Bispecific antibodies with alternatively matched interchain cycteines and uses thereof

Abstract

Engineered bispecific antibodies with a translocated interchain disulfide bond on one arm and retaining a native interchain disulfide bond on the second arm are described. Anti-CD47 / FRα bispecific antibodies and their antigen-binding fragments are also described. The nucleic acid encoding the antibody, compositions containing the antibody, and methods for preparing the antibody and using the antibody to treat or prevent diseases such as cancer and / or related complications are also described.

Description

[0001] Cross-reference to related applications

[0002] 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. Each disclosure is incorporated herein by reference in its entirety. Invention Field

[0003] This invention relates to engineered bispecific antibodies having a displaced interchain disulfide bond on one arm while retaining a native interchain disulfide bond on the second arm. These bispecific antibodies offer stability and manufacturing advantages, and can be used for therapeutic purposes. The invention relates to bispecific antibodies, nucleic acids encoding the antibody and expression vectors, recombinant cells containing the vector, and compositions comprising the antibody. Methods for preparing the antibody and methods for using the antibody to treat diseases including cancer and / or related complications are also provided.

[0004] References to sequence lists submitted electronically

[0005] This application contains a sequence list, which was submitted electronically via EFSWeb in ASCII format, with the file name "065799.30WO1 sequence list", created on November 24, 2020, and a size of 36kb. The sequence list submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety. Background Technology

[0006] Antibodies (immunoglobulins) are naturally occurring proteins that play a crucial role in protecting the body from foreign invaders such as bacteria and viruses by the immune system. In their natural structure, 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 a variable region (VH) and three constant regions (CH1, CH2, and CH3), arranged from the N-terminus to the C-terminus in the order VH, CH1, CH2, and CH3. The light chain contains a variable region (VL) and a constant region (CL), arranged from the N-terminus to the C-terminus in the order VL and CL. The binding of the heavy and light chains in each arm is commonly referred to as "pairing," and includes VH, CH1, VL, and CL. Through physical interaction, VH and VL form the antibody's binding domain against its antigen, giving Y-type antibodies two identical binding domains—one on each arm—resulting in bivalent monospecificity, a hallmark of monoclonal antibodies.

[0007] As part of the antibody structure, CH1 and CL also physically interact, involving physical contact and interchain disulfide bonds (also known as “disulfide bridges”) formed by the free thiol groups of two native cysteine ​​residues located on CH1 and CL, respectively. These interchain disulfide bonds help stabilize the overall structure formed by the heavy and light chains on each arm. In addition, intrachain disulfide bonds also form as part of the native antibody structure. The C-termini of the heavy chains (CH2 and CH3) form a tight structure, which is important for the divalent nature of the native antibody.

[0008] Monoclonal antibodies (mAbs) have long been an excellent protein therapy platform due to their high affinity for antigens, long in vivo half-life, naturally occurring stable structure, and ability to activate the immune system against drug targets, among many other advantages. However, monoclonal antibodies fail to achieve their purpose when a therapeutic strategy requires targeting two different antigens with a single antibody, such as two tumor-specific antigens on the same cancer cell. In such cases, bispecific antibodies are manufactured to target two different antigens on the same cell, with one arm binding to the first antigen and the other arm binding to the second. Although binding to each antigen is monovalent, binding to two antigens on the same cell compensates for the loss of affinity due to the loss of bivalent binding to each antigen. Compared to mAbs, bispecific antibodies offer higher selectivity because they bind more readily to cells expressing both antigens than to cells expressing only one antigen. This is particularly important for reducing safety concerns when normal cells or tissues express one of two antigens. Another advantage over mAbs is that when bispecific antibodies bind to two different cell surface antigens or soluble ligands / proteins, they can target two pathways simultaneously.

[0009] If a bispecific antibody is made from two mAbs, the product will contain two distinct arms from each mAb, each arm having a unique heavy chain and a unique light chain. During manufacturing, the expression of the bispecific antibody in the producing cells requires the expression of four different proteins: two distinct heavy chains and two distinct light chains. While the goal is to pair each HC on each arm of the bispecific antibody with its corresponding LC during manufacturing, mismatches often occur (the LC of one arm pairs with the HC of the other arm), resulting in unwanted products, making it difficult to produce and isolate the desired bispecific antibody product. Several methods have been employed to improve aspects of bispecific antibody manufacturing. One example is the identification of a common light chain through protein engineering. However, domain exchanges and numerous mutations can significantly alter the structure of native antibodies, increasing the risk of aggregation and / or reducing stability.

[0010] FRα expression is elevated in certain solid tumors such as ovarian, lung, 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 level is low in a limited number of normal human tissues (Weitman et al., Cancer Res 1992; 52:3396-3401). Consistent with this observation, phase 1 clinical trials targeting both small and large molecules of FRα have so far shown good tolerability (Cheung et al., Oncotarget 2016; 7:52553–52574). Therefore, FRα is an ideal target for cancer therapy. Furthermore, CD47, which mediates the "don't eat me" signal, is overexpressed in many tumors. Bispecific antibodies (called anti-CD47 / FRα bispecific antibodies) that bind to CD47 on one arm and FRα on the other can be used to selectively target cells expressing both antigens. Due to affinity, the binding of bispecific antibodies to two antigens on the same cell can result in increased affinity compared to either arm. Because of their lack of affinity compared to bivalent anti-CD47 mAbs, bispecific antibodies are expected to have weaker activity against cells that express only CD47 (but not FRα), which may increase the safety and / or tolerability range. Anti-CD47 / FRα bispecific antibodies can selectively block the interaction of CD47 / SIRPα with cells that co-express CD47 and FRα and activate the innate immune system against these cells, such as cancer cells. Therefore, anti-CD47 / FRα bispecific antibodies could be an effective therapy for ovarian cancer and other tumors that express significant levels of CD47 and FRα on their cell surface. Invention Overview

[0012] In one general aspect, the present invention relates to isolated bispecific antibodies or antigen-binding fragments thereof, comprising:

[0013] a. First heavy chain, H1;

[0014] b. Second chain, H2;

[0015] c. The first light chain, L1; and

[0016] d. Second light chain, L2;

[0017] Hl and Ll form the first arm, which contains a first antigen-binding domain that specifically binds to the first antigen, preferably a human-derived first antigen.

[0018] H2 and L2 form the second arm, which contains a second antigen-binding domain that specifically binds to the second antigen, preferably a human-derived second antigen.

[0019] (a) H1 contains the CH1 region of human IgG1, IgG2, IgG3, or IgG4; and

[0020] (b) L1 contains the CL region of human κ light chain or human λ light chain;

[0021] 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 NO: 15, 21, 22 or 23 of CH1 and the amino acid positions corresponding to the amino acid positions of SEQ ID NO: 19 or 24 of CL.

[0022] The amino acid substitutions or natural amino acids in the CH1 and CL regions are selected from:

[0023] (1) K133C and C220X in CH1, and F209C and C214X in CL;

[0024] (2) S131C and C220X in CH1, and P119C and C214X in CL;

[0025] (3) K133C and C220X in CH1, and K207C and C214X in CL;

[0026] (4) F170C and C220X in CH1, and S176C and C214X in CL;

[0027] (5) P171C and C220X in CH1, and S162C and C214X in CL;

[0028] (6) V173C and C220X in CH1, and Q160C and C214X in CL;

[0029] (7) F170C and C131X in CH1, and S176C and C214X in CL;

[0030] (8) P171C and C131X in CH1, and S162C and C214X in CL;

[0031] (9) V173C and C131X in CH1, and Q160C and C214X in CL;

[0032] (10) A129C and C220X in CH1, and S121C and C214X in CL;

[0033] (11) K133C and C220X in CH1, and I117C and C214X in CL;

[0034] (12) C131 in CH1, and P119C and C214X in CL;

[0035] (13) A129C and C131X in CH1, and S121C and C214X in CL;

[0036] (14) R133C and C131X in CH1, and K207C and C214X in CL;

[0037] (15) R133C and C131X in CH1, and I117C and C214X in CL;

[0038] (16) R133C and C131X in CH1, and L117C and C214X in CL;

[0039] (17) K133C and C220X in CH1, and L117C and C214X in CL;

[0040] (18) R133C and C131X in CH1, and F209C and C214X in CL;

[0041] (19) R133C and C131X in CH1, and V209C and C214X in CL; or

[0042] (20) K133C and C220X in CH1, and V209C and C214X in CL;

[0043] Where X is selected from S, A, or G.

[0044] In another general aspect, the present invention relates to isolated bispecific antibodies or antigen-binding fragments thereof, comprising:

[0045] a. First heavy chain, H1;

[0046] b. Second chain, H2;

[0047] c. The first light chain, L1; and

[0048] d. Second light chain, L2;

[0049] Hl and Ll form the first arm, which contains a first antigen-binding domain that specifically binds to the first antigen, preferably a human-derived first antigen.

[0050] H2 and L2 form the second arm, which contains a second antigen-binding domain that specifically binds to the second antigen, preferably a human-derived second antigen.

[0051] (a) H1 contains the CH1 region of human IgG1, IgG2, IgG3 or IgG4 and the heavy chain variable region (VH region);

[0052] and

[0053] (b) L1 contains the CL region of human κ light chain or human λ light chain and the light chain variable region (VL region);

[0054] The CH1, VH, CL, and VL regions contain amino acid substitutions at the amino acid residues corresponding to the amino acid positions of CH1 (SEQ ID NO: 15, 21, 22, or 23); VH (SEQ ID NO: 13); CL (SEQ ID NO: 19 or 24); and VL (SEQ ID NO: 17).

[0055] The amino acid substitutions in the CH1, VH, CL, and VL regions are selected from:

[0056] (1) C220X in CH1, G44C in VH, C214X in CL, and G101C in VL; or

[0057] (2) C131X in CH1, G44C in VH, C214X in CL and G101C in VL;

[0058] Where X is selected from S, A, or G.

[0059] In some embodiments, the first antigen-binding domain is a CD47-binding domain. In some 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.

[0060] In some embodiments, the second arm containing H2 and L2 does not contain amino acid substitutions of the first arm containing H1 and L1. In some embodiments, each of the two heavy chains H1 and H2 contains a VH region, a CH1 region, and an Fc region (containing CH2 and CH3 regions), wherein the VH region has a different amino acid sequence. In some embodiments, each of the two heavy chains H1 and H2 contains a VH region, a CH1 region, and an Fc region (containing CH2 and CH3 regions), wherein the CH1 region has a different amino acid sequence. In some embodiments, each of the two heavy chains H1 and H2 contains a VH region, a CH1 region, and an Fc region (containing CH2 and CH3 regions), wherein the Fc region has a different amino acid sequence. In some embodiments, each of the two light chains L1 and L2 contains a VL region and a CL region, wherein the CL region has a different amino acid sequence.

[0061] In some implementations, H1 and H2 form a heterodimer.

[0062] In some embodiments, isolated humanized anti-CD47 / anti-FRα bispecific antibodies or their antigen-binding fragments can block the binding of signal regulatory protein α (SIRP) to CD47 on cancer cells expressing FRα and CD47.

[0063] In some implementations, isolated humanized anti-CD47 / anti-FRα bispecific antibodies or their antigen-binding fragments can induce macrophage-mediated phagocytosis of cancer cells expressing FRα and CD47.

[0064] In some implementations, isolated humanized anti-CD47 / anti-FRα bispecific antibodies or their antigen-binding fragments are able to bind to cancer cells expressing FRα and CD47 and minimize binding to human erythrocytes (RBCs) to undetectable levels.

[0065] Also provided are isolated nucleic acids encoding isolated bispecific antibodies or antigen-binding fragments thereof of the invention disclosed herein.

[0066] Also provided are vectors containing isolated nucleic acids encoding bispecific antibodies or antigen-binding fragments thereof of the invention disclosed herein.

[0067] A host cell comprising a vector containing isolated nucleic acid encoding a bispecific antibody or an antigen-binding fragment thereof disclosed herein is also provided.

[0068] In some embodiments, a pharmaceutical composition comprising the isolated bispecific antibody or its antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier is provided.

[0069] Methods for targeting FRα and CD47 expressed on the surface of cancer cells in subjects in need are also provided, including administering a pharmaceutical composition comprising the isolated anti-CD47 / anti-FRα bispecific antibody or antigen-binding fragment of the present invention to the subject.

[0070] A method for blocking the binding of SIRPα to CD47 on cancer cells expressing FRα and CD47 is also provided in subjects in need, comprising administering to the subject a pharmaceutical composition comprising the isolated anti-CD47 / anti-FRα bispecific antibody of the present invention or an antigen-binding fragment thereof.

[0071] Also provided is a method for inducing macrophage-mediated phagocytosis of cancer cells expressing FRα and CD47 in subjects in need, comprising administering to the subject a pharmaceutical composition comprising the isolated anti-CD47 / anti-FRα bispecific antibody of the present invention or an antigen-binding fragment thereof.

[0072] Also provided is a method for minimizing to undetectable binding to human erythrocytes (RBCs) by binding cancer cells expressing FRα and CD47 to antiCD47 bispecific antibodies or antigen-binding fragments in subjects in need, comprising administering to a subject a pharmaceutical composition comprising the isolated antiCD47 / antiFRα bispecific antibody or antigen-binding fragment thereof of the present invention.

[0073] Methods for treating cancer in subjects in need are also provided, including administering the pharmaceutical composition of the invention to the subject. The cancer can be any liquid or solid cancer, for example, selected from, but not limited to, 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's 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.

[0074] A method for generating the isolated bispecific antibody or antigen-binding fragment thereof of the present invention is also provided, comprising culturing cells containing nucleic acids encoding the antibody or antigen-binding fragment under conditions for generating the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cells or culture.

[0075] A method for preparing a pharmaceutical composition comprising the isolated bispecific antibody or antigen-binding fragment thereof of the present invention is also provided, comprising combining the antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

[0076] Brief description of the attached figures

[0077] The above overview 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 specific embodiments shown in the drawings.

[0078] Figure 1A and 1B A schematic structure of a bispecific antibody comprising a right arm targeting one antigen (e.g., CD47) and a left arm targeting a second antigen (e.g., FRα) is shown. The two arms have distinct heavy chain (VH) and light chain (VL) regions; for illustrative purposes, the heavy chain (HC) and light chain (LC) of the bispecific antibody are located within the frameworks of IgG1 and κ, respectively. A toggle switch (KiH) mutation is introduced in the CH3 region of both HC regions to promote heterodimer formation. Furthermore, cysteine ​​residues are introduced into each of the two CH3 regions, respectively, to promote interchain disulfide bond formation to stabilize the heterodimer. Figure 1A As shown, by converting the natural cysteine ​​that forms the disulfide bond into serine, the natural interchain disulfide bond between CH1 and CL is eliminated (marked by dashed lines); two natural non-cysteine ​​residues on CH1 and CL are converted into cysteine, respectively, forming a new interchain disulfide bond between CH1 and CL. Figure 1B A similar strategy was used, except that the newly formed interchain disulfide bond between HC and LC in the right arm is located between the VH and VL regions. H1 and L1 are the heavy and light chains of mAb 1 arm, respectively, and H2 and L2 are the heavy and light chains of mAb 2 arm, respectively.

[0079] Figures 2A-2F The sequences of various antibody components are shown. Figure 2A-2D The VH values ​​of mAb 1 (on the heavy and light chains of human IgG1) and mAb 2 (on the heavy and light chains of human IgG1) are shown respectively. Figure 2A CH1( Figure 2B VL () Figure 2C ) and CL( Figure 2DThe sequences (VH of mAb 1 (SEQ ID NO:13); VH of mAb 2 (SEQ ID NO:14); CH1 of mAb 1 (SEQ ID NO:15); CH1 of mAb 2 (SEQ ID NO:16); VL of mAb 1 (SEQ ID NO:17); VL of mAb 2 (SEQ ID NO:18); CL of mAb 1 (SEQ ID NO:19); CL of mAb 2 (SEQ ID NO:20)) are highlighted. The CDR regions identified by the Kabat method are 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 The CH1 region of human IgG2 (SEQ ID NO: 21), IgG3 (SEQ ID NO: 22), and IgG4 (SEQ ID NO: 23) is shown in the comparison. Figure 2F The CL region of the human λ light chain (SEQ ID NO: 24) is shown. * indicates a site of known allele variation.

[0080] Figure 3A-3L 3-D modeling of the Fab region (containing VH, CH1, VL, and CL) in the 1-arm of mAb is shown to identify potential cysteine ​​knock-in sites for forming novel interchain disulfide bonds between HC and LC. Dashed lines represent native interchain disulfide bonds between HC and LC (the native interchain disulfide bond region is not included in the models of bsAbs 9, 10, 11, and 12; therefore, there are no dashed lines in these four bsAb models); solid lines represent potential new interchain disulfide bonds formed by newly knocked-in cysteine ​​residues. Figure 3A 3D model of bsAb1; Figure 3B 3D model of bsAb 2; Figure 3C , 3-D model of bsAb 3; Figure 3D 3D model of bsAb 4; Figure 3E 3D model of bsAb 5; Figure 3F 3D model of bsAb 6; Figure 3G 3D model of bsAb 7; Figure 3H 3D model of bsAb 8; Figure 3I 3D model of bsAb 9; Figure 3J 3D model of bsAb 10; Figure 3K , 3-D model of bsAb 11; Figure 3L , 3-D model of bsAb 12.

[0081] Figures 4A-4H The RP-HPLC chromatograms of mutant mAbs containing inter-chain disulfide bonds with different shifts are shown under reducing or non-reducing conditions. Figure 4I-4J This demonstrates the binding of mutant mAbs containing different translocated interchain disulfide bonds to CD47 in ELISA assays.

[0082] Figure 5A-5G The SEC (size exclusion chromatography) spectra of mutant mAbs containing different shifted interchain disulfide bonds in thermal stability studies are shown, with samples incubated at different temperatures for 5 minutes. Chromatograms corresponding to each temperature are displayed. Tables in each figure show the %AUC (area under the curve) of the different peaks for each given temperature. Figure 5A It has a mutant mAb designed with M1; Figure 5B It has a mutant mAb with M2 design; Figure 5C , with mutant mAbs designed with Z1; Figure 5D It has a mutant mAb with Z2 design; Figure 5E , with mutant mAbs designed with bsAb 10; Figure 5F , with mutant mAbs designed with bsAb 11; Figure 5G Mutant mAbs with bsAb 12 design. HMW, high molecular weight substance; MMW, medium molecular weight substance (mutant mAb); LMW, low molecular weight substance.

[0083] Figures 6A-6H The SEC (size exclusion chromatography) chromatograms of mutant mAbs containing different translocated interchain disulfide bonds in the pH stability study are shown, with samples incubated at room temperature at pH 3.0 for different durations. Chromatograms corresponding to each incubation duration (0, 1, 3, 5, and 7 hours) are displayed; the 0-hour chromatogram represents the sample not incubated at pH 3.0. The tables in each chromatogram show the %AUC (area under the curve) of the different peaks for each given incubation period. Figure 6A It has a mutant mAb designed with M1; Figure 6B It has a mutant mAb with M2 design; Figure 6C , with mutant mAbs designed with Z1; Figure 6D It has a mutant mAb with Z2 design; Figure 6E , with mutant mAbs designed with bsAb 5; Figure 6F , with mutant mAbs designed with bsAb 10; Figure 6G , with mutant mAbs designed with bsAb 11; Figure 6H Mutant mAbs with bsAb 12 design. HMW, high molecular weight substance; MMW, medium molecular weight substance (mutant mAb); LMW, low molecular weight substance.

[0084] Figures 7A-7D The image shows an SDS-PAGE image of the bispecific antibody purified using protein A chromatography. Figure 7CSDS-PAGE is performed under non-reducing conditions; Figure 7D SDS-PAGE is performed under reducing conditions.

[0085] Figure 8A-8J The graph shown illustrates the binding results of the purified bispecific antibody against protein A with two antigens (i.e., CD47 and FRα) in a bridging ELISA assay.

[0086] Figure 9A-9N The bispecific antibody purified using protein A chromatography is shown. Figures 9A-9E Or, bispecific antibodies purified by hydrophobic interaction chromatography (HIC) after using protein A chromatography. Figure 9F-9N Size exclusion chromatography (SEC) chromatograms. Figure 9A SEC map of bsAb 1; Figure 9B SEC plot of bsAb 5; Figure 9C SEC map of bsAb6; Figure 9D SEC plot of bsAb7; Figure 9E SEC plot of bsAb 8; Figure 9F SEC plot of bsAb5b(E / K); Figure 9G SEC plot of bsAb 10(E / K); Figure 9H SEC plot of bsAb 12(E / K); Figure 9I SEC plot of bsAb 5b(K / E); Figure 9J SEC plot of bsAb 10(K / E); Figure 9K SEC plot of bsAb 12(K / E); Figure 9L SEC plot of bsAb 5b; Figure 9M SEC plot of bsAb 10; Figure 9N SEC map of bsAb 12.

[0087] Figure 10A-10L The RP-HPLC chromatogram of the HIC-purified bispecific antibody under non-reducing conditions is shown. Figure 10A RP-HPLC chromatogram of bsAb 6; Figure 10B RP-HPLC chromatogram of bsAb 7; Figure 10C RP-HPLC chromatogram of bsAb 8; Figure 10D RP-HPLC chromatogram of bsAb 5b(E / K); Figure 10E RP-HPLC chromatogram of bsAb 10(E / K); Figure 10F RP-HPLC chromatogram of bsAb 12 (E / K); Figure 10GRP-HPLC chromatogram of bsAb 5b(K / E); Figure 10H RP-HPLC chromatogram of bsAb 10(K / E); Figure 10I RP-HPLC chromatogram of bsAb 12(K / E); Figure 10J RP-HPLC chromatogram of bsAb 5b; Figure 10K RP-HPLC chromatogram of bsAb 10; Figure 10L RP-HPLC chromatogram of bsAb 12.

[0088] Figure 11A-11N The RP-HPLC chromatogram of the HIC-purified bispecific antibody under reducing conditions is shown. Figure 11A RP-HPLC chromatogram of H1, H2 and L1 of control antibody #1 and bsAb 7 and purified by protein A chromatography; Figure 11B RP-HPLC chromatogram of control antibody #2 assembled with bsAb 7 at H1, H2 and L2 and purified by protein A chromatography; Figure 11C RP-HPLC chromatogram of HIC-purified bsAb 6; Figure 11D RP-HPLC chromatogram of HIC-purified bsAb 7; Figure 11E RP-HPLC chromatogram of HIC-purified bsAb 8; Figure 11F RP-HPLC chromatogram of HIC-purified bsAb 5b (E / K); Figure 11G RP-HPLC chromatogram of HIC-purified bsAb 10 (E / K); Figure 11H RP-HPLC chromatogram of HIC-purified bsAb 12 (E / K); Figure 11I RP-HPLC chromatogram of HIC-purified bsAb5b(K / E); Figure 11J RP-HPLC chromatogram of HIC-purified bsAb 10(K / E); Figure 11K RP-HPLC chromatogram of HIC-purified bsAb 12(K / E); Figure 11L RP-HPLC chromatogram of HIC-purified bsAb 5b; Figure 11M RP-HPLC chromatogram of HIC-purified bsAb 10; Figure 11N RP-HPLC chromatogram of HIC-purified bsAb 12. H1 and L1 represent the HC and LC of mAb1 (anti-CD47) arm, respectively; H2 and L2 represent the mAb2 (anti-FRα) arm, respectively. Figure 11A-11E ) or mAb 2b (anti-FRα) arm ( Figure 11F-11NHC and LC. Predicted area % represents the AUC ratio calculated based on the amino acid sequence of each chain; area % represents the AUC ratio calculated using the AUC of all four peaks for each bispecific antibody on RP-HPLC under reducing conditions.

[0089] Figure 12A-12F The graph shown illustrates the results of HIC-purified bispecific antibodies binding to two antigens (CD47 and FRα) in a bridging ELISA assay. Figure 12A Bridging ELISA data for bsAb 6, bsAb 7, and bsAb 8; Figure 12B Bridging ELISA data for bsAb 5b(E / K), bsAb 10(E / K), and bsAb 12(E / K); Figure 12C Bridging ELISA data for bsAb 5b(K / E); Figure 12D Bridging ELISA data (K / E) for bsAb 10; Figure 12E Bridged ELISA data (K / E) for bsAb 12; Figure 12F Bridging ELISA data for bsAb 5b, bsAb 10, and bsAb 12.

[0090] Figures 13A-13C The binding of the HIC-purified bispecific antibody to SK-OV-3 cells known to express CD47 and FRα was demonstrated.

[0091] Figures 14A-14C Mass spectrometry (MS) spectra of papain-digested samples of HIC-purified bispecific antibodies are shown. Fab fragments generated from both arms (mAb 1 arm and mAb 2 arm) of each bispecific antibody were identified. Figure 14A MS spectrum of bsAb 6 digested by papain; Figure 14B MS spectrum of bsAb 7 digested by papain; Figure 14C MS spectrum of bsAb 8 digested with papain.

[0092] Figure 15A-15F MS spectra of trypsin-digested samples of HIC-purified bispecific antibodies are shown. Disulfide-linked peptide fragments generated from the first arm of the mAb of each bispecific antibody were identified. Cysteine ​​residues forming disulfide bonds are shown in bold. Figure 15A MS spectrum of disulfide-linked peptide fragments generated from the mAb 1 arm of bsAb 6 digested by trypsin; Figure 15B MS spectrum of disulfide-linked peptide fragments generated from the mAb 1 arm of bsAb 7 by trypsin digestion; Figure 15CMS spectrum of disulfide-linked peptide fragments generated from the mAb 1 arm of bsAb 8 by trypsin digestion; Figure 15D MS spectrum of disulfide-linked peptide fragments generated from the mAb 1 arm of bsAb 5b digested by trypsin; Figure 15E MS spectrum of disulfide-linked peptide fragments generated from the mAb 1 arm of bsAb 10 digested by trypsin; Figure 15F MS spectrum of disulfide-linked peptide fragments generated from the mAb 1 arm of bsAb 12 by trypsin digestion.

[0093] Figures 16A-16C MS spectra of IdeZ protease-digested samples of HIC-purified bispecific antibodies are shown. (Fab')2 generated from each bispecific antibody was identified. Figure 16A MS spectrum of bsAb 6(Fab')2; Figure 16B MS spectrum of bsAb 7(Fab')2; Figure 16C MS spectrum of bsAb 8(Fab')2.

[0094] Figures 17A-17C The study demonstrated the inhibition of antibody binding to SK-OV-3 cells by F(ab')2 generated from anti-CD47 or anti-FRα parental mAb in FACS assays. Figure 17A ,bsAb 5b; Figure 17B ,bsAb 10; Figure 17C bsAb12. Anti-FRα parental mAb, the anti-FRα arm derived from this mAb was used to construct bispecific antibodies bsAb 5b, bsAb10, and bsAb 12; anti-CD47 parental mAb, the anti-CD47 arm derived from this mAb was used to construct bispecific antibodies bsAb 5b, bsAb 10, and bsAb 12; Ab, antibody. The antibody concentrations used in the assay are shown below each figure; 5,000 nM F(ab')2 was used to assess inhibitory activity.

[0095] Figures 18A-18B The sequence of binding of two antigens (CD47 and FRα) to the bispecific antibody bsAb 12 on Biacore is shown.

[0096] Figures 19A-19C MS spectrum of Fab fragment from a papain-digested sample derived from a bispecific antibody purified from protein A. Figure 19A Wild-type bispecific antibody purified from protein A; Figure 19B bsAb 10, purified protein A; Figure 19CbsAb 12, purified from protein A. Wild-type bispecific antibody, produced by co-expression of mAb1 arm and mAb 2b arm without the introduction of mutations (natural interchain disulfide bonds on both arms); H1 and L1 are the heavy and light chains of mAb 1 arm, respectively, and H2 and L2 are the heavy and light chains of mAb 2b arm, respectively.

[0097] H1 / L2 Fab and H2 / L1 Fab represent Fab fragments of heavy / light chain mismatched substances. ND, not detected.

[0098] Figure 20 MS spectrum of the Fab fragment from papain-digested bsAb 12, purified first with protein A and then by HIC purification. No mismatch species were detected. Invention Details

[0100] Various publications, articles, and patents are cited or described in the background and throughout this specification; each of these references is incorporated herein by reference in its entirety. Discussions of documents, actions, materials, devices, articles, etc., already included in this specification are for the purpose of providing context for the invention. Such discussion does not imply that any or all of these matters constitute part of the prior art relating to any disclosed or claimed invention.

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings specified in the specification.

[0102] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise.

[0103] Unless otherwise stated, any numerical value, such as concentrations or concentration ranges described herein, should be understood to be modified by the term "about" in all cases. Therefore, numerical values ​​typically include ±10% of the listed values. 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). As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within that range, including integers and fractions of values ​​within such ranges, unless the context clearly indicates otherwise.

[0104] Unless otherwise stated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalents of the particular embodiments of the invention described herein using only conventional experiments. Such equivalents are intended to be covered by the invention.

[0105] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or any other variation thereof, shall be understood to imply inclusion of the specified integers or groups of integers, but not to exclude any other integers or groups of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated to the contrary, “or” means inclusive or, not exclusive or. For example, condition A or B satisfies either: 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).

[0106] As used herein, the connecting term "and / or" between multiple listed elements is understood to include both individual and combined options. For example, in the case where 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 of these options should be understood to fall within this meaning, thus satisfying the requirement of the term "and / or" as used herein. The simultaneous applicability of multiple options is also understood to fall within this meaning, thus satisfying the requirement of the term "and / or".

[0107] As used herein, the term “consistsof” or variations such as “consist of” or “consisting of” as used throughout the specification and claims means that any of the enumerated integers or groups of integers are included, but no additional integers or groups of integers may be added to the specified method, structure or combination.

[0108] As used herein, the term "consists essentially of" or variations thereof, as used throughout the specification and claims, means to include any enumerated integers or groups of integers, and optionally any enumerated integers or groups of integers that do not substantially alter the fundamental or novel properties of the specified method, structure, or composition. See MPEP §2111.03.

[0109] As used herein, “subject” means any animal, preferably a mammal, and most preferably a human. As used herein, the term “mammal” includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., with humans being more preferred.

[0110] The words “right,” “left,” “down,” and “up” indicate directions in the attached diagram.

[0111] It should also be understood that the terms “about,” “approximately,” “generally,” “substantially,” and similar terms used herein, when referring to the size or feature of a component of a preferred invention, indicate that the described size / feature, as understood by one of ordinary skill in the art, is not a strict boundary or parameter, and does not exclude minor variations that are functionally identical or similar. At a minimum, such references containing numerical parameters will include variations that do not change the least significant figure using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0112] As used herein, the terms “different heavy chains” or “different light chains” as used throughout the specification and claims indicate that the heavy chains or light chains have sequences that are not identical to each other.

[0113] In the case 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-encoding polypeptides and polynucleotides, and CD47-encoding polypeptides and polynucleotides), the term "identical" or "identity" percentage refers to the specified percentage of two or more sequences or subsequences that are identical or have the same amino acid residues or nucleotides when compared or contrasted using one of the following sequence comparison algorithms or by visual inspection to obtain the maximum correspondence.

[0114] For sequence comparisons, typically one sequence serves as a reference sequence, which is then compared to the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, with subsequence coordinates specified if necessary, along with the sequence algorithm program parameters. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the specified program parameters.

[0115] The best sequence alignment for comparison can be performed, 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. Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by implementing these algorithms by computer (GAP, BESTFIT, FASTA, and TFASTA in the genetic software package of the Genetics Computer Group of the 575 Science Madison, PhD, Wisconsin), or by visual inspection (see, generally, Current Protocols in Molecular Biology, edited by FMAusubel et al., Greene Publishing Associates, Inc. and John Wiley & Sons). A joint venture of Wiley & Sons, Inc. (added in 1995) (Ausubel).

[0116] Examples of algorithms suitable for determining sequence identity percentages and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., (1990) J.Mol.Biol.215:403-410 and Altschul et al., (1997) Nucleic Acids Res.25:3389-3402, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. The algorithm first identifies high-scoring sequence pairs (HSPs) by recognizing short sequences of length W in the query sequence. These sequences match or satisfy a positive threshold score T when aligned with sequences of the same length in the database. T is called the neighbor word score threshold (Altschul et al., ibid.). These initial neighbor word hits act as seeds to initiate a search for longer HSPs containing them. Sequence hits are then extended in both directions along each sequence until the cumulative alignment score no longer increases.

[0117] For nucleotide sequences, a cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Sequence hits will stop expanding in each direction if: the cumulative alignment score decreases by X from its maximum reached value; the cumulative score becomes zero or lower due to the accumulation of one or more negative score residues; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expected value (E) of 10, M = 5, N = -4, and a comparison of two strands. For amino acid sequences, the BLASTP program defaults to a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0118] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One similarity metric provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in a comparison of the test nucleic acid with a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

[0119] A further indication that two nucleic acid sequences or polypeptides are substantially identical is the immune cross-reaction between the polypeptide encoded by the first nucleic acid and the polypeptide encoded by the second nucleic acid, as described below. Therefore, one polypeptide is usually substantially identical to the second polypeptide, for example, where the two peptides differ only by conserved substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0120] As used herein, the term "polynucleotide" is synonymous with "nucleic acid molecule," "nucleotide," or "nucleic acid," and refers to any polynucleotide or polydeoxynucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA as a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded or more typically double-stranded or a mixture 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 with a backbone modified for stability or other reasons. "Modified" bases include, for example, triphenylmethylated bases and uncommon bases such as inosine. DNA and RNA can be modified in a variety of ways; therefore, "polynucleotides" include polynucleotides in chemically, enzymatically, or metabolically modified forms commonly found in nature, as well as chemical forms of DNA and RNA specific to viruses and cells. "Polynucleotides" also include relatively short nucleic acid chains, often called oligonucleotides.

[0121] As used in this article, the term "vector" is a replicon in which another nucleic acid segment can be operatively inserted to induce replication or expression of that segment.

[0122] As used herein, the term "host cell" refers to a cell containing the nucleic acid molecules of the present invention. A "host cell" can be any type of cell, such as a primary cell, a cultured cell, or a cell derived from a cell line. In one embodiment, a "host cell" is a cell transfected with the nucleic acid molecules of the present invention. In another embodiment, a "host cell" is a descendant or potential descendant of such a transfected cell. The descendant of a cell may be the same as or may differ from the parent cell, for example, due to mutations or environmental influences that may occur in subsequent generations, or due to the integration of nucleic acid molecules into the host cell genome.

[0123] 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 encompasses the translation of RNA into one or more polypeptides, and further covers all naturally occurring post-transcriptional and post-translational modifications. Expressed bispecific antibodies can be present in the cytoplasm of host cells, in extracellular environments such as growth media of cell cultures, or anchored to the cell membrane.

[0124] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule composed of amino acids and can be recognized by those skilled in the art as a protein. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to amino acid polymers of any length. Polymers can be linear or branched, can contain modified amino acids, and can be broken down by non-amino acid components. The term also includes native or modified amino acid polymers; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as binding to a labeled component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids), and other modifications known in the art.

[0125] The peptide sequences described herein are written according to common 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, they represent the L-form of the amino acid unless otherwise explicitly stated.

[0126] Bispecific antibodies

[0127] This invention generally relates to isolated bispecific antibodies in which the natural cysteine ​​residues that form interchain disulfide bonds between the heavy chain CH1 region and the light chain CL region of one arm are eliminated by converting natural cysteine ​​residues to non-cysteine ​​residues (e.g., natural cysteine ​​residues to serine residues); simultaneously or subsequently, two natural non-cysteine ​​residues in the CH1 and CL regions or VH and VL regions of the same arm are converted to cysteine, thereby forming new interchain disulfide bonds between the heavy and light chains. The two natural non-cysteine ​​residues, one each in the CH1 and CL regions or one each in the VH and VL regions, are structurally modeled to determine their proximity and potential for forming interchain disulfide bonds once converted to cysteine. Amino acid substitutions cause minimal interference to the overall structure of the antigen-binding domain. The overall effect of this invention is that the natural interchain disulfide bonds between the CH1 and CL regions in one arm of the bispecific antibody are transferred to different sites with the cysteine ​​introduced by recombination, while the natural interchain disulfide bonds in the second arm remain unchanged.

[0128] This invention also generally relates to isolated bispecific antibodies, one arm having a transposed interchain disulfide bond as described above, and the other arm having a native interchain disulfide bond that produces a heterodimer, wherein one arm contains the transposed interchain disulfide bond and the second arm contains the native interchain disulfide bond. Bispecific antibodies are capable of binding two antigens. Bispecific antibodies formed from two different heavy chains (HC) and light chains (LC) are difficult to produce because the two heavy chains and two light chains tend to mispair, resulting in unwanted products that are difficult to eliminate during production; even if unwanted products from mismatches can be eliminated during purification, mismatches reduce the production efficiency of the desired bispecific antibody product. Different strategies have been attempted to reduce or eliminate HC and LC mismatches, but many of these strategies involve protein engineering, including domain exchange and mutagenesis, thus leading to increased risks of aggregation and immunogenicity. Heterodimeric bispecific antibodies can be generated with increased efficiency by introducing a "displaced interchain disulfide bond" in one arm while retaining the native interchain disulfide bond in the second arm, while simultaneously introducing a knot in the hole and a cysteine ​​mutation that forms a disulfide bond in the Fc region. Such bispecific antibodies can be generated by co-expressing two heavy chains and two light chains, including but not limited to anti-CD47 / anti-FRα bispecific antibodies.

[0129] Antibody

[0130] This invention generally relates to isolated bispecific antibodies having a displaced interchain disulfide bond on one arm and retaining a natural interchain disulfide bond on the second arm. Specifically, this invention generally relates to anti-CD47 / anti-FRα bispecific antibodies, nucleic acids encoding the antibody and expression vectors, recombinant cells containing the vectors, and compositions comprising the antibody. Methods for preparing the antibodies and methods for using the antibodies to treat diseases, including cancer, are also provided. The antibodies of this invention possess one or more desired functional properties, including but not limited to high affinity binding to FRα and CD47, high specificity for FRα and CD47, the ability to induce effector-mediated tumor cell lysis, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cell-mediated cytotoxicity (ADCC) against cells expressing FRα and / or CD47, the ability to mediate drug-conjugated recruitment, and the ability to inhibit tumor growth in subjects and animal models when administered alone or in combination with other anticancer therapies.

[0131] As used herein, the term "antibody" is used broadly and includes immunoglobulin or antibody molecules, including human, humanized, complex, and chimeric antibodies, as well as monoclonal or polyclonal 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 classified into five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) based on 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 can be any of the five major classes or corresponding subclasses. Preferably, the antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. The light chains of antibodies in vertebrate species can be classified into one of two distinct types, namely κ and λ, based on the amino acid sequence of their constant domains. Therefore, the antibodies of the present invention can contain either the κ or λ light chain constant domain. According to a particular embodiment, the antibodies of the present invention comprise heavy chain and / or light chain constant regions derived from rat or human antibodies. In addition to the constant heavy and light chain domains, the antibody also contains an antigen-binding region consisting of a light chain variable region and a heavy chain variable region, each containing three domains (i.e., complementarity-determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains can be alternatively referred to as LCDR1, LCDR2, and LCDR3, while the heavy chain variable region domains can be alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0132] Several systems exist for numbering amino acid residues in antibodies. The Kabat numbering method is based on a scheme for antibody variable regions (Elvin A. Kabat et al., Immunology-Associated Protein Sequences, 5th Edition (1991)). The EU numbering system is widely used for constant domains (including CH1, hinge, and Fc regions) (Elvin A. Kabat et al., Immunology-Associated Protein Sequences, 5th Edition (1991)).

[0133] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to FRα is substantially free of antibodies that do not bind to FRα, an isolated antibody that specifically binds to CD47 is substantially free of antibodies that do not bind to CD47, and a bispecific antibody that specifically binds to both CD47 and FRα is substantially free of antibodies that do not bind to both CD47 and FRα). Furthermore, isolated antibodies are substantially free of other cellular material and / or chemicals.

[0134] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical, except for the possibility of naturally occurring mutations present in small amounts. The monoclonal antibodies of this invention can be prepared using hybridoma methods, phage display technology, single-lymphocyte gene cloning technology, or recombinant DNA methods. For example, monoclonal antibodies can be produced from hybridomas comprising B cells obtained from transgenic nonhuman animals such as transgenic mice or rats, having a genome containing both human heavy-chain and light-chain transgenes.

[0135] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as a biantibody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibody (ds diabody), single-chain antibody molecule (scFv), single-domain antibody (sdab), scFv dimer (bivalent biantibody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelified single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. The antigen-binding fragment is capable of binding to the same antigen that the parent antibody or the parent antibody fragment binds to. According to a specific embodiment, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd fragment of the heavy chain. According to other specific embodiments, the antigen-binding fragment comprises Fab and F(ab').

[0136] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art that comprises 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 comprises a heavy chain variable region and a heavy chain constant region or comprises only a heavy chain variable region.

[0137] As used herein, the term "human antibody" refers to an antibody produced by a human being or an antibody having an amino acid sequence corresponding to that of a human-produced antibody, manufactured using any technology known in the art. The definition of a human antibody includes full-length or complete antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide.

[0138] As used in this article, the term "humanized antibody" refers to a non-human antibody that has been modified to increase its sequence homology with human antibodies, thereby retaining the antigen-binding properties of the antibody but reducing its antigenicity in the human body.

[0139] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable regions of the light and heavy chains typically correspond to the variable regions of antibodies derived from one mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and ability, while the constant regions correspond to antibody sequences derived from another mammal (e.g., human) to avoid triggering an immune response in that species.

[0140] As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has binding specificity for a first epitope, and a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, such as the same protein (or a subunit of a multimeric 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 do not overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of a multimeric 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 trispecific antibody molecule, or a tetraspecific antibody molecule.

[0141] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than 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, such as the same protein (or a subunit of a multimeric 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, such as different proteins (or different subunits of a multimeric 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 the first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises a hapten or a fragment thereof having binding specificity to the first epitope and a hapten or a fragment thereof having binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises an scFv or a fragment thereof having binding specificity to the first epitope, and an scFv or a fragment thereof having binding specificity to the second epitope.

[0142] As used herein, the term “FRα” refers to folate receptor α, also known as folate receptor 1 (FOLR1) or folate-binding protein (FBP), a glycosyl-phosphatidylinositol (GPI)-anchored membrane protein on the cell surface that has a high affinity for and transports the active form of folate, 5-methyltetrahydrofolate (5-MTF), and its derivatives into the cell (Salazar and Ratnam, Cancer Metastasis Rev 2007; 26: 141-52). FRα has become an oncology target 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: 193-198 and Boogerd et al., Oncotarget 2016; 7: 17442-17454), but its expression level is low in a limited number of normal human tissues (Weitman et al., Cancer Res 1992; 52: 3396-3401). Consistent with this observation, Phase 1 clinical trials targeting both small and large molecules of FRα have to date demonstrated good tolerability (Cheung et al., Oncotarget 2016; 7:52553–52574). Therefore, FRα is a tumor-associated / tumor-specific antigen, and anti-FRα monoclonal antibodies (mAbs) and bispecific antibodies may be potential anticancer therapies. Furthermore, FRα could be used to specifically target cancer cells with therapeutic molecules. An exemplary amino acid sequence of human FRα is represented by GenBank accession number NP_057937.1.

[0143] As used herein, the term "CD47" refers to a multi-transmembrane receptor belonging to the immunoglobulin superfamily, which has been shown to be involved in a variety of cellular processes, including cell migration, adhesion, and T cell function. CD47, also known as integrin-associated protein (IAP), ovarian cancer antigen (OA3), Rh-associated antigen, and MER6, was initially identified as a tumor antigen for human ovarian cancer and has subsequently been shown to be expressed in multiple human tumor types, including hematologic malignancies and solid tumors. The interaction between CD47 and signal regulatory protein α (SIRP), an inhibitory protein expressed on macrophages, prevents phagocytosis by CD47-expressing cells. CD47 is also expressed at low levels in almost all non-malignant cells. The term "human CD47" refers to CD47 derived from humans. An exemplary amino acid sequence of human CD47 is represented by GenBank accession number NP_001768.1.

[0144] As used herein, an antibody that "specifically binds to CD47 and / or FRα" refers to an antibody that binds to CD47 and / or FRα, preferably human CD47 and / or human FRα, with a KD of 1×10⁻⁶. -7 M or smaller, preferably 1×10-8 M or smaller, preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller, or 1×10 -10 M or less. 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). In view of this disclosure, the KD value of an antibody can be determined using methods in the art. For example, the KD of an antibody can be determined by using surface plasmon resonance, for example by using a biosensor system, such as... The system, or by using biological layer interferometry techniques such as the Octet RED96 system, can be used to determine this.

[0145] The smaller the KD value of an antibody, the higher its affinity for binding to the target antigen.

[0146] According to another specific aspect, the present invention relates to isolated bispecific antibodies or antigen-binding fragments thereof, comprising:

[0147] a. First heavy chain, H1;

[0148] b. Second chain, H2;

[0149] c. The first light chain, L1; and

[0150] d. Second light chain, L2;

[0151] Hl and Ll form the first arm, which contains a first antigen-binding domain that specifically binds to the first antigen, preferably a human-derived first antigen.

[0152] H2 and L2 form the second arm, which contains a second antigen-binding domain that specifically binds to the second antigen, preferably a human-derived second antigen.

[0153] (a) H1 contains the CH1 region of human IgG1, IgG2, IgG3, or IgG4; and

[0154] (b) L1 contains the CL region of human κ light chain or human λ light chain;

[0155] 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 NO: 15, 21, 22 or 23 of CH1 and the amino acid positions corresponding to the amino acid positions of SEQ ID NO: 19 or 24 of CL.

[0156] The amino acid substitutions or natural amino acids in the CH1 and CL regions are selected from:

[0157] (1) K133C and C220X in CH1, and F209C and C214X in CL;

[0158] (2) S131C and C220X in CH1, and P119C and C214X in CL;

[0159] (3) K133C and C220X in CH1, and K207C and C214X in CL;

[0160] (4) F170C and C220X in CH1, and S176C and C214X in CL;

[0161] (5) P171C and C220X in CH1, and S162C and C214X in CL;

[0162] (6) V173C and C220X in CH1, and Q160C and C214X in CL;

[0163] (7) F170C and C131X in CH1, and S176C and C214X in CL;

[0164] (8) P171C and C131X in CH1, and S162C and C214X in CL;

[0165] (9) V173C and C131X in CH1, and Q160C and C214X in CL;

[0166] (10) A129C and C220X in CH1, and S121C and C214X in CL;

[0167] (11) K133C and C220X in CH1, and I117C and C214X in CL;

[0168] (12) C131 in CH1, and P119C and C214X in CL;

[0169] (13) A129C and C131X in CH1, and S121C and C214X in CL;

[0170] (14) R133C and C131X in CH1, and K207C and C214X in CL;

[0171] (15) R133C and C131X in CH1, and I117C and C214X in CL;

[0172] (16) R133C and C131X in CH1, and L117C and C214X in CL;

[0173] (17) K133C and C220X in CH1, and L117C and C214X in CL;

[0174] (18) R133C and C131X in CH1, and F209C and C214X in CL;

[0175] (19) R133C and C131X in CH1, and V209C and C214X in CL; or

[0176] (20) K133C and C220X in CH1, and V209C and C214X in CL;

[0177] Where X is selected from S, A, or G.

[0178] According to another specific aspect, the present invention relates to isolated bispecific antibodies or antigen-binding fragments thereof, comprising:

[0179] a. First heavy chain, H1;

[0180] b. Second chain, H2;

[0181] c. The first light chain, L1; and

[0182] d. Second light chain, L2;

[0183] Hl and Ll form the first arm, which contains a first antigen-binding domain that specifically binds to the first antigen, preferably a human-derived first antigen.

[0184] H2 and L2 form the second arm, which contains a second antigen-binding domain that specifically binds to the second antigen, preferably a human-derived second antigen.

[0185] (a) H1 contains the CH1 region of human IgG1, IgG2, IgG3 or IgG4 and the heavy chain variable region (VH region);

[0186] and

[0187] (b) L1 contains the CL region of human κ light chain or human λ light chain and the light chain variable region (VL region);

[0188] 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 CH1 (SEQ ID NO: 15, 21, 22 or 23), VH (SEQ ID NO: 13), CL (SEQ ID NO: 19 or 24) and VL (SEQ ID NO: 17).

[0189] The amino acid substitutions in the CH1, VH, CL, and VL regions are selected from:

[0190] (1) C220X in CH1, G44C in VH, C214X in CL, and G101C in VL; or

[0191] (2) C131X in CH1, G44C in VH, C214X in CL and G101C in VL;

[0192] Where X is selected from S, A, or G.

[0193] According to another specific aspect, the first antigen-binding domain is a CD47-binding domain. In some 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.

[0194] According to another specific aspect, (a) the second arm containing H2 and L2 does not contain amino acid substitutions of the first arm containing H1 and L1; (b) each of the two heavy chains H1 and H2 contains a VH region, a CH1 region, and an Fc region (containing CH2 and CH3 regions), wherein the VH region has a different amino acid sequence; (c) each of the two heavy chains H1 and H2 contains a VH region, a CH1 region, and an Fc region (containing CH2 and CH3 regions), wherein the CH1 region has a different amino acid sequence; (d) each of the two heavy chains H1 and H2 contains a VH region, a CH1 region, and an Fc region (containing CH2 and CH3 regions), wherein the Fc region has a different amino acid sequence; (e) each of the two light chains L1 and L2 contains a VL region and a CL region, wherein 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, wherein the CL region has a different amino acid sequence.

[0195] In another specific aspect, H1 and H2 form a heterodimer.

[0196] In another specific aspect, (a) the VH region of H1 and the VL region of L1 have Q39E and Q38K substitution mutations, respectively, and the VH region of H2 and the VL region of L2 have Q39K and Q38E substitution mutations, respectively; or the VH region of H1 and the VL region of L1 have Q39K and Q38E substitution mutations, respectively, and the VH region of H2 and the VL region of L2 have Q39E and Q38K substitution mutations, respectively.

[0197] In another specific aspect, the isolated bispecific antibody or its antigen-binding fragment is an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment. In some embodiments, the anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment can block the binding of signal regulatory protein α (SIRPα) to CD47 on cancer cells expressing FRα and CD47. In some embodiments, the isolated humanized anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment can induce macrophage-mediated phagocytosis of cancer cells expressing FRα and CD47. In some embodiments, the isolated humanized anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment can bind to cancer cells expressing FRα and CD47 while minimizing binding to human erythrocytes (RBCs) to undetectable levels.

[0198] In another specific aspect, the first antigen-binding domain has the VH sequence of SEQ ID:13 and the VL sequence of SEQ ID:17, and the second antigen-binding domain has the VH sequence of SEQ ID:33 and the VL sequence of SEQ ID:35; or the first antigen-binding domain has the VH sequence of SEQ ID:13 and the VL sequence of SEQ ID:17, and the second antigen-binding domain has the VH sequence of SEQ ID:14 and the VL sequence of SEQ ID:18.

[0199] The full-length bispecific antibody of this invention can be generated, for example, by using Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies, through the introduction of substitutions at the heavy chain CH3 interface in each half-molecule to facilitate the formation of heterodimers of two antibody half-molecules with distinct specificity in vitro in a cell-free environment, or by using co-expression. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and the dissociation binding of the CH3 domain. The heavy chain disulfide bond in the hinge region of the parent monospecific antibody is reduced. The free cysteine ​​of one of the resulting parent monospecific antibodies forms an inter-heavy chain disulfide bond with the cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and recombined through dissociation binding. The CH3 domain of the Fab arm can be designed to favor heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each molecule binding to a different epitope, namely an epitope on CD47 and an epitope on FRα.

[0200] As used herein, “homodimerization” refers to the interaction between 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.

[0201] As used in this article, "heterodimerization" refers to the interaction between two heavy chains with different CH3 amino acid sequences. As used in this article, "heterodimer" refers to an antibody with two heavy chains and different CH3 amino acid sequences.

[0202] The “knob-in-hole” strategy (see, for example, PCT Publication WO2006 / 028936) can be used to generate full-length bispecific antibodies. In short, selected amino acids that form the CH3 domain interface in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. Amino acids with small side chains (pores) are introduced into the heavy chain of an antibody that specifically binds to the first antigen, while 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 both antibodies, heterodimers form due to the preferential interaction between the heavy chain with the “pore” and the heavy chain with the “knob.” Exemplary CH3 substitution pairs forming the knob and hole are (denoted as the modification position in the first CH3 domain of the first heavy chain / the 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.

[0203] Other strategies can be used, such as using electrostatic interactions to promote heavy chain heterodimerization by substituting positively charged residues on one CH3 surface and negatively charged residues on a 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 facilitated by the following substitutions (denoted as the modification position in the first CH3 domain of the first heavy chain / the 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_F405AY407V / T350V_T366L_K392L_T394W, as described in U.S. Patent Publication No. 2012 / 0149876 or U.S. Patent Publication No. 2013 / 0195849.

[0204] In addition to the methods described above, the bispecific antibody of the present invention can be generated in vitro in a cell-free environment by introducing asymmetric mutations into the CH3 region of two monospecific homodimer antibodies and forming a bispecific heterodimer antibody from the two parental monospecific homodimer antibodies under reducing conditions, thereby producing disulfide isomerization as described in PCT Patent Publication No. WO2011 / 131746. In these methods, the first and second monospecific divalent antibodies are modified to have certain substitutions in the CH3 domain, thereby promoting heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to cause disulfide isomerization of the cysteine ​​in the hinge region; thereby generating the bispecific antibody through Fab arm exchange. The incubation conditions can optionally be restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol. Preferably, the reducing agent is selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation for at least 90 minutes at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or at a pH of 5-8 (e.g., at a pH of 7.0 or 7.4) in the presence of at least 0.5 mM dithiothreitol.

[0205] In another general aspect, the present invention relates to isolated nucleic acids encoding the bispecific antibodies or antigen-binding fragments thereof of the present invention. Those skilled in the art will understand that the coding sequence of a protein can be altered without changing the amino acid sequence of the protein (e.g., substitution, deletion, insertion, etc.). Therefore, those skilled in the art will understand that the nucleic acid sequence encoding the antibodies or antigen-binding fragments thereof of the present invention can be altered without changing the amino acid sequence of the protein.

[0206] In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding a bispecific antibody or an antigen-binding fragment thereof of the present invention. Any vector known to those skilled in the art according to this disclosure may be used, such as plasmids, granules, phage vectors, or viral vectors. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any elements that establish the conventional function of the expression vector, such as a promoter, ribosome-binding element, terminator, enhancer, selection marker, and origin of replication. The promoter may be a constitutive, inducible, or repressive promoter. Many expression vectors capable of delivering nucleic acids to cells are known in the art and may be used herein to generate antibodies or antigen-binding fragments thereof in cells. Recombinant expression vectors according to embodiments of the present invention may be generated using conventional cloning techniques or artificial gene synthesis. These techniques are well known to those skilled in the art in light of this disclosure.

[0207] In another general aspect, the present invention relates to host cells comprising a vector containing isolated nucleic acid encoding a bispecific antibody or an antigen-binding fragment thereof of the present invention. 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 antigen-binding fragment thereof of the present invention. In some embodiments, the host cell is *E. coli* TG1 or BL21 cells (for expressing, for example, scFv or Fab antibodies), CHO-DG44 or CHO-K1 cells, or HEK293 cells (for expressing, for example, full-length IgG antibodies). According to specific embodiments, the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, wherein it is stably integrated into the host cell genome, thereby enabling efficient expression of the recombinant nucleic acid.

[0208] In another general aspect, the present invention relates to a method for producing the bispecific antibody or antigen-binding fragment thereof of the present invention, comprising culturing cells containing nucleic acids encoding the bispecific antibody or antigen-binding fragment thereof under conditions for producing the bispecific antibody or antigen-binding fragment thereof, and recovering the bispecific antibody or antigen-binding fragment thereof from the cells or cell culture (e.g., from the supernatant). The expressed antibody or antigen-binding fragment thereof can be harvested from the cells and purified according to conventional techniques known in the art as described herein.

[0209] Pharmaceutical Composition

[0210] In another general aspect, the present invention relates to a pharmaceutical composition comprising an isolated bispecific antibody or an antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a product comprising the antibody of the present invention and a pharmaceutically acceptable carrier. The antibodies of the present invention and compositions comprising them can also be used to prepare medicaments for the therapeutic applications mentioned herein.

[0211] As used herein, the term "carrier" means any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicles, microspheres, liposomes, or other materials well known in the art for use in pharmaceutical formulations. It should be understood that the properties of the carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceuticalally acceptable carrier" means a non-toxic material that does not interfere with the effectiveness or bioactivity of the compositions according to the invention. According to specific embodiments, in view of this disclosure, any pharmaceutically acceptable carrier suitable for antibody pharmaceutical compositions may be used in this invention.

[0212] Formulations of pharmaceutically active ingredients with pharmaceutically acceptable carriers are known in the art, for example, Remington: The Science and Practice of Pharmaceuticals (e.g., 21st edition (2005) and any subsequent editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tension modifiers, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers may be used to formulate the pharmaceutical compositions of the present invention.

[0213] In one embodiment of the 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. Liquid formulations may include solutions, suspensions, emulsions, microemulsions, gels, etc. Aqueous formulations typically contain at least 50% w / w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w / w water.

[0214] In one embodiment, the pharmaceutical composition may be formulated as an injectable agent, for example, that can be injected via an injection device (e.g., a syringe or infusion pump). For example, the injection may be delivered subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously.

[0215] In another embodiment, the pharmaceutical composition is a solid dosage form, such as a freeze-dried or spray-dried composition, which can be used as is, or a solvent and / or diluent can be added to it prior to use by a physician or patient. Solid dosage forms can include tablets, such as compressed tablets and / or coated tablets, and capsules (e.g., hard or soft gelatin capsules). For example, the pharmaceutical composition can also be in the form of sachets, sugar-coated pills, powders, granules, tablets, or powders for reconstitution.

[0216] Dosage forms can be immediately released, in which case they may contain a water-soluble or dispersible carrier, or they can be delayed-release, sustained-release, or modified-release, in which case they may contain a water-insoluble polymer with a modulated dissolution rate in a dosage form under the gastrointestinal tract or skin.

[0217] In other embodiments, the pharmaceutical composition may be delivered intranasally, intrabuccally, or sublingually.

[0218] The pH of the aqueous formulation can be between pH 3 and pH 10. In one embodiment of the invention, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is from about 3.0 to about 7.0.

[0219] In another embodiment of the invention, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, dicaprylic acid, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycyl glycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tris(hydroxymethyl)methylglycine, and tris(hydroxymethyl)aminomethane, and mixtures thereof. The buffer may be present alone or in aggregate form at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific buffers constitute alternative embodiments of the invention.

[0220] In another embodiment of the invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include: benzyl chloride, benzoic acid, benzyl alcohol, bromonitol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorophenyl ether, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidureus, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thimerosal, and mixtures thereof. The preservative may be present alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific preservatives constitute alternative embodiments of the invention.

[0221] In another embodiment of the 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), sugar alcohols (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 includes sugars. Non-limiting examples of sugars can be monosaccharides, disaccharides, or polysaccharides, or water-soluble dextran, including, for example, fructose, glucose, mannose, sorbitol, xylose, maltose, lactose, sucrose, trehalose, dextran, amylopectin, dextrin, cyclodextrin, α- and β-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethyl cellulose. Another example of an isotonic agent is a sugar alcohol, wherein 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, xylitol, and araitol. Pharmaceutical compositions comprising each of the isotonic agents listed in this paragraph constitute alternative embodiments of the invention. The isotonic agents may be present alone or in aggregates at concentrations 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 isotonic agents constitute alternative embodiments of the invention.

[0222] In another embodiment of the invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agent may be present alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific chelating agents constitute alternative embodiments of the invention.

[0223] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0224] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, wherein the stabilizer is carboxy / hydroxycellulose and its derivatives (e.g., HPC, HPC-SL, HPC-L, and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (e.g., PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (e.g., sodium chloride), sulfur-containing substances (e.g., monothioglycerol), or mercaptoacetic acid. The stabilizer may be present alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific stabilizers constitute alternative embodiments of the invention.

[0225] In a further embodiment of the 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 consisting of a water-soluble (hydrophilic) portion and a lipophilic (lipophilic) portion. Surfactants can be, for example, selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants. Surfactants can be present alone or in aggregates 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 invention.

[0226] In a further embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as EDTA and / or benzoamide hydrochloride (HCl). The protease inhibitors may be present alone or in aggregates at concentrations from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific protease inhibitors constitute alternative embodiments of the invention.

[0227] In another general aspect, the present invention relates to a method for preparing a pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof of the present invention, comprising combining the bispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

[0228] How to use

[0229] In another general aspect, the present invention relates to a method for targeting FRα and CD47, both of which are expressed on the surface of cancer cells in a subject of need, the method comprising administering to the subject a bispecific antibody against CD47 / anti-FRα of the present invention, or an antigen-binding fragment thereof, or a pharmaceutical composition thereof. The binding of the bispecific antibody or its antigen-binding fragment to FRα and / or CD47 can mediate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cytotoxicity (ADCC), or other effects leading to the death of the target cancer cells. For example, the bispecific antibody or its antigen-binding fragment may be used to recruit conjugate drugs to mediate the death of targeted cancer cells.

[0230] In another general aspect, the present invention relates to a method for blocking the binding of SIRPα to CD47 on cancer cells expressing FRα and CD47 in subjects of need, the method comprising administering to the subjects of need an anti-CD47 / anti-FRα bispecific antibody of the present invention or an antigen-binding fragment thereof or a pharmaceutical composition thereof.

[0231] In another general aspect, the present invention relates to a method for inducing macrophage-mediated phagocytosis of cancer cells expressing FRα and CD47 in subjects of need, the method comprising administering to the subjects of need an anti-CD47 / anti-FRα bispecific antibody of the present invention or an antigen-binding fragment thereof or a pharmaceutical composition thereof.

[0232] In another general aspect, the present invention relates to a method for binding cancer cells expressing FRα and CD47 to a subject in need of a humanized anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment thereof, and minimizing their binding to human erythrocytes (RBCs) to undetectable levels. The method comprises administering to the subject in need of the isolated humanized anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment thereof, or a pharmaceutical composition thereof, of the present invention. The humanized anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment of the present invention exhibits high selectivity for cancer cells and minimal to undetectable binding to human erythrocytes (RBCs).

[0233] The functional activity of bispecific antibodies binding to FRα and CD47 and their antigen-binding fragments can be characterized by methods known in the art and as described herein. Methods for characterizing bispecific antibodies binding to FRα and CD47 and their antigen-binding fragments include, but are not limited to, affinity and specificity assays, including Biacore, ELISA, FACS, and OctetRed assays. According to specific embodiments, methods for characterizing bispecific antibodies binding to FRα and CD47 and their antigen-binding fragments include those described below.

[0234] In another general aspect, the present invention relates to a method of treating cancer in a subject in need, comprising administering to the subject in need an isolated humanized anti-CD47 / anti-FRα bispecific antibody or an antigen-binding fragment thereof or a pharmaceutical composition thereof of the present invention. The cancer can be any liquid or solid cancer, for example, selected from, but not limited to, 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's 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.

[0235] According to embodiments of the present invention, the pharmaceutical composition comprises a therapeutically effective amount of the anti-CD47 / anti-FRα bispecific antibody of the present invention or an antigen-binding fragment thereof. As used herein, the term "therapeutically effective amount" refers to the amount of active ingredient or component that elicits a desired biological or medical response in a subject. Therapeuticly effective amounts can be determined empirically and conventionally according to the stated purpose.

[0236] As used herein, a therapeutically effective dose of an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment refers to the amount of an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment that modulates an immune response in a subject in need. Similarly, as used herein, a therapeutically effective dose of an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment refers to the amount of an anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment that treats, prevents or slows the progression of a disease, disorder, or condition; or reduces or completely alleviates symptoms associated with a disease, disorder, or condition.

[0237] According to a specific embodiment, the disease, disorder, or condition to be treated is cancer, preferably 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's 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 specific embodiments, the disease, disorder, or condition to be treated is an inflammatory disease, a metabolic disease, or any other disease that can be treated with bispecific antibodies.

[0238] According to the specific implementation, a therapeutically effective dose refers to a therapeutic dose 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 to be treated or related symptoms; (ii) shortening the duration of the disease, disorder, or condition to be treated or related symptoms; (iii) preventing the progression of the disease, disorder, or condition to be treated or related symptoms; (iv) causing the remission of the disease, disorder, or condition to be treated or related symptoms; (v) preventing the development or onset of the disease, disorder, or condition to be treated or related symptoms; (v (i) to prevent the recurrence of a disease, disorder, or condition to be treated or related symptoms; (vii) to reduce hospitalization of subjects with a disease, disorder, or condition to be treated or related symptoms; (viii) to shorten the length of hospitalization of subjects with a disease, disorder, or condition to be treated or related symptoms; (ix) to increase the survival rate of subjects with a disease, disorder, or condition to be treated or related symptoms; (xi) to suppress or reduce a disease, disorder, or condition to be treated or related symptoms in subjects; and / or (xii) to enhance or improve the preventive or therapeutic effect of another therapy.

[0239] The effective therapeutic dose or dosage can vary depending on various factors, such as the disease, disorder, or condition to be treated, the route of administration, the target site, the subject's physiological state (including, for example, age, weight, and health), whether the subject is human or animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Optimizing the titration of the therapeutic dose can optimize safety and efficacy.

[0240] Depending on the specific implementation, the compositions described herein are formulated to suit the intended route of administration to a subject. For example, the compositions described herein may be formulated to suit intravenous, subcutaneous, or intramuscular administration.

[0241] As used herein, the terms “treatment,” “management,” and “treatment” all mean improving or reversing at least one measurable physical parameter associated with cancer, which may not be identifiable in the subject but can be identified in the subject. The terms “treatment,” “management,” and “treatment” can also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In one specific embodiment, “treatment,” “management,” and “treatment” refer to reducing or preventing the development or onset of one or more symptoms associated with a disease, disorder, or condition (e.g., a tumor or, more preferably, cancer), or reducing its duration. In one specific embodiment, “treatment,” “management,” and “treatment” refer to preventing the recurrence of a disease, disorder, or condition. In one specific embodiment, “treatment,” “management,” and “treatment” refer to increasing the survival rate of a subject suffering from a disease, disorder, or condition. In one specific embodiment, “treatment,” “management,” and “treatment” refer to eliminating a disease, disorder, or condition in a subject.

[0242] According to a specific embodiment, a composition for treating cancer is provided. For cancer treatment, the composition can be used in combination with another treatment, 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 agents, radiotherapy, antibody-drug conjugates (ADCs), targeted therapy, or other anticancer drugs.

[0243] As used herein, in the context of administering two or more therapies to a subject, the term “combination” means the use of more than one therapy. The use of the term “combination” does not limit the order in which the therapies are administered to the subject. For example, a first therapy (e.g., the composition described herein) may be administered to the subject before (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 to, simultaneously with, or after (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 to), during, or after (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 to), during, or after, the administration of a second therapy.

[0244] Implementation

[0245] The present invention also provides the following non-limiting embodiments.

[0246] Implementation method 1 is an isolated bispecific antibody or its antigen-binding fragment, comprising:

[0247] a. First heavy chain, H1;

[0248] b. Second chain, H2;

[0249] c. The first light chain, L1; and

[0250] d. Second light chain, L2;

[0251] Hl and Ll form the first arm, which contains a first antigen-binding domain that specifically binds to the first antigen, preferably a human-derived first antigen.

[0252] H2 and L2 form the second arm, which contains a second antigen-binding domain that specifically binds to the second antigen, preferably a human-derived second antigen.

[0253] (a) H1 contains the CH1 region of human IgG1, IgG2, IgG3, or IgG4; and

[0254] (b) L1 contains the CL region of human κ light chain or human λ light chain;

[0255] 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 NO: 15, 21, 22 or 23 of CH1 and the amino acid positions corresponding to the amino acid positions of SEQ ID NO: 19 or 24 of CL.

[0256] The amino acid substitutions or natural amino acids in the CH1 and CL regions are selected from:

[0257] (1) K133C and C220X in CH1, and F209C and C214X in CL;

[0258] (2) S131C and C220X in CH1, and P119C and C214X in CL;

[0259] (3) K133C and C220X in CH1, and K207C and C214X in CL;

[0260] (4) F170C and C220X in CH1, and S176C and C214X in CL;

[0261] (5) P171C and C220X in CH1, and S162C and C214X in CL;

[0262] (6) V173C and C220X in CH1, and Q160C and C214X in CL;

[0263] (7) F170C and C131X in CH1, and S176C and C214X in CL;

[0264] (8) P171C and C131X in CH1, and S162C and C214X in CL;

[0265] (9) V173C and C131X in CH1, and Q160C and C214X in CL;

[0266] (10) A129C and C220X in CH1, and S121C and C214X in CL;

[0267] (11) K133C and C220X in CH1, and I117C and C214X in CL;

[0268] (12) C131 in CH1, and P119C and C214X in CL;

[0269] (13) A129C and C131X in CH1, and S121C and C214X in CL;

[0270] (14) R133C and C131X in CH1, and K207C and C214X in CL;

[0271] (15) R133C and C131X in CH1, and I117C and C214X in CL;

[0272] (16) R133C and C131X in CH1, and L117C and C214X in CL;

[0273] (17) K133C and C220X in CH1, and L117C and C214X in CL;

[0274] (18) R133C and C131X in CH1, and F209C and C214X in CL;

[0275] (19) R133C and C131X in CH1, and V209C and C214X in CL; or

[0276] (20) K133C and C220X in CH1, V209C and C214X in CL;

[0277] Where X is selected from S, A, or G.

[0278] Implementation method 2 is an isolated bispecific antibody or its antigen-binding fragment, comprising:

[0279] a. First heavy chain, H1;

[0280] b. Second chain, H2;

[0281] c. The first light chain, L1; and

[0282] d. Second light chain, L2;

[0283] Hl and Ll form the first arm, which contains a first antigen-binding domain that specifically binds to the first antigen, preferably a human-derived first antigen.

[0284] H2 and L2 form the second arm, which contains a second antigen-binding domain that specifically binds to the second antigen, preferably a human-derived second antigen.

[0285] (a) H1 contains the CH1 region and heavy chain variable region (VH region) of human IgG1, IgG2, IgG3, or IgG4; and

[0286] (b) L1 contains the CL region of human κ light chain or human λ light chain and the light chain variable region (VL region);

[0287] 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 CH1 (SEQ ID NO: 15, 21, 22 or 23), VH (SEQ ID NO: 13), CL (SEQ ID NO: 19 or 24) and VL (SEQ ID NO: 17).

[0288] The amino acid substitutions in the CH1, VH, CL, and VL regions are selected from:

[0289] (1) C220X in CH1, G44C in VH, C214X in CL, and G101C in VL; or

[0290] (2) C131X in CH1, G44C in VH, C214X in CL and G101C in VL;

[0291] Where X is selected from S, A, or G.

[0292] Implementation method 3 is the isolated bispecific antibody or its antigen-binding fragment from implementation method 1 or 2, wherein the first antigen-binding domain is the CD47 binding domain.

[0293] Implementation 4 is the isolated bispecific antibody or its antigen-binding fragment from Implementation 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.

[0294] Implementation method 5 is the isolated bispecific antibody or its antigen-binding fragment from any one of implementation methods 1 to 4, wherein...

[0295] (a) The second arm containing H2 and L2 does not contain amino acid substitutions in the first arm containing H1 and L1;

[0296] (b) The two heavy chains H1 and H2 each contain a VH region, a CH1 region and an Fc region (containing CH2 and CH3 regions), wherein the VH region has a different amino acid sequence;

[0297] (c) The two heavy chains H1 and H2 each contain a VH region, a CH1 region and an Fc region (containing CH2 and CH3 regions), wherein the CH1 region has a different amino acid sequence;

[0298] (d) The two heavy chains H1 and H2 each contain a VH region, a CH1 region and an Fc region (containing CH2 and CH3 regions), wherein the Fc region has a different amino acid sequence;

[0299] (e) Each of the two light chains, L1 and L2, contains a VL region and a CL region, wherein the VL regions have different amino acid sequences; and / or

[0300] (f) The two light chains L1 and L2 each contain a VL region and a CL region, wherein the CL region has a different amino acid sequence.

[0301] Implementation 6 is the isolated bispecific antibody or its antigen-binding fragment of Implementation 5, wherein H1 and H2 form a heterodimer.

[0302] Embodiment 7 is the isolated bispecific antibody or its antigen-binding fragment as described in any one of claims 1 to 6, wherein...

[0303] (a) The VH region of H1 and the VL region of L1 have Q39E and Q38K substitution mutations, respectively, and the VH region of H2 and the VL region of L2 have Q39K and Q38E substitution mutations, respectively; or

[0304] (b) The VH region of H1 and the VL region of L1 have Q39K and Q38E substitution mutations, respectively, and the VH region of H2 and the VL region of L2 have Q39E and Q38K substitution mutations, respectively.

[0305] Embodiment 8 is an isolated bispecific antibody or its antigen-binding fragment from 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 its antigen-binding fragment, wherein the first antigen-binding domain specifically binds to CD47, preferably human CD47, and the second antigen-binding domain specifically binds to folic acid receptor α (FRα), preferably human FRα.

[0306] Embodiment 9 is the isolated bispecific antibody or its antigen-binding fragment as described in any one of claims 1 to 8, wherein...

[0307] (a) The first antigen-binding domain has the VH sequence of SEQ ID:13 and the VL sequence of SEQ ID:17, and the second antigen-binding domain has the VH sequence of SEQ ID:33 and the VL sequence of SEQ ID:35; or

[0308] (b) The first antigen-binding domain has the VH sequence of SEQ ID:13 and the VL sequence of SEQ ID:17, and the second antigen-binding domain has the VH sequence of SEQ ID:14 and the VL sequence of SEQ ID:18.

[0309] Implementation 10 is the isolated bispecific antibody or its antigen-binding fragment of Implementation 8 or 9, wherein the anti-CD47 / anti-FRα bispecific antibody or its antigen-binding fragment is capable of blocking the binding of signal regulatory protein α (SIRPα) to CD47 on cancer cells that simultaneously express FRα and CD47, inducing macrophage-mediated phagocytosis of cancer cells expressing FRα and CD47, and / or binding to cancer cells expressing FRα and CD47 while minimizing binding to human erythrocytes (RBCs) to undetectable levels.

[0310] Implementation 11 is an isolated nucleic acid encoding a bispecific antibody or antigen-binding fragment of any one of Implementation 1 to 10.

[0311] Implementation method 12 is a vector containing the isolated nucleic acid of implementation method 11.

[0312] Implementation 13 is a host cell containing the vector of Implementation 12.

[0313] Embodiment 14 is a pharmaceutical composition comprising the isolated bispecific antibody or its antigen-binding fragment as described in any one of Embodiments 1 to 10 and a pharmaceutically acceptable carrier.

[0314] Implementation method 15 is a method of targeting FRα and CD47 expressed on the surface of cancer cells in a subject of need, blocking the binding of SIRPα to CD47 on cancer cells expressing FRα and CD47 in the subject of need, inducing macrophage-mediated phagocytosis of cancer cells expressing FRα and CD47 in the subject of need, minimizing binding to human erythrocytes (RBCs) to undetectable levels while binding to cancer cells expressing FRα and CD47 in the subject of need, and / or treating cancer in the subject of need, comprising administering a pharmaceutical composition to the subject, said pharmaceutical composition comprising an isolated anti-inflammatory agent of any one of implementation methods 8 to 10. CD47 / anti-FRα bispecific antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier, optionally, 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.

[0315] Embodiment 16 is a method for generating a bispecific antibody or an antigen-binding fragment thereof from any one of Embodiments 1 to 10, comprising culturing a cell containing a nucleic acid encoding a bispecific antibody or an antigen-binding fragment thereof under conditions for generating a bispecific antibody, and recovering the bispecific antibody or an antigen-binding fragment thereof from the cell or culture.

[0316] Embodiment 17 is a method for preparing a pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof from any one of Embodiments 1 to 10, comprising combining the bispecific antibody or the antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition. Example

[0317] Example 1: Construction of a bispecific antibody with altered cysteine ​​sites

[0318] Figure 1A and 1B This demonstrates a bispecific antibody with two distinct heavy chains and two distinct light chains in the heterodimer of H1H2, which can be facilitated by common methods such as knob-in-hole and charged pairs. The two native cysteine ​​residues forming an interchain disulfide bond in the H1L1 arm can be converted to non-cysteine ​​residues, thereby eliminating the native interchain disulfide bond. Figure 1A and 1B(dashed line in the text); simultaneously, two closely spaced natural non-cysteine ​​residues in the 3-D structure of H1L1 can be converted into cysteine, forming new interchain disulfide bonds ( Figure 1A and 1B (The solid line in the middle). The newly formed interchain disulfide bond can 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 in H1L1, two light chains can preferentially pair with their corresponding heavy chains. Furthermore, the asymmetric structure formed by the interchain disulfide bond shifts on the mAb 1-arm allows for better differentiation of the physical properties and potential impurities of bispecific antibodies, facilitating the purification and production of the target bispecific antibody. Multiple pairs of native non-cysteine ​​residues (also known as "knock-in sites") in H1L1 that can form new interchain disulfide bonds have been identified. The following examples show bispecific antibodies in the human IgG1 heavy chain VH and CH1 and the κ light chain VL and CL sequences. This concept can also be applied to constructing bispecific antibodies using CH1 of the human IgG2, IgG3, or IgG4 heavy chain and CL of the human λ light chain, provided conserved knock-in sites exist. Whenever the knock-in site happens to be a native cysteine ​​(not a cysteine ​​involved in forming a native interchain disulfide bond), it can be directly used to form a new interchain disulfide bond.

[0319] A humanized anti-CD47 mAb (described in International Patent Publication No. WO2019 / 217145; referred to herein as mAb 1) and two humanized anti-FRα mAbs (described in International Patent Publication No. WO2019 / 177854; referred to herein as mAb 2 and mAb2b, respectively) are used to construct anti-CD47 / anti-FRα bispecific antibodies. Figure 1A and 1BThe structure of the desired bispecific antibody is shown, with mAb 1 (anti-CD47) as the right arm and mAb 2 (anti-FRα) as the left arm (in some bispecific antibodies, mAb2b is used instead of mAb 2 as the left arm). The VH and VL regions of the bispecific antibody are fused to the constant regions of the human IgG1 heavy chain (HC) and κ light chain (LC), respectively. mAb 1HC has a T366W (EU number) mutation to form a “button”, and mAb 2HC has T366S, L368A, and Y407V mutations to form a “pore”. Therefore, the two heavy chains favor the formation of a bispecific antibody with heterodimeric HC (mAb 1HC / mAb2HC) rather than homodimeric HC (mAb 1HC / mAb 1HC or mAb 2HC / mAb 2HC). Furthermore, an S354C cysteine ​​mutation was introduced on mAb 1HC and a Y349C cysteine ​​mutation was introduced on mAb 2HC to stabilize heterodimer pairing of the heterodimer heavy chains (Merchant et al., Nat. Biotechnol. 16(7):677-81(1998)). When two HCs and two LCs were co-transfected into cells, a strategy was employed to transfer the interchain disulfide bonds between the HCs and LCs on the mAb 1 arm to facilitate the expression, purification, and / or production of the desired bispecific antibody. To achieve this, the two native cysteine ​​residues forming interchain disulfide bonds on the HCs and LCs of the mAb 1 arm were converted to serine residues, while the two native non-cysteine ​​residues on the HCs and LCs of mAb 1 were structurally modeled to determine their close proximity, so that once cysteines were introduced into these sites (referred to in this paper as "knock-in cysteine" or "replacement cysteine"), the cysteine ​​residues could form new interchain disulfide bonds. Newly formed interchain disulfide bonds through cysteine ​​substitution pairing can exist between the CH1 and CL regions, as shown in Figure 1, or between the VH and VL regions, as shown in Figure 1. Figure 1B As shown, the native interchain disulfide bonds between the CH1 and CL regions of mAb 1 are marked with dashed lines, while the newly formed interchain disulfide bonds on mAb 1 through knock-in cysteine ​​residues are marked with solid lines. No mutations were introduced into the VH, VL, CH1, or CL regions of mAb 2, therefore mAb 2 has the same native interchain disulfide bonds.

[0320] The VH, CH1, VL, and CL sequences of mAb 1 and mAb 2, and the CH1, CH1, CH1, CH1, and λCL sequences of IgG2, IgG3, IgG4, and λCL are listed in Figures 2A-2FThe sequences are shown in Table 1 (SEQ ID NOs: 13, 15, 17, and 19 for mAb 1; SEQ ID NOs: 14, 16, 18, and 20 for mAb 2; and SEQ ID NOs: 21, 22, 23, and 24 for IgG2 CH1, IgG3 CH1, IgG4 CH1, and λCL). The VH, CH1, VL, and CL sequences of mAb 2b are listed in Table 1 (SEQ ID NOs: 33, 34, 35, and 36). Anti-CD47 antibody mAb 1 was selected as the arm with modified interchain disulfide bonds. (Schrodinger Bio) (Schrodinger; New York City, NY) generated homology models with many previously solved antibody structures in the public domain, and used cysteine ​​mutation tools to... The β-carbon cutoff distance determined the possible interchain disulfide bonds. Further individual and pairwise analyses were performed on the identified pairings using mutagenesis tools to identify non-cysteine ​​to cysteine ​​mutations, which resulted in minimal interference with the overall structure and reduced interchain binding affinity for individual non-cysteine ​​to cysteine ​​mutations without significantly affecting the binding affinity of paired cysteine ​​mutations. The best candidates were selected for expression and experimental validation. Potential disulfide bonds formed by knock-in cysteine ​​sites are marked with solid lines, while native interchain disulfide bonds are marked with dashed lines, as shown below. Figure 3A-3L As shown. The models of bsAbs 9, 10, 11, and 12 do not include the native interchain disulfide bond region; therefore, there are no dashed lines in the models of these four bsAbs. Each mAb 1 arm with different knock-in cysteine ​​pairs is paired with a native mAb 2 arm to form a bispecific antibody (bsAb). Table 2 lists the amino acid substitutions on the mAb 1 arm of the mAb 1 / mAb 2 bispecific antibodies used for transposition of the interchain disulfide bond.

[0321] To evaluate the impact of the various translocated interchain disulfide pairs listed in Table 2 on mAb stability, some mutant groups (or designs) from Table 2 were introduced into both arms of mAb 1. M1, M2, Z1, and Z2, representing mutant designs from patent literature (US9,527,927 B2 and US10,344,099 B2), were also introduced into both arms of mAb 1 for comparison. Mutant mAbs were expressed and purified using protein A chromatography and hydrophobic interaction chromatography (HIC). Protein A chromatography was performed using standard methods. For HIC, the protein A purified sample buffer was exchanged for PBS, and (NH4F150)SO4 was added to a final concentration of 800 mM. The samples were loaded onto a Source 15 PHE (GE) column pre-equilibrated with 50 mM MES pH 6.0 and 1 M (NH4F150)SO4 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), with the major fraction eluted in 60% buffer B. The eluted fractions were analyzed by SDS-PAGE, and fractions containing a predominantly 145 kDa band and free of lower molecular weight impurities were combined to form purified protein. Figures 4A-4H The RP-HPLC chromatograms of purified mutant mAbs containing inter-chain disulfide bonds with different shifts are shown under reducing or non-reducing conditions. Figure 4I-4J This demonstrates the binding of mutant mAbs containing different translocated interchain disulfide bonds to CD47 in ELISA assays.

[0322] The thermal stability of the purified mutant mAb was analyzed. The mutant mAb was exchanged for DPBS (Corning, catalog number: 21-031-CM) with 1 mg / mL buffer and incubated for 5 minutes at 55°C, 60°C, 65°C, 70°C, or 75°C on a Thermo Fisher Scientific thermal cycler (Simpliamp). After incubation, the mAb was analyzed by size exclusion chromatography (SEC). Each peak was quantified to determine the percentage of antibody (medium molecular weight substance). The results are shown in... Figure 5A-5G Mutated mAbs containing the bsAb 10 and bsAb 12 mutations, respectively, exhibited better thermostability than any other mutant mAbs, including those containing the M1, M2, Z1, and Z2 mutations, respectively. These surprising findings suggest that the bsAb 10 and bsAb 12 designs are superior to any M1, M2, Z1, or Z2 designs. Thermostability is a crucial property required during antibody manufacturing and storage. Good thermostability significantly enhances antibody development potential and increases its chances of becoming a therapeutic agent.

[0323] The pH stability of the mutant mAb or its recovery from low pH incubation was also tested. Zeba was used. TMThe Spin desalting column (Thermo Fisher Scientific, catalog number: 89882) replaces 10 mg / mL mAb buffer with a buffer containing 0.1 M citrate and 0.1 M sodium chloride (pH 3.0). The mAb is diluted to 2 mg / mL in the same buffer and incubated at room temperature for 1 h, 3 h, 5 h, or 7 h, then neutralized by adding 1.0 M Tris-HCl (pH 9.0) to adjust the final pH of the sample to 6.0–7.0. After neutralization, the mAb is diluted in DPBS to 1 mg / mL for SEC analysis. Results are shown in... Figures 6A-6H The mutant mAb designed with bsAb 12 exhibited higher pH stability than any mutant mAb containing M1, M2, Z1, or Z2 mutations; it also outperformed other tested mutant mAbs in terms of pH stability. Figures 6A-6H Mutant mAbs containing the bsAb 10 design showed superior recovery from pH stability studies compared to any mutant mAb containing M1, M2, Z1, or Z2 mutations. Data indicated that the bsAb 12 and bsAb 10 designs outperformed any M1, M2, Z1, or Z2 designs in terms of pH stability or recovery from low pH incubation. Furthermore, mutant mAbs containing the bsAb 5 design exhibited superior pH stability compared to any mutant mAb containing M1, M2, Z1, or Z2 designs when incubated at pH 3.0 for 1 or 3 hours. These surprising findings suggest that the bsAb 5, 10, and 12 designs are superior to any M1, M2, Z1, or Z2 designs. Antibody pH stability, or recovery from low pH incubation, is an important property required in the manufacturing process. Good pH stability or recovery from low pH incubation, especially under low pH conditions for 3 hours, significantly improves antibody development potential and increases its chances of becoming a therapeutic.

[0324] Table 1: Sequences of mAb 1, mAb 2, human IgG2, IgG3 and IgG4 heavy chains (HC), and various regions in human λCL.

[0325]

[0326]

[0327]

[0328] Table 2: Amino acid substitutions on the mAb 1 arm of mAb 1 / mAb 2 bispecific antibody (bsAb) for transposition of interchain disulfide bonds.

[0329]

[0330]

[0331] Note: Kabat numbers are used for VH and VL zones; EU numbers are used for CH1 and CL zones.

[0332] The bispecific antibodies in the examples are located on the IgG1 HC and κLC framework (Kabat numbers for the VH and VL regions; EU numbers for the CH1 and CL regions). Several bispecific antibodies were constructed using mAb 1 and mAb 2, including bsAb 1, bsAb 2, bsAb 3, bsAb 4, bsAb 5, bsAb 6, bsAb 7, and bsAb 8 (bsAb 1 refers to a bispecific antibody with a mAb 1 arm containing the corresponding mutation in Table 2 (expected to result in repositioning (or "shifting") of the disulfide bond between the HC / LC chains), and mAb 2 as the second arm; other bispecific antibodies in this group follow the same nomenclature). No mutations were introduced in the VH, CH1, VL, or CL regions of the mAb 2 arm. The mutation G44C refers to the conversion of native glycine at residue 44 (G44) to cysteine; all other mutations follow the same nomenclature. Other bispecific antibodies were constructed using mAb 1 and mAb 2b, including bsAb 5b, bsAb 10, and bsAb 12 (bsAb 5b refers to a bispecific antibody where the mAb 1 arm contains the corresponding bsAb 5 mutation from Table 2, and mAb 2b serves as the second arm; bsAb 10 refers to a bispecific antibody where the mAb 1 arm contains the corresponding bsAb 10 mutation from Table 2, and mAb 2b serves as the second arm; bsAb 12 refers to a bispecific antibody where the mAb 1 arm contains the corresponding bsAb 12 mutation from Table 2, and mAb 2b serves as the second arm). The sequences of the CH1, CL, VH, and VL regions of the mAb 1 arm of the bispecific antibodies are listed in Table 3. Furthermore, charged amino acid pairs were introduced at Q39 (Kabat number) on HC and Q38 (Kabat number) on LC for each arm of the given bispecific antibody. These bispecific antibodies are named as follows: bsAb 5b(E / K) refers to a bispecific antibody containing Q39E (Q replaced with E; the same naming rules apply to the other mutations listed below) on the HC of the mAb 1 arm of bsAb 5bd and Q38K on the LC, and Q39K on the HC of the mAb 2b arm of bsAb 5b and Q38E on the LC; bsAb 10(E / K) and bsAb 12(E / K) follow the same naming rules. Furthermore, bsAb 5b(K / E) refers to a bispecific antibody containing Q39K on the HC of the mAb 1 arm of bsAb 5b and Q38E on the LC, and Q39E on the HC of the mAb 2b arm of bsAb 5b and Q38K on the LC; bsAb 10(K / E) and bsAb 12(K / E) follow the same naming rules. mAb 1HC has the T366W (EU number) mutation to form a “knob”, while mAb 2 or mAb 2b HC has the mutations T366S, L368A and Y407V to form a “pore”.In addition, an S354C cysteine ​​mutation was introduced on mAb1HC and a Y349C cysteine ​​mutation was introduced on mAb2 or mAb2bHC to stabilize heterodimer pairing.

[0333] Table 3: Sequences of CH1, CL, VH, and VL regions of arm 1 of the bispecific antibody mAb

[0334]

[0335]

[0336]

[0337] Note: Residues resulting from amino acid substitutions are shown in bold and underline.

[0338] Example 2: Characterization of bispecific antibodies

[0339] Simultaneous expression of two heavy chains and two light chains in the same cell leads to the expression and assembly of the desired bispecific antibody, which comprises an anti-CD47 arm and an anti-FRα arm. The bispecific antibody was purified using protein A chromatography. Some samples were further purified using hydrophobic interaction chromatography (HIC).

[0340] The bispecific antibody purified from protein A was analyzed by SDS-PAGE. Protein samples were diluted to a concentration of 1 mg / mL. For the non-reducing lanes, 3 μL of protein was added to 4.5 μL of water and 2.5 μL of 4x LDS sample buffer (Thermo NP0007; Waltham, MA) and loaded directly onto the gel. For the reducing lanes, 3 μL of protein was added to 3.5 μL of water, 2.5 μL of 4x LDS sample buffer, and 1 μL of 1M DTT, and heated at 95 °C for 3 min before loading onto the gel. Bolt 4-12% bis-tris gels (Thermo NP0323BOX) were used for all samples. Samples were electrophoresed at 180 V for 30 min and developed with Coomassie G-250. Figures 7A-7BSDS-PAGE images of mAb 1 / mAb 2 bispecific antibody samples purified using protein A chromatography are shown. Under reducing and 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 and denaturing conditions, all samples showed multiple bands, with the top band of each lane representing either intact antibody or a mixture of intact antibodies (approximately 150 kDa), although it is unclear whether this is a mixture of heterodimeric bispecific antibody (facilitated by H1H2 interaction) and homodimeric antibody. Smaller bands represent various incomplete antibodies on SDS-PAGE—for example, the second band at the top of each lane (approximately 125 kDa) may represent an antibody missing a light chain; this could originate from an intact antibody where one light chain has not formed an interchain disulfide bond, thus separating from the host antibody on SDS-PAGE. Figures 7C-7D SDS-PAGE images of mAb 1 / mAb 2b bispecific antibody samples purified using protein A chromatography are shown. Under non-reducing and denaturing conditions, all samples showed a main band corresponding to the molecular weight of the bispecific antibody. Figure 7C Under reducing and denaturing conditions, all samples showed two bands corresponding to two different heavy chain molecular weights and one band corresponding to two different light chain molecular weights. Figure 7D ).

[0341] To evaluate the binding activity of bispecific samples in a bridging ELISA assay, recombinant folate receptor 1 (Novoprotein C784; Summit, NJ) was diluted to 0.25 μg / mL in PBS and used to coat 96-well plates (Genesee Scientific 91-415F; San Diego, CA). 50 μL of antigen was added to each well and coated overnight at 4°C. The plates were blocked with TBST containing 5% BSA and washed with TBST. 50 μL of antibody was prepared in TBST containing 5% BSA and added at the specified concentration, incubated at room temperature, and washed with TBST. A second recombinant biotinylated CD47 antigen (AcroBio CD47-H82E9-258g; Newark, Delaware) was diluted to 0.05 μg / mL in TBST containing 5% BSA and added to each well, incubated at room temperature, and washed with TBST. Streptavidin HRP (JIR 016-030-084) was diluted to a concentration of 0.2 or 0.5 μg / mL in TBST solution containing 5% BSA and added to each well. The plates were incubated at room temperature and washed. The plates were then developed with TMB substrate (Thermo 34028) and quenched with stop solution (Thermo SS04). Quantification of the wells was then performed by absorbance at 450 nm. Figure 8A-8JThe results show the binding of purified bispecific antibodies to two antigens in a bridging ELISA assay. Figure 8A The data indicate that bsAb 1, 5, 6, 7 and 8 have anti-CD47 / FRα bispecific activity, confirming that each of these constructs forms the expected anti-CD47 / FRα bispecific antibody. Figure 8B-8J All bispecific antibodies tested in the study exhibited bridging ELISA activity.

[0342] To determine the purity of samples purified by protein A chromatography or by HIC following protein A chromatography, size exclusion chromatography (SEC) was used. Protein samples were diluted to a concentration of 1 mg / mL and filtered through submicron filters. 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 at a flow rate of 0.35 mL / min. Bio-Rad gel filtration standards were used as standards (Bio-Rad 1511901; Hercules, CA). Samples were quantified by measuring absorbance at 280 nm. Figures 9A-9E The SEC curve of the sample purified by protein A chromatography is shown. The appearance of the main peak at approximately 150 kDa indicates that the predominant species in the sample is a whole antibody (or antibody) with two heavy chains and two light chains. These data are consistent with... Figures 7A-7B The SDS-PAGE observations shown are consistent. Figure 9F-9N The SEC curves of the samples purified by protein A chromatography and HIC are shown.

[0343] Under non-reducing conditions, the purity of the HIC-purified bispecific antibody samples was further analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC). Figure 10A-10L The RP-HPLC curves of the bispecific antibodies are shown. One dominant species was observed in each case. RP-HPLC was also performed under reducing conditions to determine the presence of the two HCs (H1 and H2) and two LCs (L1 and L2) of the expected bispecific antibody. Control antibody samples were generated by transiently expressing H1, H2, and L1 of bsAb 7 (control antibody #1) and H1, H2, and L2 of bsAb 7 (control antibody #2), respectively, followed by purification using protein A chromatography. These samples were used as controls to identify the LC peaks on RP-HPLC. Figure 11A and 11B Combined with bsAb 6 ( Figure 11C ),bsAb 7( Figure 11D ) and bsAb 8( Figure 11E The RP-HPLC curves identified HC peaks (H1 and H2) and LC peaks (L1 and L2). Figure 11A-11E ). Figure 11F-11N The RP-HPLC curves of the bispecific antibodies constructed using mAb 1 and mAb 2b under reducing conditions are shown. Different HC and LC peaks (data not shown here) for a given bispecific antibody were identified using appropriate control transfections with one of the two arms. The area under the curve (AUC) of each HC or LC peak relative to the total AUC in the RP-HPLC curves was quantified and displayed. Figure 11C-11N The proportions of H1, L1, H2, and L2 are consistent with those of the heterodimeric bispecific antibody. These data indicate that the dominant species in the HIC-purified bispecific sample is the heterodimeric bispecific antibody, with the expected components being HC (H1 and H2) and LC (L1 and L2). Importantly, Figure 10A-10L and Figure 11A-11N Data from the study indicate that interchain disulfide bonds exist between HC and LC on the two arms of the bispecific antibody.

[0344] The ability of bispecific antibody samples purified by the same HIC to bind to both antigens was analyzed using a bridging ELISA. Consistent with the data from the previous analysis, all bispecific antibodies showed the ability to bind to both CD47 and FRα simultaneously, indicating that each arm of the bispecific antibody was correctly formed by the correct HC and LC. Figure 12A-12F The ability of bispecific antibodies to bind to SK-OV-3 cells expressing two antigens (CD47 and FRα) was tested. In this assay, SK-OV-3 cells were incubated with different concentrations of bispecific antibodies at 4°C for 15 min. Cells were then centrifuged for 5 min and washed three times with FACS buffer (HBSS supplemented with 5% BSA and 0.05% sodium azide). Cells were then incubated with Alexa Fluor 488-conjugated anti-human IgG secondary antibody (Thermo Fisher Scientific, catalog number: H10120) and incubated on ice for another 15 min. Cells were then washed twice with FACS buffer and resuspended in FACS buffer. Cells were then run via Attune NxT, and data were analyzed using Attune NxT software. In the FACS assay, the bispecific antibodies showed significant binding to SK-OV-3 cells. Figures 13A-13C ).

[0345] HIC-purified bispecific antibody samples were digested with papain under non-reducing conditions to determine whether the expected Fab fragments were generated by the bispecific antibody. Samples were concentrated to a final concentration of 5–10 mg / mL, and the buffer was replaced with papain digestion buffer (20 mM cysteine, 20 mM sodium phosphate, 10 mM EDTA, pH 7.0). Agarose-fixed papain (Thermo 20341) was pre-equilibrated in papain digestion buffer and resuspended in 50% slurry. Protein samples were added to the slurry at a 2:1 volume ratio and incubated overnight at room temperature with shaking. The supernatant was directly extracted for mass spectrometry analysis. The calculated molecular weight (mw) of each Fab fragment is shown together with its observed mw. Figures 14A-14C The data indicate that the expected Fab fragment produced by papain digestion was detected on mass spectrometry, and the expected bispecific antibody was correctly formed.

[0346] HIC-purified bispecific antibody samples were also subjected to trypsin digestion under non-reducing conditions to determine whether the calculated trypsin-digested peptide fragment from the mAb 1 arm of the expected bispecific antibody was generated. The sample was diluted to a final concentration of 1 mg / mL in 200 mM guanidine hydrochloride and heated at 95 °C for 1 min. 2x trypsin digestion buffer (NEB P8101S) was added at a 1:1 volume ratio (NEB P8101S) along with 2 μg of mass spectrometry-grade total trypsin (NEB P8101S). The resulting reaction was shaken at 37 °C for 4 h and used directly for mass spectrometry analysis. The amino acid sequence and disulfide bond sites of the disulfide-linked peptide fragment expected to be generated from the trypsin digestion of the mAb 1 arm (anti-CD47 arm) of bsAb 6 are shown in [image / image / description]. Figure 15A In the diagram, cysteine ​​residues forming disulfide bonds are shown in bold; the expected interchain disulfide bonds formed by knock-in cysteine ​​residues are also indicated. Figure 15A bsAb 7( Figure 15B ),bsAb 8( Figure 15C ),bsAb 5b( Figure 15D ),bsAb 10( Figure 15E ) and bsAb 12 ( Figure 15F Similar illustrations were also shown. Figure 15A-15F MS results showed that each bispecific antibody (bsAb) could be identified on MS.

[0347] The expected disulfide-linked peptide fragments of mAb 1 arm were obtained from trypsin-digested mAb 6, 7, 8, 5b, 10, and 12. These data further demonstrate that the knock-in cysteine ​​residues enabled the correct formation of interchain disulfide bonds in each of the bispecific antibodies bsAb6, 7, 8, 5b, 10, and 12. Figure 15A-15F ).

[0348] The HIC-purified bispecific antibody samples were also digested with IdeZ protease under non-reducing conditions to determine whether the calculated (Fab')2 moiety from the expected bispecific antibody was generated. The samples were diluted to a final concentration of 0.5 mg / mL in 1x sugar buffer 2 (NEB). IdeZ protease was added at a ratio of 80 U protease per 12.5 μg antibody. The resulting mixture was incubated at 37 °C for 4 hours and used directly for mass spectrometry analysis. Figures 16A-16C MS spectra of HIC-purified bispecific antibody samples digested with IdeZ protease are shown. (Fab')2 generated from each bispecific antibody was identified, indicating that the expected bispecific antibodies are formed via knock-in cysteine ​​residues with appropriately formed interchain disulfide bonds.

[0349] To demonstrate the role of the two arms of the bispecific antibody in binding to SK-OV-3 cells known to express two antigens, the inhibitory effect of F(ab')2 generated by anti-CD47 or anti-FRα parental mAbs on antibody-cell binding was evaluated in a FACS assay. The Ab (antibody) concentrations used in the assay are shown in the figure. Figures 17A-17C Below each graph. Using 5000nM F(ab')2( Figures 17A-17C The inhibitory effect was tested. Furthermore, the sequential binding of CD47 and FRα to immobilized bsAb 12 was detected using Biacore. Figures 18A-18B ).

[0350] Bispecific antibodies purified from protein A were digested with papain, and the resulting Fab fragments were identified on MS. Figures 19A-19C In WT bispecific antibody samples, Fab fragments were detected from both correctly paired arms and from both mismatched arms (H1 / L2 and H2 / L1). Figure 19A However, in bsAb 10 ( Figure 19B ) and bsAb 12 ( Figure 19C In the sample, only one of the two mismatched arms was detected. Meanwhile, no HC / LC mismatched species that do not form HC / LC interchain disulfide bonds were found in the bsAb 10 and bsAb 12 designs, because no LC bands were observed on non-reducing SDS-PAGE in these designs. Figure 7C Therefore, these data indicate that the bsAb 10 and bsAb 12 designs eliminated the formation of the H2 / L1 mismatched species; furthermore, the absence of H2L1 pairing also supports the conclusion that no disordered species H1L2 / H2L1 were formed. Compared with other bispecific antibody designs using a transposition HC / LC interchain disulfide bond strategy revealed in the literature, the superiority of the bsAb 10 and bsAb 12 designs lies in their prevention of both a mismatched species and the formation of a disordered species. When bsAb 12 was further purified by HIC, the mismatched H1 / L2 Fab disappeared on the MS spectrum. Figure 20 The purification process for bsAb 12 was optimized and is shown in Table 4. The yield of 77 mg of ExpiCHO cell culture was obtained from 500 mL of transient transfection, which is similar to the range of mAbs in the same transient transfection system.

[0351] Table 4: Optimization of bsAb 12 purification process

[0352]

[0353]

[0354] Table 5: Amino acid residues in the CH1 and CL regions used to form charged pairs

[0355] name CH1 CL bsAb 5CP S131 P119 bsAb 9CP A129 S121 bsAb 10CP K133 K207 bsAb 11CP K133 I117 bsAb 12CP K133 F209 CP9 G166 S114 CP10 T187 D170

[0356] Note: EU designations are used for CH1 and CL regions. CP, live pair.

[0357] To further facilitate HC and LC pairing on each arm of a given bispecific antibody and / or improve the feasibility of purification using conventional manufacturing processes, amino acid mutations are introduced at a pair of residues in the CH1 and CL regions, respectively, to form charged pairs. Any combination of each charged pair or more charged pairs can be used to construct bispecific antibodies because they increase the correct pairing of HC and LC and / or facilitate purification by introducing physical properties different from impurities (i.e., mismatched molecular species). Any charged pair or combination of 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 physical properties different from impurities (i.e., mismatched molecular species). One of the two residues used to form a charged pair can be substituted with glutamic acid (E), and the other can be substituted with lysine (K), and vice versa. For example, on a given bispecific antibody composed of H1L1 and H2L2, if “E” is introduced on H1 and “K” is introduced on L1, then “K” is introduced on H2 and “E” is introduced on L2. Besides “E” and “K”, the two residues forming the charged pair can be replaced with acidic and basic amino acids, respectively. For example, if “E” is not used, the residue that introduces “E” can also be replaced with aspartic acid (D). No mutation is required as long as the native amino acid of a given residue happens to be the intended substituted amino acid (i.e., E, D, or K). The amino acid residues used to form the charged pair in the CH1 and CL regions are listed in Table 5. Each pair of these residues was selected based on various factors, including proximity, through 3-D modeling.

[0358] Those skilled in the art will understand that changes can be made to the above embodiments without departing from their broad inventive concept. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined in this specification. sequence list <110> Vanenz Pharmaceutical Company <120> Bispecific antibodies with replaceable matching inter-chain cysteine ​​and their uses <130> 065799.30WO1 <150> US 62 / 948,953 <151> 2019-12-17 <150> US 62 / 952,747 <151> 2019-12-23 <150> US 62 / 988,144 <151> 2020-03-11 <150> US 63 / 007,996 <151> April 10, 2020 <150> US 62 / 704,973 <151> June 5, 2020 <150> US 62 / 706,511 <151> August 21, 2020 <160> 36<?? <170> PatentIn version 3.5 <210> 1 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> bsAb1 VH <400> 1[[ID=2�]] Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Ile 35 40 45 Gly Asn Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80[[ID=??]] Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 It should be noted that there is an unclear "??0000967" and "??" in the original text which might be incorrect or incomplete information. The translation is done as accurately as possible based on the available content.Ala Gly Thr Asp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 2 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> bsAb 1 CH1 <400> 2 ]>Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Ser 100 <210> 3 <211> 107 <212> PRT<00,01014><213> Artificial Sequence <220> <223> bsAb 1 VL <400> 3 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys His Ala Ser Gln Asn Ile Asn Val Trp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gln Cys Thr Lys Val Glu Ile Lys 100 105 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> bsAb 1 CL <400> 4 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser 100 105 <210> 5 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> bsAb 5 CH1 <400> 5 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Ser 100 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> bsAb 5 CL <400> 6 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Cys Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser 100 105 <210> 7 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> bsAb 6 CH1 <400> 7 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Cys Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Ser 100 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> bsAb 6 CL <400> 8 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Cys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser 100 105 <210> 9 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> bsAb 7 CH1 <400> 9 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Cys Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Ser 100 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> bsAb 7 CL <400> 10 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Cys Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser 100 105 <210> 11 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> bsAb 8 CH1 <400> 11 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Cys Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Ser 100 <210> 12 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> bsAb 8 CL <400> 12 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Cys Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser 100 105 <210> 13 <211> 114 <212> PRT <213> Artificial Sequence <220><00012​​​​​​​​​​​​​​​​​​​​Gln Gly Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Thr Asp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 14 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> mAb 2 VH <400> 14 Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Gly Met His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Met Ser Tyr Thr Pro Gly Thr Phe His Tyr Ala Asp Thr Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val His Val Gly Thr Val Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 15 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> mAb 1 CH1 <400> 15 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys 100 <210> 16 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> mAb 2 CH1 <400> 16 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 � Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys [[ID=4ᅳ]]85 90 95 Lys Val Glu Pro Lys Ser Cys 100 <210> 17 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> mAb 1 VL <400> 17 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys His Ala Ser Gln Asn Ile Asn Val Trp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 18 [[ID=​​​​​​​​​​​​​​​Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Asn Asn Asn 20 25 30 Leu His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Ala 85 90 95 Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 19 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> mAb 1 CL <400> 19 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> mAb 2 CL <400> 20 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 21 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> IgG2 CH1 <400> 21 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys 100 <210> 22 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> IgG3 CH1 <400> 22 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly 100 105 <210> 23 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> IgG4 CH1 <400> 23 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly 100 <210> 24 <211> 104 <212> PRT <213> Artificial Sequence <220> <223> lambda CL <400> 24 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 80 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95 Lys Thr Val Ala Pro Thr Glu Cys 100 <210> 25 <211> 103 <212> PRT <213> Artificial Sequence <220>[[ID=3,6]] <223> bsAb 9 CH1 <400> 25 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Cys Pro Ser Ser Lys[[ID=,42]]< / / 1 5 10 ¹5 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30< / / Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Ser 100 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> bsAb 9 CL <400> 26 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Cys Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser 100 105 <210> 27 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> bsAb 10 CH'1 <400> 27 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Cys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Note: There seems to be a possible error in the original text where it says "<223> bsAb 10 CH1" which might be "<223> bsAb 10 CH1" (with a space added for better readability). The translation is done as accurately as possible based on the provided text.Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Ser 100 <210> 28 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> bsAb 10 CL <400> 28 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln [[ID={32]]35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Cys Ser Phe Asn Arg Gly Glu Ser 100 105 <{210}> 29 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> bsAb 11 CH1 <400> 29 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Cys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Ser 100 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> bsAb 11 CL <400> 30 Arg Thr Val Ala Ala Pro Ser Val Phe Cys Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser 100 105 <210> 31 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> bsAb 12 CH1 <400> 31 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Cys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Ser 100[[ID=2*]] <210> 32 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> bsAb 12 CL <400> 32 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Cys Asn Arg Gly Glu Ser 100 105 <210> 33 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> mAb 2b VH <400> 33 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Tyr Thr His Tyr Asn Gly Met Phe 50 55 60 Lys Gly Arg Ala Ser Leu Thr Ala Asp Lys Ser Thr Ser Thr Gly Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Phe Phe Cys 85 90 95 Thr Arg His Gly Asp Phe Pro Tyr Trp Tyr Phe Asp Val Trp Gly Arg 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 34 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> mAb 2b CH1 <400> 34 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr<C 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys 100 <210> 35 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> mAb 2b VL <400> 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Leu Leu Val 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Val Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Ser Thr Pro Pro 85 9​​​​​​ <211> 107 <212> PRT <213> Artificial Sequence <220> <223> mAb 2b CL <400> 36 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims

1. An isolated bispecific antibody or its antigen-binding fragment, comprising: First chain, H1; The second chain, H2; First light chain, L1; and The second light chain, L2; H1 and L1 form the first arm, which contains a first antigen-binding domain that specifically binds to the first antigen, and H2 and L2 form the second arm, which contains a second antigen-binding domain that specifically binds to the second antigen. H1 contains the CH1 region of human IgG1, IgG2, IgG3, or IgG4; and L1 contains the CL region of human κ light chain or human λ light chain; The amino acid sequences in the CH1 region are selected from the following groups: SEQ ID NO: 15, 21, 22, 23; the amino acid sequences in the CL region are selected from the following groups: SEQ ID NO: 19, 24; and the CH1 and CL regions also contain amino acid substitutions selected from the following groups: K133C and C220X in CH1, and F209C and C214X in CL; K133C and C220X in CH1, and K207C and C214X in CL; K133C and C220X in CH1, and I117C and C214X in CL; R133C and C131X in CH1, and K207C and C214X in CL; R133C and C131X in CH1, and I117C and C214X in CL; R133C and C131X in CH1, and L117C and C214X in CL; K133C and C220X in CH1, and L117C and C214X in CL; R133C and C131X in CH1, and F209C and C214X in CL; R133C and C131X in CH1, and V209C and C214X in CL; or K133C and C220X in CH1, and V209C and C214X in CL; X is selected from S, A or G, and the CH1 and CL regions use EU designations.

2. The isolated bispecific antibody or its antigen-binding fragment according to claim 1, wherein the first antigen is a human-derived first antigen.

3. The isolated bispecific antibody or its antigen-binding fragment according to claim 1, wherein the second antigen is a human-derived second antigen.

4. The isolated bispecific antibody or its antigen-binding fragment according to claim 1, wherein the first antigen-binding domain is a CD47 binding domain.

5. The isolated bispecific antibody or its antigen-binding fragment according to claim 4, wherein the H1 further comprises a heavy chain variable region, i.e., a VH region, the VH region comprising the amino acid sequence of SEQ ID NO: 1, and the CH1 region comprising the amino acid sequence of SEQ ID NO: 2; wherein the L1 further comprises a light chain variable region, i.e., a VL region, the VL region comprising the amino acid sequence of SEQ ID NO: 3, and the CL region comprising the amino acid sequence of SEQ ID NO:

4.

6. The isolated bispecific antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein... (a) The second arm containing H2 and L2 does not contain amino acid substitutions in the first arm containing H1 and L1; (b) The two heavy chains H1 and H2 each contain a VH region, a CH1 region and an Fc region containing CH2 and CH3 regions, wherein the VH region has a different amino acid sequence; (c) The two heavy chains H1 and H2 each contain a VH region, a CH1 region and an Fc region containing CH2 and CH3 regions, wherein the CH1 region has a different amino acid sequence; (d) The two heavy chains H1 and H2 each contain a VH region, a CH1 region, and an Fc region containing CH2 and CH3 regions, wherein the Fc region has a different amino acid sequence; (e) Each of the two light chains, L1 and L2, contains a VL region and a CL region, wherein the VL regions have different amino acid sequences; and / or (f) The two light chains L1 and L2 each contain a VL region and a CL region, wherein the CL region has a different amino acid sequence.

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

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

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 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. The isolated bispecific antibody or its antigen-binding fragment according to claim 9, wherein the CD47 is human CD47.

11. The isolated bispecific antibody or its antigen-binding fragment according to claim 9, wherein the folic acid receptor α (FRα) is human FRα.

12. The isolated bispecific antibody or its antigen-binding fragment according to any one of claims 1 to 11, wherein... (a) The first antigen-binding domain has the VH sequence of SEQ ID: 13 and the VL sequence of SEQ ID: 17, and the second antigen-binding domain has the VH sequence of SEQ ID: 33 and the VL sequence of SEQ ID: 35: or (b) The first antigen-binding domain has the VH sequence of SEQ ID: 13 and the VL sequence of SEQ ID: 17, and the second antigen-binding domain has the VH sequence of SEQ ID: 14 ​​and the VL sequence of SEQ ID:

18.

13. The isolated bispecific antibody or antigen-binding fragment thereof according to claim 9 or 12, wherein the anti-CD47 / anti-FRα bispecific antibody or antigen-binding fragment thereof is capable of blocking the binding of signal regulatory protein α (SIRPα) to CD47 on cells expressing FRα and CD47, inducing macrophage-mediated phagocytosis of cancer cells expressing FRα and CD47, and / or binding to cancer cells expressing FRα and CD47 while minimizing binding to human erythrocytes (RBCs) to undetectable levels.

14. An isolated nucleic acid encoding a bispecific antibody or antigen-binding fragment as described in any one of claims 1 to 13.

15. A vector comprising the isolated nucleic acid of claim 14.

16. A host cell comprising the vector of claim 15.

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

18. A method for preparing a bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, comprising culturing a cell containing a nucleic acid encoding the bispecific antibody or antigen-binding fragment thereof under conditions for generating the bispecific antibody or antigen-binding fragment thereof, and recovering the bispecific antibody or antigen-binding fragment thereof from the cell or culture.

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

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