Bispecific antibodies against DR5 and CEACAM5 and uses thereof

Abstract

The invention provides a bispecific antibody which is specifically combined with human CEACAM5 and human DR5. The invention further provides a nucleic acid molecule for coding the antibody, an expression vector for expressing the antibody, a host cell and a preparation method thereof. The invention also provides methods of treatment using the bispecific antibodies of the invention.

Description

Technical Field

[0001] The present invention relates to the field of antibody engineering, and in particular to a bispecific antibody that recognizes DR5 and CEACAM5, as well as therapeutic uses of the antibody and a pharmaceutical composition containing the same. Background Art

[0002] Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can bind to and activate specific cell surface-anchored death receptors DR4 (TRAILR1) or DR5 (TRAILR2), subsequently causing apoptosis. Many preclinical studies have found that TRAIL signaling effectively induces apoptosis in multiple tumor cell lines, but not in most normal cells. Therefore, targeting TRAIL receptors has become an important research direction for the development of cancer therapy. DR5 is highly expressed in a variety of solid cancers (including colorectal tumors). At present, clinical studies have reported a variety of agonist drugs targeting DR5. The first generation of agonists is anti-DR5 monoclonal antibodies or recombinant protein molecules of its ligand TRAIL. Although clinical studies have found good safety, the efficacy is poor due to its inherent weak agonistic properties. The second generation of molecules is anti-DR5 multivalent antibodies. Although clinical studies have found that the efficacy has been improved, the side effects (mainly liver toxicity that is sensitive to DR5-induced apoptosis) limit the application of DR5 targeted therapeutic drugs.

[0003] CEACAM5 (also known as CEA or CD66e) is a carcinoembryonic antigen (CEA) that is expressed at low levels in normal human tissues, but is expressed at high levels in colorectal cancer, gastric cancer, pancreatic cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer and bladder cancer tissues. Therefore, CEACAM5 is regarded as a tumor biomarker and is suitable as a therapeutic target for tumor-specific targeted approaches (such as immunoconjugates).

[0004] Currently, no bispecific antibodies simultaneously targeting DR5 / CEACAM5 have entered clinical research.

[0005] Although the technology for constructing bispecific antibodies has made great progress, obtaining the desired bispecific antibodies is still a time-consuming, laborious and uncertain task. How to achieve the biological function of bispecific antibodies through different formats requires consideration and optimization of multiple parameters: such as the selection of antibodies for each target in the bispecific antibody (not all antibodies are suitable for bispecific antibodies), the valency of the antibody for each target, the structural form and conformation of each antibody part, how to arrange the layout to ensure the optimal spatial distance between each antibody, etc. For example, the study in CN 110582513A showed that the arrangement order of VH and VL in scFv has a great influence on its activity.

[0006] Here, we report an optimized bispecific antibody with high affinity for DR5 and potent apoptosis triggering that is active against multiple tumor models with a wide therapeutic window. Summary of the invention

[0007] The present invention provides a bispecific antibody that can specifically recognize DR5 and CEACAM5. The present invention also provides the therapeutic use of the antibody and a pharmaceutical composition containing the antibody.

[0008] In the first aspect, the present invention provides a bispecific antibody (also referred to as an anti-DR5×CEACAM5 bispecific antibody, an anti-DR5 / CEACAM5 bispecific antibody) that simultaneously targets DR5 and CEACAM5. In the antibody, the anti-DR5 antibody is in the form of a Fab, which maintains the activity of DR5 monoclonal antibody targeted therapy, and the anti-CEACAM5 antibody is in the form of a scFv and has been affinity optimized, so that it can better assist in improving the activity of DR5 targeted therapy. In addition, the bispecific antibody of this configuration of the present invention allows the auxiliary activity of CEACAM5 to occur only in tumor tissues that express CEACAM5, but not in normal tissues that do not express CEACAM5, thereby improving the therapeutic accuracy of the anti-DR5 / CEACAM5 bispecific antibody.

[0009] The anti-DR5 / CEACAM5 bispecific antibody of the present invention has the following advantages:

[0010] (1) It specifically binds to human and cynomolgus monkey DR5 proteins, but not mouse DR5 proteins; it specifically binds to human CEACAM5 proteins, but not cynomolgus monkey CEACAM5 proteins;

[0011] (2) does not bind to other proteins in the human CEACAM family;

[0012] (3) It can induce apoptosis of tumor cells in various solid tumors;

[0013] (4) The configuration of the anti-DR5 / CEACAM5 bispecific antibody of the present invention is the best antibody form for achieving the drug efficacy of the anti-DR5 / CEACAM5 bispecific antibody;

[0014] (5) The anti-DR5 / CEACAM5 bispecific antibody of the present invention has reduced molecular heterogeneity, high yield, high yield in a one-step purification method, and improved overall drugability;

[0015] (6) Excellent tumor inhibition effect.

[0016] In some embodiments, the present invention provides an anti-DR5 / CEACAM5 bispecific antibody, which comprises two heavy chains with the same structure of VH-CH1-Fc-scFv, and two light chains with the same structure of VL-CL, wherein the VL of the two light chains are paired with the VH of the two heavy chains to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on each heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of the Fc and has a VL CEACAM5 -Connector-VH CEACAM5 structure.

[0017] In a specific embodiment, the present invention provides an anti-DR5 / CEACAM5 bispecific antibody, which comprises two heavy chains with the same structure of VH-CH1-Fc-scFv, and two light chains with the same structure of VL-CL, wherein the VL of the two light chains are paired with the VH of the two heavy chains to form two antigen recognition sites for recognizing DR5 molecules, and the scFv on each heavy chain forms an antigen recognition site for recognizing CEACAM5 molecules, wherein the scFv is connected to the C-terminus of Fc and has a VL CEACAM5 -Connector-VH CEACAM5 The structure of which:

[0018] The VH that recognizes the DR5 molecule comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the VL that recognizes the DR5 molecule comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; and

[0019] The scFv that recognizes CEACAM5 comprises a VH that recognizes CEACAM5 (referred to herein as VH to distinguish it from a VH that recognizes DR5). CEACAM5 ) and a VL that recognizes CEACAM5 (referred to herein as VL to distinguish it from the VL that recognizes DR5) CEACAM5 ), where VH CEACAM5 It comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:11, and recognizes the VL of CEACAM5 CEACAM5 comprising LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14;

[0020] or

[0021] The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8;

[0022] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 39, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20;

[0023] or

[0024] The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:22, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:24;

[0025] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 30, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32;

[0026] or

[0027] The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:22, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:24;

[0028] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 34, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36;

[0029] or

[0030] The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28;

[0031] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 34, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36;

[0032] or

[0033] The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28;

[0034] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 30, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32;

[0035] or

[0036] The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28;

[0037] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 39, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20;

[0038] or

[0039] The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8;

[0040] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 34, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36;

[0041] or

[0042] The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8;

[0043] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 30, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32;

[0044] or

[0045] The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:38, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:40;

[0046] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1, HCDR2 and HCDR3 contained in SEQ ID NO: 39, and VL CEACAM5 It comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20.

[0047] In a specific embodiment, the present invention provides an anti-DR5 / CEACAM5 bispecific antibody, which comprises two heavy chains with the same structure of VH-CH1-Fc-scFv, and two light chains with the same structure of VL-CL, wherein the VL of the two light chains are paired with the VH of the two heavy chains to form two antigen recognition sites for recognizing DR5 molecules, and the scFv on each heavy chain forms an antigen recognition site for recognizing CEACAM5 molecules, wherein the scFv is connected to the C-terminus of Fc and has a VL CEACAM5 -Connector-VH CEACAM5 The structure of which:

[0048] The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 8;

[0049] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20;

[0050] or

[0051] The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 22, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 24;

[0052] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 34 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:36;

[0053] or

[0054] The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 22, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 24;

[0055] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 30 and recognizing the CEACAM5 molecule CEACAM5 comprising the sequence shown in SEQ ID NO:32;

[0056] or

[0057] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO: 28;

[0058] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 34 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:36;

[0059] or

[0060] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO: 28;

[0061] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 30 and recognizing the CEACAM5 molecule CEACAM5 comprising the sequence shown in SEQ ID NO:32;

[0062] or

[0063] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO: 28;

[0064] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20;

[0065] or

[0066] The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 8;

[0067] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 34 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:36;

[0068] or

[0069] The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 8;

[0070] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 30 and recognizing the CEACAM5 molecule CEACAM5 comprising the sequence shown in SEQ ID NO:32;

[0071] or

[0072] The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:38, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO:40;

[0073] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20;

[0074] or

[0075] The VH of the intact antibody that recognizes DR5 comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8; and

[0076] VH of scFv recognizing CEACAM5 CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 15 and recognizing the CEACAM5 molecule CEACAM5 comprising the sequence shown in SEQ ID NO:16;

[0077] or

[0078] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0079] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 41 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20;

[0080] or

[0081] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0082] VH that recognizes CEACAM5 molecules CEACAM5A VL comprising the sequence shown in SEQ ID NO: 43 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20;

[0083] or

[0084] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0085] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 45 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20;

[0086] or

[0087] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0088] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 47 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20;

[0089] or

[0090] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0091] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 49 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20;

[0092] or

[0093] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0094] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 51 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20;

[0095] or

[0096] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0097] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:54;

[0098] or

[0099] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0100] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:56;

[0101] or

[0102] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0103] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 It comprises the sequence shown in SEQ ID NO:58.

[0104] In one embodiment, the present invention provides an anti-DR5 / CEACAM5 bispecific antibody, comprising an antibody or antigen-binding fragment that recognizes a DR5 molecule and an antibody or antigen-binding fragment that recognizes a CEACAM5 molecule, wherein:

[0105] The VH that recognizes the DR5 molecule comprises HCDR1 as shown in SEQ ID NO: 1, HCR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the VL that recognizes the DR5 molecule comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; and

[0106] Among them, the VH that recognizes CEACAM5 molecules CEACAM5 It comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:11, and recognizes the VL of CEACAM5 CEACAM5 It comprises LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14.

[0107] In one embodiment, the present invention provides a DR5 / CEACAM5 bispecific antibody, comprising an antibody or antigen-binding fragment that recognizes a DR5 molecule and an antibody or antigen-binding fragment that recognizes a CEACAM5 molecule, wherein:

[0108] The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 7, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 8; and

[0109] VH of scFv recognizing CEACAM5 CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 15 and recognizing the CEACAM5 molecule CEACAM5 It comprises the sequence shown in SEQ ID NO:16.

[0110] In some embodiments, the present invention provides a DR5 / CEACAM5 bispecific antibody, which comprises an antibody or antigen-binding fragment that recognizes a DR5 molecule and an antibody or antigen-binding fragment that recognizes a CEACAM5 molecule, wherein the antibody that recognizes a DR5 molecule is a complete antibody, and the antigen-binding fragment that recognizes a CEACAM5 molecule is selected from scFv, scFab, Fab or Fv.

[0111] In one embodiment, in the anti-DR5 / CEACAM5 bispecific antibody provided above, the Fc region can be selected from the natural Fc region of any IgG, IgA, IgM class antibody known in the prior art, such as a common or germline IgG Fc region. In a specific embodiment, the IgG Fc region can be from human IgG1, IgG2, IgG3 or IgG4 isotype.

[0112] In a specific embodiment, the IgG Fc region is the Fc sequence of human IgG1. In a further embodiment, the Fc region comprises mutations, such as mutations that increase the stability of the bispecific antibody dimer (e.g., disulfide bridge structure), mutations that reduce effector function (e.g., L234A and L235A mutations).

[0113] In any of the above embodiments, the scFv of the anti-DR5 / CEACAM5 bispecific antibody of the present invention is connected to Fc via a linker, and the linker can be any universal flexible sequence known in the art or obtained in the future, usually a short flexible amino acid sequence. In a specific embodiment, the linker is (G3S)n (SEQ ID NO: 62) or (G4S)n (SEQID NO: 63), wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9. In a specific embodiment, the linker is selected from (G3S)3 (SEQ ID NO: 64), (G4S)4 (SEQ ID NO: 65).

[0114] In any of the above embodiments, the VH in the scFv of the bispecific antibody of the present invention CEACAM5 and VL CEACAM5 The linker is connected by a linker, which can be any universal flexible sequence known in the art or obtained in the future, usually a short flexible amino acid sequence. In a specific embodiment, the linker is (G3S)n or (G4S)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9. In a specific embodiment, the linker is selected from (G3S)3, (G4S)4.

[0115] In one embodiment, the present invention provides an anti-DR5 / CEACAM5 bispecific antibody, comprising two heavy chains with the same structure of VH-CH1-Fc-scFv, and two light chains with the same structure of VL-CL, wherein the VL of the two light chains are respectively paired with the VH of the two heavy chains to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on each heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the heavy chain comprises the sequence shown in SEQ ID NO: 17, and the light chain comprises the sequence shown in SEQ ID NO: 18.

[0116] In another embodiment, the present invention provides an anti-DR5 / CEACAM5 bispecific antibody, comprising one heavy chain with a structure of VH-CH1-Fc-scFv, one heavy chain with a structure of VH-CH1-Fc, and two light chains with a structure of VL-CL, wherein the two heavy chains have the same VH-CH1-Fc, wherein the VLs of the two light chains are paired with the VHs of the two heavy chains respectively to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on the heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of the Fc and has a VL CEACAM5 -Connector-VH CEACAM5 structure.

[0117] In a specific embodiment, the present invention provides an anti-DR5 / CEACAM5 bispecific antibody, which comprises a heavy chain with a structure of VH-CH1-Fc-scFv, a heavy chain with a structure of VH-CH1-Fc, and two light chains with a structure of VL-CL, wherein the two heavy chains have the same VH-CH1-Fc, wherein the VLs of the two light chains are paired with the VHs of the two heavy chains respectively to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on the heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of the Fc and has a VL CEACAM5 -Connector-VH CEACAM5 The structure of which:

[0118] The VH that recognizes the DR5 molecule comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the VL that recognizes the DR5 molecule comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6;

[0119] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1 as shown in SEQ ID NO: 9, HCDR2 as shown in SEQ ID NO: 10, HCDR3 as shown in SEQ ID NO: 11, and VL CEACAM5 comprising LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14;

[0120] or

[0121] The VH that recognizes the DR5 molecule comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the VL that recognizes the DR5 molecule comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6;

[0122] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 30, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32;

[0123] or

[0124] The VH that recognizes the DR5 molecule comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the VL that recognizes the DR5 molecule comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6;

[0125] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 34, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36;

[0126] or

[0127] The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28;

[0128] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1 as shown in SEQ ID NO: 9, HCDR2 as shown in SEQ ID NO: 10, HCDR3 as shown in SEQ ID NO: 11, and VL CEACAM5 comprising LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14;

[0129] or

[0130] The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28;

[0131] The scFv that recognizes CEACAM5 molecules contains VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 34, and VL CEACAM5 It comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36.

[0132] In a specific embodiment, the present invention provides an anti-DR5 / CEACAM5 bispecific antibody, which comprises a heavy chain with a structure of VH-CH1-Fc-scFv, a heavy chain with a structure of VH-CH1-Fc, and two light chains with a structure of VL-CL, wherein the two heavy chains have the same VH-CH1-Fc, wherein the VLs of the two light chains are paired with the VHs of the two heavy chains respectively to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on the heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of the Fc and has a VL CEACAM5 -Connector-VH CEACAM5 The structure of which:

[0133] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0134] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 15 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:16;

[0135] or

[0136] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0137] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 30 and recognizing the CEACAM5 molecule CEACAM5 comprising the sequence shown in SEQ ID NO:32;

[0138] or

[0139] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8;

[0140] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 34 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:36;

[0141] or

[0142] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO: 28;

[0143] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 15 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:16;

[0144] or

[0145] The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO: 28;

[0146] VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 34 and recognizing the CEACAM5 molecule CEACAM5 It comprises the sequence shown in SEQ ID NO:36.

[0147] In one embodiment, in the anti-DR5 / CEACAM5 bispecific antibody provided above, the Fc region can be selected from the natural Fc region of any IgG, IgA, IgM class antibody known in the prior art, such as a common or germline IgG Fc region. In a specific embodiment, the IgG Fc region can be from human IgG1, IgG2, IgG3 or IgG4 isotype.

[0148] In a specific embodiment, the IgG Fc region is the Fc sequence of human IgG1. In a further embodiment, the Fc region comprises mutations, such as mutations that increase the stability of the bispecific antibody dimer (e.g., disulfide bridge structure), mutations that reduce effector function (e.g., L234A and L235A mutations).

[0149] In any of the above embodiments, the scFv of the anti-DR5 / CEACAM5 bispecific antibody of the present invention is connected to Fc via a linker, and the linker can be any universal flexible sequence known in the art or obtained in the future, usually a short flexible amino acid sequence. In a specific embodiment, the linker is (G3S)n or (G4S)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9. In a specific embodiment, the linker is selected from (G3S)3, (G4S)4.

[0150] In any of the above embodiments, the VH in the scFv of the bispecific antibody of the present invention CEACAM5 and VL CEACAM5 The linker is connected by a linker, which can be any universal flexible sequence known in the art or obtained in the future, usually a short flexible amino acid sequence. In a specific embodiment, the linker is (G3S)n or (G4S)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9. In a specific embodiment, the linker is selected from (G3S)3, (G4S)4.

[0151] In a second aspect, the present invention also provides a polynucleotide (nucleic acid) encoding the anti-DR5 / CEACAM5 bispecific antibody of the present invention, and a vector comprising the polynucleotide, preferably an expression vector.

[0152] In a third aspect, the present invention provides a host cell comprising the polynucleotide or vector of the present invention. The host cell may be a prokaryotic cell or a eukaryotic cell commonly used in the art.

[0153] In a fourth aspect, the present invention provides a method for producing the anti-DR5 / CEACAM5 bispecific antibody of the present invention, comprising the steps of (i) culturing the host cell of the present invention under conditions suitable for expressing the anti-DR5 / CEACAM5 bispecific antibody of the present invention, and optionally, (ii) recovering the anti-DR5 / CEACAM5 bispecific antibody of the present invention.

[0154] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the anti-DR5 / CEACAM5 bispecific antibody of the present invention.

[0155] In one embodiment, the present invention provides a drug combination comprising the anti-DR5 / CEACAM5 bispecific antibody of the present invention and other therapeutic agents, and optional pharmaceutical excipients; preferably, the other therapeutic agents are selected from 5-fluorouracil, oxaliplatin, exotecan, and SN-38.

[0156] In a sixth aspect, the present invention provides a drug kit comprising the anti-DR5 / CEACAM5 bispecific antibody of the present invention and other therapeutic agents. Preferably, the other therapeutic agents are selected from 5-fluorouracil, oxaliplatin, exotecan, and SN-38.

[0157] In a seventh aspect, the present invention provides the use of the anti-DR5 / CEACAM5 bispecific antibody, pharmaceutical composition, and drug kit of the present invention for treating cancer. In one embodiment, the cancer is, for example, colorectal cancer, gastric cancer, lung cancer, or pancreatic cancer.

[0158] In one embodiment, the present invention provides the use of the anti-DR5 / CEACAM5 bispecific antibody, polynucleotide, vector, host cell, pharmaceutical composition or kit described in the first to third aspects and the sixth aspect in the preparation of a medicament for treating and / or diagnosing cancer. In one embodiment, the cancer is, for example, colorectal cancer, gastric cancer, lung cancer, or pancreatic cancer.

[0159] In an eighth aspect, the present invention provides a method for treating cancer, comprising administering a therapeutically effective amount of the anti-DR5 / CEACAM5 bispecific antibody, polynucleotide, vector, host cell, pharmaceutical composition or kit of the first to third and sixth aspects of the present invention to a patient in need thereof. In one embodiment, the cancer is, for example, colorectal cancer, gastric cancer, lung cancer, or pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0160] Figure 1 The antibody structures that were originally designed and constructed are shown.

[0161] Figure 2 The results of the activity test of the molecule with antibody structure 1 in inducing apoptosis of tumor cells are shown, wherein the molecule with antibody structure 1 has a stronger activity of inducing apoptosis of tumor cells than the anti-DR5 monoclonal antibody molecule.

[0162] Figure 3 shows the results of the activity test of inducing tumor cell apoptosis of molecules having antibody structure 1. Among the molecules of antibody structure 1, the molecules of anti-DR5 antibody in the Fab region and in the scFv region have stronger activity of inducing tumor cell apoptosis.

[0163] Figure 4 shows the results of the activity test of inducing tumor cell apoptosis by the molecule having antibody structure 2. The molecule having antibody structure 1 has a stronger activity of inducing tumor cell apoptosis than the molecule having antibody structure 2.

[0164] Figure 5 shows the results of the activity test of inducing tumor cell apoptosis by the molecule having antibody structure 3. The molecule having antibody structure 1 has a stronger activity of inducing tumor cell apoptosis than the molecule having antibody structure 3.

[0165] Figure 6 shows the results of the activity test of inducing tumor cell apoptosis of molecules having antibody structure 4, molecules having antibody structure 5 and molecules having antibody structure 6. The molecule having antibody structure 1 has a stronger activity of inducing tumor cell apoptosis than the molecules having antibody structure 4, molecules having antibody structure 5 and molecules having antibody structure 6.

[0166] Figure 7 The results of the activity test of anti-DR5 monoclonal antibody and its mutants in inducing apoptosis in human colon cancer Colo205 cells are shown. KMmut1 antibody can induce apoptosis in a larger proportion of tumor cells at high concentrations.

[0167] Figure 8 The results of KMmut1-HL1 activity detection in inducing apoptosis in human colon cancer HT55 cells are shown.

[0168] FIG. 9 shows the activity of KMmut1-HL1 in inducing apoptosis of various tumor cell lines in vitro.

[0169] Fig.10 The synergistic effect of KMmut1-HL1 combined with other drugs in vitro was shown.

[0170] FIG. 11 shows that KMmut1-HL1 inhibits the growth of xenograft tumor cells in mice. DETAILED DESCRIPTION

[0171] I. Definitions

[0172] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0173] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0174] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

[0175] The term "and / or" means any one of the alternatives or two or more of the alternatives.

[0176] The term "comprising" or "including" means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the combination of the elements, integers or steps described. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0177] The term "antibody" is used herein in the broadest sense to refer to a protein that contains an antigen binding site, covering natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, single-domain antibodies, complete antibodies, and antibody fragments.

[0178] The term "antibody fragment" refers to a molecule different from an intact antibody, which comprises a portion of an intact antibody and is capable of binding to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent or bispecific antibody fragments and camelid antibodies (heavy chain antibodies).

[0179] The term "Fc region" comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index.

[0180] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co. 91 pages (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity.

[0181] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy chain are usually referred to as HCDR1, HCDR2 and HCDR3, and the CDRs of the light chain are usually referred to as LCDR1, LCDR2 and LCDR, and are numbered sequentially from the N-terminus, respectively. In a given variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of antibodies and the topology of the CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics (ImmunoGeneTics), and the like. database (IMGT) (http: / / imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0182] The term "multispecific antibody" refers to an antibody having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens. A multispecific antibody is an antibody that has binding specificity for at least two different antigenic epitopes. In one embodiment, provided herein is a multispecific antibody that has binding specificity for a first antigen and a second antigen, also referred to as a "bispecific antibody."

[0183] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of the binding dissociation equilibrium constant (K D ). Affinity can be measured by common methods known in the art, including those known in the art and described herein. In one embodiment, the KD value of an antibody of the present invention that "specifically binds" an antigen as determined by biofilm thin layer interferometry (BLI) is about 1×10 -8 M or less, preferably about 1×10 -9 M or less; more preferably 1×10 -10 M or smaller.

[0184] The term "single-chain scFv antibody" or "scFv" or "single-chain scFv" refers to a single polypeptide chain comprising the heavy chain variable region (VH) and the light chain variable region (VL) of an immunoglobulin or antibody, in which the VH region and the VL region are paired to provide an antigen binding site. Usually VH and VL are connected by an artificially synthesized connecting peptide (or "linker"). scFv is suitable for use as a genetic engineering component to prepare other antigen-specific binding molecules with new properties, such as full-length antibodies, scFv-Fc, multispecific antibodies, etc.

[0185] The terms "connecting peptide", "linker" or "peptide linker" and the like are used interchangeably in this application and refer to a peptide comprising one or more consecutive amino acids, such as small amino acid residues or hydrophilic amino acid residues (e.g., glycine, serine, threonine, proline, aspartic acid, asparagine, etc.). The connecting peptide generally comprises a length of 5-50 amino acids, such as 10, 15, 20, 25, 30 amino acids in length. Those skilled in the art will appreciate that many commonly used linkers can be used in embodiments of the present invention.

[0186] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0187] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, without considering the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant progeny with the same function or biological activity that are screened or selected in the initially transformed cells.

[0188] The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0189] The term “median effective concentration (EC 50 )” refers to the concentration of a drug, antibody or toxic agent that induces a response that is 50% between baseline and maximum after a specified exposure time.

[0190] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.

[0191] The term "treat" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay disease development or to slow the progression of a disease.

[0192] The term "prevention" includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

[0193] The term "effective amount" refers to an amount or dosage of an antibody or conjugate or composition of the invention that produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in single or multiple doses. The effective amount can be readily determined by the attending physician, who is a person skilled in the art, by considering a variety of factors such as the species of the mammal; body weight, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0194] The term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody or antibody fragment or its conjugate or composition can vary according to a variety of factors such as disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to stimulate the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or its conjugate or composition are outweighed by the therapeutically beneficial effects. Relative to untreated subjects, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70%, and still more preferably at least about 80% or 90%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors.

[0195] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0196] II. Antibodies of the Invention

[0197] The "anti-DR5 / CEACAM5 bispecific antibody", "anti-CEACAM5 / DR5 bispecific antibody" and similar terms mentioned herein refer to bispecific antibodies that can bind to the targets CEACAM5 and DR5 with sufficient affinity. As shown in the Examples, not all monoclonal antibodies targeting CEACAM5 and DR5 are suitable for constructing the bispecific antibody of the present invention, and bispecific antibody molecules of different configurations have different activities. The present invention finally obtains a bispecific antibody targeting CEACAM5 and DR5 with excellent performance, and its molecular structure is as shown in FIG. Figure 1 As shown in A and 1C, the bispecific molecule comprises a full-length monoclonal antibody targeting DR5 and a scFv antibody targeting CEACAM5. The scFv molecule is fused to the heavy chain C-terminus of the above-mentioned DR5 monoclonal antibody through a linker 2, and the order of the scFv antibody from the N-terminus to the C-terminus is VL-linker 1-VH, wherein linker 2 is a (G3S)3 polypeptide and linker 1 is a (G4S)4 polypeptide.

[0198] In one embodiment, the bispecific anti-DR5 / CEACAM5 antibody of the present invention comprises an amino acid modification, such as an amino acid substitution, addition or deletion, preferably an amino acid substitution, more preferably an amino acid conservative substitution.

[0199] In one embodiment, the amino acid modification described in the present invention occurs in a region outside of the CDR (e.g., in the FR). In one embodiment, the substitution is a conservative substitution. A conservative substitution refers to the substitution of an amino acid by another amino acid within the same class, such as an acidic amino acid by another acidic amino acid, a basic amino acid by another basic amino acid, or a neutral amino acid by another neutral amino acid.

[0200] Exemplary substitutions are shown in the following table:

[0201]

[0202]

[0203] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein to produce Fc region variants. Fc region variants may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions. In a specific embodiment, the Fc region of the bispecific antibody described herein has a knob in hole and a disulfide bridge to promote and stabilize the structure of the bispecific antibody. In one embodiment, the bispecific anti-DR5 / CEACAM5 antibody of the present invention comprises amino acid replacements at positions 234 and 235 (EU numbering) of the heavy chain. In a specific embodiment, the amino acid replacements are L234A and L235A (also referred to as "LALA mutations").

[0204] The present invention also provides a single-chain scFv antibody targeting CEACAM5. In a preferred embodiment, the VH region and the VL region of the single-chain scFv antibody of the present invention are covalently linked together by a flexible linker. As known in the art, in the construction of scFv, preferably, the linker will facilitate the pairing of VH and VL, and will not interfere with the formation of a functionally effective antigen binding site for VH and VL. In a preferred embodiment, the order of the scFv antibody from N-terminus to C-terminus is VH-VL, wherein VH is connected to VL via a linker (G4S)4 polypeptide.

[0205] In certain embodiments, it may be desirable to generate antibodies engineered with cysteine, such as "thio mAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In a preferred embodiment, cysteine ​​amino acid mutations are introduced into VH44 and VL100 (Kabat numbering convention), respectively, to form interchain disulfide bridges to stabilize the scFv structure.

[0206] III. Nucleic acids of the present invention and vectors and host cells containing the same

[0207] In one aspect, the present invention provides nucleic acids encoding the above bispecific antibodies or antigen-binding fragments thereof targeting DR5 and CEACAM5. The present invention also encompasses nucleic acids that hybridize with the above nucleic acids under stringent conditions, nucleic acids that have one or more substitutions (e.g., conservative substitutions), deletions or insertions compared to the above nucleic acids, or nucleic acid sequences that have at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity compared to the above nucleic acids.

[0208] On the other hand, the present invention provides a vector comprising the above-mentioned nucleic acid. In a preferred embodiment, the vector is an expression vector. It is fully understood by those skilled in the art that the vectors commonly used in the technical field to which the present invention belongs can be applied to the present invention.

[0209] In one embodiment, the present invention provides a host cell comprising the nucleic acid or the vector.

[0210] Host cells are any type of cell system that can be used to produce antibody molecules of the present invention, including eukaryotic cells, for example, mammalian cells (e.g., CHO cells or HEK293 cells), insect cells, yeast cells; and prokaryotic cells, for example, E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0211] IV. Compositions of the Invention

[0212] In some embodiments, the present invention provides a composition comprising a bispecific antibody or antigen-binding fragment thereof targeting DR5 and CEACAM5 as described herein, preferably a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. The term "pharmaceutical excipient" refers to a diluent, adjuvant, carrier, excipient or stabilizer, etc., which is administered together with an active substance.

[0213] In one embodiment, a composition (e.g., a pharmaceutical composition) comprises an anti-DR5 / CEACAM5 bispecific antibody or an antigen-binding fragment thereof of the present invention and a combination of one or more other therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators). In a preferred embodiment, the other therapeutic agent is selected from 5-fluorouracil, oxaliplatin, exotecan, and SN-38.

[0214] The pharmaceutical composition of the present invention may also include one or more other therapeutic agents, which are required for the specific indications being treated, and include any substance effective in preventing or treating tumors (e.g., cancer) and infections (e.g., chronic infections), preferably with those therapeutic agents that do not adversely affect each other's activities. For example, it is desirable to also provide other anti-cancer active therapeutic agents, such as chemotherapeutics, cytotoxic agents, vaccines, other antibodies, anti-infective active agents, small molecule drugs or immunomodulators, etc. The therapeutic agents are suitably combined in an amount effective for the intended application. In a preferred embodiment, other therapeutic agents are selected from 5-fluorouracil, oxaliplatin, exotecan, SN-38.

[0215] V. Preparation of Antibodies of the Invention

[0216] In one embodiment, the present invention provides a method for preparing an anti-DR5 / CEACAM5 bispecific antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an anti-DR5 / CEACAM5 bispecific antibody or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the anti-DR5 / CEACAM5 bispecific antibody, and optionally isolating the anti-DR5 / CEACAM5 bispecific antibody. In a certain embodiment, the method further comprises recovering the anti-DR5 / CEACAM5 bispecific antibody from the host cell (or host cell culture medium).

[0217] The anti-DR5 / CEACAM5 bispecific antibodies of the present invention can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these are obvious to those skilled in the art. The purity of the anti-DR5 / CEACAM5 bispecific antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including molecular exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0218] VI. Uses and therapeutic methods of the antibodies of the present invention

[0219] The present application provides use of an anti-DR5 / CEACAM5 bispecific antibody in preparing a medicament for treating cancer.

[0220] In one embodiment, the therapeutically effective amount of the anti-DR5 / CEACAM5 bispecific antibody disclosed herein or a pharmaceutical composition comprising the same can be used to treat cancer expressing CEACAM5. In one embodiment, the therapeutically effective amount of the anti-DR5 / CEACAM5 bispecific antibody disclosed herein or a pharmaceutical composition comprising the same can be used to promote apoptosis of cancer cells expressing CEACAM5.

[0221] In some embodiments, the cancer is colorectal cancer, gastric cancer, lung cancer, or pancreatic cancer.

[0222] In another aspect, the present invention relates to a method for treating a tumor / cancer in a subject, comprising administering to the subject an effective amount of the anti-DR5 / CEACAM5 bispecific antibody disclosed herein, a nucleic acid encoding the same, or a pharmaceutical composition comprising the same.

[0223] In some embodiments, the cancer is colorectal cancer, gastric cancer, lung cancer, or pancreatic cancer.

[0224] The subject can be a mammal, eg, a primate, preferably, a higher primate, eg, a human (eg, a human patient having or at risk of having a cancer described herein).

[0225] In some embodiments, the treatment methods described herein further comprise administering to the subject or individual an anti-DR5 / CEACAM5 bispecific antibody disclosed herein or a pharmaceutical composition comprising the same, in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents.

[0226] The following examples are described to assist understanding of the present invention. The examples are not intended to and should not be interpreted in any way as limiting the scope of protection of the present invention.

[0227] Example

[0228] Example 1. Molecular design of anti-DR5 / CEACAM5 bispecific antibody

[0229] At present, although the prior art discloses a variety of monoclonal antibodies against DR5 or CEACAM5, no bispecific antibodies specifically targeting both have entered clinical trials. To this end, the present invention attempts to construct an anti-DR5 / CEACAM5 bispecific antibody targeting both for clinical application. As mentioned above, the construction of a druggable bispecific antibody requires consideration of multiple factors. Therefore, in the early stage of research and development, the inventor first constructed various forms of bispecific molecules based on the sequences of anti-DR5 or anti-CEACAM5 monoclonal antibodies known in the prior art, such as the attached Figure 1 As shown. The anti-DR5 antibody molecules used include conatumumab (US20190284290A1), drozitumab (US10501552B2), KMTR2 (CN100475848C), Lexatumumab (EP2016744669), Tigatuzumab (CN100506277C), LBY135 (US10501552B2), DR5mAb1 (US10501552B2), Hx01 (US20190315877) and Hx05 (US20190315877). Anti-CEACAM5 antibodies include hPR1A3 (WO2012117002), labetuzumab (US20150125386A1) and tusamitamab (CN104918958A). The above-mentioned various antibody molecules were assembled as follows Figure 1 The various bispecific antibody structures shown are shown in Table 1 for specific assembly methods.

[0230] Table 1

[0231]

[0232]

[0233]

[0234] The numbers shown in the antibody structure column in Table 1 correspond to Figure 1 The antibody structures of the corresponding numbers in .

[0235] According to the above combination, the amino acid sequences of the corresponding antibody molecules are obtained respectively, and the corresponding encoding nucleotide sequences are obtained according to conventional methods in the art, and constructed into separate expression vectors pcDNA3.1 for the heavy chain and light chain of each bispecific antibody molecule. These vectors contain corresponding elements conventionally used for expression and purification in eukaryotic cells and prokaryotic cells (e.g., regulatory elements for gene expression, selection markers) for use in subsequent experiments.

[0236] Example 2. Expression and purification of anti-DR5 / CEACAM5 bispecific antibody molecules

[0237] The expression vectors encoding each antibody molecule obtained in Example 1 were used to transfect CHO cells or 293 cells to express and purify the corresponding antibody molecules.

[0238] 2.1 Expression and purification in ExpiCHO cells

[0239] ExpiCHO cells (Invitrogen) were passaged according to the required cell volume, and the cell density was adjusted to 3.5 × 10 one day before transfection. 6 cells / ml. The cell density was tested on the day of transfection (approximately 8 to 10×10 6 cells / ml), the viability reached more than 95%, and ExpiCHO TM Expression Medium (Gibco Catalog Number: A29100-01) was used to adjust the cell density to 6 x 10 6 Take OptiPROTM SFM (Gibco catalog number: 12309-019) with a final volume of 8% (v / v) as the transfection buffer, add the corresponding amount (0.8 μg / mL cell) of the expression vector (plasmid), mix well, filter with a 0.22 μm filter membrane for sterilization, and add ExpiFectamine at 3.2 μL / mL. TMThe reagent in the CHO Transfection Kit (Gibco, Catalog No.: A29130) was used to incubate the complex formed by the transfection reagent and plasmid DNA at room temperature for 1 to 5 minutes, and then slowly added to the cells. After culturing for 18 hours at 37°C and 8% CO2, 0.6% (v / v) Enhancer and 30% (v / v) Feed were added and the culture was continued for 6 days.

[0240] The cell culture medium was centrifuged at 4000 rpm for 50 min, and the supernatant was collected and filtered through a 0.45 μM filter membrane, and purified by affinity chromatography and gel chromatography.

[0241] Affinity chromatography: The supernatant was purified using the pre-packed column Hitrap Mabselect Sure (GE, 11-0034-95). Before purification, the column was equilibrated with 5 column volumes of equilibration solution (20 mM Tris, 150 mM NaCl, pH 7.2); the collected supernatant was passed through the column, and the column was washed with 10 column volumes of equilibration solution to remove non-specific binding proteins; the column was washed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and the eluate was collected. The pH of each 1 ml of eluate was adjusted to 6.0 with 2 M Tris, and the concentration was determined.

[0242] Take 100 μg of purified protein, adjust the concentration to 1 mg / mL, and further purify the protein using a gel filtration column SW3000 (TOSOH catalog number: 18675). The gel chromatography purification operation steps are as follows: Select a Superdex 200Increase 10 / 300GL (GE Healthcare, catalog number: 28-9909-44) gel chromatography column and place it in the AKTApure system. Use 0.1M NaOH to remove endotoxins from the AKTApure system equipped with a Superdex 200Increase 10 / 300GL gel chromatography column for 2 hours, then wash the system and column with distilled water; use 2-5 column volumes of 1×PBS to balance the column until the conductivity and pH are stable; load the protein obtained by affinity chromatography, continue to elute with 1×PBS, remove impurities such as aggregates according to the ultraviolet absorption peak, and collect high-purity samples. Filter the sample with a 0.22μm membrane to determine the protein concentration.

[0243] 2.2 Expression and purification of FreeStyle 293-F cells

[0244] Expi293F cells (Thermo Fisher scientific company) were subcultured in Expi293F cell culture medium (Thermo Fisher scientific company). The cell density was checked one day before transfection and adjusted to 2×10 6 The cell density was adjusted to 3×10 cells / ml on the day of transfection. 6 cells / ml.

[0245] Take Opti-MEM medium (Gibco) with 1 / 10 of the final volume of the transfected Expi293F cells as the transfection buffer, add 10 μg of the corresponding expression vector (plasmid) per mL of transfection buffer, mix well, add 30 ug of polyethylenimine (PEI) (Polysciences, catalog number: 23966) per mL of transfection buffer, mix well, incubate at room temperature for 20 minutes, then gently pour the PEI / DNA mixture into the Expi293F cell suspension, mix well, and place in a shaker for culture under the conditions of 8% CO2, 36.5°C, and 120 rpm.

[0246] After 16-18 hours of culture, 1 / 50 of the volume of the culture after transfection, 200 g / L FEED (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a glucose solution with a final concentration of 4 g / L, and VPA (Gibco, catalog number: 11140-050) with a final concentration of 2 mM / L were added to the culture flask, gently mixed, and placed in a shaking table at 8% CO2, 36.5°C, and 120 rpm for continued culture. After continuous culture for 6 days, the culture was collected, centrifuged at 4000 rpm for 30 minutes, the cell supernatant was filtered with a 0.22 μM filter membrane, and the antibody was purified by affinity chromatography and gel chromatography as described above.

[0247] Example 3. In vitro efficacy of anti-DR5 / CEACAM5 bispecific antibody in inducing tumor cell apoptosis

[0248] The various bispecific antibody molecules expressed and purified in Example 2 were used for in vitro activity evaluation experiments. The tumor cells used in this example include human colon adenocarcinoma LoVo cells (ATCC), human colon adenocarcinoma LS174T cells (Cell Bank of the Chinese Academy of Sciences), human cecal adenocarcinoma NCI-H508 cells (Nanjing Kebai), human colon adenocarcinoma GP2d cells (Nanjing Kebai), and human rectal adenocarcinoma HT55 cells (Nanjing Kebai).

[0249] Induce tumor cell apoptosis in vitro by:

[0250] 1. After digesting the tumor cells with trypsin (Gibco, 25200-072) into a single-cell suspension, the cells were plated into a 96-well white-bottom plate (Shanghai Wohong, WHB-96-03) at a density of 1000 cells / well and cultured in a 37°C 5% CO2 incubator overnight;

[0251] 2. Dilute the antibody to be tested in a 96-well plate (Corning, 3799) in a gradient manner, add the diluted antibody to the 96-well white-bottom plate on which the tumor cells have been laid, and culture the plate in a 37°C 5% CO2 incubator for 24-72 hours;

[0252] 3. Take out the CellTiter-Glo (Promega, G7572) reagent and the tumor cell culture plate in step 2, return to room temperature, and add CellTiter-Glo to the tumor cells to be tested according to the detection method in the reagent instruction manual. Keep it at room temperature and away from light for 15-30 minutes, and read the detection value using a multi-function microplate reader (Molecular Devices, Spectra MAXi3);

[0253] 4. The relative cell viability was calculated with the control antibody Ctrl.hIgG (Merck, AG711) as 1 to reflect the activity of the antibody molecule to be tested in inducing apoptosis in vitro.

[0254] The in vitro apoptotic activity of various bispecific antibody molecules on various tumor cells can be found in Figure 2 -6. From the results, it can be seen that: 1) the anti-DR5 antibody in the form of a complete antibody and the anti-CEACAM5 antibody in the form of a scFv have Figure 1 -Antibodies with structure 1 mode (configuration) have better activity than other structures; 2) Anti-DR5 / CEACAM5 bispecific antibody molecules under the same configuration, different antibody sequences have different activities on cell apoptosis. The above results show that the structural mode (configuration) and sequence of the bispecific antibody molecule affect the activity of the bispecific antibody molecule, among which the bispecific antibody molecule KM-hPR1A3 exhibits the strongest activity of inducing cell apoptosis. In the antibody molecule KM-hPR1A3, the scFv is connected to the C-terminus of the heavy chain of the anti-DR5 antibody and has a VL-linker-VH structure.

[0255] The bispecific antibody KM-hPR1A3 has the following heavy chain and light chain sequences:

[0256] Heavy chain

[0257] QVQLVQSGAEMKKPGASVKVSCKTSGYTFTNYKINWVRQAPGQGLEWMGWMNPDTDSTGYPQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCARSYGSGSYYRDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSGGGSDIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQCLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSS(SEQ ID NO:59)

[0258] Light chain

[0259] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:60)

[0260] Example 4. Anti-DR5 / CEACAM5 bispecific antibody induces apoptosis of various tumor cells

[0261] The in vitro activity of the anti-DR5 / CEACAM5 bispecific molecule in inducing apoptosis of various tumor cells within 24 hours was detected using the KM-hPR1A3 molecule. For specific experimental methods, see Example 3. The experimental results are shown in Table 2.

[0262] Table 2

[0263]

[0264]

[0265]

[0266] It can be seen that the anti-DR5 / CEACAM5 bispecific molecule KM-hPR1A3 has a stronger activity in inducing tumor cell apoptosis than the anti-DR5 monoclonal antibody.

[0267] Example 5. Optimization of drugability of anti-DR5 / CEACAM5 bispecific antibodies

[0268] In this example, the anti-DR5 antibody sequence and the anti-CEACAM5 antibody sequence of the KM-hPR1A3 molecule having the antibody structure 1 were optimized respectively to improve the drugability of the molecule, and finally the KMmut1-HL1 molecule sequence was optimized.

[0269] 1. Optimization of drugability of anti-DR5 antibody sequence

[0270] The sequence of the anti-DR5 antibody of the present invention was modified as shown in Table 3. The modified antibody molecule was expressed and purified, and its related drug properties were detected. The data are shown in Table 4. According to the method disclosed in Example 3, the in vitro activity of the modified antibody molecule in inducing cell apoptosis was detected. The data are shown in Figure 7 .

[0271] Table 3

[0272]

[0273]

[0274] Wherein KMTR2 is the monoclonal antibody sequence disclosed in WO2002094880.

[0275] Maintaining integrity and monomer non-aggregation is critical for the processability, activity and stability of antibody products. To this end, the thermal stability of the engineered antibodies was evaluated by dynamic light scattering (DLS) and differential scanning calorimetry (DSC), as follows:

[0276] Dynamic Light Scattering (DLS) to Assess Antibody Thermal Stability

[0277] Dynamic light scattering (DLS) uses laser to illuminate small particles in Brownian motion in a solution and detects changes in the intensity of scattered light, thereby measuring the particle size distribution of the particles. Therefore, DLS can be used to detect the particle size of proteins in a solution and their stability at different temperatures or concentrations. A 1 mg / ml solution containing each mutant antibody molecule was centrifuged at 13000G / min for 5 minutes and then added to the sample plate, which was then centrifuged for 1 minute in a plate centrifuge to remove bubbles. The changes in the size of protein molecules during the continuous temperature rise were detected, and the detection settings were DLS acquisition time of 5s, acquisition times of 5 times, and experimental temperature of 25-85°C. After the experiment, the changes in sample particle size with temperature were analyzed.

[0278] Differential Scanning Calorimetry (DSC) to Assess Antibody Thermal Stability

[0279] Differential scanning calorimetry (DSC) is an analytical method that measures the energy difference between a sample and a reference substance as it changes with temperature under programmed temperature conditions. As the temperature rises, the protein structure changes, and the accompanying heat changes are recorded by the differential scanning calorimeter into the DSC curve. The Tm value of the protein is obtained by analyzing the DSC curve. Specifically, the antibody mutant samples were diluted to 0.5-1 mg / ml with PBS solution, the diluted samples and PBS buffer were degassed, and then the samples were added to the left sample plate, and PBS buffer was added to the corresponding position in the right reference plate. The starting temperature was 30°C and the equilibrium time was 10min; heating was performed at a rate of 1°C / min, and the end temperature was 90°C.

[0280] The prior art did not modify the NTS glycosylation modification site of the KMTR2 molecule. In this application, after multiple experiments, it was mutated to DTS, thereby improving the thermal stability of the antibody and reducing the heterogeneity of the bispecific antibody molecule. In addition, in order to block the interaction between the antibody Fc and the Fc receptor, the leucine at positions 234 and 235 of the Fc region of the KMTR2 antibody were mutated to alanine.

[0281] By testing the in vitro activity of the antibody in inducing tumor cell apoptosis, and evaluating the thermal stability of the antibody by dynamic light scattering (DLC) and differential scanning calorimetry (DSC), the DR5 mutant antibody KMmut1 has a higher maximum activity in inducing tumor cell apoptosis and better drugability.

[0282] Table 4

[0283]

[0284]

[0285] Note: “None” in the table means that the sequence has no relevant data due to poor activity, and subsequent drugability analysis has been abandoned.

[0286] 2. Optimization of the drugability of anti-CEACAM5 antibody sequences

[0287] After optimizing the anti-DR5 antibody modified sequence KMmut1, the sequence of the anti-CEACAM5 antibody scFv part of the bispecific antibody was further modified. The modified molecular sequence is shown in Table 5. Based on the hPR1A3 molecule, its variable region was constructed into a scFv in the form of VL-linker-VH from the N-terminus to the C-terminus. In order to stabilize the scFv structure and reduce protein aggregation, cysteine ​​amino acid mutations were introduced at H44 of VH and L100 of VL (Kabat numbering rules), respectively, to form an interchain disulfide bridge.

[0288] Table 5

[0289]

[0290]

[0291]

[0292] Then, according to the method disclosed in the example, the complete anti-DR5 / CEACAM5 bispecific antibody molecule was evaluated for in vitro activity and drugability. For specific molecular design, please refer to Table 6, and for molecular activity detection, please refer to Figure 8For the evaluation of the drugability of the molecule, please refer to Table 7. When evaluating the drugability of the molecule, in order to detect whether the order of VH and VL in the scFv molecule affects the function of the bispecific antibody molecule, a KMmut1-hPR1A3-v1 molecule was constructed in which the scFv was composed in the form of VH-linker-VL.

[0293] Table 6

[0294]

[0295] Table 7

[0296]

[0297]

[0298] After screening, the KMmut1-HL1 molecule had the best drugability, and the protein yield was improved, and the purity after one-step affinity purification of protein A was also improved. The KMmut1-hPR1A3-v1 molecule with scFv in the form of VH-linker-VL showed the worst drugability, indicating that in bispecific antibody molecules, the arrangement of VH and VL in the scFv structure has a great influence on the drugability of the antibody molecule.

[0299] After the above transformation, the preferred Figure 1 The anti-DR5 / CEACAM5 bispecific antibody molecule with structure A (also called KMmut1-HL1) has improved yield and reduced protein molecule heterogeneity compared to the parental monoclonal antibody and sequence-unmodified bispecific antibody molecules, and reduces the formation of polymers in the one-step purification method, thereby improving the overall drugability.

[0300] The anti-DR5 / CEACAM5 bispecific antibody molecule (KMmut1-HL1) is composed of two identical heavy chains and two identical light chains, and the sequences of the heavy chains and light chains are shown below:

[0301] HC:

[0302] QVQLVQSGAEMKKPGASVKVSCKTS GYTFTNYKIN WVRQAPGQGLEWMG WMNPDTDSTGYPQKFQG R

[0303] VTMTRDTSISTAYMELSSLRSEDTAVYYCAR SYGSGSYYRDYYYGMDV WGQGTTVTVSSASTKGPSVFP

[0304] LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI

[0305] CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED

[0306] PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA

[0307] KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL

[0308] TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSGGGSDIQMTQSPSSLSASVGDRVTIT

[0309] CKASAAVGNNVAWYQQKPGKAPKLLIY LASYRKR GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC HkDJ Y

[0310] TYPLFT FGCGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKAS GYTFTEFG

[0311] MN WVRQAPGQCLEWMG WINTKTGEATYVEEFKG RVTFTTDTSTSTAYMELRSLRSDDTAVYYCAR WDF AYYVEAMDY WGQGTTVTVSS(SEQ ID NO:17)

[0312] LC:

[0313] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:18)

[0314] The CDR sequences, heavy chain variable region sequences, and light chain variable region sequences contained in the KMmut1-HL1 molecule are shown in the following table, wherein the CDR in the heavy chain variable region is determined according to the AbM coding rule; and the CDR in the light chain variable region is determined according to the Kabat coding rule:

[0315]

[0316] Example 6. Affinity detection of anti-DR5 / CEACAM5 bispecific antibody molecule KMmut1-HL1

[0317] The anti-DR5 / CEACAM5 bispecific antibody molecule KMmut1-HL1 of the present invention can bind to human and cynomolgus monkey DR5 proteins, but not mouse DR5 protein; it can bind to human CEACAM5 protein, but not cynomolgus monkey CEACAM5 protein, and does not bind to other human CEACAM family proteins, and has good specificity.

[0318] The affinity (KD) of the bispecific antibody KMmut1-HL1 of the present invention for binding to DR5 and CEACAM5 was determined by biofilm thin layer interferometry (BLI). The specific method is as follows:

[0319] 1. Half an hour before the start of the experiment, according to the number of samples, take an appropriate number of AHC sensors (18-5060, Sartorius) and soak them in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20). The KMmut1-HL1 antibody and antigenic proteins human DR5 protein (TR2-H5229, Acro Biosystem), cynomolgus monkey DR5 protein (TR2-C52H7, AcroBiosystem), mouse DR5 protein (TR2-M52H5, Acro Biosystem), human CEACAM5 (CE5-H5226, AcroBiosystem) protein, cynomolgus monkey CEACAM5 protein (CE5-C52H5, Acro Biosystem), human CEACAM1 protein (CE1-H5220, Acro Biosystem), human CEACAM3 protein (11933-H08H, Sino Biological), human CEACAM6 protein (CE6-H5223, Acro Biosystem), and human CEACAM8 protein (CE8-H5224, Acro Biosystem) were diluted to 100 nM, respectively;

[0320] 2. Add SD buffer, antibody solution, and antigen to a 96-well black polystyrene microplate (Greiner, 655209). Use Fortebio Octet Red96e for detection, arrange the plate according to the sample position, and select the sensor position. The instrument setting parameters are as follows: running steps: 120s of equilibration baseline, 100s of adding solidified antigen, 120s of equilibration baseline, 100s of binding antibody, and 120s of dissociation, the speed is 1000rpm, and the temperature is 30℃. After the experiment is completed, the KD value is analyzed using ForteBioOctet analysis software. The results are shown in Table 8.

[0321] Table 8

[0322]

[0323] Example 7. Anti-DR5 / CEACAM5 bispecific antibody KMmut1-HL1 can induce apoptosis of various tumor cells

[0324] This example examines whether the excellent apoptotic tumor cell ability of the anti-DR5 / CEACAM5 bispecific antibody of the present application is generated by anchoring the bispecific antibody molecule to the tumor cells based on CEACAM5 on the tumor cells.

[0325] By replacing the anti-DR5 antibody sequence or the anti-CEACAM5 antibody sequence of the anti-DR5 / CEACAM5 bispecific antibody molecule KMmut1-HL1 of the present invention with the GP120 antibody sequence targeting the human immunodeficiency virus (HIV) envelope glycoprotein, respectively, a KMmut1-GP120 control molecule having the same structure as the KMmut1-HL1 of the present invention, targeting DR5 but not CEACAM5, and a GP120-HL1 control molecule targeting CEACAM5 but not DR5 can be obtained.

[0326] Referring to the following method for detecting apoptosis, the pro-apoptotic activities of the KMmut1-HL1 molecule of the present invention, the KMmut1-GP120 control molecule, the GP120-HL1 control molecule, the anti-human epidermal growth factor (EGFR) antibody Cetuximab and the topoisomerase I inhibitor irinotecan (Irinotecan, MCE, HY-16562) on various tumor cells were compared.

[0327] Experimental methods for inducing tumor cell apoptosis in vitro:

[0328] 1. The tumor cells: human colon cancer GP2d tumor cells (Nanjing Kebai), human colon cancer HT55 cells (Nanjing Kebai), human colorectal adenocarcinoma NCI-H508 cells (Nanjing Kebai), human gastric cancer MKN45 cells (Nanjing Kebai) and human pancreatic adenocarcinoma BxPC-3 cells (Cell Bank of the Chinese Academy of Sciences) were digested into single cell suspension with trypsin (Gibco, 25200-072), and then plated into 96-well white bottom plates (Shanghai Wohong, WHB-96-03) at a density of 1000 cells / well, and cultured in a 37°C 5% CO2 incubator overnight;

[0329] 2. The molecules to be tested were diluted in a gradient in a 96-well plate (Corning, 3799), added to the 96-well white-bottom plate with tumor cells, and cultured in a 37°C 5% CO2 incubator for 72 hours;

[0330] 3. Take out the CellTiter-Glo (Promega, G7572) reagent and the tumor cell culture plate to be tested, return to room temperature, and add CellTiter-Glo to the tumor cells to be tested according to the detection method in the reagent instruction manual. Keep it at room temperature and away from light for 15-30 minutes, and read the detection value using a multi-function microplate reader (Molecular Devices, Spectra MAXi3);

[0331] 4. The relative cell viability was calculated with the control antibody Ctrl.hIgG (Merck, AG711) as 1 to reflect the activity of the antibody molecule to be tested in inducing apoptosis in vitro.

[0332] The results showed that KMmut1-HL1 can induce apoptosis of various tumor cells in vitro, and its activity is better than that of the control molecules GP120-HL1, KMmut1-GP120, Cetuximab and irinotecan (Figure 9), indicating that CEACAM5 expressed on tumor cells further promotes the stronger apoptosis-inducing activity of DR5 antibody.

[0333] Example 8. Synergistic effect of anti-DR5 / CEACAM5 bispecific antibody KMmut1-HL1 in combination with other drugs

[0334] This example tests whether the KMmut1-HL1 molecule combined with other drugs can synergistically induce tumor cell apoptosis. The specific method is as follows:

[0335] 1. After digesting the tumor cells (human gastric cancer MKN45 cells, human colon cancer HT55 cells and human pancreatic adenocarcinoma BxPC-3 cells) with trypsin (Gibco, 25200-072) into a single cell suspension, the cells were plated into a 96-well white bottom plate (Shanghai Wohong, WHB-96-03) at a density of 1000 cells / well and cultured in a 37°C 5% CO2 incubator overnight;

[0336] 2. In a 96-well plate (Corning, 3799), KMmut1-HL1 was diluted from 100 nM to 9 concentration gradients by a 4-fold dilution method. Other drugs 5-fluorouracil (5-FU, MCE, HY-90006) was diluted from 100 nM, oxaliplatin (Oxaliplatin, MCE, HY-17371) was diluted from 100 μM, exatecan (Exatecan, MCE, HY-13631) was diluted from 1 μM, and SN-38 (MCE, HY-13704) was diluted from 100 nM to 5 concentration gradients by a 10-fold dilution method.

[0337] 3. KMmut1-HL1 and other drugs were mixed in a chessboard cross pattern to obtain a mixture system of the two drugs at various concentrations. The mixed system was added to a 96-well white-bottom plate with tumor cells, and cultured in a 37°C 5% CO2 incubator for 96 hours;

[0338] 4. Take out the CellTiter-Glo (Promega, G7572) reagent and the tumor cell culture plate to be tested, return to room temperature, and add CellTiter-Glo to the tumor cells to be tested according to the detection method in the reagent instruction manual. Keep it at room temperature and away from light for 15-30 minutes, and read the detection value using a multi-function microplate reader (Molecular Devices, Spectra MAXi3);

[0339] 5. The relative cell viability was calculated by taking the control antibody Ctrl.hIgG (Merck, AG711) as 1, and the synergistic effect score was calculated using SynergyFinder (DOI: 10.1093 / nar / gkac382).

[0340] The results are as follows Fig.10 As shown, the anti-DR5 / CEACAM5 bispecific antibody KMmut1-HL1 of the present invention can be used in combination with other drugs in vitro and have a synergistic effect.

[0341] Example 9. Anti-DR5 / CEACAM5 bispecific antibody KMmut1-HL1 can inhibit the growth of xenograft tumor cells in mice

[0342] This example evaluates the in vivo efficacy of the anti-DR5 / CEACAM5 bispecific antibody KMmut1-HL1. The specific method is as follows:

[0343] 1. NCI-H508 tumor cells (Nanjing Kebai), GP2d tumor cells (Nanjing Kebai) and HT55 tumor cells (Nanjing Kebai) were cultured according to the conventional cell subculture method. After reaching a sufficient number of cells, the cells were digested and centrifuged to collect the cells. After the cells were resuspended in PBS, they were mixed with Matrigel Matix (Corning, 356231) in equal proportions to prepare a cell concentration of 20×10 6 Cell suspension of 100 cells / ml;

[0344] 2. Take 0.2 ml of cell suspension and inoculate subcutaneously into the right abdominal area of ​​6-8 week old SPF female NOG immunodeficient mice (Viton River) to establish a tumor-bearing mouse model and monitor the growth of subsequent subcutaneous inoculations;

[0345] 3. Wait until the subcutaneous tumor volume reaches about 200mm 3 Afterwards, the tumor-bearing mice were divided into groups so that the average tumor size in each group was similar, and the mice were intraperitoneally injected with the drug to be tested, KMmut1-HL1 molecule; Cetuximab + irinotecan + 5-fluorouracil (5-FU, MCE, HY-90006); KMmut1-GP120 + GP120-HL1 control molecule (for specific dosage, see Figure 11);

[0346] 4. Administer the drug once a week for three to four weeks, monitor the tumor volume and body weight of the mice twice a week, and record the data;

[0347] 5. Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated using the following formula: V = L*W2 / 2. Body weight was measured using an electronic balance. For tumors larger than 2000 mm 3 Mice with a body weight loss of more than 20% were euthanized.

[0348] The results are shown in FIG11 . Compared with other groups, the anti-DR5 / CEACAM5 bispecific antibody KMmut1-HL1 exhibited good anti-tumor efficacy in vivo ( Fig.11A ), and even at a dose of 1 mg / kg, a better tumor inhibition effect was achieved than that of the Cetuximab+irinotecan+5-FU (10 mg / kg+10 mg / kg+10 mg / kg) group ( Figures 11B-11C ), indicating that the antibody has favorable anti-tumor activity.

[0349] Sequences used in constructing bispecific antibodies in this application

[0350]

Claims

1. An anti-DR5 / CEACAM5 bispecific antibody, comprising a complete antibody that recognizes a DR5 molecule and a scFv that recognizes a CEACAM5 molecule, wherein: A. The bispecific antibody comprises two identical heavy chains of structure VH-CH1-Fc-scFv, and two identical light chains of structure VL-CL, wherein the VL of the two light chains are paired with the VH of the two heavy chains to form two antigen recognition sites for recognizing DR5 molecules, and each scFv forms an antigen recognition site for recognizing CEACAM5 molecules, wherein the scFv is connected to the C-terminus of Fc and the scFv has VL from the N-terminus to the C-terminus. CEACAM5 -VH CEACAM5 structure; or B. The bispecific antibody comprises a heavy chain with a structure of VH-CH1-Fc-scFv, a heavy chain with a structure of VH-CH1-Fc, and two identical light chains with a structure of VL-CL, wherein the two heavy chains have the same VH-CH1-Fc, and the VLs of the two light chains are paired with the VHs of the two heavy chains to form two antigen recognition sites that recognize the DR5 molecule, and the scFv forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of the Fc and the scFv has VL from the N-terminus to the C-terminus. CEACAM5 -VH CEACAM5 structure.

2. The bispecific antibody of claim 1, wherein: A. When the bispecific antibody comprises two identical heavy chains of structure VH-CH1-Fc-scFv and two identical light chains of structure VL-CL, The VH that recognizes the DR5 molecule comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the VL that recognizes the DR5 molecule comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; and VH of scFv recognizing CEACAM5 CEACAM5 It comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:11, and recognizes the VL of CEACAM5 CEACAM5 comprising LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 39, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:22, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:24; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 30, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:22, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:24; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 34, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 34, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 30, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 39, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 34, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 30, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:38, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:40; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 contained in SEQ ID NO: 39, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20; or B. When the bispecific antibody comprises one heavy chain with a structure of VH-CH1-Fc-scFv, one heavy chain with a structure of VH-CH1-Fc, and two identical light chains with a structure of VL-CL, The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 39, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 30, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 34, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28; and VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 39, and VL CEACAM5 comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28; VH of scFv that recognizes CEACAM5 molecules CEACAM5 comprising HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 34, and VL CEACAM5 It comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:

36.

3. The bispecific antibody according to claim 1 or 2, wherein: A. When the bispecific antibody comprises two identical heavy chains of structure VH-CH1-Fc-scFv and two identical light chains of structure VL-CL, The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 7, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 8; and VH of scFv recognizing CEACAM5 CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 15 and recognizing the CEACAM5 molecule CEACAM5 comprising the sequence shown in SEQ ID NO:16; or The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 22, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO: 24; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 34 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:36; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 22, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO: 24; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 30 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:32; or The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 26, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 28; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 34 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:36; or The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 26, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 28; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 30 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:32; or The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 26, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 28; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20; or The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 34 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:36; or The VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO: 8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 30 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:32; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:38, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:40; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO:41 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 43 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 45 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 47 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 49 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 51 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:54; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 Comprising the sequence shown in SEQ ID NO:56; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL that recognizes the DR5 molecule comprises the sequence of SEQ ID NO:8; VH that recognizes CEACAM5 molecules CEACAM5 A VL comprising the sequence shown in SEQ ID NO: 39 and recognizing the CEACAM5 molecule CEACAM5 It comprises the sequence shown in SEQ ID NO:

58.

4. The bispecific antibody according to claim 1 or 2, wherein: When the bispecific antibody comprises one heavy chain with a structure of VH-CH1-Fc-scFv, one heavy chain with a structure of VH-CH1-Fc, and two identical light chains with a structure of VL-CL, The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 8; VH of scFv that recognizes CEACAM5 molecules CEACAM5 Contains the sequence shown in SEQ ID NO: 39, and VL CEACAM5 Comprising the sequence shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 8; VH of scFv that recognizes CEACAM5 molecules CEACAM5 Contains the sequence shown in SEQ ID NO: 30, and VL CEACAM5 Comprising the sequence shown in SEQ ID NO:32; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 8; VH of scFv that recognizes CEACAM5 molecules CEACAM5 Contains the sequence shown in SEQ ID NO: 34, and VL CEACAM5 Comprising the sequence shown in SEQ ID NO:36; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 28; VH of scFv that recognizes CEACAM5 molecules CEACAM5 Contains the sequence shown in SEQ ID NO: 39, and VL CEACAM5 Comprising the sequence shown in SEQ ID NO:20; or The VH that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises the sequence shown in SEQ ID NO: 28; VH of scFv that recognizes CEACAM5 molecules CEACAM5 Contains the sequence shown in SEQ ID NO: 34, and VL CEACAM5 It comprises the sequence shown in SEQ ID NO:

36.

5. An anti-DR5 / CEACAM5 bispecific antibody, comprising an antibody or an antigen-binding fragment thereof that recognizes a DR5 molecule and an antibody or an antigen-binding fragment thereof that recognizes a CEACAM5 molecule, wherein: The VH that recognizes the DR5 molecule comprises HCDR1 as shown in SEQ ID NO: 1, HCR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the VL that recognizes the DR5 molecule comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; and VH that recognizes CEACAM5 molecules CEACAM5 It comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:11, and recognizes the VL of CEACAM5 CEACAM5 It comprises LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:

14.

6. The bispecific antibody of claim 5, wherein: The VH that recognizes the DR5 molecule comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO:7, or consists of SEQ ID NO:7; and the VL that recognizes the DR5 molecule comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:8, or consists of SEQ ID NO:8; and VH that recognizes CEACAM5 CEACAM5 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as shown in SEQ ID NO: 15, or consisting of SEQ ID NO: 15; and recognizing the VL of the CEACAM5 molecule CEACAM5 Comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:16, or consisting of SEQ ID NO:

16.

7. The bispecific antibody of claim 5 or 6, wherein the antibody or antigen-binding fragment thereof that recognizes the DR5 molecule is selected from Fab, Fab', (Fab')2 or a complete antibody, and the antibody or antigen-binding fragment thereof that recognizes the CEACAM5 molecule is selected from scFv, scFab, Fab or Fv.

8. The bispecific antibody of any one of claims 5 to 7, wherein: The bispecific antibody comprises a complete antibody that recognizes the DR5 molecule and a scFv that recognizes the CEACAM5 molecule, wherein: The bispecific antibody comprises two identical heavy chains of structure VH-CH1-Fc-scFv, and two identical light chains of structure VL-CL, wherein the VL of the two light chains are paired with the VH of the two heavy chains to form two antigen recognition sites for recognizing DR5 molecules, and each scFv forms an antigen recognition site for recognizing CEACAM5 molecules, wherein the scFv is connected to the C-terminus of Fc and the scFv has VL from the N-terminus to the C-terminus. CEACAM5 -VH CEACAM5 structure; or The bispecific antibody comprises a heavy chain with a structure of VH-CH1-Fc-scFv, a heavy chain with a structure of VH-CH1-Fc, and two identical light chains with a structure of VL-CL, wherein the two heavy chains have the same VH-CH1-Fc, and the VLs of the two light chains are paired with the VHs of the two heavy chains to form two antigen recognition sites for recognizing DR5 molecules, and the scFv forms an antigen recognition site for recognizing CEACAM5 molecules, wherein the scFv is connected to the C-terminus of Fc and the scFv has VL from the N-terminus to the C-terminus. CEACAM5 -VH CEACAM5 structure.

9. The bispecific antibody of any one of claims 5 to 8, wherein: The bispecific antibody comprises a complete antibody that recognizes the DR5 molecule and a scFv that recognizes the CEACAM5 molecule, wherein the bispecific antibody comprises two identical heavy chains with a structure of VH-CH1-Fc-scFv and two light chains with a structure of VL-CL, wherein the VLs of the two light chains are paired with the VHs of the two heavy chains to form two antigen recognition sites that recognize the DR5 molecule, and the scFvs on each heavy chain form antigen recognition sites that recognize the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of the Fc and the scFv has VL from the N-terminus to the C-terminus. CEACAM5 -VH CEACAM5 structure.

10. The bispecific antibody of any one of claims 1 to 9, wherein: The antibody or antigen-binding fragment recognizing the DR5 molecule comprises an Fc region, and the Fc is an IgG Fc, such as an Fc from IgG1, IgG2, IgG3 or IgG4, preferably an Fc from human IgG1.

11. The bispecific antibody of any one of claims 1 to 10, wherein: The antibody or antigen-binding fragment recognizing the DR5 molecule comprises an Fc region, wherein the Fc region comprises mutations, such as mutations that increase the stability of the bispecific antibody dimer (such as disulfide bridge structure) and mutations that reduce effector function (such as L234A and L235A mutations).

12. The bispecific antibody of any one of claims 1 to 11, wherein: The antibody or antigen-binding fragment recognizing the DR5 molecule comprises an Fc region, and the Fc region comprises L234A and L235A mutations.

13. The bispecific antibody of any one of claims 1-12, wherein the scFv recognizing the CEACAM5 molecule comprises cysteine ​​mutations at H44 of VH and L100 of VL (Kabat numbering convention), thereby forming an interchain disulfide bridge.

14. The bispecific antibody according to any one of claims 1 to 13, wherein The antibody or antigen-binding fragment that recognizes the DR5 molecule comprises an Fc region, wherein the Fc region comprises L234A and L235A mutations; the scFv that recognizes the CEACAM5 molecule comprises cysteine ​​amino acid mutations at H44 of VH and L100 of VL (Kabat numbering rules).

15. The bispecific antibody of any one of claims 1 to 14, wherein: The antibody or antigen-binding fragment recognizing the DR5 molecule comprises an Fc region, and the scFv recognizing the CEACAM5 molecule is connected to the C-terminus of the Fc via a linker 2, wherein the linker 2 is (G3S)n or (G4S)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, or 9.

16. The bispecific antibody according to any one of claims 1 to 15, wherein In the scFv that recognizes CEACAM5 molecules, VH CEACAM5 With VL CEACAM5 Connected through linker 1, wherein linker 1 is (G3S)n or (G4S)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9.

17. The bispecific antibody of any one of claims 1 to 16, wherein: The bispecific antibody comprises a complete antibody that recognizes DR5 molecules and a scFv that recognizes CEACAM5 molecules, wherein the scFv is connected to the C-terminus of Fc via a linker 2. CEACAM5 With VL CEACAM5 Connected by linker 1, linker 2 is (G3S)3, and linker 1 is (G3S)4.

18. The bispecific antibody of any one of claims 1-17, wherein the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

17.

19. The bispecific antibody of any one of claims 1-18, wherein the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

18.

20. The bispecific antibody of any one of claims 1-19, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

18.

21. An isolated polynucleotide molecule encoding the bispecific antibody of any one of claims 1-20.

22. A vector comprising the polynucleotide molecule according to claim 21, preferably the vector is an expression vector.

23. A host cell comprising the vector of claim 22 or the polynucleotide molecule of claim 21.

24. A method for producing the anti-DR5 / CEACAM5 bispecific antibody according to any one of claims 1 to 20, comprising the steps of: (i) culturing the host cell of claim 23 under conditions suitable for expressing the bispecific antibody, optionally, (ii) recovering the anti-DR5 / CEACAM5 bispecific antibody. 25 . A pharmaceutical composition comprising the anti-DR5 / CEACAM5 bispecific antibody according to any one of claims 1 to 20, and a pharmaceutically acceptable carrier.

26. A pharmaceutical combination comprising the anti-DR5 / CEACAM5 bispecific antibody according to any one of claims 1 to 20 and other therapeutic agents, and optional pharmaceutical excipients; preferably, the other therapeutic agent is selected from 5-fluorouracil, oxaliplatin, exotecan, and SN-38.

27. Use of the anti-DR5 / CEACAM5 bispecific antibody of any one of claims 1 to 20, the pharmaceutical composition of claim 25, or the pharmaceutical combination of claim 26 in the preparation of a medicament for treating cancer.

28. The use of claim 27, wherein the cancer is selected from colorectal cancer, gastric cancer, lung cancer, pancreatic cancer.

29. A method for treating cancer in a subject, comprising administering to the subject an effective amount of the anti-DR5 / CEACAM5 bispecific antibody of any one of claims 1-20, or the pharmaceutical composition of claim 25, or the pharmaceutical combination of claim 26.

30. The method of claim 29, wherein the cancer is selected from colorectal cancer, gastric cancer, lung cancer, pancreatic cancer.

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