Bispecific antibodies against CEACAM5 and CD47

Abstract

Providing bispecific antibodies against CEACAM5 and CD47. [Solution] The present invention relates to bispecific antibodies that bind to the human carcinoembryonic antigen CEACAM5 and human CD47. The present invention also relates to polynucleotides encoding such bispecific antibodies, vectors, and host cells containing such polynucleotides. The present invention further relates to methods for selecting and producing such antibodies, and methods for using such antibodies in the treatment of diseases. The present invention also relates to the therapeutic use of bispecific antibodies in monotherapy and combination therapy.

Description

[Technical Field]

[0001] Sequence listing reference The contents of the electronically submitted sequence listing filed with this application are incorporated herein by reference in their entirety.

[0002] Field of Invention This invention relates to a bispecific antibody (CEAxCD47 bispecific antibody) that binds to the human carcinoembryonic antigen CEACAM5 (CEA) and human CD47. In addition, this invention relates to polynucleotides encoding such bispecific antibodies, vectors, and host cells containing such polynucleotides. This invention further relates to methods for selecting and producing such antibodies, and methods for using such antibodies in the treatment of diseases. This invention also relates, in particular, to the therapeutic use of CEAxCD47 bispecific antibodies in monotherapy and combination therapy with CEAxCD3 T cell bispecific antibodies (TCBs) and / or PD-1 or PD-L1 inhibitors. [Background technology]

[0003] Background of the Invention CEA belongs to the family of CEA-associated cell adhesion molecules (CEACAM), which includes 12 closely related human proteins encoded by 22 genes divided into the CEACAM and pregnancy-specific glycoprotein (PSG) subgroups on chromosome 19q13 (Beauchemin N & Arabzadeh A, Cancer Metastasis Rev. 2013). CEACAM is involved in various physiological processes, including cell-cell recognition, and modulates cellular processes ranging from tissue structure formation and neoangiogenesis to the regulation of insulin homeostasis and T cell proliferation; CEACAM has also been identified as a receptor for host-specific viruses and bacteria (Kuespert K et al., Curr Opin Cell Biol. 2006). CEA (CEACAM5 or CD66e; UniProtKB-P06731) is present in early embryonic and fetal development and maintains its expression in normal adult tissues. Its main expression sites are columnar epithelial cells and goblet cells of the colon, particularly the upper third of the crypts and the free lumen surface.

[0004] CEA is (over)expressed in epithelial tumors, including but not limited to colorectal, gastric, lung, and pancreatic cancers (Beauchemin N & Arabzadeh A, Cancer Metastasis Rev. 2013), loses its apical expression, and is distributed throughout the cell surface (Hammarstrom, Semin Cancer Biol 1999).

[0005] A method for treating CEA-expressing cancers using a combination of a human PD-1 axis antagonist and an anti-CEA / anti-CD3 bispecific antibody that reorients and activates T cells is described in U.S. Patent Application Publication 20140242079 and International Publication 2017118657 (both incorporated in their entirety by reference), and clinical results were presented at the ASCO Annual Meeting 2017 (Tabernero et al., J Clin Oncol). 35,2017(suppl;abstr 3002)).

[0006] A method for treating tumors by administering an immune checkpoint antagonist that binds to two or more different targets of the immune checkpoint pathway, and a T cell reinducer that binds to CEA and T cell surface antigens, is described in International Publication No. 2015112534. A class I antibody that binds to CEACAM5 and granulocytes is described in U.S. Patent Application Publication No. 20110064653.

[0007] Human CD47 (UniProtKB-Q08722 (CD47_human; IAP)) is a transmembrane protein that binds to its ligands, thrombospondin-1 (TSP-1) and signal regulatory protein alpha (SIRPα; CD172a; UniProtKB P78324), and can act as a "don't eat me" signal to the immune system, particularly to macrophages expressing SIRPα. Potent inhibition of SIRPα binding to CD47 on the surface of tumor cells (low IC50) is a means of increasing macrophage phagocytosis of tumor cells. CD47 is involved in various cellular processes, including apoptosis, proliferation, adhesion, and migration. Furthermore, it plays a crucial role in immune and angiogenic responses. CD47 is overexpressed in tumor cells derived from patients with both hematological and solid tumors. Antibodies against CD47 are currently documented and have shown promising preclinical and early clinical activity in various tumor entities, including hematological malignancies such as lymphoma and solid tumors, and gastric cancer (Weiskopf). K., European Journal of Cancer 76(2017)100-109; Huang Y et al., J Thorac Dis, 2017;9(2):E168-E174; Kaur et al., Antibody Therapeutics, 3(2020)179-192). Antibodies of the IgG1 subclass that bind to CD47 can cause Fc-dependent platelet depletion and a decrease in red blood cells (RBCs) and hemoglobin (see, for example, U.S. Patent Application Publication No. 20140140989). To avoid this adverse effect, International Publication No. 2017196793 describes mutant forms of the IgG4 subclass of anti-CD47 antibodies (IgG4PE with S228P and L235E mutations to reduce FcγR binding). Such anti-CD47 antibodies with significantly reduced FcγR binding and effector function do not cause such platelet depletion. Single-domain bispecific antibodies against CD47 and CD20 are von This has been described by Bommel PE et al. (Oncoimmunol. 7(2018)e386361 and Piccione EC et al., mAbs 7(2015)946-956). Dheilly E et al. (Mol. Thera. 25(2017)523-533; see also International Publication No. 2014087248) have described bispecific antibodies against CD19 and CD47.

[0008] Bispecific antibodies against CEACAM5 and CD47, comprising the common heavy chain (VH-CH1) of SEQ ID NO: 5 and the CD47 interaction variable light chain region VL of SEQ ID NO: 10, are described in International Publication No. 2019234576, European Patent No. 19213002, and U.S. Patent No. 62943726 (all of which are incorporated by reference). Bispecific antibodies against CD19 and CD47, comprising the common heavy chain of SEQ ID NO: 5 and the CD47 interaction variable light region VL of SEQ ID NO: 10, are described in International Publication No. 2014087248 (all of which are incorporated by reference). International Publication No. 2018098384 relates to bispecific antibodies that simultaneously target CD47 and CEACAM5. European Patent No. 3623388 relates to a bispecific binding molecule comprising a low-affinity tumor target arm and fusion protein for blocking the interaction between CD47 and SIRPα. International Publication No. 2018 / 057955 relates to a bispecific antibody that binds to both CD47 and mesothelin and contains a common heavy chain. International Publication No. 2019016411 relates to a bispecific antibody molecule that targets CD47 and tumor antigens.

[0009] Significant progress has been made in the treatment of hematological malignancies. This is in contrast to the progress made in the treatment of several types of advanced solid tumors. Despite some progress in the treatment of locally advanced or particularly metastatic solid tumor types, progression-free survival (PFS) and overall survival (OS) for patients with advanced cancers such as colorectal cancer, pancreatic cancer, and lung cancer remain quite limited, and there is usually no cure. Much hope has been placed on cancer immunotherapy, and there has been limited but definite success. Tumors develop mechanisms to protect their cells from destruction by other immune cells such as T effector cells and macrophages. Cancer immunotherapy-based strategies over the past decade have been somewhat successful in counteracting these tumor-protective mechanisms and reorienting T cells against cancer cells. The most notable example of such strategies is the inhibitor / activator of specific immune checkpoints. For example, checkpoint inhibitors such as PD-1 axis antagonists have been shown to reactivate T effector cells to fight certain solid tumors. However, not all solid tumor types are responsive to PD-1 axis antagonists, and even within the types of responsiveness, the percentage of patients who benefit from treatment with, for example, anti-PD-1 or PD-L1 antibodies is often considerably less than 50%. For example, less than 10% of patients with advanced colorectal cancer are candidates for treatment with PD-1 axis inhibitors (in particular, about 4% of patients with advanced colorectal cancer exhibiting microsatellite instability MSI in the cancer do benefit somewhat).

[0010] Adoptive T cell therapy using chimeric antigen receptor (CAR) T cells and treatment with T cell bispecific antibodies have yielded promising clinical results in hematological malignancies. However, clinical studies using adoptive T cell therapy, such as CAR T cells, in various solid tumors have mostly shown no response rate or only a very small response rate (e.g., Xu et al., Expert Review of Anticancer Therapy 2017, 17, 1099-1106; Greenbaum et al., Biol Blood Marrow Transplant 2020 Oct; 26(10): 1759-1769).

[0011] U.S. Patent Publication No. 20140242079, International Publication No. 2017055389, U.S. Patent Publication No. 20140242080, and Bacac et al. (Clin. Cancer Res., 22(13), 3286-97(2016)) (each incorporated in its entirety by reference) describe CEAxCD3 T cell bispecific antibodies. The T cell bispecific antibody in International Publication No. 2017055389 demonstrated significantly increased T cell activation potency / efficacy compared to cibisatamab in preclinical studies, and one of these higher-potency CEAxCD3 T cell bispecific antibodies was in clinical development (RO7172508 in NCT03539484). As used herein, “TCB2014” refers to a bispecific antibody that binds to CEA and CD3 in a 2+1 format, as described in U.S. Patent Application Publication No. 20140242080, and includes, as CDRs, the CDRs shown in SEQ ID NOs. 270-276 and 290-296 of U.S. Patent Application Publication No. 20140242080 (see also the CDRs in SEQ ID NOs. 4-10 and 24-30 of U.S. Patent Application Publication No. 20140242079, which are incorporated in their entirety by reference). Tabernero et al.'s presentation at the ASCO Annual Meeting 2017 (J Clin Oncol 35, 2017 (suppl;abstr 3002)) included Phase 1 clinical data from patients with advanced / metastatic colorectal cancer treated with the CEAxCD3 bispecific antibody RO6958688 (cibisatamab) as monotherapy and in combination with the anti-PD-L1 antibody atezolizumab. Stable disease and partial responses have been observed with cibisatamab monotherapy and in combination with the PD-L1 inhibitor atezolizumab. No new clinical data for cibisatamab CEAxCD3 have been published since 2017. One trial using Q3W 100 mg cibisatamab, the PD-L1 inhibitor atezolizumab, and pre-treatment with the B-cell killing anti-CD20 antibody obinutuzumab (to avoid the formation of anti-drug antibodies ADA, as reported for cibisatamab) was published in March 2019 (ClinicalTrials.gov identifier NCT038666339). To date, no data has been made public.Recently, a new clinical trial of Q3W 100 mg cibisatamab + atezolizumab + RO712290 in patients with advanced colorectal cancer exhibiting microsatellite stability was published (April 2021, NCT04826003). RO712290 is a bispecific fusion protein that binds to fibroblast-activating protein (FAP) and T cell costimulatory factor 4-1BB. When combined with CEAxCD3, it induces further T cell activation, resulting in increased efficacy / killing against tumor cells, but also increased toxicity, such as increased cytokine release. A further bispecific CEAxCD3 antibody, MEDI-565 (AMG211), which is a single-chain antibody, is in clinical development, and results from clinical trials using this antibody have been published (e.g., stable disease induction, see M. Pishvaian et al., Clin Colorectal Cancer. 2016 DEC;15(4)345-351).

[0012] Combining CEAxCD3 bispecific antibodies with PD-L1 inhibitors has been reported to improve efficacy. These data suggest that efficacy can be achieved with CEAxCD3 bispecific antibodies in advanced solid tumors. However, overall, monotherapy and even combinations with PD-L1 inhibitors were ineffective in most patients in clinical trials, as the majority of patients still had progressive disease. Those who responded showed up to partial responses and stable disease, but complete responses were not achieved.

[0013] One approach to enhance the efficacy of T cell bispecific antibodies, such as CEAxCD3 cibisatamab, is to combine them with a second drug that induces additional T cell activation via an agonist effect on costimulatory T cell receptors such as 4-1BB or CD28. A well-known side effect of T cell bispecific antibodies is the induction of cytokine release syndrome (CRS), which can be of a higher grade, e.g., grade 3 or even grade 5 (death). Adding a bispecific antibody targeting a T cell costimulatory receptor to a T cell bispecific antibody can lead to a significant increase in cytokine release and thus an increased risk of higher-grade CRS.

[0014] Another approach to obtain better results might involve adding a further checkpoint inhibitor or agonist, rather than simply adding a PD-1 checkpoint axis inhibitor to a T cell bispecific antibody. However, to date, there is no promising clinical data for such a combination approach in advanced solid tumors such as colorectal cancer. The limited availability of T cells within advanced solid tumors is certainly a key mechanism that limits the efficacy achievable with T cell bispecific antibodies + PD-1 axis inhibitors and / or other checkpoint inhibitors + bispecific agonists at T cell costimulatory receptors. The T-cell bispecific antibody TAAxCD3 (TAA = CEA and many other tumor-associated antigens) is highly effective in patients with hematological malignancies such as multiple myeloma, e.g., B-cell malignancies such as diffuse large B-cell lymphoma and follicular lymphoma. Clinical results for cibisatamab CEAxCD3 also show efficacy of TAAxCD3 in advanced solid tumors (see above), but at a much lower level than achieved in hematological malignancies. Adding a PD-1 axis inhibitor may add efficacy, but if any, it is limited. Adding an agonist bispecific antibody or fusion protein to a costimulatory T-cell receptor such as CD28 or 4-1BB increases efficacy in preclinical studies, but also increases toxicity, e.g., increased cytokine release. Instead of aiming for further activation of T cells, it may be more successful to add therapeutic agents that reorient tumor cells, other immune cells, especially macrophages. The present invention utilizes the bispecific antibody CEAxCD47, which reorients and activates macrophages against CEACAM5-expressing solid tumors, in 1. monotherapy and / or 2. combination therapy (particularly with a CEAxCD3 T-cell bispecific antibody), thereby increasing the tumor cell-killing effect of the CEAxCD3 bispecific antibody and avoiding the increased risk of CRS, in contrast to combinations with bispecific agonists targeting T-cell costimulatory receptors. Bispecific antibodies against CEACAM5 and CD47 are described in International Publication No. WO 2019 / 234576. One exemplary bispecific antibody described in International Publication No. WO 2019 / 234576 is K2AC22 (SEQ ID NO: 65 in International Publication No. WO 2019 / 234576 shows the light chain of the CEACAM5-binding portion of K2AC22, SEQ ID NO: 6 in International Publication No. WO 2019 / 234576 shows the common heavy chain of K2AC22, and SEQ ID NO: 10 shows the light chain of the CD47-binding portion of K2AC22). However, there is still a need for improved bispecific antibodies against CEACAM5 and CD47, such as improved antibodies that combine high efficacy with low toxicity, low immunogenicity, and favorable pharmacokinetic properties. Accordingly, an object of the present invention is to provide a novel bispecific antibody against CEACAM5 and CD47 that is more advantageous than prior art bispecific antibodies against CEACAM5 and CD47.

Prior Art Documents

Patent Documents

[0015]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0016] ​​​​​​​​​​​Beauchemin N & Arabzadeh A, Cancer Metastasis Rev. 2013

Non-Patent Document 4

Summary of the Invention

Means for Solving the Problems

[0017] The present invention provides a novel bispecific antibody having a first binding portion capable of binding to human CEACAM5 and a second binding portion capable of binding to human CD47. The bispecific antibody according to the present invention induces high phagocytic activity against tumor cells in both tumor cells that highly express CEACAM5 and tumor cells that lowly express CEACAM5. In one embodiment, the bispecific antibody induces its anti-tumor cell effects mainly through optimized phagocytosis / antibody-dependent cell phagocytosis (ADCP) by the involvement of immune cells, particularly macrophages. In one embodiment, the bispecific antibody according to the present invention shows a decrease in the ratio of binding affinity to CEACAM3 to CEACAM5 and an increase in the ratio of KD, respectively, compared to the CEACAM5-CD47 antibody K2AC22. In one embodiment, the bispecific antibody according to the present invention inhibits the binding of SIRPα to CD47 expressed on tumor cells and increases the phagocytosis of tumor cells. The disclosed bispecific antibody that specifically binds to human CEACAM5 and human CD47 is also suitable for use in the treatment of tumors, particularly solid tumors.

[0018] In one aspect, the present invention is a bispecific antibody (also further referred to as "CEAxCD47 bispecific antibody" or "bispecific antibody according to the present invention") comprising: 1. a first binding portion that specifically binds to human CEACAM5 (also further referred to as "CEA"), and a second binding portion that specifically binds to human CD47 (also further referred to as "CD47"), wherein a) the first binding portion comprises a heavy chain variable region comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3 as the heavy chain variable region, b) The first bonding region is a light chain variable region, b1) CDRL1 of sequence number 14, CDRL2 of sequence number 15, and CDRL3 of sequence number 16, or b2) CDRL1 of sequence number 17, CDRL2 of sequence number 18, and CDRL3 of sequence number 19, b3) CDRL1 of sequence number 20, CDRL2 of sequence number 21, and CDRL3 of sequence number 22, b4) CDRL1 of sequence number 23, CDRL2 of sequence number 24, and CDRL3 of sequence number 25, and b5) A light chain variable region including a set of CDRLs selected from the group consisting of CDRL1 of SEQ ID NO: 26, CDRL2 of SEQ ID NO: 27, and CDRL3 of SEQ ID NO: 28, c) The second binding portion includes a heavy chain variable region comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, as a heavy chain variable region. The present invention provides a bispecific antibody characterized by including light chain variable regions, specifically CDRL1 of SEQ ID NO: 7, CDRL2 of SEQ ID NO: 8, and CDRL3 of SEQ ID NO: 9.

[0019] The present invention includes further embodiments of this aspect.

[0020] In one embodiment, the present invention provides a bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, a) The first and second binding portions each include heavy chain variable regions, which include CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, respectively. b) The first binding portion includes a light chain variable region comprising CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16, c) The present invention relates to a bispecific antibody characterized in that the second binding portion includes a light chain variable region comprising CDRL1 of SEQ ID NO: 7, CDRL2 of SEQ ID NO: 8, and CDRL3 of SEQ ID NO: 9 as a light chain variable region.

[0021] In one embodiment, the present invention provides a bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, a) The first and second binding portions each include heavy chain variable regions, which include CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, respectively. b) The first binding portion includes a light chain variable region comprising CDRL1 of SEQ ID NO: 17, CDRL2 of SEQ ID NO: 18, and CDRL3 of SEQ ID NO: 19, c) The present invention relates to a bispecific antibody characterized in that the second binding portion includes a light chain variable region comprising CDRL1 of SEQ ID NO: 7, CDRL2 of SEQ ID NO: 8, and CDRL3 of SEQ ID NO: 9 as a light chain variable region.

[0022] In one embodiment, the present invention provides a bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, a) The first and second binding portions each include heavy chain variable regions, which include CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, respectively. b) The first binding portion includes a light chain variable region comprising CDRL1 of SEQ ID NO: 20, CDRL2 of SEQ ID NO: 21, and CDRL3 of SEQ ID NO: 22, c) The present invention relates to a bispecific antibody characterized in that the second binding portion includes a light chain variable region comprising CDRL1 of SEQ ID NO: 7, CDRL2 of SEQ ID NO: 8, and CDRL3 of SEQ ID NO: 9 as a light chain variable region.

[0023] In one embodiment, the present invention provides a bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, a) The first and second binding portions each include heavy chain variable regions, which include CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, respectively. b) The first binding portion includes a light chain variable region comprising CDRL1 of SEQ ID NO: 23, CDRL2 of SEQ ID NO: 24, and CDRL3 of SEQ ID NO: 25, c) The present invention relates to a bispecific antibody characterized in that the second binding portion includes a light chain variable region comprising CDRL1 of SEQ ID NO: 7, CDRL2 of SEQ ID NO: 8, and CDRL3 of SEQ ID NO: 9 as a light chain variable region.

[0024] In one embodiment, the present invention provides a bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, a) The first and second binding portions each include heavy chain variable regions, which include CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, respectively. b) The first binding portion includes a light chain variable region comprising CDRL1 of SEQ ID NO: 26, CDRL2 of SEQ ID NO: 27, and CDRL3 of SEQ ID NO: 28, c) The present invention relates to a bispecific antibody characterized in that the second binding portion includes a light chain variable region comprising CDRL1 of SEQ ID NO: 7, CDRL2 of SEQ ID NO: 8, and CDRL3 of SEQ ID NO: 9 as a light chain variable region.

[0025] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that the first binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region and a variable light chain region selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 as a variable light chain region, and the second binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region and the variable light chain region of SEQ ID NO: 10 as a variable light chain region.

[0026] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that the first binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region and the variable light chain region of SEQ ID NO: 32 as a variable light chain region, and the second binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region and the variable light chain region of SEQ ID NO: 10 as a variable light chain region.

[0027] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that the first binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region and the variable light chain region of SEQ ID NO: 33 as a variable light chain region, and the second binding portion includes the variable chain region of SEQ ID NO: 4 as a variable heavy chain region and the variable light chain region of SEQ ID NO: 10 as a variable light chain region.

[0028] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that the first binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region and the variable light chain region of SEQ ID NO: 34 as a variable light chain region, and the second binding portion includes the variable chain region of SEQ ID NO: 4 as a variable heavy chain region and the variable light chain region of SEQ ID NO: 10 as a variable light chain region.

[0029] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that the first binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region and the variable light chain region of SEQ ID NO: 35 as a variable light chain region, and the second binding portion includes the variable chain region of SEQ ID NO: 4 as a variable heavy chain region and the variable light chain region of SEQ ID NO: 10 as a variable light chain region.

[0030] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that the first binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region and the variable light chain region of SEQ ID NO: 36 as a variable light chain region, and the second binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region and the variable light chain region of SEQ ID NO: 10 as a variable light chain region.

[0031] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that the first binding portion comprises the heavy chain of SEQ ID NO: 5 and a light chain selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, and the second binding portion comprises the heavy chain of SEQ ID NO: 5 and a light chain having SEQ ID NO: 11.

[0032] In one embodiment, the present invention relates to a bispecific antibody characterized in that the first binding portion comprises the heavy chain of SEQ ID NO: 5 and the light chain of SEQ ID NO: 37, and the second binding portion comprises the heavy chain of SEQ ID NO: 5 and the light chain having SEQ ID NO: 11.

[0033] In one embodiment, the present invention relates to a bispecific antibody characterized in that the first binding portion comprises the heavy chain of SEQ ID NO: 5 and the light chain of SEQ ID NO: 38, and the second binding portion comprises the heavy chain of SEQ ID NO: 5 and the light chain having SEQ ID NO: 11.

[0034] In one embodiment, the present invention relates to a bispecific antibody characterized in that the first binding portion comprises the heavy chain of SEQ ID NO: 5 and the light chain of SEQ ID NO: 39, and the second binding portion comprises the heavy chain of SEQ ID NO: 5 and the light chain having SEQ ID NO: 11.

[0035] In one embodiment, the present invention relates to a bispecific antibody characterized in that the first binding portion comprises the heavy chain of SEQ ID NO: 5 and the light chain of SEQ ID NO: 40, and the second binding portion comprises the heavy chain of SEQ ID NO: 5 and the light chain having SEQ ID NO: 11.

[0036] In one embodiment, the present invention relates to a bispecific antibody characterized in that the first binding portion comprises the heavy chain of SEQ ID NO: 5 and the light chain of SEQ ID NO: 41, and the second binding portion comprises the heavy chain of SEQ ID NO: 5 and the light chain having SEQ ID NO: 11.

[0037] In one embodiment, the present invention relates to a bispecific antibody containing the same CEACAM5 binding moiety as that of the K2AC82, K2AC84, K2AC91, K2AC100, or K2AC117 bispecific antibodies. In such embodiments, the bispecific antibody is • Light chain CDRs of sequence numbers 14-16, VL of sequence number 32, and / or VLCL (K2AC82) of sequence number 37, • CDRs of sequence numbers 17-19, VL of sequence number 33, and / or VLCL (K2AC84) of sequence number 38, • CDRs of sequence numbers 20-22, VL of sequence number 34, and / or VLCL (K2AC91) of sequence number 39, • CDRs with sequence numbers 23-25, VL with sequence number 35, and / or VLCL (K2AC100) with sequence number 40, · CDRs of sequence numbers 26-28, VL of sequence number 36, and / or VLCL (K2AC117) of sequence number 41, or • Includes derivatives comprising the CDR region and / or light and heavy chains of the antibody.

[0038] In one embodiment, the constant and variable framework region arrays are human.

[0039] In one embodiment, the bispecific antibody according to the present invention is characterized in that the first and second binding portions each contain an immunoglobulin heavy chain and an immunoglobulin light chain. In one embodiment, the bispecific antibody according to the present invention is a full-length antibody. In one embodiment, the bispecific antibody according to the present invention is characterized in that it is of the human IgG1 type.

[0040] In one embodiment, the bispecific antibody according to the present invention is characterized by comprising a first binding moiety specific to CEA, which includes a lambda light chain variable domain (VL) and a lambda light chain constant (CL) domain, and a second binding moiety specific to CD47, which includes a kappa light chain variable domain (VK) and a kappa light chain constant domain (CK) (κλ bispecific antibody, κλ antibody). In such an embodiment, the second binding moiety includes the light chain of SEQ ID NO: 11 as the light chain LC (CD47 VKCK). The kappa light chain of SEQ ID NO: 11 includes the variable light chain domain of SEQ ID NO: 10 (Mab CD47 VK) as the variable light chain domain, and the constant light chain domain of SEQ ID NO: 13 (CD47 CK) as the constant light chain domain.

[0041] In one embodiment, the bispecific antibody of the present invention is in a fully human bispecific IgG (particularly IgG1) format and, in addition, is a type 1 or type 2 κλ bispecific antibody.

[0042] In one embodiment, the bispecific antibody according to the present invention is a κλ bispecific antibody and is characterized by containing a common heavy chain (cHC). In one embodiment, the common heavy chain contains the variable heavy chain domain VH of SEQ ID NO: 4. In one embodiment, the bispecific antibody according to the present invention is characterized by containing the common heavy chain VH-CH1 of SEQ ID NO: 5. In one embodiment, the bispecific antibody according to the present invention is characterized by containing the common heavy chain (VH-CH1-CH2-CH3) of SEQ ID NO: 6.

[0043] In one embodiment, the bispecific antibody according to the present invention is characterized in that the first binding site is monovalent and the second binding site is monovalent.

[0044] In one embodiment, the bispecific antibody according to the present invention competes for binding to CEACAM5 with the anti-CEACAM5 antibody SM3E, which contains VK and VH domains as the VK and VH domains in the sequences of SEQ ID NOs. 43 and 44. In one embodiment, the bispecific antibody according to the present invention does not compete for binding to CEACAM5 with cibisatamab and / or MEDI-565 (AMG 211; (MD Oberst et al., mAbs 6(2014)1571-1584)). In one embodiment, the bispecific CEAxCD47 antibody according to the present invention can be administered in parallel with the CEAxCD3 bispecific antibody cibisatamab and / or MEDI-565.

[0045] In another embodiment, the bispecific antibody according to the present invention is characterized by being sugar-modified to have an Fc region having a modified oligosaccharide. In yet another embodiment, the bispecific antibody according to the present invention is characterized by containing an Fc region that is sugar-modified to have a reduced number of fucose residues compared to the same bispecific antibody that is not sugar-modified.

[0046] In one embodiment, the bispecific antibody according to the present invention contains a reduced amount of fucose in an oligosaccharide chain(s).

[0047] In one embodiment, the bispecific antibody of the present invention is characterized in that 50% to 100% of the N-linked oligosaccharide in the Fc region is not fucosylated.

[0048] In one embodiment, the bispecific antibody of the present invention is characterized in that the amount of fucose in the oligosaccharide chain(s) of the bispecific antibody of the present invention is reduced by 80% to 100% compared to the fucose content of each antibody when the afucosylation method is not applied.

[0049] In one embodiment, the bispecific antibody is characterized in that 80% to 100% of the N-linked oligosaccharides in the Fc region are bisectified and not fucosylated. Afucosylated bispecific antibodies that generally bind to CEACAM5 and CD47, as well as their production and purification, are described in International Publication No. 2019234576, U.S. Patent No. 62 / 943,726, and European Patent No. 19213002.

[0050] In one embodiment, the bispecific antibody of the present invention is characterized by comprising one, two, or three amino acid substitutions in an Fc region selected from the group consisting of monosubstituted S239D, I332E, G236A, disubstituted I332E and G236A, S239D and I332E, S239D and G236A, and trisubstituted S329D, I332E and G236A, and a glycosylated Fc region having a reduced number of fucose residues compared to the same bispecific antibody except that it is not glycosylated.

[0051] In one embodiment, the bispecific antibody of the present invention is characterized by a ratio of KD values ​​for binding to recombinant CEACAM3 and recombinant CEACAM5 of 100-fold or more (Example 3, Table 2).

[0052] In one embodiment, the bispecific antibody of the present invention is characterized by a ratio of KD values ​​for binding to recombinant CEACAM3 and recombinant CEACAM5 of 100 to 200 times.

[0053] In one embodiment, the bispecific antibody of the present invention exhibits relative separation (discriminative binding) of binding to CEACAM5 and CEACAM3. Compared to the bispecific CEAxCD47 antibody K2AC22, binding to full-length recombinant human CEACAM5 protein increased, but binding to full-length recombinant human CEACAM3 did not increase proportionally. The KD exponent / ratio for binding to full-length CEACAM3 versus CEACAM5 shows an increase from 83 (K2AC22) to 137 (K2AC84) to 146 (K2AC100). This is equivalent to a 65% to 76% increase in discriminative binding (Example 3, Table 2).

[0054] In one embodiment, the bispecific antibody according to the present invention is characterized by concentration-dependent phagocytosis (ADCP of CEACAM5-expressing tumor cell lines by human macrophages). ADCP is measured according to the present invention as an imaging phagocytic index (EC50 and / or maximum value) with an E:T ratio of typically 1:3 (human macrophages:target cells (tumor cells); see, for example, Figure 2 and Tables 6-9 for EC50 values ​​and the maximum phagocytic index Emax). The results in Figure 2 were obtained with an E:T ratio of 1:3. Details of the assay are described in Example 7. Imaging assay based on CellInsight CX5. Unless otherwise specified, phagocytic index values ​​are measured by such imaging methods.

[0055] In one embodiment, the bispecific antibody according to the present invention is characterized by an increase of at least 8% in the maximum phagocytic index (Emax) of LoVo tumor cells compared to the phagocytic index of K2AC22. In one embodiment, the increase is 8% to 20% for LoVo tumor cells compared to the phagocytic index of K2AC22. In one embodiment, the bispecific antibody according to the present invention is characterized by an increase of at least 8% in the maximum phagocytic index of Ls174T tumor cells compared to the phagocytic index of K2AC22. In one embodiment, the increase is 8% to 25% in Ls174T tumor cells compared to the phagocytic index of K2AC22 (Example 7, Table 5). LoVo and LS174T are tumor cells with considerably low CEACAM5 expression (see Table 3 below Example 5).

[0056] In one embodiment, the bispecific antibody according to the present invention inhibits the interaction between human CD47 and human SIRPα. In one embodiment, the bispecific antibody according to the present invention inhibits the interaction between CD47 and SIRPα on MKN-45 cells with an IC50 that is 10 times or more lower than the IC50 measured for K2AC22 under the same experimental conditions. In one embodiment, the factor is between 10 and 30. In one embodiment, the bispecific antibody according to the present invention inhibits the interaction between CD47 and SIRPα on MKN-45 cells with an IC50 of 0.1 nM or less. In one embodiment, the bispecific antibody according to the present invention inhibits the interaction between CD47 and SIRPα on MKN-45 cells with an IC50 of 0.1 nM to 0.04 nM (see Example 10 and Table 12).

[0057] In one embodiment, the bispecific antibody according to the present invention is characterized by having two or more of the following characteristics: having a KD ratio of 100-fold or more for binding to recombinant CEACAM3 and recombinant CEACAM5; having relative separation of binding to CEACAM5 and CEACAM3; having concentration-dependent ADCP; having at least an 8% increase in the maximum phagocytic index (Emax) of LoVo tumor cells compared to the phagocytic index of K2AC22; and having the ability to inhibit the interaction between human CD47 and human SIRPα with an IC50 more than 10-fold lower than that of K2AC22.

[0058] The bispecific antibody K2AC22 is a bispecific antibody that binds to human CEACAM5 and human CD47, and is described in International Publication No. 2019234576. K2AC22 contains a common heavy chain of SEQ ID NO: 6, a light chain of SEQ ID NO: 42 in the CEACAM5 binding region, and a light chain of SEQ ID NO: 11 in the CD47 binding region. The CDRs of K2AC22 are shown in SEQ ID NOs: 1-3, 7-9, and 29-31 (Table 1).

[0059] In one embodiment, the bispecific antibody according to the present invention is characterized by binding to recombinant human CD47 with a binding affinity (KD) of 100 nM to 600 nM, and in another embodiment, it is characterized by binding with a binding affinity (measured by biolayer interferometry) of 100 nM to 500 nM.

[0060] In one embodiment, the bispecific antibody according to the present invention is characterized by binding to recombinant human CEACAM5 with a KD of 2 nM to 10 nM (Example 3, Table 2). In one embodiment, the bispecific antibody according to the present invention has a binding affinity (lower KD) that is 10 to 50 times higher, and in one embodiment, 20 to 50 times higher, than the bispecific antibody K2AC22 of the current state of the art (Example 3, Table 2).

[0061] In one embodiment, the bispecific antibody according to the present invention is useful for combined treatment with a CEAxCD3 T cell bispecific antibody such as cibisatamab.

[0062] In one embodiment, the bispecific antibody according to the present invention is characterized by its specific binding to CEACAM5, but does not compete with TCB2014 and cibisatamab for binding to CEACAM5 on tumor cells, such as MKN-45 and LS174T (Example 8).

[0063] In one embodiment, the bispecific antibody according to the present invention is characterized in that the bispecific antibody TCB2014, which binds to human CEACAM5 and CD3ε (shown above) at a concentration of 300 nM, does not shift the EC50 of the binding curve of the bispecific antibody according to the present invention to MKN-45 cells, or, in another embodiment, to LS174 T cells, by more than 3 times, in one embodiment toward higher concentrations (Example 8 and Figure 5). In such cases, the bispecific antibody according to the present invention and TCB2014 are defined as "non-competitive" and are considered to be able to bind to CEA simultaneously without significantly interfering with the binding to CEA. In such cases, the bispecific antibody according to the present invention and TCB2014 are defined as "non-competitive" and are considered to be able to bind to CEA simultaneously without significantly interfering with the binding to CEA, and therefore, even when therapeutic levels of both drugs are simultaneously present in the blood and / or tumor tissue, they can develop without interfering with their effects on phagocytosis (CEAxCD47) and on T cell activation (TCB2014 and cibisatamab). This facilitates the combined treatment of TCB2014 or cibisatamab with the CEAxCD47 bispecific antibody of the present invention.

[0064] In one embodiment, the CEAxCD47 bispecific antibody of the present invention, combined with the CEAxCD3 bispecific antibody TCB2014, exhibits at least additive or even synergistic % toxicity of tumor cells in an assay involving, for example, LoVo or LS174T tumor cells and human macrophages and T cells derived from the same volunteer human donor.

[0065] In one embodiment, the CEAxCD47 bispecific antibody of the present invention, combined with the CEAxCD3 bispecific antibody TCB2014, exhibits at least additive or even synergistic killing of tumor cells in an assay involving, for example, LoVo or LS174T tumor cells and human macrophages and T cells derived from the same volunteer human donor.

[0066] The present invention further provides an expression vector comprising one or more polynucleotides encoding a bispecific antibody according to the present invention.

[0067] The present invention further provides a host cell containing an expression vector according to the present invention.

[0068] The present invention relates to a method for producing a bispecific antibody, a) Culturing host cells containing an expression vector encoding the bispecific antibody under conditions that enable the production of the antibody of the present invention, and b) Isolating the antibody that can specifically bind to CEACAM5 and CD47. The present invention further provides a method characterized by including the following:

[0069] The second polypeptide encoding the antibody of the present invention may be a single polypeptide encoding each of the two distinct light chains and the common heavy chain, or separate polypeptides encoding each of the light chains and heavy chains separately. The expression vector may also be one, two, or three vectors, each expressing two distinct light chains and the common heavy chain.

[0070] The present invention provides a method for inducing cytolysis of tumor cells, further comprising contacting the tumor cells with the bispecific antibody of the present invention. The tumor cells are preferably human tumor cells in a patient. In one embodiment of the method for inducing cytolysis of tumor cells, the tumor cells are colorectal cancer cells, NSCLC (non-small cell lung cancer) cells, gastric cancer cells, pancreatic cancer cells, breast cancer cells, or other tumor cells expressing CEACAM5.

[0071] The present invention further provides a method for treating a subject having cancer expressing CEACAM5, comprising administering a therapeutically effective amount of the bispecific antibody of the present invention to the subject.

[0072] The present invention further provides a method for increasing the survival time of a subject having cancer expressing CEACAM5, comprising administering a therapeutically effective amount of the bispecific antibody of the present invention to the subject. Further embodiments of the present invention are the method of the present invention, characterized in that the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, or breast cancer.

[0073] The present invention further provides a method for treating a subject having cancer expressing CEACAM5, comprising administering a therapeutically effective amount of the bispecific antibody according to the present invention to the subject. A further embodiment of the present invention is a method characterized by administering the bispecific antibody according to the present invention to a human subject in combination with chemotherapy or radiotherapy.

[0074] The present invention further provides the use of the bispecific antibody of the present invention in the manufacture of a pharmaceutical for treating subjects having cancer expressing CEACAM5. Further embodiments of the present invention are the bispecific antibody of the present invention for the manufacture of a pharmaceutical, wherein the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, and breast cancer.

[0075] The present invention further provides a bispecific antibody for use separately or sequentially in combination with a second bispecific antibody, which includes a third binding site that specifically binds to human CEACAM5 and a fourth binding site that specifically binds to human CD3ε, in the treatment of subjects having cancer expressing CEACAM5. A further embodiment of the present invention is a bispecific antibody for use separately or sequentially in combination with TCB2014 or cibisatamab in the treatment of subjects having cancer expressing CEACAM5.

[0076] A further embodiment of the present invention is a bispecific antibody of the present invention for use in combination with the second bispecific antibody, either separately or sequentially, in the treatment of subjects having cancer expressing CEACAM5.

[0077] A further embodiment of the present invention is a bispecific antibody for use of the present invention, characterized in that the bispecific antibody of the present invention and a second bispecific antibody are administered alternately to the subject at intervals of 6 to 15 days.

[0078] A further embodiment of the present invention is a bispecific antibody for use of the present invention, characterized in that the bispecific antibody of the present invention and a second bispecific antibody are administered simultaneously to the subject at intervals of 6 to 15 days.

[0079] A further embodiment of the present invention is a first bispecific antibody of the present invention for use in the present invention, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47 of the present invention, characterized in that the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, and breast cancer.

[0080] Further embodiments of the present invention provide a method for treating human patients diagnosed with tumors (cancer), particularly solid tumors, particularly solid tumors expressing CEA, particularly colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer and breast cancer, comprising administering to the human patient an effective amount of the bispecific antibody of the present invention and the second bispecific antibody against CEACAM5 and CD3 (in one embodiment, TCB2014, in one embodiment, cibisatamab), followed by The patient is administered the second anti-CEAxCD3 antibody in a dose of 0.1 to 10 mg / kg, in a further embodiment 0.5 to 10 mg / kg, in a further embodiment 1 to 2 mg / kg, for example weekly or over a period of 4 to 12 weeks (q2w), and after these 4 to 12 weeks, and after waiting for a further 2, 3, or 4 elimination half-lives of the anti-CEAxCD3 antibody, the patient is administered the antibody of the present invention in a dose of 0.1 to 20 mg / kg. The patient is administered the antibody of the present invention to the patient for a further 12 weeks, for example, q1, q2w, q3w, or q4w if necessary, and thereafter, if necessary, the cycle of administering the CEAxCD3 bispecific antibody, followed by the administration of the bispecific antibody according to the present invention is repeated, and the cycle is repeated again if necessary. This method includes [something].

[0081] Since the CEAxCD3 bispecific antibody and CEAxCD47 bispecific antibody of the present invention are non-competitive, the two bispecific antibodies may also be administered in a manner in which the patient experiences therapeutically effective plasma and tissue concentrations of both bispecific antibodies in parallel ("simultaneous method"), for example, by administering to the patient at approximately the same time doses of the CEAxCD3 bispecific antibody at 0.1–10 mg / kg, in a further embodiment 0.5–10 mg / kg, in a further embodiment 1–2 mg / kg and the CEAxCD47 bispecific antibody of the present invention at 3–30 mg / kg, in a further embodiment 1–10 mg / kg, followed by one or more combined administrations of these at a frequency of q1w, q2w, q3w, or q4w as needed. The term "q1w" means administration once a week. q2w means administration every two weeks, and so on.

[0082] For safety reasons, in one embodiment, it may be necessary to initiate treatment with the second antibody CEAxCD3 without adding the bsAb of the present invention, and to initiate co-administration of the two bsAbs only after the typical cytokine release syndrome (CRS) for CEAxCD3 has ended (usually after two or three doses of TAAxCD3 antibody).

[0083] The present invention further provides a pharmaceutical composition comprising the antibody of the present invention and a pharmaceutically acceptable excipient or carrier.

[0084] The present invention further provides pharmaceutical compositions comprising the antibody of the present invention for use as a pharmaceutical. In one such embodiment, the present invention provides a pharmaceutical composition comprising the antibody of the present invention for use as a pharmaceutical in the treatment of solid tumor disorders. In one embodiment, the pharmaceutical composition comprises the antibody of the present invention for use as a pharmaceutical in the treatment of colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer, or breast cancer.

[0085] The present invention relates to a composition comprising a bispecific antibody of the present invention for use separately or sequentially in combination with TCB2014 or cibisatamab as defined above in the treatment of subjects having cancer expressing CEACAM5, wherein the second bispecific antibody at a concentration of 300 nM does not shift the EC50 of the binding curve of the bispecific antibody of the present invention to MKN-45 and / or LS174 T cells by more than threefold in the direction of higher concentrations in one embodiment. Further embodiments of the present invention relate to a composition of the present invention characterized in that the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, or breast cancer.

[0086] The present invention further provides the use of the antibody of the present invention for the manufacture of pharmaceutical compositions.

[0087] The present invention further provides the use of the antibody of the present invention with a pharmaceutically acceptable excipient or carrier for the manufacture of pharmaceutical compositions.

[0088] The present invention further provides the use of the antibodies of the present invention for the manufacture of pharmaceuticals in the treatment of solid tumor disorders. Further embodiments of the present invention include the use of the antibodies of the present invention in the treatment of colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer, or breast cancer.

[0089] Another aspect of the present invention provides a method for inducing cytolysis of tumor cells, comprising contacting the tumor cells with a bispecific antibody of any of the above embodiments. In some embodiments, the tumor cells are colorectal cancer cells, NSCLC (non-small cell lung cancer), gastric cancer cells, pancreatic cancer cells, or breast cancer cells. In one embodiment, cytolysis is induced by antibody-dependent phagocytosis and / or antibody-dependent cell-mediated cytotoxicity of the bispecific antibody according to the present invention.

[0090] Another aspect of the present invention provides a method for treating a subject having cancer that abnormally expresses CEACAM5, comprising administering to the subject a therapeutically effective amount of a bispecific antibody of any of the above embodiments.

[0091] Another aspect of the present invention provides a method for treating a subject having cancer that abnormally expresses CEACAM5, comprising administering to the subject a therapeutically effective amount of a bispecific antibody of any of the above embodiments in combination with a bispecific antibody that binds to human CEA and human CD3. Since the CEAxCD3 bispecific antibody and the CEAxCD47 bispecific antibody according to the present invention are non-competitive or minimally competitive, they can be administered not only sequentially but also in parallel (simultaneously), which can be very beneficial because tumor cell killing via T cell engagement by the CEAxCD3 bispecific antibody and simultaneously via macrophage engagement by the CEAxCD47 bispecific antibody according to the present invention may be additive or even synergistic, meaning that administering both drugs in parallel increases efficacy.

[0092] Another aspect of the present invention provides a method for increasing progression-free survival and / or overall survival in a subject having a cancer that abnormally expresses CEACAM5, comprising administering to the subject a therapeutically effective amount of a bispecific antibody of any of the above embodiments. In one embodiment, the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, or breast cancer, or another cancer that expresses CEACAM5.

[0093] In certain embodiments of these methods, the bispecific antibodies according to the present invention are administered in combination with chemotherapy or radiotherapy. In one embodiment, the subject is a patient with colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, breast cancer, or another cancer that expresses CEACAM5.

[0094] Another aspect of the present invention provides a method for treating a subject having cancer that abnormally expresses CEACAM5, comprising administering to the subject a therapeutically effective amount of a bispecific antibody of any of the above embodiments in combination with a bispecific antibody against human CEA and human CD3 epsilon.

[0095] Another aspect of the present invention provides a method for increasing progression-free survival and / or overall survival in subjects having cancer that abnormally expresses CEACAM5, comprising administering to the subjects a therapeutically effective amount of a bispecific antibody of any of the above embodiments. In one embodiment, the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, or breast cancer.

[0096] In certain embodiments of these methods, the bispecific antibodies according to the present invention are administered in combination with chemotherapy or radiotherapy. In one embodiment, the subject is a cancer patient having colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, breast cancer, or another cancer that expresses CEACAM5.

[0097] Another embodiment of the present invention provides the use of the bispecific antibody of the present invention for any of the above treatment methods. In one embodiment, the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, and breast cancer. [Brief explanation of the drawing]

[0098] [Figure 1-1]Concentration-dependent binding of five CD47xCEACAM5 bispecific antibodies (K2AC82, K2AC84, K2AC91, K2AC100, and K2AC117) according to the present invention compared with the current state of bispecific CEAxCD47 antibody K2AC22. These figures also show the binding of the corresponding anti-CD47 monovalent antibody, unrelated hIgG1 control (hIgG1, line below the line for CD47 monovalent) and bivalent anti-CD47 mAb (hB6H12, dotted line) in six CEACAM5-expressing cancer cell lines: (Figure 1A) SK-CO-1 cells, (Figure 1B) MKN-45 cells, (Figure 1C) HPAF-II cells, (Figure 1D) SNU-C1 cells, (Figure 1E) Ls174T cells, and (Figure 1F) LoVo cells. The EC50 of the bispecific antibody according to the present invention is lower than that of K2AC22, and the maximum binding (MFI) of the bispecific antibody according to the present invention is higher than that of K2AC22. [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0099] [Figure 2-1] Concentration-dependent phagocytosis of LoVo cancer cells induced by two CEACAM5xCD47 bispecific antibodies (K2AC84 and K2AC100) according to the present invention, compared to the current state of the CEACAM5xCD47 bispecific antibody K2AC22. These figures also show phagocytosis induced by the corresponding anti-CD47 monovalent antibody and isotype control hIgG1. Figures 2A–2F show data obtained using macrophages derived from PBMCs (peripheral blood mononuclear cells) of six different human donors ((Figure 2A) Donor 862, (Figure 2B) Donor 872, (Figure 2C) Donor 873, (Figure 2D) Donor 863, (Figure 2E) Donor 874, and (Figure 2F) Donor 866). In most donors, phagocytosis induced by the two antibodies according to the present invention is superior to that induced by K2AC22. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0100] [Figure 3-1]Concentration-dependent phagocytosis of CEACAM5-expressing cancer cells induced by 4-5 afucosylated CEACAM5xCD47 bispecific antibodies according to the present invention (K2AC82 afuco, K2AC84 afuco, K2AC91 afuco, K2AC100 afuco, and K2AC177 afuco) compared with the current state of the afucosylated CEACAM5xCD47 bispecific antibody K2AC22. These figures also show phagocytosis induced by the corresponding anti-CD47 monovalent antibody and hIgG1 isotype control (hIgG1). Figure 3A shows data obtained using macrophages (peripheral blood mononuclear cells) derived from PBMCs of two human donors (donor (D)830, donor (D)831) with MKN-45 as the target cell, and Figure 3B shows data obtained using macrophages (donor (D)831, donor (D)833) derived from two human donors with SNU-C1 as the target cell. With respect to the donors, the phagocytic activity induced by the antibody according to the present invention is superior to that of K2AC22. [Figure 3-2] Same as above.

[0101] [Figure 4] CD47 / SIRPα blocking activity by five CEACAM5xCD47 bispecific antibodies (K2AC82, K2AC84, K2AC91, K2AC100, and K2AC177) according to the present invention, compared to the current state-of-the-art CEACAM5xCD47 bispecific antibody K2AC22. This figure also shows the CD47 / SIRPa blocking activity by the corresponding anti-CD47 monovalent antibody. Two negative controls were added for comparison, and either an hIgG1 isotype control (hIgG1) was added or no Ab was added. The bivalent mAb hB6H12 was added as a positive control. All five CEACAM5xCD47 bispecific antibodies according to the present invention (K2AC82, K2AC84, K2AC91, K2AC100, and K2AC177) showed improved blocking activity compared to the current state-of-the-art CEACAM5xCD47 bispecific antibody K2AC22.

[0102] [Figure 5]Concentration-dependent binding of the CD47xCEACAM5 bispecific antibody K2AC100 to the cell surface of CEACAM5-expressing cells MKN-45 in the presence of anti-CEACAM5 mAbs, TCB2014 and TCB2017. K2AC100 was directly labeled with a fluorescent dye, and its binding to MKN-45 cells alone (dark line, dark circle) was tracked in the presence of 300 nM TCB2014 (dark line, dark triangle) or 30 nM TCB2017 (dark line, black diamond). A negative control (Ctrl) was used (IgG1 in the presence of TCB2014 or TCB2017). [Modes for carrying out the invention]

[0103] Unless otherwise specifically defined below, terms are used herein in the manner they are commonly used in the art.

[0104] The antibody according to the present invention has one or more beneficial properties among the following properties: - Ratio of KD values ​​for binding to CEACAM3 versus CEACAM5, - The maximum phagocytic index (Emax) in low-CEA-expressing tumor cells, and / or - Inhibition of SIRPα binding to CD47 on the surface of tumor cells (low IC50).

[0105] The antibodies according to the present invention surprisingly exhibit beneficial binding ratios to CEACAM3 versus CEACAM5, as shown in Example 3 and Table 2. For example, K2AC100 shows a binding affinity (KD) that is 25 times higher (lower KD) to CEACAM5 compared to K2AC22, but surprisingly, it shows a binding affinity (KD) that is only 14 times higher (lower KD) to CEACAM3. Similarly, K2AC84 shows a binding affinity (KD) that is 46 times higher to CEACAM5 compared to K2AC22, but surprisingly, it shows a binding affinity (KD) that is only 28 times higher to CEACAM3. Therefore, the ratio of the KD value for binding to CEACAM3 to the KD value for binding to CEACAM5 is 83 for K2AC22, 146 for K2AC100, and 137 for K2AC84.

[0106] While some family members, such as CEACAM5 or CEACAM6, are expressed by epithelial cells, other family members, such as CEACAM3 (CGM1 or CD66d; UniProtKB-P40198), are exclusively expressed on human granulocytes, for example, a cell type involved in the elimination of bacterial infections (Kuespert K et al., Curr Opin Cell Biol. 2006; Pils S et al., Int J Med Microbiol. 2008). Despite the high sequence homology between CEACAM5 and CEACAM3, CEACAM3 does not support cell-cell adhesion, in contrast to other members of the CEACAM family, but rather mediates opsonin-independent recognition and elimination of a limited set of Gram-negative bacteria, including Neisseria gonorrhoeae, Hemophilus influenzae, and Moraxella catarrhalis (Kuroki et al., J. Biol. Chem. 1991; Pils S et al., Int J Med Microbiol. 2008). CEACAM3 has been discussed as a phagocytic receptor of the innate immune system (Schmitter et al., J Exp Med. 2004). According to our knowledge, bispecific antibodies against CEACAM5 and CD47, if they also bind significantly to CEACAM3, would have adverse effects on neutrophil granulocytes and could reduce neutrophil counts, i.e., induce neutropenia through increased phagocytosis. This could increase the risk of developing potentially life-threatening bacterial infections without immediate medical intervention. High binding affinity is characterized by a low KD (knockout date). The distribution of CEA-targeted bispecific antibodies between CEACAM5 and CEACAM3 is determined by the ratio of their binding affinities to these two CEACAM family members. A high ratio of KD for binding to CEACAM3 to KD for binding to CEACAM5 means less binding of bispecific antibodies to CEACAM3 compared to binding to CEACAM5, which would be beneficial.

[0107] The antibody according to the present invention, surprisingly, in one embodiment, exhibits a beneficial maximum phagocytic index (Emax) in low-CEA-expressing tumor cells (such as LoVo cell lines) compared to the phagocytic index of K2AC22 in each cell line. As shown in Table 5, the bispecific antibody of the present invention shows that the maximum value of the phagocytic index curve in LoVo cells (4000 CEACAM5 on the cell surface) is 8.5–17% higher compared to the state-of-the-art bispecific antibody K2AC22. In the case of LS174T cells (26000 CEACAM5 on the cell surface), the maximum phagocytic index is 8.7–20.6% higher for the antibody of the present invention compared to K2AC22 (Table 5). In cells with higher CEACAM5 expression, such as SNU-C1 or MKN-45, the increase in Emax is lower.

[0108] Therefore, a higher percentage of patients could be successfully treated with the bispecific antibody according to the present invention.

[0109] As disclosed in Examples 5 and 11, CEA expression in malignant cells can vary significantly with respect to RNA expression or the counting of cell surface CEA molecules. The CEA-expressing cancer cell lines used to study the phagocytic activity of the bispecific antibodies of the present invention express an average of 108,000 CEA targets on the cell surface (Example 5, Table 3). Organoids derived from fresh tumor tissue of cancer patients (colorectal and lung) were investigated by the method described in Example 11. The average expression of CEACAM5 in these major organoids was found to be 28,000 CEACAM5 targets per cell, i.e., approximately four times lower than the average expression on the cell lines, as shown in Table 3. Therefore, bispecific antibodies that are improved for phagocytosis in malignant cells with lower CEACAM5 expression may be advantageous for use in tumor therapy. Thus, given the heterogeneous and / or considerably low expression in, for example, lung adenocarcinoma, colorectal cancer, and other CEACAM5-expressing tumors, such patients can be successfully treated with the CEAxCD47 bispecific antibodies of the present invention.

[0110] The antibody according to the present invention, surprisingly, exhibits beneficial inhibition (low IC50) of SIRPα binding to CD47 on the surface of tumor cells compared to antibody K2AC22, as shown in Example 10 and Figure 4. The interaction between SIRPα on macrophages and CD47 on tumor cells inhibits phagocytosis in tumor cells, meaning that effective inhibition of this interaction increases phagocytosis.

[0111] As used herein, the term "Emax" represents the maximum activity of a compound. For example, in a cell killing assay, Emax represents the elimination / killing of cancer cells by macrophages (e.g., labeled with calcein AM, see Example 7) within a given time frame. This is presumably clinically important because the total number of tumor-infiltrating macrophages is limited: for example, if twice the number of tumor cells are eliminated per time interval, this is equal to half the number of macrophages that would need to be present to eliminate the same number of tumor cells at once.

[0112] As used herein, the term "EC50" represents the compound concentration at which half of the maximum activity (Emax / 2) is achieved. Low EC50 is useful when it is necessary to inject a smaller amount of compound, and therefore useful, for example, to achieve lower manufacturing costs compared to higher EC50 and / or potentially lower side effect rates. Thus, Emax and EC50 describe different aspects of compound activity. For two compounds with equivalent Emax, EC50 becomes important because the same therapeutic effect can be achieved at a lower concentration, thus requiring a smaller amount of drug and potentially resulting in a lower rate of side effects.

[0113] As used herein, the terms “antigen-binding moiety” and “binding moiety” refer, in their broadest sense, to the portion of an antibody that specifically binds to an antigenic determinant such as CEA, CD47, and CD3.

[0114] More specifically, as used herein, the binding moieties that bind to membrane-bound human carcinoembryonic antigen (CEA, same as CEACAM5) or CD47 specifically bind to CEA or CD47, more specifically to the cell surface or membrane-bound CEA or CD47. Thus, each binding moiety binds to either CEA or CD47. "Specifically binds," "specific to," and "bound to" means that the binding is selective to the antigen and can be distinguished from undesirable or nonspecific interactions. In some embodiments, the degree of binding of an antitarget antibody to an unrelated, non-target protein is about 10 times, preferably less than >100 times, than the binding of the antibody to the target, as measured, for example, by biolayer interferometry, e.g., Octet®, surface plasmon resonance (SPR), e.g., Biacore®, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS). The targets are the proteins discussed herein, e.g., CEA, CD47, and CD3ε.

[0115] The phrases "specifically binds to CEA and CD47," "bound to CEA and CD47," and "specific to CEA and CD47" refer, in one embodiment, to an antibody capable of binding to target CEA and CD47 with sufficient affinity to be useful as a therapeutic agent in targeting tumor cells expressing CEACAM5 and CD47, such as a bispecific antibody. References to binding to MKN-45, SNU-C1, LS174T, SK-CO-1, HPAF-II, and / or LoVo cells having specific EC50 values ​​refer to EC50 values ​​measured by flow cytometry (see Example 6).

[0116] As used herein, the term “antibody” refers to an antibody comprising two heavy chains and two light chains. In one embodiment, the antibody is a full-length antibody. As used herein, the term “antibody heavy chain” refers to an antibody heavy chain consisting of a variable region and a constant region as defined for a full-length antibody. As used herein, the term “antibody light chain” refers to an antibody light chain consisting of a variable region and a constant region as defined for a full-length antibody.

[0117] The term "full-length antibody" refers to an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains." A "full-length antibody heavy chain" is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant heavy chain domain 1 (CH1), antibody hinge region (HR), antibody heavy chain constant domain 2 (CH2), and antibody heavy chain constant domain 3 (CH3), from the N-terminus to the C-terminus, and is abbreviated as VH-CH1-HR-CH2-CH3. A "full-length antibody light chain" is a polypeptide consisting of an antibody light chain variable domain (VL) and antibody light chain constant domain (CL), from the N-terminus to the C-terminus, and is abbreviated as VL-CL. The antibody light chain constant domain (CL) can be κ (kappa) or λ (lambda). The two full-length antibody domains are linked to each other via polypeptide disulfide bonds between the CL domain and the CH1 domain and between the hinge regions of the full-length antibody heavy chains. Typical examples of full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE. In one embodiment, the full-length antibody of the present invention is of the human IgG1 type, and in a further embodiment, it contains one or more amino acid substitutions in the Fc moiety as defined below and / or is glycosylated with a polysaccharide chain bound to Asn297. The full-length antibody of the present invention comprises two binding sites, each formed by a pair of VH and VL, one of which binds to CEA and the other to CD47.

[0118] Where used herein and referred to above, “complementarity-determining regions” (CDRs) refer to non-adjacent antigen-binding sites (also known as antigen-binding regions) found within the variable regions of both heavy-chain and light-chain polypeptides. CDRs are also referred to as “hypervariable regions” (HVRs), and the term is used herein interchangeably with “CDR” in reference to the portion of the variable region that forms an antigen-binding region. This particular region is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th This is described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), which is incorporated herein by reference. The appropriate amino acid residues containing a CDR as defined by Kabat are listed in the following sequence listings. The exact residue number containing a particular CDR varies depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues contain a particular CDR by considering the variable region amino acid sequence of the antibody. As used herein, the term "containing CDRL1 of SEQ ID NO: x" means that the CDRL1 region of the variable light chain referred to is that of SEQ ID NO: x (containing CDRL1 of SEQ ID NO: x as CDRL1). This also applies to other CDRs. Unless otherwise indicated, HVR residues are numbered and named "CDR" herein according to Kabat et al. above, and references to the numbering of other specific amino acid residue positions in the bispecific antibodies according to the present invention also follow the Kabat numbering system.

[0119] As used herein, the terms “Fc region” and “Fc domain” refer to the C-terminal region of the IgG heavy chain; in the case of IgG1 antibodies, the C-terminal region includes -CH2-CH3 (see above). While the boundaries within the Fc region of the IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226 to the carboxyl terminus. The constant region is well known in the art and has been described, for example, by Kabat, EA (see, e.g., Johnson, G., and Wu, TT, Nucleic Acids Res. 28(2000) 214-218; Kabat, EA et al., Proc. Natl. Acad. Sci. USA 72(1975) 2785-2788).

[0120] IgG molecules carry two N-linked oligosaccharides in their Fc region, one on each heavy chain. As any glycoprotein, antibodies are produced as a collection of glycoforms that share the same polypeptide backbone but have different oligosaccharides bound to the glycosylation site. Antibodies with reduced fucose content in the glycan moiety exhibit higher antibody-dependent cell-mediated cytotoxicity (ADCC) activity compared to conventionally fucosylated antibodies (Niwa R et al., Cancer Res, 64, 2127-33, 2004). Cell lines in which both alleles of the gene involved in fucose addition (α1,6-fucosyltransferase; FUT8) are knocked out are described in U.S. Patents 6,946,292, 7,425,446, and 8,067,232 (each incorporated in its entirety by reference). Using such cell lines, the bispecific antibodies according to the present invention can be produced using a glycan moiety with reduced fucose content and increased ADCC and antibody-dependent cell-mediated phagocytosis (ADCP). Another technique that can be used to produce antibodies with reduced fucose content is described in U.S. Patent No. 8,642,292 (incorporated herein by reference). This technique is designed to constitute the stable incorporation of a heterologous bacterial enzyme into antibody-producing cell lines, such as CHO cell lines. This means blocks the de novo synthesis of fucose from D-mannose. Furthermore, when the producing cells are cultured in a fucose-free medium, the result is the production of antibodies with stable levels of afucosylation. Exemplary methods for producing and purifying the afucosylated bispecific antibodies of the present invention are described in Examples 9 (1. and 2.).

[0121] Mutations within the Fc domain can also alter the binding properties of the Fc domain to different Fc receptors (International Publication Nos. 2004063351, 2004099249, 2005018669, 2005063815, 2005110474, 2005056759, International Publication Nos. 200506759) Publication No. 2005092925, International Publication No. 2005018572, International Publication No. 2006019447, International Publication No. 2006116260, International Publication No. 2006023420, International Publication No. 2006047350, International Publication No. 2006085967, International Publication No. 2006105338, International Publication No. 2007021841, International Publication No. 2 International Publication No. 007008943, International Publication No. 2007024249, International Publication No. 2007041635, International Publication No. 2007048077, International Publication No. 2007044616, International Publication No. 2007106707, International Publication No. 2008022152, International Publication No. 2008140603, International Publication No. 2008036688, International Publication No. 200 International Publication No. 8091798, International Publication No. 2008091954, International Publication No. 2008092117, International Publication No. 2008098115, International Publication No. 2008121160, International Publication No. 2008150494, International Publication No. 2010033736, International Publication No. 2014113510 (each incorporated herein by reference in its entirety).

[0122] The term “epitope” includes any polypeptide determinant that can specifically bind to an antibody. In certain embodiments, the “epitope” includes a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, phosphoryl, or sulfonyl, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features. The epitope is the target region to which the antibody binds. In one embodiment, the bispecific antibody of the present invention binds to the N-terminal domain of CEACAM5 (an Ig-like V-type domain of amino acids 35-144, UniProtKB-P06731). The binding site of the CEAxCD47 bispecific antibody to CEACAM5 is achieved via epitope binning. In epitope binning, antibodies are tested in a pairwise combinatorial manner and antibodies competing for the same binding region are grouped together in a bin. The competitive testing is carried out herein, according to the state of the art and as described herein, using anti-CEA antibodies. In one embodiment, the bispecific antibody of the present invention competes with the reference antibody SM3E for binding to CEACAM5. Competition is measured by an assay in which biotinylated human CEACAM5 at a concentration of 0.5 μg / ml is immobilized and incubated with serial dilutions of the reference (67 nM to 0.09 nM). 0.1 μg / ml of the CEAxCD47 bispecific antibody of the present invention is added at room temperature for 1 hour. The plate is washed and the bound CEAxCD47 bispecific antibody is detected.

[0123] As used herein, the term “common heavy chain (cHC)” refers to a polypeptide comprising, from N-terminus to C-terminus, an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), and is abbreviated as VH-CH-HR-CH2-CH3. A common heavy chain suitable for the bispecific antibody of the present invention is the heavy chain of the anti-CD47 antibody described in International Publication Nos. 2012023053, 2013088259, 2014087248, and 2016156537 (each of which is incorporated in whole by reference). In one embodiment, the common heavy chain of the bispecific antibody according to the present invention includes, as a heavy chain CDR, CDRH1 of SEQ ID NO. 1, CDRH2 of SEQ ID NO. 2, and CDRH3 of SEQ ID NO. 3. In one embodiment, the cHC of the bispecific antibody according to the present invention includes the VH region of SEQ ID NO: 4 as the heavy chain variable region VH. In one embodiment, the Fab portion of the common heavy chain cHC of the bispecific antibody according to the present invention is SEQ ID NO: 5 (VH-CH1). In one embodiment, the common heavy chain cHC of the bispecific antibody according to the present invention is SEQ ID NO: 6 (VH-CH1-CH2-CH3). SEQ ID NO: 6 is a heavy chain further containing the IgG1 Fc portion. In one embodiment, the antibody according to the present invention is a κλ bispecific antibody containing a cHC (κλ isomer).

[0124] "The κλ body format is a bispecific antibody indistinguishable from a standard IgG molecule, enabling affinity purification of bispecific antibodies with characteristics indistinguishable from a standard monoclonal antibody (see, for example, International Publication No. 2013088259 and International Publication No. 2012023053), and promises no or low potential for immunogenicity in patients."

[0125] The bispecific antibodies of the present invention, which include a common heavy chain, can be prepared, for example, according to International Publication No. 2012023053 (which is incorporated in its entirety by reference). The method described in International Publication No. 2012023053 produces bispecific antibodies that are structurally identical to human immunoglobulins. This type of molecule consists of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant kappa domain, and a second light chain variable region fused to a constant lambda domain. One binding site exhibits specificity to CEA, and the other site exhibits specificity to CD47, with the heavy chain and each light chain contributing to each, respectively. The light chain variable regions may be of the lambda or kappa family and are preferably fused to the constant lambda and kappa domains, respectively. This is preferred to avoid the formation of non-natural polypeptide binding. However, for the first specificity, the bispecific antibodies of the present invention can also be obtained by fusing a kappa light chain variable domain to a constant lambda domain, or for the second specificity, by fusing a lambda light chain variable domain to a constant kappa domain. In this case, the other light chain is always either entirely kappa (VL and CL) or entirely lambda (the so-called hybrid format of kappa-lambda bispecific antibodies). The bispecific antibody described in International Publication No. 2012023053 is a "κλ body". This κλ-body format is preferred over previous formats, for example, those containing amino acid crosslinks or other non-natural elements, because it allows for affinity purification of bispecific antibodies indistinguishable from standard IgG molecules that have characteristics indistinguishable from standard monoclonal antibodies.

[0126] As used herein, the terms “CEA” and “CEACAM5” refer to human carcinoembryonic antigen (CEA, CEACAM-5 or CD66e; UniProtKB-P06731), which is a cell surface glycoprotein and tumor-associated antigen (Gold and Freedman, J Exp. Med., 121:439-462, 1965; Berinstein NL, J Clin Oncol., 20:2197-2207, 2002). As used herein, the term “CEACAM3” refers to human CEACAM3 (UniProtKB-P40198 (CEAM3_HUMAN)), which is also a member of the carcinoembryonic antigen-associated cell adhesion molecule (CEACAM) family. Further information and information on other members of the CEA family can be found at http: / / www.uniprot.org.

[0127] In one embodiment, the bispecific antibody according to the present invention is not competitive with TCB2014. The bispecific anti-CEACAM5 × anti-CD3ε antibody cibisatamab is described by Bacac et al. (Clin. Cancer Res., 22(13), 3286-97(2016)). The antibody chain of TCB2014 is described in U.S. Patent Application Publication No. 20140242079 (SEQ ID NOs. 1, 2, 21, 22, 23 and 27 of U.S. Patent Application Publication No. 20140242079 (the whole of which is incorporated by reference)). Further bispecific CEAxCD3 Mab (TCB2017) is a molecule B "2+1" having charge modifications (VH / VL exchange in the CD3 binder, charge modification in the CEA binder, humanized CEA binder). It is described in International Publication No. 2017055389 as “IgG CrossFab, inverted” (see Sequence IDs 34, 36-38 of International Publication No. 2017055389 (which is incorporated in its entirety by reference)). As used herein, in one embodiment, “bispecific CEA × CD3 antibody” refers to antibody TCB2014, cibisatamab, or antibody TCB2017.

[0128] Cibisatamab, TCB2014, and TCB2017 bind to the CEACAM5 epitope located proximal to the cell membrane. In contrast, the CEAxCD47 bispecific antibody of the present invention binds to the distal epitope of the cell membrane near the N-terminus of CEACAM5 and does not compete with cibisatamab, TCB2014, and TCB2017 for binding to CEACAM5.

[0129] As used herein, the terms “specifically binds to CD47,” “bounds to CD47,” and “CD47-binding moiety” refer to specificity to human CD47 in the context of the bispecific antibodies of the present invention. Human CD47 is a multi-pass transmembrane protein comprising three extracellular domains (amino acids 19-141, 198-207, and 257-268; see UniProtKB-Q08722). As used herein, “binding affinity to CD47” is quantitatively measured (KD) by biolayer interferometry (Octet Technology) and / or surface plasmon resonance (Biacore Technology). In one embodiment, binding of the bispecific antibody according to the present invention to CD47 occurs via one or more of the extracellular domains.

[0130] In one embodiment, the second binding portion of the antibody according to the present invention (which specifically binds to human CD47) is characterized by a light chain containing CDRL1 of SEQ ID NO: 7, CDRL2 of SEQ ID NO: 8, and CDRL3 of SEQ ID NO: 9 as light chain CDRs, and a heavy chain containing CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3 as heavy chain CDRs. In one embodiment, the second binding portion of the antibody according to the present invention (which specifically binds to human CD47) is characterized by the kappa light chain variable region of SEQ ID NO: 10. In one embodiment, the second binding portion of the antibody according to the present invention (which specifically binds to human CD47) is characterized by the kappa light chain of SEQ ID NO: 11. In one embodiment, the second binding portion of the antibody according to the present invention (which specifically binds to human CD47) is characterized by the heavy chain variable region of SEQ ID NO: 4. In one embodiment, the second binding portion of the antibody according to the present invention (which specifically binds to human CD47) is characterized by the heavy chain of SEQ ID NO: 5. In one embodiment, the second binding portion of the antibody according to the present invention (which specifically binds to human CD47) is characterized by the heavy chain of SEQ ID NO: 6.

[0131] As used herein, the term “characterized by the heavy chain of SEQ ID NO: 5” refers to the VH-CH1 portion of the heavy chain that is the Fab portion of the antibody according to the present invention, as shown in Table 1. Such a heavy chain may also include further portions as hinge regions CH2, CH3 according to general knowledge, and may be any antibody format such as the F(ab')2 format. The preferred format is the common heavy chain format as described above.

[0132] As used herein, the terms “specifically binds to CEA,” “bound to CEA,” and “CEA-binding moiety” refer to the binding of the bispecific antibody of the present invention to recombinant human CEACAM5, wherein the antibody binds to recombinant human CEACAM3 at a KD value of 100 times or greater compared to the KD value of binding to recombinant human CEACAM5. As used herein, the term “KD” refers to the equilibrium dissociation constant between the bispecific antibody of the present invention and its antigen CEACAM5 or CEACAM3, specified in nM, and can be measured, for example, by surface plasmon resonance and / or biolayer interferometry (Example 3).

[0133] Binding to CEA (CEACAM5) on cells is measured using different tumor cell lines such as LoVo, LS174T, MKN-45, SNU-C1, SK-CO-1, and HPAF-II. The concentration of the antibody according to the present invention varies within an appropriate range with respect to the EC50 and Emax values ​​obtained for binding to the cells defined above. The binding curves of the bispecific antibody of the present invention are shown in Figures 1A to 1F, and the EC50 and Emax values ​​are listed in Table 4.

[0134] As used herein, the term “membrane-bound human CEA” refers to human carcinoembryonic antigen (CEA) bound to the membrane portion of a cell or to the surface of a cell, particularly to the surface of tumor cells.

[0135] As used herein, the terms “bispecific antibody conjugating to human CEA and human CD3” and “CEAxCD3 Mab” mean a bispecific antibody conjugating to human CEACAM5 and CD3ε. Such antibodies are, for example, cibisatamab, “TCB2014” and “TCB2017”. As used herein, “TCB2014” refers to a bispecific antibody conjugating to CEA and CD3 as described in U.S. Patent Application Publication No. 20140242079 (which is incorporated in its entirety by reference) as SEQ ID NOs: 1, 2, 21, and 22. As used herein, “TCB2017” refers to molecule B in the “2+1 IgG CrossFab, inverted” format having charge modifications (VH / VL exchange in the CD3 conjugate, charge modification in the CEA conjugate, humanized CEA conjugate); SEQ ID NOs: 34, 36-38 of International Publication No. 2017055389 (which is incorporated in its entirety by reference). Further CEAxCD3 mabs are described in International Publication Nos. 2007071426, 2013012414, 2015112534, 2017118675, U.S. Patent Application Publication Nos. 20140242079 and 2017055389 (each incorporated in its entirety by reference). Another CEAxCD3 mab is cibisatamab (formerly RO6958688) (see, for example, Bacac et al., Clin. Cancer Res., 22(13), 3286-97(2016)). In one embodiment, the CEAxCD47 mab according to the present invention is non-competitive and / or does not bind to the same human CEACAM5 epitope as TCB2014 or TCB2017.

[0136] As used herein, "CD3ε" and "CD3" refer to human CD3ε (UniProtKB-P07766(CD3E_HUMAN)). The terms "antibody against CD3ε(CD3)" and "anti-CD3ε(CD3) antibody" refer to antibodies that specifically bind to CD3ε. In one embodiment, the antibody against CD3ε specifically binds to the same epitope as the anti-CD3 antibody SP34 (BD Biosciences catalog number 565983).

[0137] In one embodiment, the bispecific antibodies of the present invention do not compete with TCB2014 and / or TCB2017 for binding to CEA presented on MKN-45 and / or LS174T cells. Therefore, TCB2014 at concentrations of 300 nM (TCB2014) or 30 nM (TCB2017) does not shift the EC50 of the phagocytic index curve of the bispecific antibodies of the present invention against MKN-45 and / or LS174T cells by more than threefold in the direction of higher concentrations in one embodiment.

[0138] The 300 nM TCB2014 concentration was selected because this concentration was measured in the plasma of patients treated with therapeutically effective doses of cibisatamab, such as 100 mg iv (see Melero et al., ASCO 2017, Abstract 2549 and Poster No. 41, Abstract in Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017) 2549-2549 for cibisatamab PK data; see Tabernero et al., J.Clin.Oncol. 35, 2017 (suppl.Abstr. 3002) for their respective clinical results). Currently active recruitment studies of cibisatamab in combination with PD-L1 inhibitors administer 100 mg of cibisatamab (ClinicalTrials.gov identifier: NCT03866239). TCB2017 is in preclinical investigation and is approximately 10 to 100 times potent than TCB2014 (as measured by binding affinity or tumor cell lysis in T cell-dependent cytotoxic TDCC assays; see International Publication No. 2017055389). Therefore, the EC50 shift of the phagocytic curve of the bispecific antibody of the present invention by TCB2017 was tested with 30 nM TCB2017.

[0139] Binding competition can be determined by flow cytometry-based measurement of the binding curve to MKN-45 cells and determination of the EC50 of this binding curve. Non-competition means that when 300 nM of TCB2014 is added to the assay, the EC50 shifts less than threefold in one embodiment toward higher concentrations. 300 nM is within the therapeutic active dose / plasma concentration range for the CEAxCD3 bispecific antibody (TCB2014) (J. Tabernero et al., J. Clin. Oncol. 35, 2017 (suppl. Abstr. 3002)). Non-competition with TCB2017 means that when 30 nM of TCB2017 is added to the assay, the EC50 shifts less than threefold.

[0140] As used herein, the term “non-competitive” means that a second antibody at a concentration of 300 nM (TCB2014) or 30 nM (TCB2017) (a bispecific antibody against CEAxCD3ε, e.g., TCB2014 or TCB2017) does not shift the EC50 of the binding curve of the bispecific antibody of the present invention to MKN-45 cells by more than 3 times, in one embodiment toward higher concentrations.

[0141] As used herein, the term “ADCP” refers to antibody-dependent cytophagocytosis. As used herein, phagocytosis, EC50 value of phagocytosis, maximum value of phagocytosis, and phagocytosis index according to the present invention refer to phagocytosis measured by “imaging” in tumor cell lines such as LoVo, LS174T, SNU-C1, and / or MKN-45. A suitable imaging method using incubation with an effector (macrophage):target (tumor) cell ratio of, for example, 1:1 or 1:3, and the “phagocytosis index” (imaging-determined ADCP) as a readout is described in Example 7. As used herein, “phagocytosis of the bispecific antibody” means phagocytosis caused / induced by the antibody.

[0142] The terms "human IgG" and "hIgG" refer to human antibody isotypes. When used in an experimental setting, these terms refer to commercially available, clinical-grade, homogeneous preparations of human immunoglobulin IgG (e.g., available from Bio-Rad) that do not specifically bind to CD47 and CEACAM5.

[0143] Therapeutic applications and methods using anti-CEA antigen-binding molecules The CEACAMxCD47 bispecific antibody of the present invention is optimized for the treatment of solid tumors, primarily by macrophage-mediated phagocytosis of tumor cells and also by ADCC, either in monotherapy or in combination therapy with a CEAxCD3 T cell bispecific antibody and / or a PD-1 axis antagonist such as cibisatamab, TCB2014, or TCB2017. The antibody of the present invention and the CEAxCD3 T cell bispecific antibody may be administered as described below.

[0144] In certain embodiments, the disease or solid tumor is a cancer that expresses or even overexpresses CEACAM5, but is not limited to the group of colorectal tumors, non-small cell lung tumors, gastric tumors, pancreatic tumors, and breast tumors. In certain embodiments, the tumor is a colorectal tumor. In certain embodiments, the tumor is a gastric tumor or gastroesophageal junction tumor. In certain embodiments, the tumor is a gastric tumor / gastroesophageal junction tumor expressing CEACAM5 and HER-2. In certain embodiments, the tumor is a lung tumor. All methods of use, uses, combinations, etc. for the therapeutic uses described herein are embodiments for the treatment of these tumors / diseases in particular.

[0145] The inventors recognize that the antibodies of the present invention may exhibit low or no ADA-forming ability, or loss of drug exposure, due to neutralization or loss of efficacy of anti-drug antibodies (ADAs).

[0146] In one embodiment, the present invention provides a method for treating carcinomas (cancer, tumors, e.g., human carcinomas), particularly CEACAM5-expressing tumors, in vivo. This method comprises administering to a subject a pharmaceutically effective amount of a composition containing the bispecific antibody of the present invention. "Subject" means a human subject, in one embodiment, a patient suffering from cancer / tumor / carcinoma.

[0147] CEACAM5 expression can be found in various tumor entities, particularly colorectal carcinoma, pancreatic adenocarcinoma, gastric cancer, non-small cell lung cancer, and breast cancer. In healthy normal glandular epithelium of the gastrointestinal tract, CEACAM5 is primarily expressed in a biased pattern on the apical surface of cells. This biased expression pattern limits accessibility by systemically administered anti-CEA monospecific or bispecific antibodies, thus limiting potential toxicity to healthy tissue. Together with the low affinity CD47 binding of the antibodies of the present invention, this leads to a lack of or limited phagocytosis of such normal cells by the antibodies of the present invention. In cells of gastrointestinal and other malignant tumors, this biased expression pattern is lost. CEACAM5 is expressed equally across the entire cell surface of cancer cells, meaning that cancer cells have much better access to the antibodies of the present invention than normal healthy cells, and can be selectively killed by the CEAxCD47 bispecific antibodies of the present invention, or by the above combination. CEACAM5 expression in cancer cells is predominantly higher than in non-malignant cells.

[0148] In one embodiment, the bispecific antibody of the present invention may be used as monotherapy for the treatment of progressive solid tumors, in one embodiment, CEACAM5-expressing tumors. In one embodiment, the bispecific antibody of the present invention is used in combination with a CEAxCD3 Mab, either concurrently, separately, or sequentially. In one embodiment, the bispecific antibody of the present invention is used in combination with a CEAxCD3 Mab and / or a PD-1 axial antagonist, either concurrently, separately, or sequentially. In one embodiment, the bispecific antibody of the present invention is used in combination with a PD-1 axial antagonist, either concurrently, separately, or sequentially. Such PD-1 axial antagonists are described, for example, in International Publication No. 2017118675. Such combinations attack solid tumors by macrophages and T cells. One CEAxCD3 Mab is in clinical development (see cibisatamab; ClinicalTrials.gov Identifier:NCT03866239). MEDI-565 was in clinical development, but active clinical trials could not be identified on clinicaltrials.gov. In one embodiment, the antibody TCB2014 or cibisatamab is used as a bispecific antibody against CEA and CD3.

[0149] The binder for CEA used in TCB2014 and cibisatamab is derived from the anti-CEA antibody PR1A3 (see, for example, European Patent No. 2681244). This antibody binds to the so-called B3 domain of CEA, which is located close to the cell membrane. TCB2014 has a low nM binding affinity for CEA and exhibits efficacy at high doses (40-600 mg per patient; see, e.g., J. Tabernero et al., J. Clin. Oncol. 35, 2017 (suppl. Abstr. 3002)). At the highest dose, almost all CEA targets on the cell surface are occupied by TCB2014. In the inventors' knowledge, combinations of cibisatamab, TCB2014, or TCB2017 with CEAxCD47 can simultaneously generate therapeutic plasma levels of both drugs, achieving best results (additive or even synergistic) when both drugs bind to different, non-overlapping epitopes and are non-competitive against the CEA antigen.

[0150] As used herein, the term “combined, simultaneous, separate or sequential combination” of the antibody of the present invention and a second bispecific antibody conjugating to human CEA and human CD3ε means any separate or together administration of the two antibodies (or, in the case of the combination of the antibody of the present invention and CEAxCD3 Mab and a PD-1 axis antagonist, three antibodies), for example, two or three antibodies being administered as part of an appropriate dosing regimen designed to obtain the benefits of combination therapy in separate, sequential, simultaneous, combined, time-delayed, or alternating administrations. Thus, two or three antibodies may be administered as part of the same pharmaceutical composition or in any of separate pharmaceutical compositions. The antibody of the present invention may be administered before, simultaneously with, or after the administration of the second bispecific antibody, or in some combination thereof. If the antibody of the present invention is administered to a patient at repeat intervals, the second bispecific antibody may be administered as a single dose before, simultaneously with, or after each administration of the antibody of the present invention, or in some combination thereof, or at different intervals in relation to treatment with the antibody of the present invention, or before, at any point in between, or after the treatment process with the antibody of the present invention. In one embodiment, the antibody of the present invention and the second bispecific antibody are administered alternately with an interval of 6 to 15 days between the administrations of the antibody of the present invention and the second antibody. In such an alternate administration, the first dose may be the antibody of the present invention or the second antibody.

[0151] The term "PD-1 axis antagonist" refers to an anti-PD-1 antibody or anti-PD-L1 antibody. Examples of anti-PD-1 antibodies include pembrolizumab (Keytruda®, MK-3475), nivolumab, pizilizumab, lambrolizumab, MEDI-0680, PDR001, and REGN2810. Examples of anti-PD-1 antibodies include 5 International Publication No. 200815671, International Publication No. 2013173223, International Publication No. 2015026634, U.S. Patent No. 7521051, U.S. Patent No. 8008449, U.S. Patent No. 8354509, International Publication No. 2009114335, International Publication No. 2015026634, International Publication No. 2008156712, International Publication No. 20150 These are described in International Publication No. 26634, International Publication No. 2003099196, International Publication No. 2009101611, International Publication No. 2010 / 027423, International Publication No. 2010 / 027827, International Publication No. 2010 / 027828, International Publication No. 2008 / 156712 and International Publication No. 2008 / 156712 (each of which is incorporated in its entirety by reference).

[0152] Anti-PD-L1 antibodies include, for example, atezolizumab, MDX-1 105, durvalumab, and avelumab. Anti-PD-L1 antibodies are described, for example, in International Publication No. 2015026634, International Publication No. 2013 / 019906, International Publication No. 2010077634, U.S. Patent No. 8383796, International Publication No. 2010077634, International Publication No. 2007005874, and International Publication No. 2016007235 (each of which is incorporated in its entirety by reference).

[0153] With regard to the combined administration of the antibody of the present invention and a second bispecific antibody, both compounds may exist in one single dosage form or in separate dosage forms, for example, in two different or identical dosage forms.

[0154] If the antibody of the present invention and the second antibody do not compete with respect to CEACAM5, in one embodiment, both antibodies may be administered simultaneously if desired by the physician. If the antibody of the present invention and the second antibody do compete with respect to CEACAM5, in one embodiment, both antibodies may be administered alternately.

[0155] The antibodies of the present invention are typically administered to a patient in a dosing regimen that provides the most effective treatment (in terms of both efficacy and safety) for the cancer being treated, as is known in the art. Preferably, tumor cells are attacked simultaneously by T cells and macrophages, and in order to achieve the full therapeutic potential of this approach, the CEAxCD3 and CEAxCD47 bispecific antibodies according to the present invention must be non-competitive with respect to binding to CEA on the cell surface.

[0156] As described above, the amount of antibody administered and the timing of administration of the antibody of the present invention may depend on the type of patient being treated (e.g., sex, age, weight) and symptoms, the severity of the disease or symptoms being treated, and the route of administration. For example, the antibody of the present invention and the second antibody may be administered to a patient in doses ranging from 0.1 to 100 mg / kg body weight / day / week, as a single dose, in divided doses, or by continuous infusion. In one embodiment, the antibody of the present invention and the second antibody are each administered to a patient in doses ranging from 0.1 to 30 mg / kg. In some cases, dose levels below the lower limit of the above range may be appropriate, while in other cases, higher doses may be used without causing any adverse side effects.

[0157] As used herein, the term “antibody half-life” refers to the elimination half-life of the antibody as measured by a standard pharmacokinetic assay. The antibody of the present invention and the second bispecific antibody against CEA and CD3 have elimination half-lives of 3 to 14 days.

[0158] In another aspect, the present invention also relates to the use of the bispecific antibodies of the present invention in the treatment of diseases, particularly cell proliferation disorders in which CEACAM5 is expressed, especially in which CEACAM5 is abnormally expressed (e.g., overexpressed or expressed in different patterns on the cell surface) compared to normal tissue of the same cell type. Such disorders include, but are not limited to, colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, gastroesophageal cancer, pancreatic cancer, and breast cancer. CEACAM5 expression levels can be determined by a variety of modern methods (e.g., via immunohistochemical assays, immunofluorescence assays, immunoenzyme assays, ELISA, flow cytometry, radioimmunoassays, etc.).

[0159] In one embodiment, the bispecific antibody of the present invention may be used to target cells expressing CEACAM5 in vivo or in vitro. The bispecific antibody of the present invention is particularly useful in preventing tumor formation through induction of ADCP and ADCC in tumor cells, eradicating tumors, and inhibiting tumor growth or metastasis. The bispecific antibody of the present invention may be used to treat any tumor expressing CEACAM5. Specific malignancies that can be treated with the bispecific antibody of the present invention include, but are not limited to, colorectal cancer, non-small cell lung cancer, gastric cancer, gastroesophageal junction cancer, pancreatic cancer, and breast cancer.

[0160] The bispecific antibodies of the present invention are discussed below and may be administered to humans in pharmaceutically acceptable dosage forms, such as intravenously as a bolus, or by continuous infusion over a period of time via intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intra-sacral, intrathecal, oral, topical, or inhalation routes, and are administered to mammals, preferably humans. The bispecific antibodies of the present invention may also be appropriately administered via intratumoral, peritumoral, intralesional, or perilesional routes to exert local and systemic therapeutic effects.

[0161] Regarding the treatment of a disease, the appropriate dosage of the bispecific antibody of the present invention depends on the type of disease to be treated, the severity and course of the disease, previous treatments, the patient's medical history and response to the antibody, and the discretion of the attending physician. The bispecific antibody of the present invention is administered appropriately to the patient in a single dose or over a series of treatments. The present invention provides a method for selectively killing tumor cells (also referred to herein as cancer cells) that express CEACAM5.

[0162] The method described above involves the interaction of the bispecific antibody of the present invention with the tumor cells. These tumor cells may originate from human cancers, including colorectal carcinoma, non-small cell lung carcinoma (NSCLC), gastric carcinoma, gastroesophageal junction carcinoma, pancreatic carcinoma, and breast carcinoma.

[0163] In another embodiment, the present invention relates to the use of the bispecific antibodies of the present invention for the manufacture of pharmaceuticals for treating diseases relating to abnormal CEACAM5 expression. In certain embodiments, the disease is a cancer that expresses or even overexpresses CEACAM5, including, but not limited to, colorectal tumors, non-small cell lung tumors, gastric tumors, gastroesophageal junction tumors, pancreatic tumors, and breast tumors. In certain embodiments, the tumor is a colorectal tumor.

[0164] Composition, formulation, dosage, and route of administration In one embodiment, the present invention relates to a pharmaceutical composition comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier. The present invention further relates to the use of such a pharmaceutical composition in methods for treating diseases such as cancer, or in the manufacture of pharmaceuticals for treating diseases such as cancer. Specifically, the present invention relates to a method for treating a disease, more specifically for treating cancer, which comprises administering a therapeutically effective amount of the pharmaceutical composition of the present invention.

[0165] In one embodiment, the present invention encompasses pharmaceutical compositions, combinations, and methods for treating human carcinomas, tumors, as defined above. For example, the present invention includes a pharmaceutical composition for use in the treatment of human carcinomas, comprising a pharmaceutically effective amount of the antibody of the present invention and a pharmaceutically acceptable carrier.

[0166] The bispecific antibody compositions of the present invention may be administered using conventional methods of administration, including, but not limited to, intravenous, intraperitoneal, oral, intralymphatic, or direct intratumoral administration. Intravenous or subcutaneous administration is preferred.

[0167] In one aspect of the present invention, a therapeutic formulation containing the bispecific antibody of the present invention is prepared for storage in the form of a lyophilized or liquid formulation by mixing the antibody of the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dosage and concentration used. Formulations to be used for in vivo administration must be sterile. This can be easily achieved by filtration with a sterile filtration membrane. The most effective mode of administration and administration regimen for the pharmaceutical composition of the present invention depends on the severity and course of the disease, the patient's symptoms and response to treatment, and the judgment of the treating physician. Therefore, the dosage of the composition may be a uniform dose or may be adapted to the individual patient, for example, body weight. Nevertheless, the effective dose of the composition of the present invention may generally be in the range of 0.1 to 30 mg / kg.

[0168] The bispecific antibodies of the present invention have a molecular weight of 150 kDa / mol. In one embodiment, they possess an Fc moiety. The elimination half-life in patients is in the range of 3 to 14 days. This half-life, while not limited, allows for administration once daily, once weekly, or once every two weeks or even once every four weeks.

[0169] The bispecific antibodies of the present invention and their respective compositions may be in a variety of dosage forms, including, but are not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymer microcapsules or microvesicles, liposomes, and injections or infusions. The preferred form depends on the mode of administration and therapeutic application.

[0170] The composition comprising the bispecific antibody of the present invention is formulated, administered, and given in a manner consistent with good medical practice. Factors to consider in this context include the specific disease or disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disease or disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to the physician.

[0171] product In another aspect of the present invention, a product is provided containing a material useful for treating, preventing and / or diagnosing the above-mentioned disorder. The product comprises a container and a label or accompanying information on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, and infusion bags. The container may be formed from a variety of materials, such as glass or plastic. The container may hold a composition that is effective by itself or in combination with another composition for treating, preventing and / or diagnosing a symptom and may have a sterile access port (for example, the container may be an infusion bag or vial with a stopper that can be punctured with a subcutaneous needle). At least one activator in the composition is the bispecific antibody of the present invention. The label or accompanying information indicates that the composition is used to treat a selected symptom. The product may also comprise (a) a first container in which the composition contains the bispecific antibody of the present invention; and (b) a second container in which the composition contains further cytotoxic agents or other therapeutic agents. The product in this embodiment of the present invention may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively, the product may further include a second (or third) container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0172] Further embodiments of the invention 1. A bispecific antibody comprising a first binding site that specifically binds to human CEACAM5 and a second binding site that specifically binds to human CD47, a) The first binding portion includes a heavy chain variable region which includes CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, b) The first bonding portion is defined as a light chain variable region, b1) CDRL1 of sequence number 14, CDRL2 of sequence number 15, and CDRL3 of sequence number 16, or b2) CDRL1 of sequence number 17, CDRL2 of sequence number 18, and CDRL3 of sequence number 19, b3) CDRL1 of sequence number 20, CDRL2 of sequence number 21, and CDRL3 of sequence number 22, b4) CDRL1 of sequence number 23, CDRL2 of sequence number 24, and CDRL3 of sequence number 25, and b5) A light chain variable region including a set of CDRLs selected from the group consisting of CDRL1 of SEQ ID NO: 26, CDRL2 of SEQ ID NO: 27, and CDRL3 of SEQ ID NO: 28, c) The second binding portion includes a heavy chain variable region comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, as a heavy chain variable region. A bispecific antibody characterized by including a light chain variable region comprising CDRL1 of SEQ ID NO: 7, CDRL2 of SEQ ID NO: 8, and CDRL3 of SEQ ID NO: 9. 2. The bispecific antibody according to Embodiment 1, characterized in that the first binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region, and a variable light chain region selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 as a variable light chain region, and the second binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region, and the variable light chain region of SEQ ID NO: 10 as a variable light chain region. 3. The bispecific antibody according to Embodiment 1, characterized in that the first binding portion includes a heavy chain containing SEQ ID NO: 5, and a light chain selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, and the second binding portion includes the heavy chain of SEQ ID NO: 5 and a light chain having SEQ ID NO: 11. 4. A bispecific antibody according to any one of the prior embodiments, characterized in that it contains the common heavy chain of SEQ ID NO: 6 as the heavy chain. 5. A bispecific antibody according to any one of the embodiments described above, wherein the first binding site is monovalent and the second binding site is monovalent. 6. A bispecific antibody according to any one of the embodiments described above, wherein the constant and variable framework region sequences are human. 7. A bispecific antibody according to any one of the preceding embodiments, wherein the light chain of the first binding portion is a lambda light chain (VLCL) and the light chain of the second binding portion is a kappa light chain (VKCK). 8. A bispecific antibody of the human IgG1 type, as described in any one of the embodiments described above. 9. The bispecific antibody according to any one of the preceding embodiments, wherein the antibody comprises a sugar-modified Fc region such that the number of fucose residues is reduced compared to the same bispecific antibody that has not been sugar-modified. 10. The bispecific antibody according to any one of the prior embodiments, characterized in that the bispecific antibody competes with the anti-CEACAM5 antibody SM3E, which includes the variable light chain region and the variable heavy chain region of SEQ ID NOs. 43 and 44, for binding to CEACAM5. A bispecific antibody according to any one of the prior embodiments, characterized by binding to recombinant human CEACAM5 with a binding affinity (KD) of 11.2 to 10 nM. A bispecific antibody according to any one of the prior embodiments, characterized by binding to human recombinant CD47 with a binding affinity of 12,100 nM to 600 nM. A bispecific antibody according to any one of the prior embodiments, characterized by a ratio of the KD values ​​for binding to recombinant CEACAM3 and recombinant CEACAM5 of 13.100 times or more. A bispecific antibody according to Embodiment 13, characterized by a ratio of the KD values ​​for binding to recombinant CEACAM3 and recombinant CEACAM5 of 14.100 to 200 times. 15. A bispecific antibody according to any one of the prior embodiments, characterized by an increase of at least 8% in the maximum phagocytic index of LoVo tumor cells compared to the phagocytic index of the bispecific antibody K2AC22. 16. A bispecific antibody according to Embodiment 15, characterized by an 8% to 20% increase in the maximum phagocytic index for LoVo tumor cells. 17. A bispecific antibody according to any one of the preceding embodiments, characterized by an increase of at least 8% in the maximum phagocytic index of Ls174T tumor cells compared to the phagocytic index of K2AC22. 18. A bispecific antibody according to Embodiment 17, characterized by an 8% to 25% increase in the maximum phagocytic index for Ls174T tumor cells. 19. A bispecific antibody according to any one of the prior embodiments, characterized in that it inhibits the interaction between CD47 and SIRPα on MKN-45 cells with an IC50 that is 10 times or more lower than the IC50 measured for K2AC22 under the same experimental conditions. 20. A bispecific antibody according to Embodiment 19, characterized by inhibiting the interaction between CD47 and SIRPα on MKN-45 cells with an IC50 of 10 to 30 times. 21. A bispecific antibody according to any one of the prior embodiments, characterized by inhibiting the interaction between CD47 and SIRPα on MKN-45 cells with an IC50 of 0.1 nM or less. 22. A bispecific antibody according to Embodiment 21, characterized by inhibiting the interaction between CD47 and SIRPα on MKN-45 cells with an IC50 of 0.1 nM to 0.04 nM. 23. A bispecific antibody according to any one of the prior embodiments, characterized in that it does not compete with cibisatamab for binding to CEACAM5. 24. A bispecific antibody according to any one of the preceding embodiments, characterized in that the first bispecific antibody, at a concentration of 300 nM, does not cause the second bispecific antibody, which binds to human CEACAM5 and CD3ε, to shift the EC50 of the binding curve of the first bispecific antibody to MKN-45 cells or LS174T cells by more than three times toward higher concentrations. 25. The bispecific antibody according to Embodiment 24, wherein the second bispecific antibody is TCB2014 or cibisatamab. 26. An isolated polynucleotide or a group of polynucleotides encoding a bispecific antibody as described in any one of the preceding embodiments. 27. An expression vector comprising one or more polynucleotides as described in Embodiment 26. 28. Host cells containing the expression vector described in Embodiment 27. 29. A method for producing a bispecific antibody according to any one of Embodiments 1 to 25, comprising: a) culturing the host cells described in Embodiment 28 under conditions that enable the production of the bispecific antibody; and b) isolating the antibody. 30. A bispecific antibody according to any one of Embodiments 1 to 25, for use in the treatment of human cancer. 31. A bispecific antibody for use according to Embodiment 30, characterized in that the cancer is a solid tumor. 32. A bispecific antibody for use according to Embodiment 30, characterized in that the cancer is colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer, breast cancer, or another CEACAM5-expressing cancer. 33. A bispecific antibody according to any one of Embodiments 1 to 25, for use in the manufacture of a pharmaceutical product for treating subjects having cancer expressing CEACAM5. 34. A bispecific antibody according to any one of Embodiments 1 to 25, for use in the treatment of a subject having cancer expressing CEACAM5, in combination with a second bispecific antibody comprising a third binding portion that specifically binds to human CEACAM5 and a fourth binding portion that specifically binds to human CD3ε, either simultaneously, separately, or sequentially. 35. The bispecific antibody according to any one of Embodiments 1 to 25, for use in simultaneous, separate, or sequential combinations, wherein the second bispecific antibody is TCB2014 or cibisatamab. 36. A bispecific antibody for use according to Embodiment 34 or 35, characterized in that the bispecific antibody according to the present invention and the second bispecific antibody are administered simultaneously to the subject at intervals of 6 to 15 days. 37. A pharmaceutical composition comprising a bispecific antibody according to any one of Embodiments 1 to 25 and a pharmaceutically acceptable excipient or carrier. 38. A pharmaceutical composition according to Embodiment 37 for use as a pharmaceutical product. 39. A pharmaceutical composition according to Embodiment 37 or Embodiment 38 for use as a pharmaceutical in the treatment of solid tumors. 40. A pharmaceutical composition according to any one of Embodiments 37 to 39, for use as a pharmaceutical in the treatment of colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer, or breast cancer. 41. A method for treating a subject having cancer expressing CEACAM5, comprising administering to the subject a therapeutically effective amount of a bispecific antibody described in any one of Embodiments 1 to 25 or a pharmaceutical composition described in any one of Embodiments 37 to 40. 42. The method according to Embodiment 41, wherein the cancer is a human cancer. 43. The method according to Embodiment 41 or 42, wherein the subject is a patient. 44. The method according to any one of Embodiments 41 to 43, characterized in that the cancer is colorectal cancer cells, NSCLC (non-small cell lung cancer) cells, gastric cancer cells, pancreatic cancer cells, breast cancer cells, or other tumor cells expressing CEACAM5. 45. The method according to any one of embodiments 41 to 44, wherein the bispecific antibody is administered in combination with chemotherapy or radiotherapy. 46. ​​A method for treating a human patient having a tumor, comprising administering an effective amount of a CEACAM5 × CD47 bispecific antibody described in any one of Embodiments 1 to 25 and a second bispecific antibody against CEACAM5 and CD3. 47. The method according to Embodiment 46, wherein the CEACAM5 × CD47 bispecific antibody and the CEACAM5 and CD3 antibodies are not competitive. 48. The method according to embodiment 46 or 47, wherein the antibody is administered simultaneously. 49. The method according to any one of Embodiments 41 to 48, wherein the patient is administered one or more doses of the bispecific antibody described in any one of Embodiments 1 to 25, ranging from 0.01 mg / kg to 10 mg / kg. 50. The method according to any one of Embodiments 46 to 48, wherein the patient is administered one or more doses of the CEACAM5 × CD3 bispecific antibody in a dose of 0.01 mg / kg to 10 mg / kg and one or more doses of the CEACAM5 × CD47 bispecific antibody in a dose of 1 mg / kg to 20 mg / kg. 51. The method according to any one of Embodiments 46 to 50, wherein the second antibody is TCB2014 or cibisatamab. 52. A method for extending the survival time of a subject having cancer expressing CEACAM5, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody described in any one of Embodiments 1 to 25. 53. The method according to Embodiment 52, characterized in that the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, or breast cancer. 54. The method according to any one of Embodiments 52 or 53, wherein the bispecific antibody is administered in combination with chemotherapy and / or radiotherapy. 55. The method according to any one of Embodiments 52 to 54, wherein the patient is administered one or more doses of the bispecific antibody described in any one of Embodiments 1 to 25, ranging from 0.01 mg / kg to 10 mg / kg. [Table 1-1] [Table 1-2] [Examples]

[0173] Example 1: Cloning, expression, and purification of human CEACAM5; source of huCEACAM3 and huCD47. The sequence corresponding to the complete extracellular domain (ECD) of CEACAM5 was subcloned into a pEAK8 mammalian expression vector (Edge Biosystems, Gaithersburg, Md.). The vector was modified to introduce Avitag® (Avidity, Denver Colo.) and a hexahistidine tag, a human Fc region, or a mouse Fc region to the C-terminus. The constructs were validated by DNA sequencing. Recombinant soluble proteins were purified by IMAC (Immobilized Metal Ion Affinity Chromatography), FcXL, or CaptureSelect® IgG-Fc(ms) affinity matrix. Human CEACAM3 and biotinylated CEACAM3 are available from ACROBiosystems, Newark, USA (Thermo Ffisher Scientific). Human CD47 and biotinylated CD47 can be produced as described in International Publication No. 2019234576, or are available from ACROBiosystems, Newark, USA.

[0174] Example 2: Expression and purification of bispecific antibodies containing lambda and kappa light chains Co-expression can be achieved in different ways, such as transfection of multiple vectors, each expressing one of the chains to be co-expressed, or by using a vector that drives the expression of multiple genes. The vector pNovi κHλ was previously generated to enable co-expression of one heavy chain, one kappa light chain, and one lambda light chain, as described in U.S. Patent Application Publication 2012 / 0184716 and International Publication 2012 / 023053 (which are each incorporated herein by reference in their entirety). Driving the expression of three genes by the human cytomegalovirus promoter (hCMV), the vector also contains a glutamine synthetase gene (GS) that enables the selection and establishment of stable cell lines. For transient expression in mammalian cells, the VL genes of anti-hCEACAM5 IgGλ or anti-hCD47 IgGκ were cloned into the vector pNovi κHλ. Peak cells or CHO cells are cultured in a suitable flask containing an appropriate number of cells and a suitable amount of culture medium (containing fetal bovine serum). Transfect the cells with plasmid DNA using Lipofectamine 2000) according to the manufacturer's instructions. During production, measure the antibody concentration in the supernatant of the transfected cells using OctetRED96. Depending on the antibody concentration, collect the supernatant 5-7 days after transfection and clarify by centrifugation at 1300g for 10 minutes. The purification process consists of three affinity steps. First, wash the FcXL affinity matrix (Thermo Fisher Scientific) with PBS and then add it to the clarified supernatant. After overnight incubation at +4°C, centrifuge the supernatant at 2000g for 10 minutes, save the flow-through, and wash the resin twice with PBS. Then transfer the resin to an Amicon Pro column and use a solution containing 50mM glycine at pH 3.0 for elution. Several elution fractions are generated, pooled, and desalted against PBS using a 50kDa Amicon® ultracentrifuge filter unit (Merck KGaA, Darmstadt, Germany).Using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, Del.), the eluted product containing total human IgG from the supernatant is quantified and incubated with an appropriate amount of Kappa Select Affinity Matrix (GE Healthcare) at room temperature and 20 rpm for 15 minutes. The incubation, resin recovery, elution, and desalting steps are carried out as described above. The final affinity purification step is performed using Lambda Fab Select Affinity Matrix (GE Healthcare), applying the same process as the two previous purifications. The final product is quantified using NanoDrop. The purified bispecific antibodies are analyzed by electrophoresis under denaturing and reducing conditions. Manufacturer (Agilent. Use the Agilent 2100 Bioanalyzer with the Protein 80 kit, as described by Technologies, Santa Clara, Calif., USA. Mix 4 μL of purified sample with sample buffer supplemented with dithiothreitol (DTT; Sigma Aldrich, St. Louis, Mo.). Heat the sample at 95°C for 5 minutes, then load it onto the chip. Test all samples for endotoxin contamination using the Limulus Amebocyte Lysate test (LAL; Charles River Laboratories, Wilmington, Mass.).

[0175] Example 3: KD measurement. a) Experimental procedure for measuring the KD of Ab against recombinant human CEACAM5 (Octet) The affinity of the CD47xCEACAM5 bispecific antibody of the present invention for recombinant soluble human CEACAM5 against the anti-human CEACAM5 arm was determined using biolayer interferometry (BLI) technology. An OctetRED96 instrument and a Protein A biosensor (Sartorius) were used. Measurements were performed at 30°C. After hydration, preconditioning, and baseline steps in kinetic buffer (PBS, 0.002% Tween® 20, 0.01% BSA, Kathon; Sartorius), 0.5 μg / mL of κλ in kinetic buffer was loaded onto the biosensor for 5 minutes. The biosensor was then immersed in serial dilutions of recombinant human CEACAM5 extracellular domain (ECD) soluble protein (proprietary) at 2-fold dilutions, starting at 50 nM. The association and dissociation phases were monitored for 600 seconds each. The biosensor was regenerated using 10 mM glycine pH 1.7. Standard acquisition rates were applied (averaged at 5.0 Hz, 20). Curves were processed without inter-step correction using reference well subtraction and Y-alignment on baseline. Affinity was measured by applying a 1:1 global fitting model to the complete association and dissociation steps. The binding affinity (KD) of the bispecific antibodies of the present invention to recombinant human CD47 was determined by the same experimental procedure. The KD of exemplary bispecific antibodies of the present invention to CEACAM5, as determined by this procedure, is shown in Table 2 below.

[0176] b) Experimental procedure for measuring the KD of Ab against recombinant human CEACAM3 (Octet) The affinity of the CD47xCEACAM5 bispecific antibody of the present invention for recombinant soluble human CEACAM3 in the anti-human CEACAM5 arm was determined using biolayer interferometry (BLI) and the OctetRED96 instrument. His-tagged recombinant huCEACAM3 (R&D Systems, #9868-CM) was captured using a HIS1K biosensor (Sartorius) loaded with anti-His-tagged antibody. Measurements were performed at 30°C. After hydration, preconditioning, and baseline steps in kinetic buffer (PBS, 0.002% Tween® 20, 0.01% BSA, Kathon; Sartorius), 5 μg / mL of recombinant huCEACAM3 in kinetic buffer was loaded onto the biosensor for 5 minutes. The biosensor was then immersed in serial dilutions of the κλ form at 2-fold dilutions, starting at 667 nM. The association and dissociation phases were monitored for 60 and 120 seconds, respectively. The biosensor was regenerated using 10 mM glycine pH 1.7. Standard acquisition rates were applied (averaged at 5.0 Hz, 20). Curves were processed using double-reference subtraction, Y-alignment on baseline, and inter-step correction. Affinity was measured by applying a 1:1 global fitting model during the first 5 seconds of the complete association and dissociation steps. The KD of the exemplary bispecific antibody of the present invention for CEACAM3 determined by this procedure is shown in Table 2 below. [Table 2]

[0177] Example 4: Epitope binning of CD47xCEACAM5 bispecific antibody by competition with reference antibody SM3E. Epitope binning is a competitive immunoassay used to characterize the binding of the antibody of the present invention, for example, the binding of the first binding portion of the bispecific antibody of the present invention to a related bivalent anti-CEA (target protein) antibody. A competitive blockade profile of an antibody that binds to a target protein is created against a novel antibody that also binds to this target protein, and whose binding epitope is already established / published. Competition with this reference antibody indicates that the antibodies have the same or closely located epitopes and that they "binn" together. The ability of the CD47xCEACAM5 bispecific antibody of the present invention to compete with a CEACAM5 reference antibody is tested by ELISA against recombinant human CEACAM5 using a reference antibody derived from SM3E (US Patent Application Publication No. 20050147614) having a mouse Fc region (mAb produced using a standard method). SM3E binds more to the distal cell membrane portion of the N-terminus of CEA.

[0178] Biotinylated human CEACAM5 is coated at 0.5 μg / ml in a streptavidin-coated 96-well plate and incubated for 1 hour with a serially diluted reference mAb (0.09 nM to 67 nM) or an unrelated mAb containing the mouse Fc region. The CD47xCEACAM5 bispecific antibody of the present invention is added at 0.1 μg / ml at room temperature for 1 hour. The plate is washed, and the conjugated CD47xCEACAM5 bispecific antibody is detected with anti-human IgG(Fc)-HRP (Jackson ImmunoResearch). After washing, the plate is cleared using Amplex Red reagent. Synergy The fluorescence signal is measured using an HT plate reader (Biotek).

[0179] Competitive experiments were conducted using the CD47×CEACAM5 bispecific antibody of the present invention. Binding to K2AC82, K2AC84, K2AC91, K2AC100, and K2AC117 was reduced by 80% or more by the respective competitive (i.e., tool) antibody. The CD47xCEACAM5 bispecific antibody is identified herein as competitive with the SM3E antibody if the binding of the bispecific antibody is reduced by 80% or more at the highest concentration of the reference tool antibody. The CD47xCEACAM5 bispecific antibody is identified as non-competitive with the tool antibody if, when comparing the results with and without the tool antibody, the binding to CEACAM5 is reduced by less than 20%.

[0180] Example 5: Determination of target densities (i.e., numbers) of CEACAM5 and CD47 on the cell surface of six different cancer cell lines. The target density (i.e., number) of CEACAM5 and CD47 on the cell surface of six different cancer cell lines was measured. The cell lines tested were human gastric adenocarcinoma cells (MKN-45, DSMZ ACC 409), human colorectal cancer cells (SK-CO-1 (ATCC;HTB-39), SNU-C1 (ATCC;CRL-5972), Ls174T (ATCC;CL-188), and LoVo (ATCC;CCL-229)), or pancreatic adenocarcinoma cells (HPAF-II, ATCC, CRL-1997).

[0181] QIFIKIT® (Agilent Dako) was used for the quantification of cell surface antigens by flow cytometry using an indirect immunofluorescence assay. QIFIKIT® consists of a set of six bead populations coated with different but clearly defined amounts of mouse monoclonal antibodies (Mab). The beads mimic cells labeled with a specific primary mouse monoclonal antibody. Different cell specimens can be labeled with different primary antibodies and then quantified using the same calibration bead set.

[0182] Cells were cultured in their respective adaptation media, detached with trypsin-EDTA (Sigma Aldrich), centrifuged (3 min, 350 g), resuspended in cold FACS buffer (PBS, 2% BSA-Sigma Aldrich), and filtered through 0.22 μm (Stericup, Millipore) to obtain 3.106 cells / mL. 3.10 5 Cells were plated onto a V-bottom plate. 1 μL of FcγR blocking reagent was added to each well, and the plate was incubated at 4°C for 10 minutes. 10 μL of primary antibody against human CEACAM5 (#sc-23928; mIgG1 (Santa Cruz)) and human CD47 (internal production; B6H12; mouse skeleton) at a final concentration of 20 μg / mL was added to the cells, and the plates were incubated at 4°C for 30 minutes. The cells were washed twice with 200 μL of PBS BSA 2% and centrifuged at 400 g for 3 minutes. 100 μL of beads (QIFIKIT® setting or calibration) were washed with the cells and treated similarly. 100 μL of secondary antibody from the kit (1 / 50 in PBS BSA 2%) was added to each well, and the plates were incubated at 4°C for 30–45 minutes. The cells were centrifuged (3 minutes, 400 g at 4°C), the supernatant was discarded, and the cells were washed twice. After the final centrifugation, cells were resuspended in 130 μL of CellFix and acquired using a CytoFlex cytometer (Beckman Coulter). Analysis was performed using FlowJo software, and geometric means were exported to an Excel file. Linear regression was performed using MFI values ​​from calibration beads. The antibody-binding capacity (ABC) of the cells was extrapolated from this regression line. Specific antibody-binding capacity (sABC) was obtained by subtracting the ABC for one of the specific stains from the isotype control. The data from this analysis are shown in Table 3 below. [Table 3]

[0183] Example 6: Measurement of binding of a bispecific antibody to CEAxCD47 against a CEACAM5-expressing cancer cell line (EC50 and maximum binding Emax). The binding of CD47xCEACAM5 bispecific antibodies was tested in CEACAM5-expressing human gastric adenocarcinoma cells (e.g., MKN-45), CEACAM5-expressing human colorectal cancer cells (SK-CO-1, SNU-C1, Ls174T, LoVo), and CEACAM5-expressing pancreatic adenocarcinoma cells (HPAF-II).

[0184] Cells were harvested, counted, and checked for viability. 3x10 cells were then placed in FACS buffer (PBS 2% BSA, 0.1% NaN3). 6 The cells were resuspended at a concentration of cells / ml. 100 μl of the cell suspension was distributed into a V-bottom 96-well plate (3 × 10⁶ cells). 5 Cells / well). The supernatant was removed by centrifugation at 1300 rpm, 4°C for 3 minutes. Then, the antibody of the present invention was added to the wells in increasing concentrations and incubated at 4°C for 15 minutes. The cells were washed twice with cold FACS buffer and re-incubated at 4°C for a further 15 minutes with PE (R-phycoerythrin) conjugate mouse anti-human IgG Fc secondary antibody (SouthernBiotech, 1:100 pre-diluted in FACS buffer). The cells were washed twice with cold FACS buffer and resuspended in 300 μl of FACS buffer containing 1:15000 dilution of SytoxBlue (Life Technologies). Fluorescence, specifically mean fluorescence activity (MFI), was determined using a Cytoflex (Millipore) flow cytometer. Binding curves and EC50 and Emax values ​​were obtained and calculated using GraphPad Prism7 software. The data from this analysis are shown in Table 4 below. [Table 4] * MFI - Mean Fluorescence Intensity N / A - Not applicable - No available data on this Ab in this cell line.

[0185] The data in Table 4 shows that all bispecific antibodies according to the present invention exhibit significantly lower EC50 and significantly higher Emax compared to K2AC22.

[0186] As shown in Table 4, the bispecific antibody according to the present invention binds to SK-CO1 cells with an EC50 value of 10 - 30 nM, to MKN-45 cells with an EC50 value of 5 - 15 nM, to HPAF-II cells with an EC50 value of 5 - 15 nM, to SNU-C1 cells with an EC50 value of 1 - 10, to LS174T cells with an EC50 value of 3 - 15 nM, and / or to LoVo cells with an EC50 value of 15 - 25 nM.

[0187] Also, as shown in Table 4, the bispecific antibody according to the present invention has an Emax value of 0.5 - 1.5 (MFIx10 6 ) for SK-CO1 cells, an Emax value of 1 - 2 (MFIx10 6 ) for MKN-45 cells, an Emax value of 0.5 - 1.5 (MFIx10 6 ) for HPAF-II cells, an Emax value of 0.2 - 0.6 (MFIx10 6 ) for SNU-C1 cells, an Emax value of 0.05 - 0.2 (MFIx10 6 ) for LS174T cells and / or an Emax value of 0.2 - 0.5 (MFIx10 6 ) for LoVo cells.

[0188] Example 7: Measurement of phagocytosis (phagocytosis index) of antibody-dependent cell phagocytosis (ADCP). The phagocytic in vitro activity of the CEACAM5xCD47 bispecific antibody of the present invention was evaluated using six CEACAM5-expressing cancer cell lines (MKN-45, SK-CO-1, SNU-C1, Ls174T, LoVo and HPAF-II). K2AC22 was evaluated for comparison using the same cell lines and experimental procedures.

[0189] The assay depends on an imaging-based method using the CellInsight CX5 High Content Screening Platform. The readout evaluated is the phagocytosis index, which is defined as the average number of target cells engulfed by 100 macrophages.

[0190] 1. Macrophage Preparation: Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats derived from different healthy donors (5-7 different donors, depending on the cell line) using a Ficoll gradient. Macrophages were generated by culturing PBMCs for 7 days in complete medium (RPMI1640, 10% heat-inactivated fetal calf serum [Invitrogen]), 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, 25 mg / mL gentamicin (all from Sigma-Aldrich), and 50 mM 2-mercaptoethanol (Thermo Fisher Scientific) in the presence of 20 ng / mL human macrophage colony-stimulating factor (M-CSF) (PeproTech). Next, non-adherent cells were eliminated during the differentiation stage (+1 day) by changing the cell culture medium, and adherent cells corresponding to macrophages were isolated using cell dissociation buffer (Sigma-Aldrich). For cytometry-based ADCP experiments, these cells were washed with complete medium on the day of use (day 7, 8, or 9). For cell imaging-based ADCP, macrophages were isolated on day 6 using cell dissociation buffer and seeded at 30,000 cells / well in 96 optical plates (Costar).

[0191] 2. Evaluation of phagocytic activity (CellInsight®-based assay) Macrophages (stained with calcein red-orange) attached to microplate wells were co-incubated with calcein AM-labeled target tumor cells in a 1:3 effector:target cell ratio at 37°C for 30 minutes (MKN45 and SNU-C1) or 2.5 hours (LoVo and Ls174T) in the presence of different concentrations of test antibodies. At the end of the incubation period, the supernatant was replaced with complete culture medium, and the microplates were imaged using the CellInsight® CX5 high-content screening platform. 1500 macrophages per well were acquired and analyzed. Phagocytosis was demonstrated as a double-positive event (macrophage + target tumor cell), and the phagocytic index was calculated using CellInsight® manufacturer's software.

[0192] All results shown in Figure 2 and Tables 5, 6, 7, 8, and 9 were obtained in four CEACAM5-expressing cancer cell lines (MKN-45, SNU-C1, Ls174T, and LoVo). The effector cell to target / tumor cell ratio was 1:3. [Table 5]

[0193] All five bispecific antibodies according to the present invention showed better binding compared to K2AC22 (lower EC50 and higher Emax; see Example 6, Table 4). Surprisingly, the percentage increase in the maximum achieved phagocytic index Emax ADCP of the antibodies of the present invention compared to K2AC22 was strongest in the low-CEACAM5 expressing cell lines LoVo and Ls174T.

[0194] These results were obtained in experiments using macrophages from different human donors. Data from such experiments are shown in Table 6 (for MKN-45 cells), Table 7 (for SNU-C1 cells), Table 8 (for Ls174T cells), and Table 9 (for LoVo cells). [Table 6] [Table 7] [Table 8] [Table 9]

[0195] Example 8: Measurement of competition for binding to CEACAM5 between the bispecific antibody of the present invention and other therapeutic antibodies that bind to CEACAM5. The binding assay to cells expressing CEACAM5 was performed as described in Example 6. This assay can be used to measure the shift in the binding curves of the bispecific antibodies of the present invention to MKN-45 and LS174T cancer cell lines when a CEAxCD3 bispecific antibody such as cibisatamab or TCB2014 is added to the binding assay. If a 300 nM antibody shifts the binding curve of the bispecific antibodies of the present invention by less than 3-fold, the antibody is considered non-competitive.

[0196] In this experiment, the concentration-dependent binding of the CD47xCEACAM5 bispecific antibody K2AC100 was measured in the presence of either TCB2014 or TCB2017 anti-CEACAM5 mAb. This binding was measured on the cell surface of CEACAM5-expressing MKN-45 cells. K2AC100 was directly labeled with a fluorescent dye, and its binding to MKN-45 cells alone (dark line, dark circle) was tracked in the presence of 300 nM TCB2014 (dark line, dark triangle) or 30 nM TCB2017 (dark line, black diamond). A negative control (Ctrl) was used (IgG1 in the presence of TCB2014 or TCB2017). The results of this experiment are shown in Figure 5. These data indicate that there was no or minimal shift in the binding curve of the CEAxCD47 bispecific antibody K2AC100 to MKN-45 tumor cells when 300 nM TCB2014 was added. Therefore, the K2AC100 antibody does not compete with the TCB2014 and TCB2017 antibodies in terms of CEACAM5 binding.

[0197] Example 9: Production of the afucosylated bispecific antibody of the present invention. Tables 10 and 11 show the results of phagocytosis (EC50 and Emax) of two cell lines (MKN-45 and SNU-C1) with the afucosylated version of the bispecific antibody of the present invention. The afucosylated version of the bispecific antibody of the present invention was produced and purified by the following method.

[0198] 1. Production A CHO pool transfected with plasmids for each of the bispecific antibodies of the present invention (see Example 2 for the plasmids for each vector) was placed in a Thomson Erlen device with 0.3 × 10 cells. 6 Live cells were inoculated at a concentration of cells / mL, with working volumes of 700 mL and 100 mL respectively for the production of fucosylated and afucosylated antibodies. All pools were operated in fed-batch mode for 15 days using CDACF medium CDCHO and a suitable supply regime. For the production of afucosylated antibodies, a bolus of 200 μM fucose inhibitor (1,3,4-tri-O-acetyl-2-deoxy-2-fluoro-L-fucose) was added during the fed-batch process on days 0, 5, 8, and 11, based on the afucosylation strategy described by Rillahan et al., Nature Chem. Biol. 2012 Jul;8(7):661-8 and based on European Patent Application Publication No. 2282773. After 15 days of fed-batch culture, the supernatant of the bispecific antibody pools of the present invention containing fucosylated or afucosylated antibodies was collected. The recovered CHO pool supernatant was clarified using the Sartoclear Dynamics® Lab V Cell Harvesting Sartorius system (see supplier instructions).

[0199] 2. Purification The purification of the fucosylated and afucosylated bispecific antibodies of the present invention was completed by a three-step affinity purification process. To use a column with an appropriate volume of affinity matrix, the antibody concentration in the supernatant of the bispecific antibody pool was measured using OctetRED96 before the start of purification. Each clarified CHO pool supernatant containing the fucosylated or afucosylated bispecific antibody was loaded onto a MabSelect SuRe (MSS) column (GE Healthcare) without prior preparation to remove most of the cell culture contaminants. The MSS eluate was then treated with a low pH hold to inactivate the virus and neutralized to pH 6 using Tris 1M pH9. The MSS eluate was then loaded onto a LambdaFabSelect (LFS) column (GE Healthcare) to remove mono-specific κ (mono-κ). The LFS eluate was then pH adjusted to pH 6. The LFS was loaded onto a Capto L (CL) column (GE Healthcare) to remove mono-specific λ (mono-λ). The CL eluate was pH adjusted before storage. Next, the final material was concentrated, diafiltration into the final formulation buffer, and the concentration was adjusted using Nanodrop. The fucosylated and afucosylated bispecific antibodies were divided equally and stored at -80°C until delivery. The purified bispecific antibodies were analyzed for size by electrophoresis under denaturing and reducing conditions using an Agilent 2100 Bioanalyzer with the Protein 80 kit as described by the manufacturer (Agilent Technologies, Santa Clara, Calif., USA). Agglutination levels were assessed by size exclusion chromatography (SEC-UPLC) using an ACQUITY UPLC H-Class Bio System (Waters). Charge variant analysis of the purified bispecific antibodies was performed by isoelectric focusing (IEF) using a Multiphor II electrophoresis system (GE Healthcare). Using a throughput microchip-CE method with LabChip GXII Touch (Perkin Elmer), the relative distribution of N-bound bifurcated glycans of fucosylated and afucosylated K2AC5 and K2AC22 antibodies was determined.Limulus Amebocyte Lysate Test (LAL; Charles). All antibodies were tested for endotoxin contamination using River Laboratories (Wilmington, Mass). The afucosylation bispecific antibody of the present invention showed >70% afucosylation.

[0200] Using these afucosylated CEAxCD47 bispecific antibodies, we obtained the results shown in Tables 10 and 11, and Figures 3A and 3B.

[0201] 3. Other methods for producing the afucosylated bispecific antibody of the present invention 3.1. By using FUT8-negative cell lines Alternatively, according to the inventors' knowledge, the afcosylated bispecific antibody of the present invention can also be produced by the following method.

[0202] The materials and methods are as described in Naoko Yamane-Ohnuki et al., Biotech.Bioeng.;87(2004)614-622.

[0203] Isolation of Chinese hamster FUT8 cDNA Following the inventors' knowledge, total RNA was isolated from CHO / DG44 cells using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed using oligo-dT with the Superscript first-strand synthesis system (Invitrogen, Carlsbad, CA) for reverse transcription polymerase chain reaction (RT-PCR). Primers designed from mouse FUT8 cDNA (Hayashi, 2000; DNA Seq 11:91-96) were used. 5V-GTCTGAAGCATTATGTGTTGAAGC-3V (Sequence ID 45) and 5V-GTGAGTACATTCATTGTACTGTG-3V (Sequence No. 46) Using PCR, we amplified Chinese hamster FUT8 cDNA from single-stranded CHO / DG44 cell cDNA.

[0204] Targeting constructs for the FUT8 locus In accordance with the inventors' knowledge, the FUT8 gene is targeted for disruption in CHO / DG44 cells using two substitution vectors, pKOFUT8Neo and pKOFUT8Puro. To establish a targeting construct, a 9.0kb fragment of the FUT8 gene containing the first coding exon is isolated by screening the CHO-K1 cell E genome library (Stratagene, La Jolla, CA) using Chinese hamster FUT8 cDNA as a probe. The 234bp segment containing the translation initiation site is replaced with a neomycin resistance gene (Neor) cassette or a puromycin resistance gene (Puror) cassette from plasmid pKOSelectNeo or pKOSelectPuro(Lexicon,TX), respectively, adjacent to the loxP site. The diphtheria toxin gene (DT) cassette from plasmid pKOSelectDT(Lexicon) is inserted into the 5V homologous region. The resulting targeting constructs, pKOFUT8Neo and pKOFUT8Puro, contained a 1.5kb 5V homologous sequence and a 5.3kb 3V homologous sequence. Prior to transfection, the targeting constructs were linearized at a unique SalI site.

[0205] Transfection and screening of homologous recombinants According to the inventors' knowledge, 4Ag of linearized pKOFUT8Neo was electroporated into subconfluent CHO / DG44 cells (1.6 10⁶) at 350V and 250AF using Bio-Rad GenePulser® II. After electroporation, transfectants were selected using 600Ag / mL G418 (Nacalai Tesque, Kyoto, Japan). The following primers were used: 5V-TTGTGTGACTCTTAACTCTCAGAG-3V (Sequence ID 47) and 5V-GAGGCCACTTGTGTAGCGCCAAGTG-3V (Sequence No. 48) Using a previously reported modified microextraction method (Ramirez-Solis et al., 1992; Anal Biochem 201:331-335), genomic PCR is performed in a 96-well plate.

[0206] Homologous recombinants were identified using 1.7kb fragments obtained by genomic PCR, and the following primers were used: 5V-GTGAGTCCATGGCTGTCACTG-3V (Sequence ID 49) and 5V-CCTGACTTGGCTATTCTCAG-3V (Sequence ID 50) Confirmation is performed by Southern blot analysis using a 221 bp fragment amplified using [the appropriate method].

[0207] Hemizygous clones are subjected to a second round of homologous recombination using linearized pKOFUT8Puro, as described above, and drug selection with 15Ag / mL puromycin (Sigma-Aldrich, St. Louis, MO). The Cre-recombinase expression vector pBS185 (Invitrogen) is electroporated onto the identified homozygous disruptors to remove drug resistance gene cassettes derived from both FUT8 alleles.

[0208] Production of monoclonal antibodies by FUT8(-) cells According to the inventors' knowledge, an expression vector encoding the bispecific antibody of the present invention is electroporated into a FUT8(−) cell line and selected in a medium lacking hypoxanthine and thymidine. Confluent transfectants are cultured for one week in Ex-Cell® 301 medium (JRH Biosciences, Lenexa, KS). The antibody is purified from the culture supernatant using MabSelect™ (Amersham Biosciences, Piscataway, NJ). Further purification steps can be anion / cation exchange chromatography, size exclusion chromatography, and in particular purification using the above kappa or lambda selection resin.

[0209] 3.2. Recovery of extracellular fucose from the production cell medium plus enzymatic intervention in intracellular fucose biosynthesis Preferably, according to the inventors' knowledge, the afucosylated bispecific antibody of the present invention can also be produced according to the following method / technique and as described in US Patent No. 8,642,292. This technique is designed to constitute stable incorporation of a heterologous bacterial enzyme into an antibody-producing cell line such as a CHO cell line. Thereby, de novo synthesis of fucose from D-mannose is blocked. Further, when the production cells are cultured in a medium free of fucose, as a result, an antibody having a stable level of afucosylation is produced.

[0210] In eukaryotic cells, fucose is generated via two pathways a) from the extracellular space or lysosomes via the salvage pathway and b) by de novo synthesis of fucose from D-mannose in the de novo synthesis pathway of fucose.

[0211] The salvage pathway can be completely blocked by removing fucose from the culture medium. The de novo biosynthesis pathway can be blocked by converting the intermediate GDP-4-keto-6-deoxy-D-mannose in this pathway to GDP-D-rhamnose instead of GDP-4-keto-6-deoxy-D-galactose. This is achieved by stably integrating the gene encoding RMD into the producing cell line and introducing the bacterial enzyme GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) into the producing cell line, respectively. Even when the amount of RMD expressed in the producing cell line is quite low, it completely blocks the de novo synthesis pathway of the producing cells.

[0212] This technique is designed for the production of the afucosylated antibodies of the present invention and for existing producing cell lines that already produce the antibodies of the present invention and are engineered to produce antibodies with a 80% - 100% reduction in fucose content, and is used to construct producing cell lines, such as CHO cell lines.

[0213] All the results shown in FIGS. 3A and 3B and Tables 10 and 11 were obtained with two CEACAM5-expressing cancer cell lines (MKN-45 and SNU-C1). The effector cell to target / tumor cell ratio is 1:3. These results were obtained from experiments using macrophages obtained from three different human donors. The data obtained from such experiments are shown in Table 10 (for MKN-45 cells) and Table 11 (for SNU-C1 cells).

Table 10

Table 11

[0214] Example 10 Blocking the interaction between SIRPα and CD47 on tumor cells Experimental setup for measuring the SIRPα inhibitory efficacy (IC50) of the bispecific antibodies of the present invention:

[0215] A cell-based assay monitoring the interaction between soluble SIRPα and human CD47 expressed on the surface of MKN-45 cells, as described below, was used to detect blocking activity. Concentration-response experiments using the bispecific antibody according to the present invention enabled the determination of inhibition curves (see Figure 4) and IC50 values ​​(see Table 12).

[0216] MKN-45 cancer cells expressing both CD47 and CEACAM5 were stained with CFSE violet to enable detection of the cells by an imaging system (CX5). Briefly, 3,000 stained MKN-45 cells / well were seeded into a 384 optical-well plate (Costar) and incubated for 50 minutes with the bispecific antibody of the present invention at increased concentrations (1.9 pM to 333 nM, 4 replicates). Subsequently, a fixed concentration of SIRPα-mouse Fc, pre-mixed with anti-mouse IgG-Fc AF647 conjugated antibody (diluted to Jackson Immunoresearch 1:2000), was added at a final concentration of 50 ng / mL. After 3 hours and 30 minutes of incubation, images of the fluorescence signal emitted by the detected conjugated SIRPα on the plate were acquired using an imaging system (CX5, Thermo Fisher). The fluorescence signal (mean fluorescence intensity MFI) was plotted according to the dose range tested, and IC50 was calculated using software (Prism, GraphPad). The results are shown in Table 12. [Table 12]

[0217] Example 11: a. Organoid procedure to obtain CEACAM5 expression in cancer cells from fresh samples derived from cancer patients (Qifikit data) and b. Organoid procedure to obtain phagocytic data Organoids derived from patient primary samples were prepared as single-cell suspensions by standard methods (enzymatic digestion and / or mechanical dissociation). 10 μL of anti-human CEACAM5 primary antibody (#sc-23928; mIgG1 (Santa Cruz); final concentration 20 μg / mL) was added to the cells and incubated at 4°C for 30 minutes. The cells were washed and centrifuged. 100 μL of beads (QIFIKIT® setting or calibration) were washed with the cells and treated similarly. 100 μL of secondary antibody from the kit (1 / 50 in PBS BSA 2%) was added to each well and incubated at 4°C for 30–45 minutes. The cells were centrifuged, the supernatant discarded, and washed twice. After the final centrifugation, the cells were resuspended and acquired by cytometry. Analysis was performed using specific software, and geometric means were exported to an Excel file. Linear regression was performed using MFI values ​​from the calibration beads. The antibody-binding capacity (ABC) of cells was extrapolated from this regression line. Specific antibody-binding capacity (sABC) was obtained by subtracting the ABC for one specific stain from the isotype control.

[0218] The average expression level of CEACAM5 in these major organoids was found to be 28,000 CEACAM5 targets per cell, which is approximately four times lower than the average expression level in the cell lines listed in Table 5.

[0219] When the bispecific antibody of the present invention and macrophages derived from human donors are added, concentration-dependent phagocytosis / phagocytic index can also be tested using organoids derived from primary samples of cancer patients (see Example 7). Using the same method, according to our knowledge, the combination of the bispecific antibody of the present invention and the CEAxCD3 bispecific antibody can also be tested when human donor-derived T cells are added.

[0220] Example 12: Antitumor activity: Tissue section culture According to our findings, the antitumor activity of the bispecific antibodies according to the present invention can be evaluated as monotherapy and in combination therapy, respectively, in tumor tissue section cultures derived from patients diagnosed with CEA-expressing tumors (see Soennichsen et al., Clinical Colorectal Cancer 2018).

[0221] 1. Culture and treatment of tissue sections Fresh tumor tissue samples are cut and handled as previously published (Soennichsen et al., Clinical Colorectal Cancer 2018). Briefly, immediately after surgical excision and initial macroscopic pathological evaluation, tumor samples are cut into 350 μm sections using a tissue chopper. The tissue section diameters are then standardized by using a 3 mm coring tool. Three tissue sections are randomly pooled and placed on a membrane insert and cultured in a 6-well plate. The sections are incubated under standardized conditions of 37°C and 5% CO2. After pre-culturing in standard cell culture medium, the section triplets are exposed to the bispecific antibodies according to the present invention, either individually or in combination (e.g., using PD-L1 inhibitors), for up to 120 hours. After compound exposure, tumor sections are fixed overnight with 4% paraformaldehyde.

[0222] 2. Dyeing Paraformaldehyde-fixed sections are embedded in paraffin and processed into 5 μm sections. Hematoxylin and eosin (HE) staining are performed to assess histopathological aspects and the proportion of tumor cells. Overall cell count, tumor cell count, and proliferation are analyzed by immunofluorescence staining. In short, the paraffin sections are deparaffinized. After antigen recovery, the sections are washed with 0.3% PBS / Triton® X and blocked with 5% normal goat serum for 30 minutes. Primary antibodies against cytokeratin (AE1b3), Ki67, and cleaved PARP are each diluted in 0.5% bovine serum albumin and incubated overnight at 4°C. The sections are rinsed with 0.3% phosphate-buffered saline / Triton® X and labeled with secondary antibodies. Nuclei are stained with Hoechst 33342. Further staining (e.g., for CEA expression) may be included.

[0223] 3. Data Analysis Using a fluorescence microscope, five images (20x magnification) are taken per tissue section from fluorescently stained sections. The number of positive pixels is determined using a stain-specific segmentation algorithm for Hoechst 33342, cytokeratin, Ki67, and cleavage PARP staining. The proliferating / apoptotic tumor area is calculated by analyzing pixels with Ki67 / cleavage PARP-positive nuclei surrounded by cytokeratin-positive pixels. For all images, the total cell count (Hoechst-positive), tumor cell count (Hoechst-positive and cytokeratin-positive), and proliferating tumor cell count (Hoechst-, cytokeratin-, and Ki67-positive / cleavage PARP) are calculated. The tumor cell count is normalized to the total cell count, and the proliferating tumor cell count is normalized to the tumor cell count to account for different tumor cell fractions per image. The mean intersection value is then calculated from a single image value. The mean value for each condition is calculated using the mean intersection value.

[0224] Example 13: In vivo antitumor activity. According to our findings, the antitumor activity of the bispecific antibodies according to the present invention can be evaluated similarly in transgenic mice as monotherapy and in combination therapy.

[0225] 1. Preparation and growth test of cell lines For example, an hCEACAM5(Tg)hCD47(Tg)mCD47(ko) cell line based on the mouse colon cancer cell lines CT26 or MC38 is prepared. Knockout (KO) of the endogenous mouse CD47 gene is performed by using CRISPR / Cas9, and then KO clones are isolated by cell sorting. After transfecting the KO clones with a cassette that drives the expression of both hCD47 and hCEACAM5 using an internal ribosome entry site (IRES), clones that have been engineered are isolated, for example, based on overall expression levels and expression ratios. Three validated clones are selected and then their engraftment / tumorigenicity is tested in vivo for the selection of the final clone.

[0226] 2. In vivo antitumor activity Mouse strains of the BALB / cJGpt background expressing human CD3e (T001550 heterozygous BALB / c-hCD3ET / Wt mice) and human CD47 / human SIRPα (T037264 homozygous BALB / c-hCD47 / hSIRPα mice) are available from GemPharmatech. Alternatively, mouse strains of the C57BL / 6 / Bcgen background expressing human CD3e (homozygous B-hCD3E mice) and human CD47 / human SIRPα (homozygous B-hSIRPα / hCD47 mice) are available from Biocytogen. The two mouse strains are crossed to obtain triple humanized hCD3e / hSIRPa / hCD47 mice, and their progeny are used in subsequent experiments to test the bispecific antibody according to the present invention either as a single agent or in combination treatment.

[0227] Triple-humanized hCD3e / hSIRPa / hCD47 mice are inoculated on day 0 with either the CT26-hCEACAM5(Tg)hCD47(Tg)mCD47(ko) cell line (BALB / c background) or the MC38-hCEACAM5(Tg)hCD47(Tg)mCD47(ko) cell line (C57BL / 6 background). A moderate tumor size in the cohort is, for example, 200 mm. 3 Once the tumor volume of a single mouse reaches, for example, 3000 mm², treatment with the bispecific antibodies according to the present invention, either as a monotherapy or in combination, is appropriate. 3 Treatment is initiated as an intravenous bolus at intervals of, for example, 2 treatments / week until it exceeds a certain threshold or exceeds one of the pre-specified animal protection endpoints and / or care endpoints. Tumor volume and body weight are measured three times a week. Tumor volume is measured in mm using the following formula. 3 Given by: TV = 0.5a × b², where a and b are the long and short diameters of the tumor, respectively. The present invention provides, for example, the following items: (Item 1) A bispecific antibody comprising a first binding site that specifically binds to human CEACAM5 and a second binding site that specifically binds to human CD47, a) The first binding portion includes a heavy chain variable region which includes CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, b) The first bonding portion is defined as a light chain variable region, b1) CDRL1 of sequence number 14, CDRL2 of sequence number 15, and CDRL3 of sequence number 16, or b2) CDRL1 of sequence number 17, CDRL2 of sequence number 18, and CDRL3 of sequence number 19, b3) CDRL1 of sequence number 20, CDRL2 of sequence number 21, and CDRL3 of sequence number 22, b4) CDRL1 of sequence number 23, CDRL2 of sequence number 24, and CDRL3 of sequence number 25, and b5) A light chain variable region including a set of CDRLs selected from the group consisting of CDRL1 of SEQ ID NO: 26, CDRL2 of SEQ ID NO: 27, and CDRL3 of SEQ ID NO: 28, c) The second binding portion includes a heavy chain variable region comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, as a heavy chain variable region. A bispecific antibody characterized by including a light chain variable region comprising CDRL1 of SEQ ID NO: 7, CDRL2 of SEQ ID NO: 8, and CDRL3 of SEQ ID NO: 9. (Item 2) The bispecific antibody according to item 1, characterized in that the first binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region, and a variable light chain region selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 as a variable light chain region, and the second binding portion includes the variable heavy chain region of SEQ ID NO: 4 as a variable heavy chain region, and the variable light chain region of SEQ ID NO: 10 as a variable light chain region. (Item 3) The bispecific antibody according to item 1, characterized in that the first binding portion includes a heavy chain containing SEQ ID NO: 5 and a light chain selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, and the second binding portion includes a heavy chain region of SEQ ID NO: 5 and a light chain having SEQ ID NO: 11. (Item 4) A bispecific antibody according to any one of the preceding items, characterized in that it contains the common heavy chain of SEQ ID NO: 6 as the heavy chain. (Item 5) A bispecific antibody as described in any one of the above items, wherein the first binding site is monovalent and the second binding site is monovalent. (Item 6) A bispecific antibody as described in any one of the above items, wherein the constant and variable framework region sequences are human. (Item 7) A bispecific antibody according to any one of the preceding items, wherein the light chain of the first binding portion is a lambda light chain (VLCL) and the light chain of the second binding portion is a kappa light chain (VKCK). (Item 8) A bispecific antibody of the human IgG1 type, as described in any one of the items above. (Item 9) The bispecific antibody according to any one of the preceding items, wherein the antibody includes a sugar-modified Fc region such that the number of fucose residues is reduced compared to the same bispecific antibody that has not been sugar-modified. (Item 10) The bispecific antibody according to any one of the preceding items, characterized in that the bispecific antibody competes with the anti-CEACAM5 antibody SM3E, which includes the variable light chain region and the variable heavy chain region of SEQ ID NOs. 43 and 44, for binding to CEACAM5. (Item 11) A bispecific antibody as described in any one of the preceding items, characterized by binding to recombinant human CEACAM5 with a binding affinity (KD) of 2 to 10 nM. (Item 12) A bispecific antibody as described in any one of the preceding items, characterized by binding to human recombinant CD47 with a binding affinity of 100 nM to 600 nM. (Item 13) A bispecific antibody according to any one of the preceding items, characterized by a ratio of the KD values ​​for binding to recombinant CEACAM3 and recombinant CEACAM5 of 100 times or more. (Item 14) A bispecific antibody as described in item 13, characterized by a ratio of the KD values ​​for binding to recombinant CEACAM3 and recombinant CEACAM5 of 100 to 200 times. (Item 15) A bispecific antibody as described in any one of the preceding items, characterized by an at least 8% increase in the maximum phagocytic index of LoVo tumor cells compared to the phagocytic index of the bispecific antibody K2AC22. (Item 16) A bispecific antibody, as described in item 15, characterized by an 8% to 20% increase in the maximum phagocytic index for LoVo tumor cells. (Item 17) A bispecific antibody as described in any one of the preceding items, characterized by at least an 8% increase in the maximum phagocytic index of Ls174T tumor cells compared to the phagocytic index of K2AC22. (Item 18) A bispecific antibody, as described in item 17, characterized by an 8% to 25% increase in the maximum phagocytic index for Ls174T tumor cells. (Item 19) A bispecific antibody according to any one of the preceding items, characterized by inhibiting the interaction between CD47 and SIRPα on MKN-45 cells with an IC50 that is 10 times or more lower than the IC50 measured for K2AC22 under the same experimental conditions. (Item 20) A bispecific antibody as described in item 19, characterized by inhibiting the interaction between CD47 and SIRPα on MKN-45 cells with an IC50 of 10 to 30 times. (Item 21) A bispecific antibody according to any one of the preceding items, characterized by inhibiting the interaction between CD47 and SIRPα on MKN-45 cells with an IC50 of 0.1 nM or less. (Item 22) A bispecific antibody as described in item 21, characterized by inhibiting the interaction between CD47 and SIRPα on MKN-45 cells with an IC50 of 0.1 nM to 0.04 nM. (Item 23) A bispecific antibody as described in any one of the preceding items, characterized by not competing with cibisatamab for binding to CEACAM5. (Item 24) A bispecific antibody according to any one of the preceding items, characterized in that the first bispecific antibody, at a concentration of 300 nM, does not cause the second bispecific antibody, which binds to human CEACAM5 and CD3ε, to shift the EC50 of the binding curve of the first bispecific antibody to MKN-45 cells or LS174T cells by more than three times toward higher concentrations. (Item 25) The bispecific antibody described in item 24, wherein the second bispecific antibody is TCB2014 or cibisatamab. (Item 26) An isolated polynucleotide or group of polynucleotides encoding a bispecific antibody as described in any one of the preceding items. (Item 27) An expression vector containing one or more polynucleotides as described in item 26. (Item 28) Host cells containing the expression vector described in item 27. (Item 29) A method for producing a bispecific antibody according to any one of items 1 to 25, comprising: a) culturing the host cells described in item 28 under conditions that enable the production of the bispecific antibody; and b) isolating the antibody. (Item 30) A bispecific antibody described in any one of items 1 to 25, for use in the treatment of human cancer. (Item 31) A bispecific antibody for use as described in item 30, characterized in that the cancer is a solid tumor. (Item 32) A bispecific antibody for use as described in item 30, characterized in that the cancer is colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer, breast cancer, or another CEACAM5-expressing cancer. (Item 33) A bispecific antibody as described in any one of items 1 to 25, for use in the manufacture of a pharmaceutical product for treating subjects with cancer expressing CEACAM5. (Item 34) A bispecific antibody according to any one of items 1 to 25, for use in combination with a second bispecific antibody comprising a third binding site that specifically binds to human CEACAM5 and a fourth binding site that specifically binds to human CD3ε, in the treatment of a patient with cancer expressing CEACAM5, either simultaneously, separately, or sequentially. (Item 35) A bispecific antibody according to any one of items 1 to 25, for use in simultaneous, separate, or sequential combinations, wherein the second bispecific antibody is TCB2014 or cibisatamab. (Item 36) A bispecific antibody for use according to item 34 or 35, characterized in that the bispecific antibody according to the present invention and the second bispecific antibody are administered simultaneously to the subject at intervals of 6 to 15 days. (Item 37) A pharmaceutical composition comprising a bispecific antibody described in any one of items 1 to 25 and a pharmaceutically acceptable excipient or carrier. (Item 38) A pharmaceutical composition as described in item 37, for use as a medicine. (Item 39) A pharmaceutical composition as described in item 37 or item 38, for use as a pharmaceutical in the treatment of solid tumors. (Item 40) A pharmaceutical composition according to any one of items 37 to 39, for use as a pharmaceutical in the treatment of colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer, or breast cancer. (Item 41) A method for treating a subject having cancer that expresses CEACAM5, comprising administering to the subject a therapeutically effective amount of a bispecific antibody described in any one of items 1 to 25 or a pharmaceutical composition described in any one of items 37 to 40. (Item 42) The method described in item 41, wherein the cancer is a human cancer. (Item 43) The method according to item 41 or 42, wherein the subject is a patient. (Item 44) The method according to any one of items 41 to 43, characterized in that the cancer is colorectal cancer cells, NSCLC (non-small cell lung cancer) cells, gastric cancer cells, pancreatic cancer cells, breast cancer cells, or other tumor cells expressing CEACAM5. (Item 45) The method according to any one of items 41 to 44, wherein the bispecific antibody is administered in combination with chemotherapy or radiotherapy. (Item 46) A method for treating a human patient having a tumor, comprising administering an effective amount of a CEACAM5 × CD47 bispecific antibody described in any one of items 1 to 25 and a second bispecific antibody against CEACAM5 and CD3. (Item 47) The method according to item 46, wherein the CEACAM5 × CD47 bispecific antibody and the CEACAM5 and CD3 antibodies are not competitive. (Item 48) The method according to item 46 or 47, wherein the antibody is administered simultaneously. (Item 49) The method according to any one of items 41 to 48, wherein the patient is administered one or more doses of a bispecific antibody described in any one of items 1 to 25, ranging from 0.01 mg / kg to 10 mg / kg. (Item 50) The method according to any one of items 46 to 48, wherein the patient is administered one or more doses of the CEACAM5×CD3 bispecific antibody in a dose of 0.01 mg / kg to 10 mg / kg and one or more doses of the CEACAM5×CD47 bispecific antibody in a dose of 1 mg / kg to 20 mg / kg. (Item 51) The method according to any one of items 46-50, wherein the second antibody is TCB2014 or cibisatamab. (Item 52) A method for extending the survival time of a subject having cancer expressing CEACAM5, the method comprising administering a therapeutically effective amount of a bispecific antibody described in any one of items 1 to 25 to the subject. (Item 53) The method according to item 52, characterized in that the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, or breast cancer. (Item 54) The method according to either item 52 or 53, wherein the bispecific antibody is administered in combination with chemotherapy and / or radiotherapy. (Item 55) The method according to any one of items 52 to 54, wherein the patient is administered one or more doses of a bispecific antibody described in any one of items 1 to 25, ranging from 0.01 mg / kg to 10 mg / kg.

Claims

[Claim 1] The invention described in the specification.

Images