb1 arms of the mAb1 / mAb2 bispecific antibodies for the shifted interchain disulfide bond are listed in Table 2.

[0146] To evaluate the effect of the various shifted interchain disulfide bond pairs listed in Table 2 on mAb stability, some of the mutant groups (i.e., designs) listed in Table 2 were introduced into mAb1 on both arms. For comparison, M1, M2, Z1, and Z2, representing mutant designs provided in the parent patents (US Patent No. 9,527,927 and US Patent No. 10,344,099), were also introduced into mAb1 on both arms. Mutant mAbs were expressed and purified using protein A chromatography and hydrophobic interaction chromatography (HIC). Protein A chromatography was performed using conventional methods. In HIC, the protein A purified sample was buffered with PBS and (NH4)2SO4 was added to a final concentration of 800 mM. The sample was placed in a Source 15PHE column (GE) that had been pre-equilibriumized and pre-packed with 50 mM MES pH 6.0 and 1 M (NH4)2SO4 buffer. Samples were eluted using a linear or stepwise gradient (Buffer A: 50 mM MES pH 6.0, 1 M (NH4)2SO4; Buffer B: 50 mM MES pH 6.0, 10% glycerol) (the main fraction was eluted with 60% Buffer B). The eluted fractions were analyzed by SDS-PAGE, and the fraction containing mainly 145 kDa bands and free of low molecular weight impurities was pooled as purified protein. Figures 4A-4H show the RP-HPLC profiles of purified mutant mAbs containing differently shifted interchain disulfide bonds under reducing and non-reducing conditions. Figures 4I-4J show the binding of mutant mAbs containing differently shifted interchain disulfide bonds to CD47 in ELISA assays.

[0147] The thermal stability of the purified mutant mAbs was analyzed. The mutant mAbs were buffer-exchanged into PBS (Corning, Cat: 21-031-CM) at 1 mg / mL and incubated for 5 minutes at 55°C, 60°C, 65°C, 70°C or 75°C using a Thermo Cycler (Simpliamp, Thermo Fisher). After incubation, the mAbs were analyzed by SEC (size exclusion chromatography). Each peak was quantified to determine the percentage of the antibody (medium molecular weight species). The results are shown in Figures 5A - 5G. The mutant mAbs containing the bsAb10 and bsAb12 mutations, respectively, were more thermostable than any of the other mutant mAbs (such as those containing the M1, M2, Z1 and Z2 mutations, respectively). These surprising results indicate that the bsAb10 and bsAb12 designs are superior to any of the M1, M2, Z1 or Z2 designs. The thermal stability of an antibody is an important attribute required during the manufacturing process and storage. Good thermal stability greatly expands the developability of an antibody and increases its potential to become a therapeutic agent.

[0148] The pH stability or recovery rate from incubation at low pH of mutant mAbs was tested. Using a Zeba® spin-type desalting column (Thermo Fisher, Cat: 89882), 10 mg / mL of mAb was buffer-exchanged with a buffer containing 0.1 M citrate and 0.1 M sodium chloride (pH 3.0). The mAb was diluted to 2 mg / mL with the same buffer and incubated at room temperature for 1, 3, 5, or 7 hours. The sample was then neutralized with 1.0 M Tris-HCl (pH 9.0) to adjust the final pH to 6.0-7.0. After neutralization, the mAb was added to DPBS and diluted to 1 mg / mL, and SEC analysis was performed. The results are shown in Figures 6A-6H. Mutant mAbs, including the bsAb12 design, showed higher pH stability than any of the mutant mAbs containing M1, M2, Z1, or Z2 mutations. This was also good compared to the remaining mutant mAbs tested for pH stability (Figures 6A-6H). Recovery rates from pH stability studies of mutant mAbs, including the bsAb10 design, were better than those of any mutant mAb, including the M1, M2, Z1, or Z2 mutants. The data indicate that the bsAb12 and bsAb10 designs are superior to any of the M1, M2, Z1, or Z2 designs in terms of pH stability or recovery from low pH incubation. Furthermore, when incubated at pH 3.0 for 1 or 3 hours, the pH stability of mutant mAbs, including the bsAb5 design, was better than that of any of the mutant mAbs, including the M1, M2, Z1, or Z2 designs. These surprising findings indicate that the bsAb5, 10, and 12 designs are superior to any of the M1, M2, Z1, or Z2 designs. pH stability or recovery from low pH incubation of antibodies is a critical attribute required in the manufacturing process. Good pH stability or recovery rates from low pH incubation (especially under conditions of 3 hours at low pH) greatly expands the potential for antibody development and increases its likelihood of becoming a therapeutic agent.

[0149] Table 1: mAb1; mAb2; heavy chains (HC) of human IgG2, IgG3, and IgG4; and sequences of various regions within human λCL; [Table 1] TIFF0007900284000002.tif157161

[0150] Table 2: Amino acid substitutions on the mAb1 arm of mAb1 / mAb2 bispecific antibody (bsAb) for shifted interchain disulfide bonds [Table 2] Note: Kabat numbering is used for VH and VL regions, while EU numbering is used for CH1 and CL regions.

[0151] The bispecific antibodies in the examples are located on the HC and κLC framework of IgG1 (Kabat numbering for the VH and VL regions; EU numbering for the CH1 and CL regions). Using mAb1 and mAb2, several bispecific antibodies were constructed, including bsAb1, bsAb2, bsAb3, bsAb4, bsAb5, bsAb6, bsAb7, and bsAb8 (bsAb1 refers to a bispecific antibody having mAb1 arm containing the corresponding mutation predicted to result in a rearrangement (or "shift") of the HC / LC interchain disulfide bond, as described in Table 2, and mAb2 as a second arm. Other bispecific antibodies in this group are named according to the same naming convention). No mutations were introduced on the VH, CH1, VL, or CL regions of the mAb2 arm. Mutation G44C refers to the conversion of the native glycine (G44) at position 44 to cysteine. All other mutations employ the same naming convention. Using mAb1 and mAb2, other bispecific antibodies including bsAb5b, bsAb10, and bsAb12 were constructed (bsAb5b refers to a bispecific antibody having a mAb1 arm containing the corresponding bsAb5 mutation from Table 2 and mAb2 as a second arm; bsAb10 refers to a bispecific antibody having a mAb1 arm containing the corresponding bsAb10 mutation from Table 2 and mAb2b as a second arm; bsAb12 refers to a bispecific antibody having a mAb1 arm containing the corresponding bsAb12 mutation from Table 2 and mAb2b as a second arm). The sequences of the CH1, CL, VH, and VL regions of the mAb1 arm of the bispecific antibodies are listed in Table 3. Furthermore, charged amino acid pairs were introduced at position Q39 (Kabat numbering) on ​​the HC and Q38 (Kabat numbering) on ​​the LC of each arm of the given bispecific antibodies. These bispecific antibodies were named as follows: bsAb 5b(E / K) refers to a bispecific antibody of bsAb5b that has Q39E (Q changed to E: the same naming convention applies to the other mutations below) on the HC of the mAb1 arm and Q38K on the LC, and also has Q39K on the HC of the mAb2b arm and Q38E on the LC.bsAb10(E / K) and bsAb12(E / K) follow the same naming convention. Furthermore, bsAb5b(K / E) refers to a bispecific antibody of bsAb5b that has Q39K on the HC and Q38E on the LC of the mAb1 arm, and also has Q39E on the HC and Q38K on the LC of the mAb2b arm. bsAb10(K / E) and bsAb12(K / E) follow the same naming convention. mAb1 HC has the T366W (EU numbering) mutation and forms a "bump," while mAb2 or mAb2b HC has the T366S, L368A, and Y407V mutations and forms a "depression." Furthermore, the heterodimer pairing was stabilized by introducing the S354C cysteine ​​mutation into mAb1 HC and the Y349C cysteine ​​mutation into mAb2 or mAb2b HC.

[0152] Table 3: Sequences of the CH1, CL, VH, and VL regions of the mAb1 arm of bispecific antibodies [Table 3] TIFF0007900284000005.tif23162 Note: Residues resulting from amino acid substitutions are shown in bold and underlined.

[0153] Example 2 [Characterization of bispecific antibodies] By simultaneously expressing two heavy chains and two light chains within the same cell, the expression and assembly of desired bispecific antibodies, including anti-CD47 and anti-FRα arms, occurred. The bispecific antibodies were purified using protein A chromatography. Several samples were further purified using hydrophobic interaction chromatography (HIC).

[0154] Bispecific antibodies purified using protein A chromatography were analyzed by SDS-PAGE. Protein samples were diluted to a concentration of 1 mg / mL. In the non-reducing lane, 3 μL of protein was added to 4.5 μL of water and 2.5 μL of 4× LDS sample buffer (Thermo NP0007; Waltham, Massachusetts) and loaded directly onto the gel. In the reducing lane, 3 μL of protein was added to 3.5 μL of water, 2.5 μL of 4× LDS sample buffer, and 1 μL of 1 M DTT, heated at 95°C for 3 minutes, and loaded onto the gel. Bolt 4-12% bis-tris gels were used for all samples (Thermo NP0323BOX). Samples were subjected to electrophoresis at 180 V for 30 minutes and visualized using a Coomassie G-250. Figures 7A-7B show SDS-PAGE images of mAb1 / mAb2 bispecific antibody samples purified using protein A chromatography. Under reducing / denaturing conditions, all samples showed two bands (one corresponding to the heavy chain (approximately 50 kDa) and the other to the light chain (approximately 25 kDa)). Under non-reducing / denaturing conditions, all samples showed multiple bands. The top band in each lane represents either a single intact antibody or a mixture of antibodies (approximately 150 kDa), although it is unclear whether this is a mixture of heterodimeric bispecific antibodies (promoted by H1H2 interaction) and homodimeric antibodies. The smaller bands represent various antibodies that are not intact in SDS-PAGE. For example, the second band from the top in each lane (approximately 125 kDa) likely represents an antibody that has lost one light chain. This likely originates from an intact antibody in which one light chain did not form an interchain disulfide bond, thus causing the light chain to be separated from the main antibody in SDS-PAGE. Figures 7C-7D show SDS-PAGE images of mAb1 / mAb2b bispecific antibody samples purified using protein A chromatography. Under non-reducing / denaturing conditions, all samples showed one major band corresponding to the molecular weight of the bispecific antibody (Figure 7C). Under reducing / denaturing conditions, all samples showed two bands corresponding to the molecular weights of two different heavy chains and one band corresponding to the molecular weights of two different light chains (Figure 7D).

[0155] To evaluate the binding activity of bispecific samples by bridging ELISA assay, recombinant folate receptor 1 (Novoprotein C784; Summit, New Jersey) was diluted to 0.25 μg / ml in PBS and used to coat 96-well plates (Genesee Scientific 91-415F; San Diego, California). 50 μL of antigen was added per well and coated overnight at 4°C. The plates were blocked with 5% BSA in TBST and washed with TBST. 50 μL of antibody in 5% BSA in TBST was added at the indicated concentration, incubated at room temperature, and washed with TBST. Secondary recombinant biotin-labeled CD47 antigen (AcroBio CD47-H82E9-258g; Newark, Delaware), diluted to 0.05 μg / mL in 5% BSA in TBST, was added to each well, incubated at room temperature, and washed with TBST. Streptoavidin HRP (JIR 016-030-084), diluted to 0.2 or 0.5 μg / mL in 5% BSA in TBST, was added to each well, incubated at room temperature, and washed. The plates were developed with TMB substrate (Thermo34028), stopped with stop solution (Thermo SS04), and the wells were quantified by absorbance at 450 nm. Figures 8A-8J show the results of the binding of purified bispecific antibodies to both antigens in the bridging ELISA assay. The data in Figure 8A shows that bsAb1, 5, 6, 7, and 8 have anti-CD47 / FRα bispecific activity, confirming that each of these constructs formed the intended anti-CD47 / FRα bispecific antibody. All bispecific antibodies tested in Figures 8B-8J showed bridging ELISA activity.

[0156] Size exclusion chromatography (SEC) was used to measure the purity of samples purified by protein A chromatography, or by HIC following protein A chromatography. Protein samples were diluted to a concentration of 1 mg / mL and filtered through a submicron filter. 5 μL of protein was injected into an AdvanceBio SEC column (300 mm, 2.7 μm, 300A, Agilent PL1580-5301). DPBS was used as the mobile phase and flowed at 0.35 mL / min. Bio-rad gel filtration standard was used as the standard solution (Bio-rad 1511901; Hercules, California). Samples were quantified by measuring absorbance at 280 nm. Figures 9A–9E show the SEC profiles of samples purified by protein A chromatography. The appearance of a main peak of approximately 150 kDa suggests that the major species in the sample was a complete antibody (i.e., an antibody) with two heavy chains and two light chains. These data are consistent with the SDS-PAGE observations shown in Figures 7A-7B. Figures 9F-9N show the SEC profiles of samples purified by HIC following protein A chromatography.

[0157] Furthermore, the purity of the HIC-purified bispecific antibody samples was analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC) under non-reducing conditions. Figures 10A–10L show the RP-HPLC profiles of the bispecific antibodies. In each case, there is one major species. RP-HPLC was also performed under reducing conditions to determine the presence of both LCs (L1 and L2) as well as both HCs (H1 and H2) of the intended bispecific antibodies. Control antibody samples were prepared by transiently expressing bsAb7 H1, H2, L1 (control antibody #1) and bsAb7 H1, H2, L2 (control antibody #2), respectively, followed by purification by protein A chromatography. These samples served as controls for identifying LC peaks on RP-HPLC (Figures 11A and 11B). The HC peaks (H1 and H2) and LC peaks (L1 and L2) were identified in combination with the RP-HPLC profiles of bsAb6 (Figure 11C), bsAb7 (Figure 11D), and bsAb8 (Figure 11E) (Figures 11A-11E). Figures 11F-11N show the RP-HPLC profiles of bispecific antibodies constructed using mAb1 and mAb2b under reducing conditions. Different HC and LC peaks were identified for a given bispecific antibody using an appropriate control transfection with one of the two arms (data not shown). The area under the curve (AUC) of each HC or LC relative to the total AUC in the RP-HPLC profile was quantified and is shown in Figures 11C-11N. The ratios of H1, L1, H2, and L2 are consistent with the ratios of heterodimer bispecific antibodies. These data indicate that the dominant species in the HIC-purified bispecificity samples was a heterodimer bispecificity antibody with HC(H1 and H2) and LC(L1 and L2) in the intended composition. Importantly, the data in Figures 10A–10L and 11A–11N suggested the presence of interchain disulfide bonds between HCs on each of the two arms of the bispecificity antibody, and between HCs and LCs.

[0158] The same HIC-purified bispecific antibody samples were analyzed for their ability to bind to both antigens using bridging ELISA. Consistent with the analytical data above, all bispecific antibodies were shown to be able to bind simultaneously to both CD47 and FRα, suggesting that each arm of the bispecific antibody was properly formed with the correct HC and LC (Figures 12A-12F). The bispecific antibodies were tested for their binding ability to SK-OV-3 cells expressing both antigens (CD47 and FRα). In the assay, SK-OV-3 cells were incubated with bispecific antibodies at various concentrations at 4°C for 15 minutes. The cells were then centrifuged for 5 minutes and washed three times with FACS buffer (HBSS supplemented with 5% BSA and 0.05% sodium azide). Next, the cells were incubated with Alexa Fluor 488 conjugate anti-human IgG secondary antibody (ThermoFisher, catalog #: H10120) and incubated for a further 15 minutes on ice. The cells were then washed twice with FACS buffer and resuspended in FACS buffer. Subsequently, the cells were passed through Attune NxT, and the data was analyzed using Attune NxT software. The bispecific antibody showed significant binding to SK-OV-3 cells in the FACS assay (Figures 13A-13C).

[0159] HIC-purified bispecific antibody samples were subjected to papain digestion under non-reducing conditions to determine whether the Fab fragments predicted from the bispecific antibodies were generated. The samples were concentrated to a final concentration of 5–10 mg / mL and rebuffered with papain digestion buffer (20 mM cysteine, 20 mM sodium phosphate, 10 mM EDTA, pH 7.0). Agarose-immobilized papain (Thermo 20341) was pre-equilibrated with papain digestion buffer and resuspended as a 50% slurry. Protein samples were added to this slurry in a 2:1 volume ratio and shaken overnight at room temperature. The supernatant was extracted and directly analyzed by mass spectrometry. Calculated molecular weights (mw) of each Fab fragment are shown along with their observed mw values ​​(Figures 14A–14C). The data indicate that the expected Fab fragments generated by papain digestion were detected by mass spectrometry, demonstrating the proper formation of the intended bispecific antibodies.

[0160] Furthermore, HIC-purified bispecific antibody samples were subjected to trypsin digestion under non-reducing conditions to determine whether the calculated trypsin-digested peptide fragments from the intended bispecific antibody mAb1 arm were generated. The samples were diluted to a final concentration of 1 mg / mL with 200 mM guanidine HCl and heated at 95°C for 1 minute. 2x trypsin digestion buffer was added in a 1:1 volume ratio (NEB P8101S), and 2 μg total trypsin ultra (NEB P8101S) was added. The resulting reaction mixture was shaken at 37°C for 4 hours and used directly for analysis by mass spectrometry. The amino acid sequence and disulfide binding site of the disulfide-crosslinked peptide fragment predicted to be generated from the trypsin digestion of the mAb1 arm (anti-CD47 arm) of bsAb6 are shown in Figure 15A. Cysteine ​​residues forming disulfide bonds are shown in bold, and the expected interchain disulfide bonds formed by knock-in cysteine ​​are also shown (Figure 15A). Similar illustrations are shown for bsAb7 (Figure 15B), bsAb8 (Figure 15C), bsAb 5b (Figure 15D), bsAb10 (Figure 15E), and bsAb12 (Figure 15F), respectively. The MS results in Figures 15A-15F suggest that disulfide-bridged peptide fragments expected from the trypsin-digested mAb1 arm can be identified by MS in each of the bispecific antibodies (bsAb6, bsAb7, bsAb8, bsAb5b, bsAb10, and bsAb12). These data further demonstrate that the knock-in cysteine ​​enabled the proper formation of interchain disulfide bonds in each of the bispecific antibodies bsAb6, bsAb7, bsAb8, bsAb5b, bsAb10, and bsAb12 (Figures 15A to 15F).

[0161] The HIC-purified bispecific antibody sample was also subjected to IdeZ protease digestion under non-reducing conditions to determine whether the computationally predicted (Fab')2 moiety from the intended bispecific antibody was generated. The sample was diluted to a final concentration of 0.5 mg / mL with 1× glycobuffer 2 (NEB). IdeZ protease was added at a rate of 80 U protease per 12.5 μg antibody. The resulting mixture was incubated at 37 °C for 4 hours and used directly for analysis by mass spectrometry. Figures 16A - 16C show the MS profiles of the IdeZ protease-digested samples of the HIC-purified bispecific antibodies. The (Fab')2 generated from each bispecific antibody was identified, indicating that the intended bispecific antibody with properly formed interchain disulfide bonds by the knock-in cysteine was formed.

[0162] To show the contribution of both arms of the bispecific antibody in binding to SK-OV-3 cells, which are known to express both antigens, the inhibition of antibody binding to cells by F(ab')2 generated from anti-CD47 or anti-FRα parental mAbs was evaluated by FACS assay. The Ab (antibody) concentrations used in the assay are shown below each graph in Figures 17A - 17C. The inhibitory effect was tested using 5,000 nM F(ab')2 (Figures 17A - 17C). Furthermore, the sequential binding of CD47 and FRα to immobilized bsAb 12 was detected using Biacore (Figures 18A - 18B).

[0163] Purified bispecific antibodies for protein A were digested with papain, and the resulting Fab fragments were identified by MS (Figures 19A-19C). In the WT bispecific antibody samples, Fab fragments from both correctly paired arms were detected, as well as from two mismatched arms (H1 / L2 and H2 / L1) (Figure 19A). However, in the bsAb10 (Figure 19B) and bsAb12 (Figure 19C) samples, only one of the two mismatched arms was detected. On the other hand, in the bsAb10 and bsAb12 designs, no LC bands were observed in these designs on non-reducing SDS-PAGE, indicating that there were no HC / LC mismatch species that did not form HC / LC interchain disulfide bonds (Figure 7C). Therefore, these data indicate that the bsAb10 and bsAb12 designs eliminated the formation of H2 / L1 mismatch species. Furthermore, the absence of H2L1 pair formation supports the conclusion that scrambled species H1L2 / H2L1 was not formed. Compared to other bispecific antibody designs using shifted HC / LC interchain disulfide bond strategies described in the literature, the bsAb10 and bsAb12 designs are superior in that they prevent the formation of one mismatched species and scrambled species. Further purification of protein A-purified bsAb 12 by HIC resulted in the disappearance of mismatched H1 / L2Fab in the MS profile (Figure 20). The purification process for bsAb12 was optimized and is shown in Table 4. A yield of 77 mg was obtained from 500 mL of transiently transfected ExpiCHO cell culture, which is within the same range as the yield of mAbs from the same transient transfection system.

[0164] Table 4: Optimization of the bsAb12 purification process [Table 4]

[0165] Table 5: Amino acid residues in the CH1 and CL regions for charge pair formation [Table 5] Note: EU numbering is used for CH1 and CL regions. CP stands for charge pair.

[0166] To further promote HC and LC pairing on each arm of a given bispecific antibody and / or to enhance the feasibility of purification using conventional manufacturing processes, amino acid mutations are introduced into a pair of residues on the CH1 and CL regions, respectively, to form charged pairs. Each charged pair, or any combination of two or more charged pairs, can be used to construct bispecific antibodies because it increases correct HC and LC pairing and / or facilitates purification by introducing a physical property that is differentiated from impurities (i.e., mismatched molecular species). Any charged pair or any combination of such charged pairs in Table 5 can be combined with other charged pairs (known in the literature or newly designed) to achieve and / or enhance correct HC / LC pairing and / or facilitate purification by introducing a physical property that is differentiated from impurities (i.e., mismatched molecular species). One of the two residues for charged pair formation can be substituted with glutamic acid (E) and the other with lysine (K), and vice versa. For example, in a given bispecific antibody consisting of H1L1 and H2L2, if "E" is introduced into H1 and "K" into L1, then "K" will be introduced into H2 and "E" into L2. The two residues for forming the charge pair can be replaced with acidic amino acids and basic amino acids other than "E" and "K," respectively. For example, the residue into which "E" is introduced can be replaced with aspartic acid (D) if "E" is not used. If the natural amino acid of a given residue happens to be the intended substitution amino acid (i.e., E, D, or K), then mutation is not necessary. Table 5 shows the amino acid residues of the CH1 and CL regions for forming charge pairs. Each pair of these residues is selected based on various factors, including proximity, using 3D modeling.

[0167] Those skilled in the art will recognize that modifications can be made to the above embodiments without departing from the broader concept of the invention. Therefore, the present invention is understood to be not limited to the specific embodiments disclosed, but to encompass modifications within the spirit and scope of the invention as defined herein. The present invention includes, for example, the following embodiments: [1] An isolated bispecific antibody or its antigen-binding fragment, a. First heavy chain H1; b. Second heavy chain H2; c. First light chain L1; and d. Second light chain L2 Includes, H1 and L1 form a first arm containing a first antigen-binding domain that specifically binds to a first antigen, preferably a first antigen of human origin, and H2 and L2 form a second arm that includes a second antigen-binding domain that specifically binds to a second antigen, preferably a second antigen of human origin. (a) H1 comprises the CH1 region of human IgG1, IgG2, IgG3, or IgG4, and (b) L1 includes the CL region of the human κ light chain or human λ light chain, The CH1 and CL regions contain amino acid substitutions or native amino acids at the amino acid residues corresponding to the amino acid positions of SEQ ID NOs. 15, 21, 22, or 23 for CH1, and at the amino acid residues corresponding to the amino acid positions of SEQ ID NOs. 19 or 24 for CL. The amino acid substitutions or native amino acids in the CH1 and CL regions are: (1) K133C and C220X for CH1, and F209C and C214X for CL; (2) S131C and C220X of CH1, and P119C and C214X of CL; (3) K133C and C220X for CH1, and K207C and C214X for CL; (4) F170C and C220X for CH1, and S176C and C214X for CL; (5) P171C and C220X of CH1, and S162C and C214X of CL; (6) V173C and C220X for CH1, and Q160C and C214X for CL; (7) F170C and C131X of CH1, and S176C and C214X of CL; (8) P171C and C131X of CH1, and S162C and C214X of CL; (9) V173C and C131X of CH1, and Q160C and C214X of CL; (10) A129C and C220X of CH1, and S121C and C214X of CL; (11) K133C and C220X of CH1, and I117C and C214X of CL; (12) C131 of CH1, and P119C and C214X of CL; (13) A129C and C131X of CH1, and S121C and C214X of CL; (14) R133C and C131X of CH1, and K207C and C214X of CL; (15) R133C and C131X of CH1, and I117C and C214X of CL; (16) R133C and C131X of CH1, and L117C and C214X of CL; (17) K133C and C220X of CH1, and L117C and C214X of CL; (18) R133C and C131X of CH1, and F209C and C214X of CL; (19) R133C and C131X of CH1, and V209C and C214X of CL; or (20) CH1's K133C and C220X, and CL's V209C and C214X Selected from, Here, X is selected from S, A, or G. Isolated bispecific antibodies or their antigen-binding fragments. [2] An isolated bispecific antibody or its antigen-binding fragment, a. First heavy chain, H1; b. The second heavy chain, H2; c. First light chain, L1; and d. Second light chain, L2 Includes, H1 and L1 form a first arm containing a first antigen-binding domain that specifically binds to a first antigen, preferably a first antigen of human origin, and H2 and L2 form a second arm that includes a second antigen-binding domain that specifically binds to a second antigen, preferably a second antigen of human origin. (a) H1 comprises the CH1 region and the heavy chain variable region (VH region) of human IgG1, IgG2, IgG3, or IgG4, and (b) L1 includes the CL region and the variable light chain region (VL region) of the human κ light chain or human λ light chain, The CH1 region, VH region, CL region, and VL region include amino acid substitutions at amino acid residues corresponding to the amino acid positions of SEQ ID NOs. 15, 21, 22, or 23 for CH1, SEQ ID NOs. 13 for VH, SEQ ID NOs. 19 or 24 for CL, and SEQ ID NOs. 17 for VL. The amino acid substitutions in the CH1 region, VH region, CL region, and VL region are as follows: (1) C220X for CH1, G44C for VH, C214X for CL, and G101C for VL; or (2) CH1 C131X, VH G44C, CL C214X, and VL G101C Selected from, Here, X is selected from S, A, or G. Isolated bispecific antibodies or their antigen-binding fragments. [3] An isolated bispecific antibody or antigen-binding fragment thereof as described in [1] or [2], wherein the primary antigen-binding domain is a CD47-binding domain. [4] An isolated bispecific antibody or antigen-binding fragment thereof as described in [3], wherein the VH region comprises the amino acid sequence of SEQ ID NO: 1, the CH1 region comprises the amino acid sequence of SEQ ID NO: 2, the VL region comprises the amino acid sequence of SEQ ID NO: 3, and the CL region comprises the amino acid sequence of SEQ ID NO: 4. [5](a) The second arm containing H2 and L2 does not contain the amino acid substitutions of the first arm containing H1 and L1, (b) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the VH region has a different amino acid sequence; (c) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the CH1 region has a different amino acid sequence; (d) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the Fc region has a different amino acid sequence; (e) Each of the two light chains L1 and L2 includes a VL region and a CL region, where the VL region has a different amino acid sequence; and / or (f) Each of the two light chains L1 and L2 contains a VL region and a CL region, where the CL region has a different amino acid sequence. An isolated bispecific antibody or its antigen-binding fragment as described in any of [1] to [4]. [6] The isolated bispecific antibody or antigen-binding fragment thereof described in [5], wherein H1 and H2 form a heterodimer. [7](a) The VH region of H1 and the VL region of L1 have the substitution mutations Q39E and Q38K, respectively, and the VH region of H2 and the VL region of L2 have the substitution mutations Q39K and Q38E, respectively; or (b) The VH region of H1 and the VL region of L1 have the substitution mutations Q39K and Q38E, respectively, and the VH region of H2 and the VL region of L2 have the substitution mutations Q39E and Q38K, respectively. An isolated bispecific antibody or its antigen-binding fragment as described in any of [1] to [6]. [8] The isolated bispecific antibody or antigen-binding fragment thereof is an anti-CD47 / anti-FRα bispecific antibody or antigen-binding fragment thereof, wherein the first antigen-binding domain specifically binds to CD47, preferably human CD47, and the second antigen-binding domain specifically binds to folate receptor α (FRα), preferably human FRα, as described in any of [1] to [7]. [9](a) The first antigen-binding domain has the VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 17, and the second antigen-binding domain has the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 35; or (b) The first antigen-binding domain has the VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 17, and the second antigen-binding domain has the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 18 An isolated bispecific antibody or its antigen-binding fragment as described in any of [1] to [8].

[10] Anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment, Blocking the binding of signal regulatory protein α (SIRPα) to CD47 on cancer cells expressing both FRα and CD47, Inducing macrophage-mediated phagocytosis in cancer cells expressing both FRα and CD47, and / or Binding to human red blood cells (RBCs) is minimal to undetectable, while binding to cancer cells expressing both FRα and CD47. It is possible [8] or [9] an isolated bispecific antibody or its antigen-binding fragment. Isolated nucleic acids encoding a bispecific antibody or antigen-binding fragment as described in any of

[11] [1] to

[10] . A vector containing the isolated nucleic acid described in

[12]

[11] . Host cells containing the vector described in

[13]

[12] . A pharmaceutical composition comprising an isolated bispecific antibody or its antigen-binding fragment as described in any of

[14] [1] to

[10] , and a pharmaceutically acceptable carrier.

[15] A method for targeting FRα and CD47, both of which are expressed on the surface of cancer cells, in a subject that requires the same. A method for blocking the binding of SIRPα to CD47 on cancer cells expressing both FRα and CD47 in subjects requiring it, A method for inducing macrophage-mediated phagocytosis in cancer cells expressing both FRα and CD47 in subjects requiring it, A method for binding to cancer cells expressing both FRα and CD47, while binding to human red blood cells (RBCs) is minimal to undetectable in the target population, and / or A method of treating cancer in those who require it, The administration of a pharmaceutical composition comprising an isolated anti-CD-47 / anti-FRα bispecific antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier as described in any of [8] to

[10] , Cancer is optionally selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors. method. A method for producing a bispecific antibody or its antigen-binding fragment according to any one of

[16] [1] to

[10] , comprising culturing cells containing nucleic acids encoding a bispecific antibody or its antigen-binding fragment under conditions for producing a bispecific antibody or its antigen-binding fragment, and recovering the bispecific antibody or its antigen-binding fragment from the cells or culture. A method for producing a pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof as described in any of

[17] [1] to

[10] , comprising compounding the bispecific antibody or the antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

Claims

1. An isolated bispecific antibody or its antigen-binding fragment, a. First heavy chain H1; b. Second heavy chain H2; c. First light chain L1; and d. Second light chain L2 Includes, H1 and L1 form a first arm containing a first antigen-binding domain that specifically binds to the first antigen, and H2 and L2 form a second arm containing a second antigen-binding domain that specifically binds to the second antigen. (a) H1 comprises the CH1 region of human IgG1, IgG2, IgG3, or IgG4, and (b) L1 includes the CL region of the human κ light chain or human λ light chain, The sequence of the CH1 region of H1 is selected from the group consisting of sequence numbers 15, 21, 22, and 23, and the sequence of the CL region of L1 is selected from sequence number 19 or 24. The CH1 and CL regions are, (1) K133C and C220X of CH1, and F209C and C214X of CL; or (2) K133C and C220X of CH1, and K207C and C214X of CL; Further includes amino acid substitutions selected from (positions based on EU numbering), Here, X is selected from S, A, or G. The second arm, containing H2 and L2, does not contain the amino acid substitutions of the first arm, containing H1 and L1, and maintains the natural interchain disulfide bond. Isolated bispecific antibodies or their antigen-binding fragments.

2. An isolated bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein the first antigen is of human origin.

3. An isolated bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the second antigen is of human origin.

4. An isolated bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the first antigen-binding domain is a CD47-binding domain.

5. An isolated bispecific antibody or antigen-binding fragment thereof according to claim 4, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 1 and the VL region comprises the amino acid sequence of SEQ ID NO:

3.

6. (b) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the VH region has a different amino acid sequence; (c) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the CH1 region has a different amino acid sequence; (d) Each of the two heavy chains H1 and H2 comprises a VH region, a CH1 region, and an Fc region (including CH2 and CH3 regions), where the Fc region has a different amino acid sequence; (e) Each of the two light chains L1 and L2 includes a VL region and a CL region, where the VL region has a different amino acid sequence; and / or (f) Each of the two light chains L1 and L2 contains a VL region and a CL region, where the CL region has a different amino acid sequence. An isolated bispecific antibody or its antigen-binding fragment according to any one of claims 1 to 5.

7. The isolated bispecific antibody or antigen-binding fragment thereof according to claim 6, wherein H1 and H2 form a heterodimer.

8. (a) The VH region of H1 and the VL region of L1 have the substitution mutations Q39E and Q38K, respectively, and the VH region of H2 and the VL region of L2 have the substitution mutations Q39K and Q38E, respectively; or (b) The VH region of H1 and the VL region of L1 have the Q39K and Q38E substitution mutations, respectively, and the VH region of H2 and the VL region of L2 have the Q39E and Q38K substitution mutations, An isolated bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. The isolated bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the isolated bispecific antibody or antigen-binding fragment thereof is an anti-CD47 / anti-FRα bispecific antibody or antigen-binding fragment thereof, wherein the first antigen-binding domain specifically binds to CD47 and the second antigen-binding domain specifically binds to folate receptor α (FRα).

10. An isolated bispecific antibody or antigen-binding fragment thereof according to claim 9, wherein the first antigen-binding domain specifically binds to human CD47.

11. An isolated bispecific antibody or antigen-binding fragment thereof according to claim 9 or 10, wherein the second antigen-binding domain specifically binds to human FRα.

12. (a) The first antigen-binding domain has the VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 17, and the second antigen-binding domain has the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 35; or (b) The first antigen-binding domain has the VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 17, and the second antigen-binding domain has the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO:

18. An isolated bispecific antibody or its antigen-binding fragment according to claim 1.

13. Anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment, Blocking the binding of signal regulatory protein α (SIRPα) to CD47 on cancer cells expressing both FRα and CD47, Inducing macrophage-mediated phagocytosis in cancer cells expressing both FRα and CD47, and / or Binding to human red blood cells (RBCs) is minimal to undetectable, while binding to cancer cells expressing both FRα and CD47. It is possible An isolated bispecific antibody or antigen-binding fragment thereof according to any one of claims 9 to 11.

14. Isolated nucleic acid encoding a bispecific antibody or antigen-binding fragment according to any one of claims 1 to 13.

15. A vector comprising the isolated nucleic acid described in claim 14.

16. A host cell comprising the vector according to claim 15.

17. A pharmaceutical composition comprising an isolated bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 13, and a pharmaceutically acceptable carrier.

18. A pharmaceutical composition comprising an isolated anti-CD-47 / anti-FRα bispecific antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier, as described in any one of claims 9 to 11, A method for targeting FRα and CD47, both of which are expressed on the surface of cancer cells, in a target that requires this. A method for blocking the binding of SIRPα to CD47 on cancer cells expressing both FRα and CD47 in subjects requiring it, A method for inducing macrophage-mediated phagocytosis in cancer cells expressing both FRα and CD47 in subjects requiring it, A method for binding to cancer cells expressing both FRα and CD47, while binding to human red blood cells (RBCs) is minimal to undetectable in the target population, and / or A method of treating cancer in those who need it. It is intended for use in the following context: The method comprises administering the pharmaceutical composition to a target.

19. The pharmaceutical composition according to claim 18, wherein the cancer is selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

20. A method for producing a bispecific antibody or its antigen-binding fragment according to any one of claims 1 to 13, comprising culturing cells containing nucleic acids encoding a bispecific antibody or its antigen-binding fragment under conditions for producing a bispecific antibody or its antigen-binding fragment, and recovering the bispecific antibody or its antigen-binding fragment from the cells or culture.

21. A method for producing a pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 13, comprising compounding the bispecific antibody or an antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

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