Combination therapy of bispecific antibodies against ceacam5 and CD47 and bispecific antibodies against ceacam5 and CD3

Abstract

The present invention relates to bispecific antibodies that bind to human carcino-embryonic antigen CEACAM5 and human CD47 for the treatment of cancer with bispecific antibodies that bind to human carcino-embryonic antigen CEACAM5 and human CD3 epsilon, such combinations and their use in the treatment of disease.

Description

[0001] Reference sequence list

[0002] The contents of the electronically submitted Sequence Listing filed with this application are incorporated by reference in its entirety. Field of the Invention

[0003] The present invention relates to a bispecific antibody that binds to human carcinoembryonic antigen 5 (CEACAM5, CEA) and human CD47 (CEAxCD47 bispecific antibody) as described below for use in combination with a bispecific antibody that binds to CEA and human CD3ε (CEAxCD3 bispecific antibody) as described below for treating cancer, such combinations and their use in disease treatment. Background of the Invention

[0005] CEA belongs to the CEA-related cell adhesion molecule (CEACAM) family, which includes 12 closely related proteins in humans encoded by 22 genes, which are assigned to CEACAM and pregnancy-specific glycoprotein (PSG) subgroups on chromosome 19q13 (Beauchemin N & Arabzadeh A, Cancer Metastasis Rev. 2013). CEACAM is involved in a variety of physiological processes, such as intercellular recognition, and modulates cellular processes, which range from the formation of tissue architecture and new blood vessel formation 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) exists as early as embryonic and fetal development, and its expression is maintained in normal adult tissues. Its main expression sites are in the columnar epithelial cells and goblet cells of the colon, specifically, in the upper third of the crypt and the free luminal surface.

[0006] CEA is (over)expressed in tumors of epithelial origin, including but not limited to colorectal, gastric, lung and pancreatic cancers (reviewed in Beauchemin N & Arabzadeh A, Cancer Metastasis Rev. 2013). It loses its apical expression, resulting in distribution throughout the cell surface ( Semin Cancer Biol 1999). Given its frequent observation of overexpression in CEA-positive tumors and the distribution of CEA throughout the surface of tumor cells, CEA is an interesting target for immunotherapeutic attack on cancer cells, while sparing normal cells of the same tissue.

[0007] US20140242079 and WO2017118657 (each of which is incorporated by reference in its entirety) mention methods for treating cancers expressing CEA by combining a human PD-1 axis antagonist with an anti-CEA / anti-CD3 bispecific antibody (cibisatamab) for T cell redirection and activation, and clinical results have been presented at the 2017 ASCO Annual Meeting (Tabernero et al., J Clin Oncol 35, 2017 (suppl; abstr 3002)). The only ongoing clinical trial recruiting cibisatamab in June 2022 is a trial in combination with a FAP-4-1BBL bispecific antibody, which can induce additional activation of T cells by stimulating the co-stimulatory receptor 4-1BB on T cells (ClinicalTrial.gov identifier NCT04826003). Patients were pretreated with the B cell depleting agent obinutuzumab to avoid the formation of anti-drug antibodies (ADA) and subsequent exposure loss. In the above clinical trials of cerbituximab, ADA formation and exposure loss of cerbituximab were observed in monotherapy and in combination with PD-L1 inhibitors.

[0008] WO2015112534 mentions such a method for treating tumors, which comprises administering an immune checkpoint antagonist that binds to two or more different targets of the immune checkpoint pathway, and a T cell redirector that binds to CEA and a T cell surface antigen. US20110064653 mentions a class I antibody that binds to CEACAM5 and granulocytes.

[0009] Human CD47 (UniProtKB-Q08722 (CD47_HUMAN; IAP)) is a transmembrane protein that binds the 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, especially to macrophages expressing SIRPα. Potent inhibition (low IC50) of SIRPα binding to CD47 on the surface of tumor cells is a measure to increase the phagocytosis of tumor cells by macrophages. CD47 is involved in a range of cellular processes, including apoptosis, proliferation, adhesion and migration. In addition, it plays a key role in immune and angiogenic responses. CD47 is overexpressed in tumor cells of patients with both hematological malignancies and solid tumors. Antibodies against CD47 have been described in the prior art and have shown promising preclinical and early clinical activity in different tumor entities, including hematological malignancies such as lymphomas, and solid tumors such as 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). IgG1 subclass antibodies that bind to CD47 can cause platelet depletion (thrombocytopenia) and a decrease in the number of red blood cells (RBC, anemia) in an Fc-dependent manner (see, e.g., US20140140989). To avoid this adverse effect, a mutant form of the IgG4 subclass of anti-CD47 antibodies (IgG4PE, with S228P mutation and L235E mutation to reduce FcγR binding) is described in WO2017196793. This anti-CD47 antibody with severely reduced FcγR binding and effector function does not cause such platelet depletion. von Bommel PE et al. (Oncoimmunol.7 (2018) e386361) and Piccione EC et al. (mAbs 7 (2015) 946-956) describe single-domain bispecific antibodies against CD47 and CD20. Dheilly E et al. (Mol.Thera.25 (2017) 523-533; see also WO2014087248) describe bispecific antibodies against CD19 and CD47.

[0010] Bispecific antibodies against CEACAM5 and CD47 are described in WO2019234576, EP19213002 and US62943726 (incorporated by reference in their entirety), comprising a common heavy chain of SEQ ID NO: 5 (VH-CH1) and a CD47-interactive variable light chain region VL of SEQ ID NO: 10. Bispecific antibodies against CD19 and CD47 are described in WO2014087248 (incorporated by reference in its entirety), comprising a common heavy chain of SEQ ID NO: 5 and a CD47-interactive variable light chain region VL of SEQ ID NO: 10. WO2018098384 relates to bispecific antibodies for co-targeting CD47 and CEACAM5. EP3623388 relates to a bispecific binding molecule comprising a tumor targeting arm and a fusion protein that blocks the interaction between CD47 and SIRPα with low affinity. WO 2018 / 057955 relates to bispecific antibodies that bind to CD47 and mesothelin, comprising a common heavy chain. WO2019016411 relates to bispecific antibody molecules that target CD47 and tumor antigens.

[0011] Despite some progress in the treatment of locally advanced tumors, or in particular, metastatic solid cancer types, the significant increase in progression-free survival (PFS) and / or overall survival (OS) induced by new anticancer drugs in patients with advanced cancers (such as colorectal cancer, pancreatic cancer, lung cancer, etc.) remains quite limited and generally not curative. Great hopes have been placed on cancer immunotherapy, and some but limited success has been achieved. Tumors develop mechanisms that protect them from destruction by T-effector cells and other immune cells (such as macrophages). Over the past (several) decades, strategies based on cancer immunotherapy have achieved some success in counteracting these tumor-protective mechanisms and redirecting T cells to fight cancer cells. The most prominent example of this strategy is the inhibitors / activators of certain immune checkpoints. For example, checkpoint inhibitors such as PD-1 axis antagonists have been shown to reactivate T-effector cells to fight certain solid cancers. However, not all solid tumor types are responsive to PD-1 axis antagonists, and even among those that are responsive, generally less than 50% of patients derive relevant benefit from treatment with, for example, anti-PD-1 or PD-L1 antibodies. For example, less than 10% of patients with advanced colorectal cancer are eligible for treatment with such PD-1 axis inhibitors (especially only about 4% of patients with advanced colorectal cancer whose cancer exhibits microsatellite instability (MSI)) see some benefit.

[0012] Adoptive T cell therapy with chimeric antigen receptor (CAR) T cells and therapy with T cell bispecific antibodies have provided promising clinical results in hematological malignancies. However, clinical studies of adoptive T cell therapy (such as CAR T cells) in various solid tumors have mostly shown no or only mild response rates (e.g. Xu et al., Expert Review of Anticancer therapies 2017, 17, 1099-1106; Greenbaum et al., Biol Blood Marrow Transplant 2020 October; 26(10): 1759-1769).

[0013] US20140242079, WO2017055389, US20140242080, WO2007071426, WO2013012414, WO2015112534, WO2017118675, US20140242079 and Bacac et al. (Clin. Cancer Res., 22(13), 3286-97 (2016)) describe CEAxCD3 T cell bispecific antibodies.

[0014] The T cell bispecific antibody TAAxCD3 (TAA stands for Tumor associated antigen, such as CEA and many other antigens) is very effective in patients with hematological malignancies (such as multiple myeloma) and B cell malignancies (such as diffuse large B cell lymphoma, follicular lymphoma, etc.). Clinical results of cerbituximab CEAxCD3 show that TAAxCD3 is also effective in advanced solid tumors (see above), but it is far less effective than, for example, CD20xCD3 or BCMAxCD3 in hematological malignancies (Moreau P1., N Engl J Med.,. 2022June 5. doi:10.1056 / NEJMoa2203478). Adding PD-1 axis inhibitors may increase efficacy, but if there is any, it is only limited. Adding bispecific antibodies or fusion proteins that agonize co-stimulatory T cell receptors (such as CD28 or 4-1BB) can increase efficacy in preclinical testing, but it will also increase toxicity, such as increased cytokine release. Instead of targeting additional activation of T cells, adding therapeutic agents that redirect other immune cells, particularly macrophages, to tumor cells may be more successful. The present invention relates to a combination therapy of a bispecific antibody CEAxCD47 that redirects and activates macrophages against solid tumors expressing CEACAM5, in particular together with a CEAxCD3 T cell bispecific antibody, to increase the tumor cell killing effect of the CEAxCD3 bispecific antibody and avoid the risk of increasing cytokine release syndrome (CRS) and potential T cell exhaustion, unlike a combination with a bispecific agonist against a T cell co-stimulatory receptor.

[0015] Bispecific antibodies against CEACAM5 and CD47 are described in WO2019234576 and WO2021110647. One bispecific antibody described in WO2019234576 is K2AC22 (SEQ ID NO: 65 of WO2019234576 shows the light chain of the CEACAM5 binding portion of K2AC22, SEQ ID NO: 5 of WO2019234576 shows the common heavy chain of K2AC22, and SEQ ID NO: 11 shows the light chain of the CD47 binding portion of K2AC22). In WO2019234576, for K2AC22, it has been shown that combination with CEAxCD3 (called CEA-TCB) increases tumor cell killing. As far as we know, the CEA-TCB is Cerbituximab or at least CEAxCD3, which has the same basic structure (2+1 format) as Cerbituximab and binds to the same CEA epitope as Cerbituximab. One object of the present invention is to provide a method for treating or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of a bispecific antibody against CEACAM5 and CD47, in combination with a bispecific antibody against CEACAM5 and CD3. One object of the present invention is an advantageous combination of a CEAxCD47 bispecific antibody and a CEAxCD3 bispecific antibody. The present invention further describes such a combination.

[0016] Bispecific antibodies against CEACAM5 and CD3 used in the combinations and methods according to the present invention are described in WO2021053587. Bispecific antibodies against CEACAM5 and CD47 used in the combinations and methods according to the present invention are described in PCT / IB2021 / 061983 (WO2022130348). Summary of the invention

[0017] In one aspect, the present invention provides a bispecific antibody against CEACAM5 and CD47 (further also referred to as CEAxCD47 bispecific antibody) as described below in combination with a bispecific antibody against CEACAM5 and CD3ε (CEAxCD3 bispecific antibody) as described below for use in the treatment of cancer.

[0018] In one aspect, the present invention provides a method for treating or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of a bispecific antibody against CEACAM5 and CD47 as described below (further also referred to as CEAxCD47 bispecific antibody) in combination with a bispecific antibody against CEACAM5 and CD3ε as described below (CEAxCD3 bispecific antibody). In one aspect, the present invention provides such a combination. The CEAxCD47 bispecific antibody used in the method or combination according to the present invention induces high phagocytic activity against tumor cells, both against tumor cells expressing high amounts of CEACAM5 and against tumor cells expressing low amounts of CEACAM5. In one embodiment, the CEAxCD47 bispecific antibody induces its anti-tumor effect mainly through optimized phagocytosis / antibody-dependent cellular phagocytosis (ADCP) due to the participation of immune cells (particularly macrophages). In one embodiment, the CEAxCD47 bispecific antibody used according to the present invention shows a reduced CEACAM3 to CEACAM5 binding affinity ratio, or an increased KD ratio, relative to the CEACAM5xCD47 antibody K2AC22. In one embodiment, the CEAxCD47 bispecific antibody used according to the present invention inhibits the binding of SIRPα to CD47 expressed on tumor cells and increases the phagocytosis of tumor cells. In one embodiment, the CEACAM5xCD3 bispecific antibody used according to the present invention is a κλ bispecific antibody that completely retains the sequence and structure of human IgG antibodies, thereby causing a lower risk of immunogenicity leading to ADA formation and potential exposure loss.

[0019] The combination according to the invention is also suitable for use in the treatment of tumors, in particular solid tumors.

[0020] In one aspect, the present invention provides:

[0021] (A) A first bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 (further also referred to as "CEA") and a second binding portion that specifically binds to human CD47 (further named "CD47"), characterized in that:

[0022] (a) the first binding moiety comprises a heavy chain variable region comprising CDRH1 of SEQ ID NO:23, CDRH2 of SEQ ID NO:24, and CDRH3 of SEQ ID NO:25, and a light chain variable region comprising CDRL1 of SEQ ID NO:35, CDRL2 of SEQ ID NO:36, and CDRL3 of SEQ ID NO:37,

[0023] (b) the second binding moiety comprises a heavy chain variable region comprising CDRH1 of SEQ ID NO:23, CDRH2 of SEQ ID NO:24, and CDRH3 of SEQ ID NO:25, and a light chain variable region comprising CDRL1 of SEQ ID NO:29, CDRL2 of SEQ ID NO:30, and CDRL3 of SEQ ID NO:31 as the light chain variable region for use in combination with the following items to treat cancer,

[0024] (B) a second bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε (further referred to as "CD3"), characterized in that:

[0025] (a) the first binding moiety comprises a heavy chain variable region comprising CDRH1 of SEQ ID NO:2, CDRH2 of SEQ ID NO:3, and CDRH3 of SEQ ID NO:4, and a light chain variable region comprising CDRL1 of SEQ ID NO:18, CDRL2 of SEQ ID NO:19, and CDRL3 of SEQ ID NO:20, and

[0026] The second binding moiety comprises a heavy chain variable region comprising CDRH1 of SEQ ID NO:2, CDRH2 of SEQ ID NO:3, and CDRH3 of SEQ ID NO:4, and a light chain variable region comprising CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7, and CDRL3 of SEQ ID NO:8.

[0027] In one aspect, the invention includes such methods of treatment.

[0028] In one aspect, the present invention provides:

[0029] (A) a first bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, characterized in that:

[0030] (a) the first binding moiety comprises a heavy chain variable region of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 38, and

[0031] (b) the second binding moiety comprises the heavy chain variable region of SEQ ID NO: 26,

[0032] and the light chain variable region of SEQ ID NO:32,

[0033] For use in the treatment of cancer in combination with

[0034] (B) a second bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, characterized in that:

[0035] (a) the first binding moiety comprises a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5, and

[0036] (b) the second binding moiety comprises the heavy chain variable region of SEQ ID NO: 1, and

[0037] The light chain variable region of SEQ ID NO:21.

[0038] In one aspect, the invention includes such methods of treatment.

[0039] In one aspect, the present invention provides:

[0040] (A) a first bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, characterized in that:

[0041] (a) the first binding moiety comprises a heavy chain region of SEQ ID NO: 27 and a light chain of SEQ ID NO: 39, and

[0042] (b) the second binding moiety comprises a heavy chain region of SEQ ID NO: 27 and a light chain of SEQ ID NO: 33,

[0043] For use in the treatment of cancer in combination with

[0044] (B) a second bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, characterized in that:

[0045] (a) the first binding moiety comprises a heavy chain region of SEQ ID NO: 10 and a light chain of SEQ ID NO: 22, and

[0046] (b) The second binding moiety comprises the heavy chain region of SEQ ID NO:10 and the light chain of SEQ ID NO:9.

[0047] In one aspect, the invention includes such methods of treatment.

[0048] In one aspect, the invention provides the following combinations:

[0049] (A) a first bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, characterized in that:

[0050] (A) a first bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 (further also referred to as “CEA”) and a second binding portion that specifically binds to human CD47 (further also referred to as “CD47”), characterized in that:

[0051] (a) the first binding moiety comprises a heavy chain variable region having CDRH1 of SEQ ID NO:23, CDRH2 of SEQ ID NO:24, and CDRH3 of SEQ ID NO:25, and a light chain variable region having CDRL1 of SEQ ID NO:35, CDRL2 of SEQ ID NO:36, and CDRL3 of SEQ ID NO:37,

[0052] (b) the second binding moiety comprises a heavy chain variable region having CDRH1 of SEQ ID NO:23, CDRH2 of SEQ ID NO:24, and CDRH3 of SEQ ID NO:25, and a light chain variable region having CDRL1 of SEQ ID NO:29, CDRL2 of SEQ ID NO:30, and CDRL3 of SEQ ID NO:31 as the light chain variable region, and

[0053] (B) a second bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε (further referred to as "CD3"), characterized in that:

[0054] (a) the first binding moiety comprises a heavy chain variable region having CDRH1 of SEQ ID NO:2, CDRH2 of SEQ ID NO:3, and CDRH3 of SEQ ID NO:4, and a light chain variable region having CDRL1 of SEQ ID NO:18, CDRL2 of SEQ ID NO:19, and CDRL3 of SEQ ID NO:20, and

[0055] The second binding moiety comprises a heavy chain variable region having CDRH1 of SEQ ID NO:2, CDRH2 of SEQ ID NO:3, and CDRH3 of SEQ ID NO:4, and a light chain variable region having CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7, and CDRL3 of SEQ ID NO:8.

[0056] In one aspect, the invention provides the following combinations:

[0057] (A) a first bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, characterized in that:

[0058] (a) the first binding moiety comprises a heavy chain variable region of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 38, and

[0059] (b) the second binding moiety comprises a heavy chain variable region of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 32, and

[0060] (B) a second bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, characterized in that:

[0061] (a) the first binding moiety comprises a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5, and

[0062] (b) The second binding moiety comprises the heavy chain variable region of SEQ ID NO:1 and the light chain variable region of SEQ ID NO:21.

[0063] In one aspect, the invention provides the following combinations:

[0064] (A) a first bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, characterized in that:

[0065] (a) the first binding moiety comprises a heavy chain region of SEQ ID NO: 27 and a light chain of SEQ ID NO: 39, and

[0066] (b) the second binding moiety comprises the heavy chain region of SEQ ID NO: 27

[0067] and the light chain of SEQ ID NO:33, and

[0068] (B) a second bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, characterized in that:

[0069] (a) the first binding moiety comprises a heavy chain region of SEQ ID NO: 15 and a light chain of SEQ ID NO: 22, and

[0070] (b) The second binding moiety comprises the heavy chain region of SEQ ID NO:15 and the light chain of SEQ ID NO:9.

[0071] The present invention includes further embodiments of the aspects:

[0072] In one embodiment, the constant region and variable framework region sequences of both antibodies are human.

[0073] In one embodiment, both antibodies are characterized in that each of the first and second binding moieties comprises an immunoglobulin heavy chain and an immunoglobulin light chain. In one embodiment, both antibodies are full-length antibodies. In one embodiment, the bispecific antibody is characterized in that it is of human IgG1 type.

[0074] In one embodiment the two antibodies are characterized in that the first binding moiety is monovalent and the second binding moiety is monovalent.

[0075] In another embodiment, the first antibody used according to the invention is characterized in that it is glycoengineered to have an Fc region with modified oligosaccharides. In another embodiment, the first bispecific antibody according to the invention is characterized in that it comprises an Fc region that has been glycoengineered to have a reduced number of fucose residues compared to the same bispecific antibody that has not been glycoengineered.

[0076] In one embodiment, the first antibody used according to the invention is characterized by a ratio of the KD value for binding to recombinant CEACAM3 to that for binding to recombinant CEACAM5 of 100 times or more (Example 3, Table 2).

[0077] In one embodiment, the first antibody used according to the invention is characterized by a ratio of KD values ​​for binding to recombinant CEACAM3 to binding to recombinant CEACAM5 of 100-fold to 200-fold.

[0078] In one embodiment, the first antibody used in the present invention has a relative uncoupling of binding (differential binding) to CEACAM5 and CEACAM3. Although the binding to the full-length recombinant human CEACAM5 protein was increased compared to the bispecific CEAxCD47 antibody K2AC22, the binding to the full-length recombinant human CEACAM3 was not proportionally increased. The quotient / ratio of KD for binding to full-length CEACAM3 and CEACAM5 showed an increase from 83 (K2AC22) to 146 (K2AC100). This corresponds to a 65% to 76% increase in differential binding (Example 3, Table 2).

[0079] In one embodiment, the first antibody used in the present invention is characterized by concentration-dependent phagocytosis (ADCP of human macrophages on tumor cell lines expressing CEACAM5). According to the present invention, ADCP is measured as a phagocytic index (EC50 and / or maximum value) determined by imaging, typically with an E:T ratio of 1:3 (human macrophages: target cells (tumor cells); for example, see Tables 6 to 9 for EC50 values ​​and maximum index of phagocytosis Emax). The details of the assay are described in Example 7; the imaging assay is based on CellInsight CX5. If not otherwise specified, the phagocytic index value is measured by such an imaging method.

[0080] In one embodiment, the first antibody used in the present invention is characterized by an increase of at least 8% in the maximum value of the phagocytic index (Emax) of LoVo tumor cells compared to the phagocytic index of K2AC22. In one embodiment, for LoVo tumor cells, the increase is 8% to 20%. In one embodiment, the bispecific antibody according to the present invention is characterized by an increase of at least 8% in the maximum value of the phagocytic index of Ls174T tumor cells compared to the phagocytic index of K2AC22. In one embodiment, for Ls174T tumor cells, the increase is 8% to 25%. (Example 7, Table 5). LoVo and LS174T are tumor cells with relatively low expression of CEACAM5 (see Table 3 below in Example 5).

[0081] In one embodiment, the first antibody used in the present invention inhibits the interaction between human CD47 and human SIRPα. In one embodiment, the first antibody used in the present invention inhibits the interaction between CD47 and SIRPα on MKN-45 cells, and its IC50 is 10 times or more lower than the IC50 measured for K2AC22 under the same experimental conditions. In one embodiment, the multiple is 10 to 30. In one embodiment, the first antibody used in the present invention inhibits the interaction between CD47 and SIRPα on MKN-45 cells at an IC50 of 0.1nM or less. In one embodiment, the first antibody used in the present invention inhibits the interaction between CD47 and SIRPα on MKN-45 cells at an IC50 of 0.1nM to 0.04nM (see Example 10 and Table 12).

[0082] In one embodiment, the first antibody used in the present invention is characterized by having two or more of the following properties: a ratio of the KD value for binding to recombinant CEACAM3 to recombinant CEACAM5 of 100 times or more, relatively uncoupled binding to CEACAM5 and CEACAM3, concentration-dependent ADCP, an increase of at least 8% in the maximum phagocytic index (Emax) for LoVo tumor cells compared to the phagocytic index of K2AC22, and the ability to inhibit the interaction between human CD47 and human SIRPα with an IC50 that is more than 10 times lower than that of K2AC22.

[0083] The bispecific antibody K2AC22 is a bispecific antibody that binds to human CEACAM5 and human CD47, as described in Table 1 of WO2019234576. K2AC22 comprises a common heavy chain of SEQ ID NO: 5 of WO2019234576, a light chain of SEQ ID NO: 65 of WO2019234576 in the CEACAM5 binding portion, and a light chain of SEQ ID NO: 11 in the CD47 binding portion; the CDRs of K2AC22 are shown in SEQ ID NOs: 1-3, 7-9 and 34-36 of WO2019234576.

[0084] In one embodiment, the first antibody used in the invention is characterized by binding to recombinant human CD47 with a binding affinity (KD) of 100 nM to 600 nM, and in one embodiment, binds with a binding affinity of 100 nM to 500 nM (measured by biomembrane interferometry).

[0085] In one embodiment, the first antibody used in the present invention is characterized by binding to recombinant human CEACAM5 with a KD of 2nM to 10nM (Example 3, Table 2). In one embodiment, the first antibody used in the present invention has a 10-fold to 50-fold higher binding affinity (lower KD) compared to the bispecific antibody K2AC22 of the prior art, and in one embodiment 20-fold to 50-fold (Example 3, Table 2).

[0086] In one embodiment, the first antibody is characterized by specific binding to CEACAM5 but does not compete with the second antibody used according to the invention for binding to CEACAM5.

[0087] The present invention also provides a method for inducing cell lysis of a tumor cell, the method comprising contacting the tumor cell with a bispecific antibody combination according to the present invention. The tumor cell is a human tumor cell, in one embodiment in a patient. In one embodiment of the method for inducing cell lysis of a tumor cell, the tumor cell is a colorectal cancer cell, a NSCLC (non-small cell lung cancer) cell, a gastric cancer cell, a pancreatic cancer cell, a breast cancer cell or other tumor cell expressing CEACAM5.

[0088] The present invention also provides a method of treating a subject suffering from a cancer expressing CEACAM5, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody combination according to the present invention.

[0089] The present invention also provides a method for improving the survival time of a subject suffering from a cancer expressing CEACAM5, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody combination according to the present invention. A further embodiment of the present invention is such a 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.

[0090] The present invention also provides a method for treating a subject suffering from a cancer expressing CEACAM5, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody combination according to the present invention. A further embodiment of the present invention is such a method of the present invention, characterized in that the bispecific antibody combination according to the present invention is administered to a human subject in combination with chemotherapy or radiotherapy.

[0091] The present invention also provides the use of the bispecific antibody combination according to the present invention for the manufacture of a medicament for treating a subject suffering from a cancer expressing CEACAM5. A further embodiment of the present invention is the use of the bispecific antibody combination according to the present invention for the manufacture of a medicament according to the present invention, characterized in that the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer and breast cancer.

[0092] The present invention also provides the use of the bispecific antibody combination according to the present invention for simultaneous, separate or sequential administration in the treatment of a subject with a cancer expressing CEACAM5. In one embodiment, the first bispecific antibody is administered first, followed by the combination of the first and second bispecific antibodies. In one embodiment, the second bispecific antibody is administered first, followed by the combination of the first and second bispecific antibodies.

[0093] A further embodiment of the present invention is a bispecific antibody combination according to the present invention for the use according to the present invention, characterized in that the first bispecific antibody according to the present invention and the second bispecific antibody according to the present invention are administered alternately to the subject at intervals of 2 to 15 days. In one embodiment, the alternating therapy starts with the first antibody. In one embodiment, the alternating therapy starts with the second bispecific antibody according to the present invention.

[0094] A further embodiment of the present invention is the bispecific antibody combination according to the present invention for the use according to the present invention, characterized in that the first bispecific antibody and the second bispecific antibody are administered to the subject simultaneously at an interval of 2 to 15 days.

[0095] A further embodiment of the present invention is the bispecific antibody combination according to the present invention for the use according to the present invention, characterized in that the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer and breast cancer.

[0096] A further embodiment of the present invention is a method for treating a human patient diagnosed with a tumor (cancer, in particular a solid tumor, in particular a solid tumor expressing CEACAM5, in particular solid cancers of colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer and breast cancer), said method comprising administering to said human patient an effective amount of a bispecific antibody combination according to the present invention, said method comprising, in sequence:

[0097] administering to the patient a dose of 0.1 to 10 mg / kg, in further embodiments 0.5 to 10 mg / kg, in further embodiments 10 to 30 mg / kg and 0.1 to 1 mg / kg of the first bispecific antibody, in further embodiments 1 to 10 mg / kg of the second bispecific antibody, e.g. once a week for 4 to 12 weeks, or q2w for 4 to 12 weeks, and after the 4 to 12 weeks waiting for an additional 2 or 3 or 4 elimination half-lives of the second bispecific antibody, administering to the patient the first bispecific antibody at a dose of 0.1 to 20 mg / kg,

[0098] The patient is administered the first antibody q1, q2w, q3w, or optionally q4w for, e.g., more than 12 weeks, waiting for 2 or 3 or 4 elimination half-lives of the first bispecific antibody, and then optionally repeating the administration cycle of the second bispecific antibody, followed by administration of the first bispecific antibody, and optionally repeating the cycle again, and so on.

[0099] Since the CEAxCD3 bispecific antibody (AB73) and the CEAxCD47 bispecific antibody (K2AC100) do not compete for binding to CEACAM5, the two bispecific antibodies can also be administered in the following manner ("simultaneous manner"): the patient simultaneously experiences therapeutically effective plasma and tissue concentrations of the two bispecific antibodies in parallel, for example, a dose of 0.1 to 10 mg / kg, in a further embodiment 0.5 to 10 mg / kg, in a further embodiment 10 to 20 mg / kg of the first bispecific antibody and a dose of 0.1 to 10 mg / kg, in a further embodiment 1 to 10 mg / kg of the second bispecific antibody according to the invention are administered to the patient at about the same time, followed by one or more administrations of these combinations at a frequency of q1w or q2w or q3w or optionally q4w. The term "q1w" means administration once a week; q2w means administration once every two weeks, and so on.

[0100] For safety reasons, it may be desirable in one embodiment to start the treatment with the second bispecific antibody without adding the first bispecific antibody, followed subsequently by simultaneous administration of the two bispecific antibodies.

[0101] The present invention also provides a pharmaceutical composition comprising the bispecific antibody combination according to the present invention and a pharmaceutically acceptable excipient or carrier.

[0102] The present invention also provides the use of a pharmaceutical composition comprising the bispecific antibody combination according to the present invention as a medicament. In one such embodiment, the present invention provides the use of a pharmaceutical composition comprising the bispecific antibody combination according to the present invention as a medicament for treating solid tumor conditions. In one embodiment, the pharmaceutical composition includes the bispecific antibody combination according to the present invention, and is used as a medicament for treating colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer or breast cancer.

[0103] The present invention also provides a composition comprising the bispecific antibody combination according to the present invention for simultaneous, separate or sequential combination use in treating a subject suffering from a cancer expressing CEACAM5.

[0104] The present invention also provides use of the bispecific antibody combination according to the present invention for preparing a pharmaceutical composition.

[0105] The present invention also provides use of the bispecific antibody combination according to the present invention and a pharmaceutically acceptable excipient or carrier for preparing a pharmaceutical composition.

[0106] The present invention also provides the use of the bispecific antibody combination according to the present invention for the manufacture of a medicament for the treatment of solid tumor conditions. A further embodiment of the present invention is the use of the bispecific antibody combination according to the present invention in the treatment of colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer or breast cancer and other cancers expressing CEACAM5.

[0107] Another aspect of the present invention provides a method for inducing cell lysis of tumor cells, the method comprising targeting the tumor cells with the bispecific antibody combination described in 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, the cell lysis is induced by antibody-dependent cellular phagocytosis and / or antibody-dependent cell-mediated cytotoxicity of the bispecific antibody combination according to the present invention. In one embodiment, the tumor cell lysis is induced by macrophage-induced phagocytosis and T cell activation.

[0108] Another aspect of the present invention provides a method for treating a subject suffering from cancer overexpressing CEACAM5, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody combination according to any of the above embodiments.

[0109] Another aspect of the present invention provides a method for treating a subject suffering from a cancer that overexpresses CEACAM5, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody combination as described in any of the above embodiments. Since the CEAxCD3 bispecific antibody and the CEAxCD47 bispecific antibody are not competitive or only minimally competitive, they can be administered not only sequentially but also in parallel (simultaneously), which is likely to be an advantage, because the killing effect of the CEAxCD3 bispecific antibody according to the present invention through the involvement of T cells and the CEAxCD47 bispecific antibody according to the present invention through the involvement of macrophages on tumor cells may be additive or even synergistic, which means that if the two bispecific antibodies are administered simultaneously, the therapeutic effect will be improved.

[0110] Another aspect of the present invention provides a method for increasing progression-free survival and / or overall survival in a subject suffering from a cancer that overexpresses CEACAM5, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody combination 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 any other cancer expressing CEACAM5.

[0111] In certain embodiments of these methods, the bispecific antibody combination according to the invention is administered in combination with chemotherapy or radiotherapy. In one embodiment, the subject is a patient with colorectal cancer, or lung cancer, or gastric cancer, or pancreatic cancer, or breast cancer, or other cancers expressing CEACAM5.

[0112] Another aspect of the present invention provides a method for treating a subject suffering from cancer overexpressing CEACAM5, the method comprising administering to the subject a therapeutically effective amount of a combination of the bispecific antibody of any of the above embodiments and a bispecific antibody against human CEA and human CD3ε.

[0113] Another aspect of the present invention provides a method for increasing progression-free survival and / or overall survival in a subject with a cancer that overexpresses CEACAM5, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody combination 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.

[0114] In certain embodiments of these methods, the bispecific antibody combination according to the invention is administered in combination with chemotherapy or radiotherapy. In one embodiment, the subject is a cancer patient suffering from colorectal cancer or lung cancer or gastric cancer or pancreatic cancer or breast cancer or other cancer expressing CEACAM5.

[0115] Another embodiment of the invention provides the use of a bispecific antibody combination according to the invention for any of the above methods of treatment. 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 DESCRIPTION OF THE DRAWINGS

[0116] Figure 1 . Killing achieved in hybrid assays (MKN-45 cells).

[0117] Figure 1 Concentration response curves for AB73 (CEAxCD3 bispecific antibody) in monotherapy and in combination with 0.1, 1 and 10 μg / ml K2AC100 (CEAxCD47 bispecific antibody) for MKN-45 cells are shown. AB73 monotherapy, even at the highest concentration, achieved only slightly more than 30% killing. Addition of 0.1 μg / ml K2AC100 increased the maximum killing to about 40%, and addition of 1 or 10 μg / kg increased the killing to about 80%. Compared with the killing of AB73 monotherapy, the killing of the combination began at a much lower AB73 concentration; hIgG1 = control (see Example 11 for a description of the mixed assay).

[0118] Figure 2. Data from Figure 1 Results shown are from the same hybrid assay study.

[0119] Figure 2A The % killing achieved by 1 μg / ml K2AC100 with 0.6, 0.12, or 0.024 μg / ml AB73 as monotherapy and in combination is shown; Figure 2B Results using 10 μg / ml K2AC100 instead of 1 μg / ml are shown; higher % killing was always achieved with the combination compared to the two monotherapy treatments.

[0120] Figure 3 . Killing achieved in hybrid assays (LS-174T cells).

[0121] Figure 3 Concentration response curves of AB73 in monotherapy and in combination with 0.1, 1 or 10 μg / ml K2AC100 are shown. Monotherapy with AB73 already achieved about 70% killing at the highest concentration tested. Addition of K2AC100 (0.1, 1, 10 μg / ml) shifted the concentration response curve of AB73 to the left, achieving a maximum of about 80% killing; hIgG1 = control (see Example 11 for a description of the mixed assay).

[0122] Figure 4. Data from Figure 3 Results shown are from the same hybrid assay study.

[0123] Figure 4A Shown are the % killing achieved in monotherapy with 1 μg / ml K2AC100 or in combination with 0.08, 0.016, or 0.0032 μg / ml AB73; Figure 4B The use of 10 μg / ml K2AC100 instead of 1 μg / ml and Figure 4A Results at the same concentration of AB73. The combination always achieved higher % killing compared to the two monotherapy treatments.

[0124] Figure 5 . Killing achieved in hybrid assays (LS-174T cells).

[0125] Figure 5 Concentration response curves are shown for K2AC100 monotherapy and in combination with 5 μg / ml AB73. At the highest concentration tested, monotherapy with K2AC100 already achieved more than 90% killing. Addition of AB73 (5 μg / ml) shifted the concentration response curve of K2AC100 to the left; hIgG1 = control (see Example 11 for a description of the mixed assay).

[0126] Figure 6 .Data from Figure 5 Results shown are from the same hybrid assay study.

[0127] Figure 6 Shown are the % killing achieved in monotherapy with 5 μg / ml AB73 and in combination with 5 μg / ml AB73 and 0.4 or 0.08 μg / ml K2AC100; the combinations always achieved higher % killing compared to the two monotherapy treatments.

[0128] Detailed description of the invention

[0129] Due to potential side effects such as cytokine release syndrome CRS (reported for TAAxCD3), it would be beneficial if the same maximal killing of tumor cells could be achieved with lower drug concentrations compared to higher dose therapy using only one of the bispecific antibodies. Unexpectedly, the combination according to the present invention showed the following advantages:

[0130] In a mixed assay using human macrophages and PBMC / T cells from the same donor, the combination according to the invention significantly increased the maximal killing of tumor cells.

[0131] In a mixed assay using human macrophages and PBMC / T cells from the same donor, the maximum killing of tumor cells (such as MKN-45 cells) by the combination according to the present invention increased from about 30% killing in monotherapy to about 80% killing in combination form ( Figure 1 , Example 11).

[0132] In a mixed assay using human macrophages and PBMC / T cells from the same donor, the combination according to the present invention showed a higher % killing of tumor cells (such as LS-174T and / or MKN-45) at all tested concentrations compared to monotherapy with the same concentration (Figures 2, 4, 6, Example 11).

[0133] In a mixed assay using human macrophages and PBMC / T cells from the same donor, the combination of the CEAxCD47 bispecific antibody of the present invention and the CEAxCD3 bispecific antibody showed a high % killing (>80%) with either of the bispecific antibodies as monotherapy and in combination, but if lower concentrations were tested, the % killing in combination was more advantageous compared to monotherapy (>80%). Figure 5 and Figure 6 , Example 11).

[0134] Unless otherwise defined, the terms used herein are the same as commonly used in the art.

[0135] As used herein, the terms "as used", "as for use" or "for use according to the present invention" mean "as for use in the combination or in the method according to the present invention".

[0136] As used herein, the term "AB73L3-1 / N" features a bispecific CEAxCD3 antibody comprising common heavy chain CDRs of SEQ ID NOs: 2, 3, and 4, and light chain CDRs of SEQ ID NOs: 18, 19, and 20 in the first binding portion to CEA, and light chain CDRs of SEQ ID NOs: 6, 7, and 8 in the second binding portion to CD3. The AB73 portion represents the first binding portion (anti-CEACAM5 binding portion) of the antibody, and L3-1 represents the second binding portion (anti-CD3 binding portion) of the antibody. In one embodiment, AB73L3-1 / N comprises a common heavy chain of SEQ ID NO: 15 (IgG1 with L234A+L235A+P329A mutations), a light chain of SEQ ID NO: 22 in the first binding portion, and a light chain of SEQ ID NO: 9 in the second binding portion.

[0137] The term "AB73" used herein refers to the AB73L3-1 / N antibody, and is a κλCEAxCD3 bispecific antibody.

[0138] As used herein, the term "K2AC100" features a bispecific CEAxCD47 antibody comprising common heavy chain CDRs of SEQ ID NOs: 23, 24 and 25, and light chain CDRs of SEQ ID NOs: 35, 36 and 37 in a first binding portion to CEA, and light chain CDRs of SEQ ID NOs: 29, 30 and 31 in a second binding portion to CD47. AC100 represents the first binding portion of the antibody (the anti-CEACAM5 binding portion), and K2 represents the second binding portion of the antibody (the anti-CD47 binding portion).

[0139] In one embodiment, K2AC100 comprises a common heavy chain of SEQ ID NO:28 (IgG1 WT), a light chain of SEQ ID NO:39 in a first binding moiety to CEA, and a light chain of SEQ ID NO:32 in a second binding moiety to CD47.

[0140] The term "first antibody" as used herein refers to a bispecific antibody against CEACAM5 and CD47 (CEAxCD47 bispecific antibody; CEAxCD47 antibody, K2AC100) as defined herein. The term "second antibody" as used herein refers to a bispecific antibody against CEACAM5 and CD3 (CEAxCD3 bispecific antibody; CEAxCD3 antibody; AB73) as defined herein. The term "two antibodies" or "the (multiple) antibodies" as used herein refers to "the first antibody and the second antibody".

[0141] As used herein, the terms "antibody according to the invention" and "antibody used according to the invention" refer to "the antibody for use in the combination or method according to the invention".

[0142] The terms "combination therapy, co-administration, combined administration, combined use, use in combination, combination of the first and second antibodies" as used herein refer to the first and second antibodies being used, formulated, administered simultaneously or subsequently. Details of such uses, treatments and combinations are as follows.

[0143] The CEAxCD47 antibodies used according to the present invention have one or more of the following beneficial properties:

[0144] The ratio of the KD values ​​for binding to CEACAM3 compared to binding to CEACAM5,

[0145] Increased maximum phagocytic index (Emax) in tumor cells with low CEA expression, and / or

[0146] Inhibits the binding of SIRPα to CD47 on the surface of tumor cells with low IC50.

[0147] Quite unexpectedly, the first bispecific antibody had relatively uncoupled binding (differential binding) to CEACAM5 and CEACAM3, and increased binding to CEACAM5 did not result in a proportional increase in binding to CEACAM3 (Example 3 and Table 2). For example, K2AC100 exhibited a 25-fold higher binding affinity (low KD) for CEACAM5 compared to the binding affinity (KD) of K2AC22, but surprisingly, only a 14-fold higher binding affinity (low KD) for CEACAM3. Thus, for K2AC22, the ratio of the KD value for binding to CEACAM3 to the KD value for binding to CEACAM5 was 83, but for K2AC100 it was 146.

[0148] While several family members like CEACAM5 or CEACAM6 are expressed by epithelial cells, others, such as CEACAM3 (CGM1 or CD66d; UniProtKB-P40198) are expressed exclusively on human granulocytes, a cell type that is, for example, involved in the clearance 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, in contrast to other members of the CEACAM family, CEACAM3 does not support cell-cell adhesion, but rather mediates opsonin-independent recognition and clearance of a restricted 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 is discussed as a phagocytic receptor of the innate immune system (Schmitter et al., J Exp Med. 2004). According to the present inventors' knowledge, bispecific antibodies against CEACAM5 and CD47, if they also have comparable binding to CEACAM3, will have an adverse effect on neutrophils and may reduce the number of neutrophils, i.e. induce neutropenia through increased phagocytosis. This may increase the risk of developing bacterial infections, which may be life-threatening if no immediate medical intervention is performed, particularly in cancer patients whose immune systems are often deficient. A high binding affinity is characterized by a low KD. The distribution of bispecific antibodies targeting CEA between CEACAM5 and CEACAM3 depends on the ratio of binding affinities to these two CEACAM family members. A high ratio of KD for binding to CEACAM3 to KD for binding to CEACAM5 means that the bispecific antibody binds less to CEACAM3 than to CEACAM5, which would be beneficial.

[0149] Quite unexpectedly, the CEAxCD47 bispecific antibody showed an increase in the Emax of phagocytosis in the cell lines LS174T and LoVo, which express low CEACAM5, compared to K2AC22, while there was no increase in the Emax for the cell line SNU-C1, which expresses high CEACAM5. Thus, the first antibody surprisingly showed a beneficial increase in the maximum value of the phagocytic index (Emax) in low CEA expressing tumor cells (such as the LoVo cell line) compared to the increase in the phagocytic index achieved with K2AC22 in the corresponding cell lines. According to Table 5, the first antibody showed an increase in the maximum value of the phagocytic index curve of LoVo cells (4000 CEACAM5 on the cell surface) that was 8.5% to 17% higher compared to the bispecific antibody K2AC22 of the prior art. For LS174T cells (26000 CEACAM5 on the cell surface), the maximum value of the phagocytic index of the antibodies according to the present invention increased by 8.7% to 20.6% higher compared to K2AC22 (Table 5). In cells with higher expression of CEACAM5 (such as SNU-C1 or MKN-45), there was less increase or even no increase in Emax compared to K2AC22.

[0150] Thus, a higher proportion of patients can be successfully treated with the bispecific antibodies according to the invention.

[0151] As disclosed in Examples 5 and 11, CEA expression in malignant cells can vary significantly in terms of RNA expression or cell surface CEA molecule counts. The CEA-expressing cancer cell lines used to study the phagocytic activity of the bispecific antibody CEAxCD47 expressed an average of 108,000 CEA targets on the cell surface (Example 5, Table 3). Organoids (colorectal and lung) from fresh tumor tissues of cancer patients have been studied by the method described in Example 10. The average expression of CEACAM5 in these primary organoids was found to be 28,000 CEACAM5 targets per cell, which is about 4 times lower than the average expression of the cell lines shown in Table 3. Therefore, the first antibody shows improved phagocytosis of malignant cells that express CEACAM5 less. Therefore, this may be beneficial for tumor treatment. In view of heterogeneity and / or relatively low expression, such as in lung adenocarcinoma, colorectal cancer and other tumors expressing CEACAM5, such patients can be successfully treated with the CEAxCD47 bispecific antibody described in the present invention.

[0152] The first antibody surprisingly showed beneficial inhibition (low IC50) of SIRPα binding to CD47 on the surface of tumor cells compared to antibody K2AC22, as shown in Example 9 and Figure 4. The interaction between SIRPα on macrophages and CD47 on tumor cells inhibits the phagocytosis of the tumor cells, which means that effective inhibition of this interaction increases phagocytosis.

[0153] The term "Emax" as used herein describes the maximum activity of a compound. For example, in a cell killing assay, Emax describes the % elimination / killing of cancer cells by macrophages in a given time frame at a concentration that is already saturating (e.g., labeled with calcein AM, see Example 7). Since the total number of tumor-infiltrating macrophages is limited, this is considered to be of high clinical importance: for example, if twice the number of tumor cells are eliminated per time interval, this is equivalent to halving the number of macrophages required to eliminate the same number of tumor cells each time.

[0154] The term "EC50" as used herein describes the concentration of a compound at half the maximum activity (Emax / 2). A lower EC50 helps meet the need to infuse a lower amount of a compound, thereby achieving lower production costs and / or potentially but not necessarily lower side effect rates, for example, compared to a higher EC50. Therefore, Emax and EC50 describe different aspects of compound activity. For two compounds with comparable Emax, the EC50 becomes very important because if the same therapeutic effect can be achieved at a lower concentration, less drug amount can be given, and potentially a lower incidence of side effects.

[0155] As used herein, the terms "antigen binding portion" and "binding portion" refer in their broadest sense to the portion of an antibody that specifically binds to an antigenic determinant, such as CEA, CD47, and CD3. Thus, the binding portion comprises the six CDRs of the heavy and light chains, which are part of the light and heavy variable chains, and part of the light and heavy chains.

[0156] More specifically, as used herein, the binding moiety that links the membrane-bound human carcinoembryonic antigen binding moiety (CEA, which is the same as CEACAM5) to CD47 or CD3 is capable of specifically binding to CEA, CD47 or CD3, more specifically to cell surface or membrane-bound CEA, CD47 or CD3. Thus, each binding moiety will bind to CEA, CD47 or CD3. "Specifically binds," "specific for," "binds to..." means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. In some embodiments, the degree of binding of the anti-target antibody to an unrelated, non-target protein is about 10 times weaker than the binding of the antibody to the target, preferably >100 times, as determined, for example, by biomembrane interferometry (e.g. ), by surface plasmon resonance (SPR) (e.g. ), enzyme-linked immunosorbent assay (ELISA) or flow cytometry (FACS). The target is the protein discussed in this article, such as CEA, CD47 and CD3ε.

[0157] The phrases "specifically bind to CEA and CD47", "bind to CEA and CD47", "specific for CEA and CD47" refer in one embodiment to an antibody, such as a bispecific antibody, that is capable of binding to the targets CEA and CD47 with sufficient affinity so that the antibody can be used as a therapeutic agent in targeting tumor cells expressing CEACAM5 and CD47. Reference to binding to MKN-45, SNU-C1, LS174T, SK-CO-1, HPAF-II and / or LoVo cells with a specific EC50 value refers to the EC50 value measured by flow cytometry (see Example 6).

[0158] As used in this article, 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 in this article, 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 in this article, 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).

[0159] 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), an antibody heavy chain constant 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) in the N-terminal to C-terminal direction, 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 an antibody light chain constant domain (CL) in the N-terminal to C-terminal direction, 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 connected together via inter-polypeptide disulfide bonds between the CL domain and the CH1 domain and between the hinge region of the full-length antibody heavy chain. Examples of typical full-length antibodies are natural antibodies, such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD and IgE. The full-length antibody according to the present invention is of human IgG1 type in one embodiment, and in a further embodiment comprises one or more amino acid substitutions (as defined below) in the Fc portion and / or is sugar-modified on the polysaccharide chain attached to Asn297. The full-length first antibody according to the present invention comprises two binding parts, each formed by a pair of VH and VL, wherein one binding part binds to CEA and the other binding part binds to CD47. The full-length second antibody according to the present invention comprises two binding parts, each formed by a pair of VH and VL, wherein one binding part binds to CEA and the other binding part binds to CD3.

[0160] As used herein and mentioned above, "complementarity determining region" (CDR) describes the non-contiguous antigen binding sites (also referred to as antigen binding regions) found within the variable regions of both heavy and light chain polypeptides. CDR is also referred to as "hypervariable region" (HVR), which term is used interchangeably with the term "CDR" herein to refer to the portion of the variable region that forms the antigen binding region. This particular region has been described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991); incorporated herein by reference. Suitable amino acid residues comprising the CDR defined by Kabat are shown below in the sequence listing table. The exact number of residues comprising a specific CDR will vary depending on the sequence and size of the CDR. Once the variable region amino acid sequence of an antibody is provided, one skilled in the art can routinely determine which residues constitute a specific CDR. As used herein, "CDRL1 comprising SEQ ID NO: x" means that the CDRL1 region of the variable light chain referred to is SEQ ID NO: x (CDRL1 comprising SEQ ID NO: x as CDRL1). This is also applicable to other CDRs. Unless otherwise indicated, HVR residues are numbered herein according to Kabat et al. (as above) and are designated as "CDRs", and references to other specific amino acid residue positions in the bispecific antibodies according to the invention are also numbered according to the Kabat numbering system.

[0161] As used herein, the term "Fc region", "Fc domain" refers to the C-terminal region of the IgG heavy chain; in the case of an IgG1 antibody, the C-terminal region comprises -CH2-CH3 (see above). Although the boundaries of the Fc region of the IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is generally determined to extend from the amino acid residue at position Cys226 to the carboxyl terminus. Constant regions are well known in the art and are described, for example, by Kabat, EA (see, for example, 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).

[0162] An IgG molecule carries two N-linked oligosaccharides in its Fc region, one on each heavy chain. As with any glycoprotein, antibodies are produced as a group of sugar forms that share the same polypeptide backbone but have different oligosaccharides attached to glycosylation sites. Antibodies with reduced fucose content in the glycan moiety show higher antibody-dependent cellular toxicity (ADCC) activity than normally fucosylated antibodies (Niwa R et al., Cancer Res, 64, 2127-33, 2004). In US6946292, US7425446, US8067232 (the entire contents of each of which are incorporated by reference), a cell line of two alleles of the gene (α1,6-fucosyltransferase; FUT8) responsible for fucose addition is described. The bispecific antibody according to the present invention produced using such a cell line has a reduced fucose content in its glycan moiety, and has increased ADCC and antibody-dependent cellular phagocytosis (ADCP). Another technique that can be used to produce antibodies with reduced fucose content is described in US8642292 (incorporated herein by reference). The technique aims to configure a heterologous bacterial enzyme to be stably integrated into an antibody producer cell line (such as a CHO cell line or other). By such measures, the de novo synthesis of fucose from D-mannose is blocked. If the production cells are further cultured in a fucose-free medium, antibodies with a stable level of afucosylation will be produced. Example 9 (1. and 2.) describes an exemplary method for producing and purifying the afucosylated bispecific antibodies of the present invention.

[0163] Mutations within the Fc domain can also alter the binding properties of the Fc domain to different Fc receptors (WO2004063351, WO2004099249, WO2005018669, WO2005063815, WO2005110474, WO2005056759, WO2005092925, WO2005018572, WO2006019447, WO2006116260, WO2006023420, WO2006047350, WO2006085967, WO2006105338, WO2007021841, WO20070 08943, WO2007024249, WO2007041635, WO2007048077, WO2007044616, WO2007106707, WO2008022152, WO2008140603, WO2008036688, WO2008091798, WO2008091954, WO2008092117, WO2008098115, WO2008121160, WO2008150494, WO2010033736, WO2014113510 (the entire contents of each of which are incorporated by reference)).

[0164] The term "epitope" includes any polypeptide determinant that can specifically bind to an antibody. In certain embodiments, an "epitope" includes a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group or a sulfonyl group, which in certain embodiments may have a particular three-dimensional structural feature, and / or a particular charge feature. An epitope is a region of a target that is bound by an antibody. In one embodiment, the first bispecific antibody binds to the N-terminal domain of CEACAM5 (Ig-like V-type domain of amino acids 35-144, UniProtKB-P06731). The binding localization of the bispecific antibody to CEACAM5 is achieved via epitope binning. In epitope binning, antibodies are tested in paired combinations, and antibodies competing for the same binding region are grouped together in bins. Competition testing is performed herein with anti-CEA antibodies according to the prior art and as described herein. In one embodiment, the first bispecific antibody of the present invention competes with the reference antibody SM3E for binding to CEACAM5. Competition was measured by an assay in which biotinylated human CEACAM5 was immobilized at a concentration of 0.5 μg / ml and incubated with serial dilutions of the reference (from 67 nM to 0.09 nM). The bispecific antibodies of the invention were added at 0.1 μg / ml for 1 hour at room temperature. The plates were washed and bound CEAxCD47 or CEAxCD3 bispecific antibodies were detected.

[0165] As used herein, the term "common heavy chain (cHC)" refers to a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant 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) in the N-terminal to C-terminal direction, abbreviated as VH-CH-HR-CH2-CH3. Common heavy chains suitable for bispecific antibodies according to the present invention are heavy chains described in WO2012023053, WO2013088259, WO2014087248, and WO2016156537 (each of which is incorporated by reference in its entirety). In one embodiment, the common heavy chain of the first antibody comprises CDRH1 of SEQ ID NO: 23, CDRH2 of SEQ ID NO: 24, and CDRH3 of SEQ ID NO: 25 as heavy chain CDRs. In one embodiment, the cHC of the bispecific antibody according to the present invention comprises the VH region of SEQ ID NO: 26 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: 27 (VH-CH1). In one embodiment, the common heavy chain cHC of the bispecific antibody according to the present invention is SEQ ID NO: 28 (VH-CH1-CH2-CH3). SEQ ID NO: 28 is a heavy chain that additionally includes an IgG1 Fc portion. In one embodiment, the antibody according to the present invention is a κλ bispecific antibody (κλ body) comprising a cHC.

[0166] The κλ body format enables affinity purification of bispecific antibodies with characteristics indistinguishable from standard monoclonal antibodies (see, e.g., WO2013088259, WO2012023053), which ensures low or no immunogenic potential in patients.

[0167] The bispecific antibodies used according to the present invention, comprising a common heavy chain, can be prepared, for example, according to WO2012023053 (the entire contents of which are incorporated by reference). This type of molecule consists of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant κ domain, and a second light chain variable region fused to a constant λ domain. One binding site exhibits specificity for CEA, and another site exhibits specificity for CD47, each contributed by a heavy chain and a respective light chain. The light chain variable region can be of the λ or κ family, and is preferably fused to the λ or κ constant domain, respectively. This is preferred in order to avoid the generation of non-natural polypeptide connections. However, the bispecific antibodies of the present invention can also be obtained by fusing the κ light chain variable domain with the constant λ domain for the first specificity or fusing the λ light chain variable domain with the constant κ domain for the second specificity. Then, the other light chain is always completely κ (VL and CL) or completely λ (thereby referred to as a κλ bispecific antibody hybrid form). The bispecific antibodies described in WO 2012023053 are "κλ bodies". This κλ-body format allows affinity purification of bispecific antibodies, which are indistinguishable from standard IgG molecules and have characteristics indistinguishable from standard monoclonal antibodies, and are therefore advantageous over previous formats (including, for example, amino acid bridges or other non-physiological elements).

[0168] As used herein, the terms "CEA", "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 (CD66c; UniProtKB-P40198 (CEAM3_HUMAN)), which is also a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Further information and information about other members of the CEA family can be found at http: / / www.uniprot.org.

[0169] As used herein, the terms "binds specifically to CD47", "binds to CD47" and "CD47 binding portion" in the context of the bispecific antibodies according to the invention refer to specificity for human CD47. Human CD47 is a multi-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 by biomembrane interferometry (Octet technology) and / or surface plasmon resonance (Biacore technology) (KD). In one embodiment, the binding of the first bispecific antibody according to the invention to CD47 occurs via one or more of said extracellular domains.

[0170] As used herein, the term "heavy chain characterized by SEQ ID NO: 27" refers to the VH-CH1 portion of the heavy chain as shown in Table 1, which is the Fab portion of the antibody according to the present invention. According to common knowledge, such a heavy chain may also include parts such as hinge regions, CH2, CH3, etc., and may be in any antibody format, such as F(ab')2 format. The preferred format is the common heavy chain format described above.

[0171] As used herein, the terms "specifically bind to CEA", "bind to CEA", "CEA binding portion" refer to the binding of the bispecific antibody according to the present invention to recombinant human CEACAM5, wherein the KD value of the antibody binding to recombinant human CEACAM3 is 100 times or higher compared to the KD value of binding to recombinant human CEACAM5. The term "KD" as used herein refers to the equilibrium dissociation constant between the bispecific antibody according to the present invention and its antigen CEACAM5 or CEACAM3, which is stated in nM and can be measured, for example, by surface plasmon resonance and / or biomembrane interferometry (Example 3).

[0172] Different tumor cell lines (such as LoVo, LS174T, MKN-45, SNU-C1, SK-CO-1, HPAF-II) were used to measure the binding to CEA (CEACAM5) on cells. The concentration of the first antibody according to the present invention was varied within an appropriate range for the EC50 value and Emax value for binding to cells as defined above. Table 4 lists the EC50 and Emax for binding of K2AC22 and K2AC100 to various tumor cell lines.

[0173] As used herein, the term "membrane-bound human CEA" refers to human carcinoembryonic antigen (CEA) bound to the membrane portion of a cell or the surface of a cell (particularly, the surface of a tumor cell).

[0174] As used herein, "CD3ε" and "CD3" refer to human CD3ε (UniProtKB-P07766 (CD3E_HUMAN)). The terms "antibodies against CD3ε (CD3)", "anti-CD3ε (CD3) antibodies" refer to antibodies that specifically bind to CD3ε. In one embodiment, antibodies against CD3ε specifically bind to the same epitope as anti-CD3 antibody SP34 (BD Biosciences, catalog number 565983).

[0175] As used herein, the term "ADCP" refers to antibody-dependent cellular phagocytosis. As used herein, according to the present invention, "phagocytosis", "EC50 value of phagocytosis", "maximum value of phagocytosis" and "phagocytosis index" refer to phagocytosis measured by "imaging" with tumor cell lines (such as, for example, LoVo, LS174T, SNU-C1 and / or MKN-45). Suitable imaging methods are described in Example 7, wherein incubation is performed with, for example, a 1:1 or 1:3 effector cell (macrophage): target (tumor) cell ratio and a "phagocytosis index" is used as a readout (ADCP determined by imaging). As used herein, "phagocytosis of the bispecific antibody" means phagocytosis caused / induced by the antibody.

[0176] The terms "human IgG" and "hIgG" refer to human antibody isotypes. When used in an experimental setting, these terms refer to a commercially available clinical grade homogenous preparation of human immunoglobulin IgG that does not specifically bind to CD47 and CEACAM5 (eg, available from Bio-Rad).

[0177] "Antibody K2AC22" used herein refers to the antibody disclosed in WO2019234576. The antibody comprises a common heavy chain (SEQ ID NO: 5 in WO2019234576), a light chain that binds to CEACAM5 (SEQ ID NO: 65 in WO2019234576) and a light chain that binds to CD47 (SEQ ID NO: 11 in WO2019234576).

[0178] Therapeutic applications and methods using anti-CEA antigen binding molecules

[0179] The combination therapy according to the invention is optimized for the treatment of solid tumors primarily through phagocytosis of tumor cells mediated by macrophages, but also through ADCC, in one embodiment in combination with PD-1 axis antagonists. The antibodies used according to the invention can be administered as described below.

[0180] In a specific embodiment, the disease or solid tumor is a cancer that expresses or even overexpresses CEACAM5, including but not limited to the group of colorectal tumors, non-small cell lung tumors, gastric tumors, pancreatic tumors and breast tumors. In a specific embodiment, the tumor is a colorectal tumor. In a specific embodiment, the tumor is a gastric tumor or a gastroesophageal junction tumor. In a specific embodiment, the tumor is a gastric tumor / gastroesophageal junction tumor expressing CEACAM5. In a specific embodiment, the tumor is a lung tumor. In particular, all therapeutic applications, methods of use, uses, combinations, etc. described herein are embodiments for treating these tumors / diseases.

[0181] The inventors recognized that, given the IgG-like structure and organization, the antibodies according to the invention show no or only low anti-drug antibody (ADA) formation potential or, correspondingly, no or only low loss of drug exposure and subsequent potency due to neutralizing ADA.

[0182] In one embodiment, the present invention provides a method for treating cancer (cancer, tumor, such as human cancer) in vivo, in particular a tumor expressing CEACAM5. The method comprises administering to a subject a pharmaceutically effective amount of a composition according to the present invention. "Subject" means a human subject, in one embodiment, a patient suffering from cancer / tumor / cancer.

[0183] CEACAM5 expression can be found in various tumor entities (especially in colorectal cancer, pancreatic cancer, gastric cancer, non-small cell lung cancer, breast cancer, etc.). In healthy, normal glandular epithelium in the gastrointestinal tract, CEACAM5 is mainly expressed on the apical surface of the cells in a polarized pattern. This polarized expression pattern limits the accessibility of anti-CEA monospecific or bispecific antibodies administered systemically alone, and therefore limits potential toxicity to healthy tissues. The administration of the second antibody together with the first antibody that binds to CD47 with low affinity results in the use of the present invention having no or only limited killing / phagocytic effect on such normal cells. This polarized expression pattern no longer exists in cells of the gastrointestinal tract and other malignancies. CEACAM5 is expressed equally on the entire cell surface of cancer cells. This means that cancer cells are much more accessible to the first and second antibodies than to normal, healthy cells, and can be selectively killed by the combination according to the present invention. The expression of CEACAM5 in cancer cells is mostly higher than that in non-malignant cells.

[0184] In one embodiment, the combination according to the invention is used in combination simultaneously, separately or sequentially. In one embodiment, the combination is used in combination with a PD-1 axis antagonist in combination simultaneously, separately or sequentially. For example, such a PD-1 axis antagonist is described in WO2017118675. Such a combination allows the attack of cancer cells in solid tumors by macrophages and T cells.

[0185] As used herein, the "combination", "simultaneous, separate or sequential combination" of the first and second antibodies refers to any administration of the two antibodies (or three antibodies in the case of the combination and a PD-1 axis antagonist), either separately or together, wherein the two or three antibodies are administered as part of an appropriate dosage regimen designed to obtain the benefits of the combination therapy, for example, in separate, sequential, simultaneous, concurrent, temporally staggered or alternating administrations. Thus, the two or three antibodies may be administered as part of the same pharmaceutical composition or in separate pharmaceutical compositions. The first antibody may be administered before, simultaneously or after, or some combination thereof, of the administration of the second bispecific antibody. When the first antibody according to the invention is administered to a patient at repeated intervals, the second bispecific antibody may be administered before, simultaneously or after, or some combination thereof, of each administration of the first antibody, or at intervals different with respect to the treatment with the first antibody, or in a single dose before, during, or after the course of treatment with the first antibody. In one embodiment, the first antibody and the second antibody are administered in an alternating manner, in one embodiment, with an interval of 6 to 15 days between the administration of the first antibody and the second antibody. In such alternating administration, the first dose can be the first antibody or the second antibody.

[0186] The term "PD-1 axis antagonist" refers to an anti-PD-1 antibody or an anti-PD-L1 antibody. An anti-PD-1 antibody is, for example, pembrolizumab ( MK-3475), nivolumab, pidilizumab, lambrolizumab, MEDI-0680, PDR001, and REGN2810. Anti-PD-1 antibodies are described in, e.g., WO200815671, WO2013173223, WO2015026634, US7521051, US8008449, US8354509, WO2009114335, WO2015026634, WO2008156712, WO2015026634, WO2003099196, WO2009101611, WO2010 / 027423, WO2010 / 027827, WO2010 / 027828, WO2008 / 156712, and WO2008 / 156712 (each of which is incorporated by reference in its entirety).

[0187] Anti-PD-L1 antibodies are, for example, atezolizumab, MDX-1 105, durvalumab and avelumab. Anti-PD-L1 antibodies are described, for example, in WO2015026634, WO2013 / 019906, WO2010077634, US8383796, WO2010077634, WO2007005874 and WO2016007235 (the entire contents of each of which are incorporated by reference).

[0188] With regard to the combined administration of the first and second antibodies, the two compounds may be present in one single dosage form or in separate dosage forms, for example in two different or identical dosage forms.

[0189] The first and second antibodies are not competitive with respect to CEACAM5 binding and therefore, if the physician desires, the two antibodies can be administered simultaneously.

[0190] Typically, the first and second antibodies will be administered to the patient in a dosage regimen that provides the most appropriate treatment for the cancer in terms of both efficacy and safety, as known in the art. Preferably, tumor cells are attacked simultaneously by T-cells and macrophages to realize the full therapeutic potential of the method. Therefore, the CEA-CD3 and CEAxCD47 bispecific antibodies according to the present invention must be non-competitive in binding to CEA on the cell surface.

[0191] As described above, the amount of the antibody administered and the timing of administration of the antibody can depend on the type (e.g., sex, age, weight) and condition of the patient being treated, the severity of the disease or condition being treated, and the route of administration. For example, the first and second antibodies can be administered to the patient at a dosage of 0.1 to 100 mg / kg body weight per day or per week, in a single dose or divided into multiple doses or administered by continuous infusion. In one embodiment, each of the antibodies is administered to the patient at a dosage of 0.1 to 30 mg / kg. In some cases, a dosage level below the lower limit of the above range may be sufficient, while in other cases a larger dosage may be used without causing any harmful side effects.

[0192] As used herein, the term "half-life of an antibody" refers to the elimination half-life of the antibody as measured in a common pharmacokinetic assay. The first and second bispecific antibodies have an elimination half-life of 3-14 days.

[0193] In another aspect, the present invention also relates to the use of a combination according to the invention in the treatment of a disease, in particular a cell proliferative disorder in which CEACAM5 is expressed, in particular a cell proliferative disorder in which CEACAM5 is overexpressed (e.g., overexpressed on the cell surface or expressed in a different pattern) relative 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 different prior art methods (e.g., via immunohistochemical assays, immunofluorescence assays, immunoenzymatic assays, ELISA, flow cytometry, radioimmunoassays, etc.).

[0194] In one aspect, the combination of the invention can be used to target cells expressing CEACAM5 in vivo or in vitro. The combination is particularly useful for eradicating tumors and inhibiting tumor growth or metastasis by inducing ADCP and ADCC of tumor cells. The combination can be used to treat any tumor expressing CEACAM5. Specific malignant diseases that can be treated with the combination of the invention include, but are not limited to, colorectal cancer, non-small cell lung cancer, gastric cancer, gastroesophageal junction cancer, pancreatic cancer, and breast cancer.

[0195] The combination is administered to humans in a pharmaceutically acceptable dosage form as described below, including administration to humans via intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, topical routes as an intravenous bolus or by continuous infusion over a period of time. The combination is also suitably administered via intratumoral, peritumoral, intralesional or perilesional routes to exert local as well as systemic therapeutic effects.

[0196] For the treatment of disease, the appropriate dosage of the first and second antibodies will depend on the type of disease to be treated, the severity and course of the disease, previous therapy, the patient's clinical history and response to the antibodies, and the judgment of the attending physician. The first and second antibodies are suitably administered to the patient at one time or over a series of treatments. The present invention provides methods for selectively killing tumor cells (also referred to herein as cancer cells) that express CEACAM5.

[0197] The method comprises the interaction of the first and second bispecific antibodies with the tumor cells. These tumor cells can be from human cancers, including colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, gastroesophageal junction cancer, pancreatic cancer and breast cancer.

[0198] In another aspect, the present invention relates to the use of the first and second antibodies for the preparation of a medicament for treating a disease involving overexpression of CEACAM5. In a specific embodiment, the disease is a cancer expressing or even overexpressing CEACAM5, including but not limited to colorectal tumors, non-small cell lung tumors (NSCLC), gastric tumors, gastroesophageal junction tumors, pancreatic tumors and breast tumors. In a specific embodiment, the tumor is a colorectal tumor.

[0199] Composition, formulation, dosage and route of administration

[0200] In one aspect, the present invention relates to a pharmaceutical composition comprising the first and second antibodies and a pharmaceutically acceptable carrier. The present invention further relates to the use of such a pharmaceutical composition in a method for treating a disease (such as cancer) or in the preparation of a medicament for treating a disease (such as cancer). In particular, the present invention relates to a method for the treatment of a disease, and more specifically, a method for the treatment of cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention.

[0201] In one aspect, the present invention includes pharmaceutical compositions, combinations and methods for treating human cancers, tumors as defined above. For example, the present invention includes a pharmaceutical composition for use in treating human cancers, comprising a pharmaceutically effective amount of the first and second antibodies and a pharmaceutically acceptable carrier.

[0202] The bispecific antibody compositions of the present invention can be administered by conventional administration methods, including but not limited to intravenous, intraperitoneal, oral, intralymphatic or direct intratumoral administration. Intravenous or subcutaneous administration is preferred.

[0203] In one aspect of the invention, a therapeutic formulation comprising the first and second antibodies is prepared for storage by mixing the antibodies having the desired degree of purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or a liquid formulation. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dose and concentration employed. The formulation to be used for in vivo administration must be sterile. This is easily accomplished by filtration through a sterile filtration membrane. The most effective mode of administration and dosage regimen for the pharmaceutical composition of the present invention depends on the severity and course of the disease, the patient's condition and response to treatment, and the judgment of the treating physician. Therefore, the dosage of the composition can be a flat dose, or it can be adjusted according to individual patients, for example, according to body weight or body weight per square meter of body surface. However, the effective dose of the composition of the present invention will generally be in the range of 0.1 to 30 mg / kg.

[0204] The molecular weight of the first and second antibodies is on the order of 150 kDa / Mol. In one embodiment, they carry an Fc portion. The elimination half-life in the patient is in the range of 3 to 14 days. This half-life allows, but is not limited to, administration once a day, once a week, once every two weeks, or even once every 4 weeks.

[0205] The compositions of the present invention can be in a variety of dosage forms, including but not limited to liquid solutions or suspensions, tablets, pills, powders, polymeric microcapsules or microbubbles, liposomes, and injectable or infusible solutions. The preferred form depends on the mode of administration and therapeutic application.

[0206] The compositions will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors for consideration in this context include the specific disease or condition being treated, the specific person being treated, the clinical condition of the individual patient, the cause of the disease or condition, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners.

[0207] Products

[0208] In another aspect of the invention, an article of manufacture comprising materials useful for treating, preventing and / or diagnosing the conditions described above is provided. The article of manufacture comprises a container and a label or package insert on or connected to the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be made from a variety of materials such as glass or plastic. The container holds a composition that is effective for treating, preventing and / or diagnosing the condition alone or in combination with another composition, and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a bispecific antibody of the invention. The label or package insert indicates that the composition is used to treat the selected condition. In one embodiment, the article of manufacture may comprise: (a) a first container containing the first antibody and a second container containing the second antibody. In one embodiment, the article of manufacture may contain both antibodies in one container. In addition, the article of manufacture may comprise another container containing an additional cytotoxic agent or other therapeutic agent. In this embodiment of the invention, the article of manufacture may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container, which contains a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further comprise other materials that are desirable from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

[0209] Table 1: Sequence Listing

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219] SEQ ID NOs: 1 to 23 refer to antibody AB73 and SEQ ID NOs: 24 to 39 refer to K2AC100. Example

[0220] Example 1: Cloning, expression and purification of human CEACAM5; origin of huCEACAM3 and huCD47.

[0221] The sequence corresponding to the complete extracellular domain (ECD) of CEACAM5 was subcloned into the pEAK8 mammalian expression vector (Edge Biosystems, Gaithersburg, Md.). The vector was modified to introduce the Avitag at the C-terminus. TM (Avidity, Denver Colo.) and hexahistidine tag, human Fc region or mouse Fc region. Constructs were verified by DNA sequencing. Immobilized metal ion affinity chromatography (IMAC), FcXL or CaptureSelect TM IgG-Fc(ms) affinity matrix was used to purify recombinant soluble proteins; human CEACAM3 and biotinylated CEACAM3 can be obtained from ACROBiosystems (Thermo Fisher Scientific), Newark, USA. Human CD47 and biotinylated CD47 can be produced as described in WO2019234576 and can also be obtained from ACROBiosystems, Newark, USA.

[0222] Example 2: Expression and purification of bispecific antibodies carrying λ and κ light chains.

[0223] Simultaneous expression can be achieved in different ways, such as transfection of multiple vectors, each expressing a chain to be co-expressed, or using vectors that drive the expression of multiple genes. The vector pNoviκHλ was previously generated to allow the co-expression of a heavy chain, a κ light chain, and a λ light chain, as described in US2012 / 0184716 and WO 2012 / 023053 (each of which is incorporated by reference in its entirety). The expression of the three genes is driven by the human cytomegalovirus promoter (hCMV), and the vector also contains a glutamine synthetase gene (GS) that can select and establish a stable cell line. The VL gene of anti-hCEACAM5IgGλ or anti-hCD47 IgGκ is cloned in the vector pNoviκHλ for transient expression in mammalian cells. pEAK cells or CHO cells are cultured in a suitable culture flask with an appropriate cell number and culture medium volume (containing fetal bovine serum). Plasmid DNA is transfected into the cells using Lipofectamine 2000 according to the manufacturer's instructions. The antibody concentration in the supernatant of transfected cells was measured using OctetRED96 during the production process. Depending on the antibody concentration, the supernatant was collected 5 to 7 days after transfection and clarified by centrifugation at 1300g for 10 minutes. The purification process consisted of three affinity steps. First, the FcXL affinity matrix (Thermo Fisher Scientific) was washed with PBS, and then the FcXL affinity matrix was added to the clarified supernatant. After incubation at +4°C overnight, the supernatant was centrifuged at 2000g for 10 minutes, the flow-through was stored and the resin was washed twice with PBS. The resin was then transferred to an Amicon TM Pro column and eluted with 50 mM glycine at pH 3.0.

[0224] Several elution fractions were generated, pooled, and analyzed using a 50 kDa Amicon TMUltracentrifugal filter unit (Merck KGaA, Darmstadt, Germany) is desalted for PBS. The eluted product containing total human IgG from the supernatant is quantitatively contained using Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, Del.), and incubated with an appropriate volume of κ selection affinity matrix (GE Healthcare) at room temperature and 20rpm for 15 minutes. As previously described, incubation, resin recovery, elution and desalting steps are performed. The final affinity purification step uses λ Fab selection affinity matrix (GE Healthcare) to carry out, using the same process as the first two purifications. The final product is quantified using Nanodrop. Purified bispecific antibodies are analyzed by electrophoresis under denaturing and reducing conditions. Agilent 2100 bioanalyzer is used together with Protein 80 kits, as described by manufacturers (Agilent Technologies, Santa Clara, Calif., the U.S.). 4 μL of purified sample was mixed with sample buffer supplemented with dithiothreitol (DTT; Sigma Aldrich, St. Louis, Mo.). The samples were heated at 95°C for 5 minutes and then loaded onto the chip. All samples were tested for endotoxin contamination using the Limulus amebocyte lysate assay (LAL; Charles River Laboratories, Wilmington, Mass., USA).

[0225] Example 3: KD measurement.

[0226] a) Experimental procedure for measuring the KD of Abs against recombinant human CEACAM5 (Octet)

[0227] The affinity of the anti-human CEACAM5 arm of the CD47xCEACAM5 bispecific antibody of the present invention to recombinant soluble human CEACAM5 was determined using biomembrane interferometry (BLI) technology. An OctetRED96 instrument and a protein A biosensor (Sartorius) were used. The measurements were performed at 30°C. After hydration, pretreatment, and a baseline step in a kinetic buffer (PBS, 0.002% Tween 20, 0.01% BSA, Kathon; Sartorius), the biosensor was loaded with 0.5 μg / mL of κλ bodies in the kinetic buffer for 5 minutes. The biosensor was then immersed in a serial dilution of a recombinant human CEACAM5 extracellular domain (ECD) soluble protein (produced in-house), starting from 50 nM with a dilution factor of 2x. The binding phase and the dissociation phase were each monitored for 600 seconds. The biosensor was regenerated using 10 mM glycine at pH 1.7. A standard acquisition rate (5.0 Hz, average 20) was applied. The curves were processed by subtracting the reference wells and aligning the Y on the baseline without inter-step correction. Affinity was measured using a 1:1 global fitting model throughout the binding 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 the exemplary bispecific antibodies of the present invention to CEACAM5 determined by the procedure is shown in Table 2 below.

[0228] b) Experimental procedure for measuring the KD of Abs against recombinant human CEACAM3 (Octet)

[0229] The affinity of the anti-human CEACAM5 arm of the CD47xCEACAM5 bispecific antibody of the present invention to recombinant soluble human CEACAM3 was determined using biomembrane interferometry (BLI) technology and the OctetRED96 instrument. The HIS1K biosensor (Sartorius) loaded with anti-His tag antibody was used to capture recombinant huCEACAM3 (R&D Systems, #9868-CM) with a His tag. The measurement was performed at 30°C. After hydration, pretreatment and a baseline step in kinetic buffer (PBS, 0.002% Tween 20, 0.01% BSA, Kathon; Sartorius), the biosensor was loaded with 5 μg / mL of recombinant huCEACAM3 in Kinetic buffer for 5 minutes. Then, the biosensor was immersed in a serial dilution of κλ body, starting from 667nM, with a dilution factor of 2. The binding phase and dissociation phase were monitored for 60 seconds and 120 seconds, respectively. The biosensor was regenerated using 10 mM glycine at pH 1.7. A standard acquisition rate (5.0 Hz, 20 averages) was applied. The curves were processed by double reference subtraction, Y alignment on the baseline, and inter-step correction. Affinity was measured using a 1:1 global fit model throughout the binding step and the first 5 seconds of the dissociation step. The KD of the exemplary bispecific antibodies of the present invention to CEACAM3 determined by the described procedure is shown in Table 2 below.

[0230] Table 2. Binding affinity (KD; nM) of the anti-CEACAM5 arms of the CEACAM5xCD47 bispecific antibodies K2AC100 and K2AC22 (comparison) measured by Octet.

[0231]

[0232]

[0233] Example 4: Epitope binning of CEACAM5xCD47 bispecific antibody by competition with reference antibody SM3E.

[0234] Epitope binning is a competitive immunoassay used to characterize the binding of an antibody according to the invention, or, for example, the binding of a related bivalent anti-CEA (target protein) antibody of the first binding portion of a bispecific antibody according to the invention. A competitive blocking spectrum of new antibodies binding to the target protein is created for antibodies that also bind to the target protein and for which the binding epitope has been established / published. Competition with the reference antibody indicates that the antibodies have the same or topologically closely related epitopes, so they are "binned" together. The ability of the CD47xCEACAM5 bispecific antibody of the invention to compete with the CEACAM5 reference antibody was tested by ELISA on recombinant human CEACAM5 using a reference antibody derived from SM3E (US20050147614) carrying a mouse Fc region (mAb produced using standard methods). SM3E binds more to the N-terminal, cell membrane distal part of CEA.

[0235] Biotinylated human CEACAM5 was coated in a 96-well plate coated with streptavidin at 0.5 μg / ml and incubated for 1 hour with serial dilutions of reference mAb (from 0.09 nM to 67 nM) or with an unrelated mAb carrying a mouse Fc region. The CD47xCEACAM5 bispecific antibody of the present invention was added at 0.1 μg / ml at room temperature for 1 hour. The plate was washed and the bound CD47xCEACAM5 bispecific antibody was detected with anti-human IgG (Fc)-HRP (Jackson ImmunoResearch). After washing, the plate was developed with Amplex Red reagent. The fluorescence signal was measured on a Synergy HT plate reader (Biotek).

[0236] Competition experiments were performed with the CD47xCEACAM5 bispecific antibodies of the present invention. The corresponding competitive (i.e., tool) antibody reduced the binding of K2AC100 by 80% or more. When the binding of the bispecific antibody was reduced by 80% or more at the highest concentration of the reference tool antibody, the CD47xCEACAM5 bispecific antibody was identified herein as being competitive with the SM3E antibody. If the binding to CEACAM5 was reduced by less than 20% when comparing the results with and without the addition of the tool antibody, the CD47xCEACAM5 bispecific antibody was identified as not being competitive with the tool antibody.

[0237] Example 5: Quantification of target density (ie number of molecules) of CEACAM5 and CD47 on the cell surface of six different cancer cell lines.

[0238] The cell lines are human gastric adenocarcinoma cells (MKN-45, DSMZ ACC 409), human colorectal carcinoma cells (SK-CO-1 (ATCC; HTB-39); SNU-C1 (ATCC; CRL-5972); Ls174T (ATCC; CL-188) and LoVo (ATCC; CCL-229)) or pancreatic carcinoma cells (HPAF-II, ATCC, CRL-1997).

[0239] use Cell surface antigens were quantified by indirect immunofluorescence assay using flow cytometry (Agilent Dako). Consists of a series of 6 bead populations coated with different but defined amounts of mouse monoclonal antibodies (Mab). The beads simulate cells labeled with specific primary mouse monoclonal antibodies. Different cell samples can be labeled with different primary antibodies and then quantified using the same set of calibration beads.

[0240] Cells were cultured in their adapted medium, detached with trypsin-EDTA (Sigma Aldrich), centrifuged (3 min, 350 g) and resuspended in cold FACS buffer (PBS, 2% BSA - from Sigma Aldrich), filtered through 0.22 μm (Stericup, Millipore)) to obtain 3.106 cells / mL. 3.10 of each sample were added. 5 Cells were plated in V-bottom plates. 1 μL of FcγR blocking reagent was added to each well and the plates were incubated at 4°C for 10 minutes. 10 μL of primary antibodies anti-human CEACAM5 (#sc-23928; mIgG1 (Santa Cruz)) and anti-human CD47 (produced in-house; B6H12; mouse backbone) were added to the cells at a final concentration of 20 μg / mL and incubated at 4°C for 30 minutes. Cells were washed twice with 200 μL of PBS BSA 2% and centrifuged at 400 g for 3 minutes. 100 μL of beads (from The cells were washed with 100 μL of secondary antibody (1 / 50 in PBS BSA 2%) from the kit and treated the same. 100 μL of secondary antibody (1 / 50 in PBS BSA 2%) from the kit was added to each well and incubated at 4°C for 30 to 45 minutes. The cells were centrifuged (3 min, 400 g at 4°C) to discard the supernatant and washed twice. After the final centrifugation, the cells were resuspended in 130 μL CellFix and collected on a CytoFlex flow cytometer (Beckman Coulter). Analysis was completed using FlowJo software, and the geometric mean was exported to an Excel file. Linear regression was performed using the MFI values ​​from the calibration beads. The antibody binding capacity (ABC) of the cell was inferred from the regression line. Specific antibody binding capacity (sABC) was obtained by subtracting the ABC of the isotype control from the ABC of the specific staining. The data from the analysis are shown in Table 3 below.

[0241] Table 3. Target density of CEACAM5 and CD47 on the cell surface of 6 cancer cell lines.

[0242] source <![CDATA[CEACAM5(x10 3 )]]> <![CDATA[CD47(x10 3 )]]> SK-CO-1 Colorectal 257 105 MKN-45 Stomach 155 135 HPAF-II pancreas 120 114 SNU-C1 Colorectal 85 68 LS174T Colorectal 26 57 LoVo Colorectal 4 25

[0243] Example 6: Measurement of binding (EC50 and maximum binding (Emax)) of CEAxCD47 bispecific antibodies to cancer cell lines expressing CEACAM5.

[0244] The binding of the CD47xCEACAM5 bispecific antibody can be tested on human gastric adenocarcinoma cells expressing CEACAM5 (e.g. MKN-45), human colorectal cancer cells expressing CEACAM5 (SK-CO-1, SNU-C1, Ls174T and LoVo), and pancreatic cancer cells expressing CEACAM5 (HPAF-II).

[0245] Cells were harvested, counted, checked for viability, and plated at 3 × 10 6 The cells were resuspended in FACS buffer (PBS 2% BSA, 0.1% NaN3) at 100 μl. 100 μl of the cell suspension was distributed in a V-bottom 96-well plate (3×10 5Cells / well). The supernatant was removed by centrifugation at 4°C, 1300rpm for 3 minutes. Then the antibody according to 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 incubated for another 15 minutes at 4°C with mouse anti-human IgG Fc secondary antibodies conjugated with PE (R-phycoerythrin) (Southern Biotech, pre-diluted at 1:100 in FACS buffer). The cells were washed twice with cold FACS buffer and resuspended in 300 μl FACS buffer containing SytoxBlue (Life Technologies) diluted 1:15000. Fluorescence or 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 the analysis are shown in Table 4 below.

[0246] Table 4. EC50 (nM) and E(k) of K2AC100 and K2AC22 binding to human cancer cell lines expressing CEACAM5 and CD47. max (MFI).

[0247]

[0248] *MFI - mean fluorescence intensity

[0249] N / A - Not Applicable - No data available for this Ab in this cell line

[0250] K2AC100 showed significantly lower EC50 values ​​and higher Emax values ​​compared to K2AC22.

[0251] K2AC100 binds to SK-CO1 cells with an EC50 value of 10 to 30 nM, binds to MKN-45 cells with an EC50 value of 5 to 15 nM, binds to HPAF-II cells with an EC50 value of 5 to 15 nM, binds to SNU-C1 cells with an EC50 value of 1 to 10, binds to LS174T cells with an EC50 value of 3 to 15 nM, and / or binds to LoVo cells with an EC50 value of 15 to 25 nM.

[0252] The Emax value of K2AC100 binding to SK-CO1 cells was 0.5 to 1.5 (MFI x10 6 ), and the Emax value of binding to MKN-45 cells was 1 to 2 (MFI x 10 6 ), and the Emax value of binding to HPAF-II cells was 0.5 to 1.5 (MFI x 10 6), and the Emax values ​​for binding to SNU-C1 cells were 0.2 to 0.6 (MFI x 10 6 ), and the Emax value of binding to LS174T cells was 0.05 to 0.2 (MFI x 10 6 ), and / or binding to LoVo cells with an Emax of 0.2 to 0.5 (MFI x 10 6 ).

[0253] Example 7: Phagocytosis (phagocytic index) of antibody-dependent cellular phagocytosis (ADCP) was measured respectively.

[0254] The in vitro phagocytic activity of K2AC100 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.

[0255] The assay relies on an imaging-based approach utilizing the CellInsight CX5 high-content screening platform. The readout assessed was the phagocytic index, defined as the average number of target cells engulfed by 100 macrophages.

[0256] 1. Preparation of Macrophages

[0257] Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from different healthy donors (5 to 7 different donors, depending on the cell line) by Ficoll gradient centrifugation. PBMCs were cultured for 7 to 9 days to generate macrophages in complete medium (RPMI 1640, 10% heat-inactivated fetal bovine serum (Invitrogen)), 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, 25 mg / mL gentamycin (all from Sigma-Aldrich) and 50 mM 2-mercaptoethanol (ThermoFisher Scientific) in the presence of 20 ng / mL human macrophage colony stimulating factor (M-CSF, PeproTech). Subsequently, non-adherent cells during the differentiation phase (day +1) were removed by replacing the cell culture medium, and adherent cells representing macrophages were detached using cell dissociation buffer (Sigma-Aldrich) and washed in complete medium on the day of use (day +7, day +8, or day +9) for cytometry-based ADCP experiments. For cell imaging-based ADCP, macrophages were detached using cell dissociation buffer on day +6 and seeded in 96-well optical plates (Costar) at 30,000 per well.

[0258] 2. Evaluation of phagocytic activity (based on CellInsight TM Determination of

[0259] Macrophages adhered to microplate wells (stained with calcein red-orange) were incubated with calcein AM-labeled target tumor cells at a 1:3 effector cell:target cell ratio in the presence of different concentrations of the tested antibodies at 37°C for 30 min (MKN45 and SNU-C1) or 2.5 h (LoVo and Ls174T). At the end of the incubation period, the supernatant was replaced with complete medium and the cells were analyzed using CellInsight TM CX5 high-content screening platform images microplates. 1500 macrophages are acquired and analyzed per well. Phagocytosis is demonstrated as a double positive event (macrophage + target tumor cell), and the phagocytic index is calculated by CellInsight TM Manufacturer's software calculations.

[0260] All results shown in Figure 2 and Tables 5, 6, 7, 8, 9 were obtained using four CEACAM5-expressing cancer cell lines (MKN-45, SNU-C1, Ls174T, LoVo); the effector cell to target / tumor cell ratio was 1:3.

[0261] Table 5. Percent increase in the maximum phagocytic index assessed for K2AC100 relative to K2AC22.

[0262] CEACAM5 levels K2AC100 MKN-45 155,000 1.8 SNU-C1 85,000 0 Ls174T 26,000 14.4 LoVo 4,000 18.6

[0263] K2AC100 showed better binding (lower EC50 and higher Emax, see Example 6, Table 4) than K2AC22. Surprisingly, the % increase in the ADCP maximum phagocytic index Emax obtained by K2AC100 compared to K2AC22 was strongest in the cell lines LoVo and Ls174T that lowly express CEACAM5.

[0264] The results were obtained in experiments using macrophages obtained from different human donors. The data obtained from the 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).

[0265] Table 6. EC50 (μg / mL) and E50 (μg / mL) of phagocytic activity of CEACAM5xCD47 bispecific antibodies K2AC100 and K2AC22 (comparison) using MKN-45 human cancer cell line as target and 7 different macrophage donors (D). max in vitro evaluation.

[0266]

[0267] Table 7. EC50 and E50 of the phagocytic activity of the CEACAM5xCD47 bispecific antibodies K2AC100 and K2AC22 using the SNU-C1 human cancer cell line as a target and 5 different macrophage donors (D). max in vitro evaluation.

[0268]

[0269]

[0270] Table 8. EC50 and E50 of phagocytic activity of CEACAM5xCD47 bispecific antibodies K2AC22 and K2AC100 using 5 different macrophage donors in Ls174T human cancer cell line max .

[0271]

[0272]

[0273] Table 9. EC50 and E50 of phagocytic activity of CEACAM5xCD47 bispecific antibodies K2AC22 and K2AC100 using 6 different macrophage donors in LoVo human cancer cell line max .

[0274]

[0275] Example 8: Generation of afucosylated bispecific antibodies of the invention.

[0276] Tables 10 and 11 show the results (EC50 and Emax) of the phagocytosis of the defucosylated forms of the bispecific antibodies of the present invention on two cell lines (MKN-45 and SNU-C1). The defucosylated forms of the bispecific antibodies of the present invention have been produced and purified by the following method:

[0277] 1. Generate

[0278] CHO pools transfected with plasmids of the corresponding bispecific antibodies of the present invention (for the corresponding vectors or plasmids, see Example 2) were cultured at 0.3×10 6The viable cell concentration of cells / mL was inoculated in a Thomson erlen apparatus with a working volume of 700mL or 100mL for the production of fucosylated and defucosylated antibodies, respectively. All pools were run in batch feeding mode using CDACF medium CDCHO and adaptive feeding schemes for 15 days. In order to produce defucosylated antibodies, according to the defucosylation strategy described by Rillahan et al. (Rillahan et al., Nature Chem. Biol. 2012 July; 8 (7): 661-8) and according to EP2282773, 200 μM fucose inhibitor (1,3,4-tri-O-acetyl-2-deoxy-2-fluoro-L-fucose) was added at days 0, 5, 8 and 11 during the batch feeding process. After 15 days of batch feeding culture, the supernatant of the bispecific antibody pool of the present invention containing fucosylated or defucosylated antibodies was collected. Sartoclear was used to generate defucosylated antibodies. The harvest of CHO pool supernatant was clarified using the Lab V Cell Harvesting Sartorius system (see supplier's instructions).

[0279] 2. Purification

[0280] The purification of the fucosylated and defucosylated bispecific antibodies of the present invention is a three-step affinity purification process. Before starting purification, the antibody concentration in the supernatant of the bispecific antibody pool is measured using OctetRED96 so that a column with an affinity matrix of appropriate volume can be used. Each clarified CHO pool supernatant containing fucosylated or defucosylated bispecific antibodies is loaded onto a MabSelect SuRe (MSS) column (GE Healthcare) without prior adjustment to remove most cell culture contaminants. The MSS eluate is then treated by low pH maintenance to inactivate viruses and neutralized to pH 6 with Tris 1M pH9. The MSS eluate is then loaded onto a LambdaFabSelect (LFS) column (GE Healthcare) to remove monospecific κ (single κ). The pH of the LFS eluate is then adjusted at a pH of 6. The LFS is loaded onto a Capto L (CL) column (GE Healthcare) to remove monospecific λ (single λ). The CL eluate was pH adjusted before storage. The final material was then concentrated and diafiltered into the final formulation buffer, the concentration of which was adjusted using a Nanodrop. The fucosylated and defucosylated bispecific antibodies were aliquoted and stored at -80 °C until use. The size of the purified bispecific antibodies was analyzed by electrophoresis under denaturing and reducing conditions using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA) and a Protein 80 kit according to the manufacturer's instructions. Aggregation 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 achieved by isoelectric focusing (IEF) using a Multiphor II electrophoresis system (GE Healthcare). The relative distribution of N-linked complex bi-branched glycoforms of fucosylated and defucosylated K2AC5 and K2AC22 antibodies was determined using a throughput microchip-CE method on a LabChip GXII Touch (Perkin Elmer). All antibodies were tested for endotoxin contamination using Limulus amebocyte lysate (LAL; Charles River Laboratories, Wilmington, Mass). The defucosylated bispecific antibodies described herein showed >70% defucosylation.

[0281] The defucosylated CEAxCD47 bispecific antibodies have been used to obtain Tables 10 and 11 and Figure 3Results shown in A and 3B.

[0282] 3. Other methods for producing the defucosylated bispecific antibodies of the present invention

[0283] 3.1 By using FUT8-negative producer cell lines

[0284] Alternatively, according to the knowledge of the inventors, the defucosylated bispecific antibody according to the present invention can also be produced according to the following method:

[0285] Materials and methods were according to Naoko Yamane-Ohnuki et al., Biotech. Bioeng.; 87 (2004) 614-622.

[0286] Isolation of Chinese hamster FUT8 cDNA

[0287] According to the knowledge of the inventors, using Total RNA was isolated from CHO / DG44 cells using the QIAQUICK ELISA Kit (Qiagen, Hilden, Germany) and reverse transcribed using the Superscript First-Strand Synthesis System for Reverse Transcription Polymerase Chain Reaction (RT-PCR) (Invitrogen, Carlsbad, CA) with oligo-dT. Chinese hamster FUT8 cDNA was amplified from single-stranded CHO / DG44 cell cDNA by PCR using the following primers:

[0288] 5V-GTCTGAAGCATTATGTGTTGAAGC-3V (SEQ ID NO:44) and

[0289] 5V-GTGAGTACATTCATTGTACTGTG-3V (SEQ ID NO: 45), designed from murine FUT8 cDNA (Hayashi, 2000; DNA Seq 11: 91-96).

[0290] Targeting constructs for the FUT8 locus

[0291] According to the present inventors' knowledge, two replacement vectors, pKOFUT8Neo and pKOFUT8Puro, were used to target the FUT8 gene in CHO / DG44 cells for disruption. A 9.0 kb fragment of the FUT8 gene containing the first coding exon was isolated by screening a CHO-K1 cell E-genomic library (Stratagene, LaJolla, CA) using Chinese hamster FUT8 cDNA as a probe to establish a targeting construct. A 234 bp segment containing the translation start site was replaced with a neomycin resistance gene (Neor) cassette or a puromycin resistance gene (Puror) cassette from plasmids pKOSelectNeo or pKOSelectPuro (Lexicon, TX), respectively, flanked by loxP sites. A diphtheria toxin gene (DT) cassette from plasmid pKOSelectDT (Lexicon) was inserted into the 5V homology region. The resulting targeting constructs, pKOFUT8Neo and pKOFUT8Puro, included 1.5 kb of 5V homology sequence and 5.3 kb of 3V homology sequence. Prior to transfection, the targeting constructs were linearized at a unique SalI site.

[0292] Transfection and screening of homologous recombinants

[0293] According to the knowledge of the inventors, using Bio-Rad II Subconfluent CHO / DG44 cells (1.6 106) were electroporated with 4Ag of linearized pKOFUT8Neo at 350V and 250AF. After electroporation, transfectants were selected with 600Ag / mL G418 (Nacalai Tesque, Kyoto, Japan). Genomic PCR was performed in 96-well plates by a modified microextraction method reported previously (Ramirez-Solis et al., 1992; Anal Biochem 201: 331-335) using the following primers:

[0294] 5V-TTGTGTGACTCTTAACTCTCAGAG-3V (SEQ ID NO:40) and

[0295] 5V-GAGGCCACTTGTGTAGCGCCAAGTG-3V (SEQ ID NO: 41).

[0296] Homologous recombinants were identified by a 1.7 kb fragment obtained using genomic PCR and confirmed by Southern blot analysis using a 221 bp fragment amplified with the following primers:

[0297] 5V-GTGAGTCCATGGCTGTCACTG-3V (SEQ ID NO:42) and 5V-CCTGACTTGGCTATTCTCAG-3V (SEQ ID NO:43).

[0298] As previously described, hemizygous clones were subjected to a second round of homologous recombination using linearized pKOFUT8Puro and drug selection with 15Ag / mL puromycin (Sigma-Aldrich, St. Louis, MO). Identified homozygous disruptors were electroporated with the Cre recombinase expression vector pBS185 (Invitrogen) to remove the drug resistance gene cassette from both FUT8 alleles.

[0299] Monoclonal antibody production by FUT8(-) cells

[0300] According to the present inventors' knowledge, the FUT8(-) cell line was electroporated with an expression vector encoding the bispecific antibody according to the present invention and selected in a medium lacking hypoxanthine and thymidine. Confluent transfectants were plated in The cells were cultured in 301 medium (JRH Biosciences, Lenexa, KS) for 1 week. TM The antibodies are purified from the culture supernatant using Amersham Biosciences, Piscataway, NJ. Further purification steps may be anion / cation exchange chromatography, size exclusion chromatography, and especially purification using kappa or lambda selective resins as described above.

[0301] 3.2. Recovery of extracellular fucose from producer cell culture media and enzymatic intervention of intracellular fucose biosynthesis

[0302] Preferably, and according to the present inventors' knowledge, the defucosylated bispecific antibodies of the present invention may also be produced according to the methods / techniques described below and as described in US8642292. The techniques are designed to stably integrate heterologous bacterial enzymes into antibody producer cell lines (such as CHO cell lines or other cell lines). In this way, the de novo synthesis of fucose from D-mannose is blocked. In addition, if the production cells are cultured in a fucose-free medium, antibodies with a stable level of afucosylation will be produced.

[0303] In eukaryotic cells, fucose is produced via two pathways:

[0304] a) from the extracellular space or lysosomes, via salvage pathways, and

[0305] b) By de novo synthesis of fucose from D-mannose in the de novo synthesis pathway of fucose.

[0306] The omission of fucose from the culture medium can completely block the salvage pathway. The de novo biosynthetic pathway can be blocked by converting the intermediate GDP-4-keto-6-deoxy-D-mannose of the pathway into GDP-D-rhamnose instead of GDP-4-keto-6-deoxy-D-galactose. This is achieved by adding the bacterial enzyme GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) to the production cell line or by stably integrating the gene encoding RMD into the production cell line. Even relatively small amounts of RMD expressed in the production cell line completely block the de novo synthesis pathway of the production cell.

[0307] The technology will be used to construct production cell lines, such as CHO-based cell lines, which are designed to produce the defucosylated antibodies described in the present invention, as well as existing production cell lines that already produce the antibodies described in the present invention, which are modified to produce antibodies with 80% to 100% reduced fucose content.

[0308] Figure 3 All results shown in A and 3B and Tables 10 and 11 were obtained with 2 CEACAM5 expressing cancer cell lines (MKN-45 and SNU-C1) at a ratio of effector cells to target / tumor cells of 1:3. The results were obtained from experiments using macrophages obtained from 3 different human donors. The data obtained from the experiments are shown in Table 10 (for MKN-45 cells) and Table 11 (for SNU-C1 cells).

[0309] Table 10. EC50 (μg / mL) and E50 (μg / mL) of the phagocytic activity of defucosylated CEACAM5xCD47 bispecific antibodies (defucosylated K2AC100 antibody and defucosylated K2AC22 antibody (comparison)) in vitro evaluation using MKN45 human cancer cell line as target and 2 different macrophage donors (D). max (Maximum Index of Phagocytosis).

[0310]

[0311]

[0312] Table 11. EC50 (μg / mL) and E50 (μg / mL) of the phagocytic activity of defucosylated CEACAM5xCD47 bispecific antibodies (defucosylated K2AC100 antibody and defucosylated K2AC22 antibody (comparison)) in vitro using the SNU-C1 human cancer cell line as a target and 2 different macrophage donors (D). max .

[0313]

[0314] Example 9

[0315] Blocking the interaction between SIRPα and CD47 on tumor cells.

[0316] Experimental setup for measuring the SIRPα inhibitory potency (IC50) of the bispecific antibodies of the present invention:

[0317] A cell-based assay monitoring the interaction of soluble SIRPα with 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 antibodies according to the invention allowed the determination of inhibition curves (see Figure 4) and IC50 values ​​(see Table 12).

[0318] MKN-45 cancer cells expressing both CD47 and CEACAM5 were stained with CFSE purple for detecting, for example, cell division by an imaging system (CX5). In short, 3,000 stained MKN-45 cells per well were inoculated in a 384-well optical plate (Costar) and incubated for 50 minutes with increasing concentrations of the bispecific antibody of the present invention (1.9pM to 333nM, 4 replicates). Then, a fixed concentration of SIRPα-mouse Fc premixed with anti-mouse IgG-Fc AF647 conjugated antibody (Jackson Immunoresearch diluted 1:2000) was added to a final concentration of 50ng / mL. After incubation for 3H30, the plate was collected with an imaging system (CX5, Thermo Fisher), and the fluorescence signal emitted by the SIRPα detected by the imaging system-specific software was recorded. The fluorescence signal (mean fluorescence intensity MFI) was plotted according to the tested dose range, and IC50 was calculated by software (Prism, GraphPad).

[0319] The results are shown in Table 12:

[0320] Table 12. IC50 (nM) measured with the CD47 / SIRPα blocking assay for the CEACAM5xCD47 bispecific antibodies K2AC100 and K2AC22 (using MKN-45 as hCD47 expressing cells).

[0321] Antibody Name <![CDATA[SIRPα inhibition potency (nM) # > K2AC22 1.2 K2AC100 0.09

[0322] Example 10: Organoid procedure used for a. CEACAM5 expression in cancer cells obtained from fresh samples of cancer patients (Qifikit data) and b. obtaining phagocytosis data

[0323] Organoids from primary patient 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 (from The cells were washed with a set of 100 μL of secondary antibody (1 / 50 in PBS BSA 2%) from the kit and treated the same. 100 μL of secondary antibody (1 / 50 in PBS BSA 2%) from the kit was added to each well and incubated at 4°C for 30 to 45 minutes. The cells were centrifuged, the supernatant was discarded and washed twice. After the last centrifugation, the cells were resuspended and evaluated using a flow cytometer. The analysis was completed using specific software and the geometric mean was exported to an Excel file. Linear regression was performed using the MFI values ​​from the calibration beads. The antibody binding capacity (ABC) of the cells was inferred from the regression line. The specific antibody binding capacity (sABC) was obtained by subtracting the ABC of the isotype control from the ABC of the specific staining.

[0324] The average expression of CEACAM5 in the primary organoids was found to be 28,000 CEACAM5 targets per cell, i.e. approximately 4-fold lower than the average expression of the cell lines in Table 5.

[0325] If bispecific antibodies according to the invention and macrophages from human donors are added, organoids derived from primary samples of cancer patients can also be used to study concentration-dependent phagocytosis / phagocytic index (see Example 7). By using the same method, according to the knowledge of the inventors, if T cells from human donors are also added, the combination of bispecific antibodies according to the invention and CEAxCD3 bispecific antibodies can be studied.

[0326] Example 11: Mixed Killing Assay by Combination of CEAxCD3 and CEAxCD47

[0327] Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats (healthy donors). Aliquots of the PBMC were cryopreserved in a culture medium containing 90% FCS and 10% cryoprotectant and used as a source of T cells for the mixed killing assay. Further aliquots of the PBMC were used to prepare monocyte-derived macrophages.

[0328] In short, 10 7 PBMCs were seeded at 175 cm2 In a culture flask, in medium containing 10% FCS depleted of complement and supplemented with 10% AB+ human serum and 20 ng / mL human M-CSF. After incubation for 24 hours at 37°C, 5% CO2, the medium was removed to remove floating cells and replaced with medium supplemented with 20 ng / mL-1 human M-CSF alone. After incubation for an additional 2 days, half of the medium was replaced with fresh medium supplemented with human M-CSF.

[0329] After 6 days of differentiation, the derived macrophages were plated in clean flat-bottom 96-well plates and incubated at 37° C. Two days after plating, frozen PBMCs from the corresponding macrophage donors were thawed and added to the macrophage plates.

[0330] The target tumor cells (e.g., MKN45 or LS174T or other CEA-positive tumor cells) are stained with Cell-Trace Violet and conditioned with the combination of the present invention. The conditioned target cells are added to a plate containing macrophages and corresponding autologous PBMCs. The plate is incubated at 37°C for 48 hours (or 72 hours).

[0331] After the incubation period, the floating cells in the supernatant were collected, the adherent cells were detached with trypsin and recovered by centrifugation. The floating cells and adherent cells recovered from the same well were pooled and stained with a labeled anti-CD14 antibody to identify the macrophages during cytometry analysis. At the end of the staining, a viability marker (sytox green) was added to remove dead cells from the analysis.

[0332] The cells were analyzed by flow cytometry (Cytoflex / Beckman). The events obtained were standardized to analyze the same number of events under each assay condition. The number of live target cells was calculated as: live target cells=total cell tracking purple positive live cells-CD14 / cell tracking purple double positive live cells. The killing percentage of each condition was calculated as (1-(live target cells in the treatment well / live target cells in the negative control well)) x 100%.

[0333] The combination according to the invention was investigated in 2 different settings:

[0334] 1. Concentration response curves of AB73 were established without K2AC100 and in combination with defined concentrations of K2AC100 (see Figure 1 ).

[0335] 2. Concentration response curves of K2AC100 were established without AB73 and in combination with AB73L3-1 / N at defined concentrations (see Figure 5 ).

[0336] The results are as follows Figure 1 , 2, 3, 4, 5 and 6.

[0337] Figure 1 Results from an experiment run in setup #1 are shown. AB73 achieved approximately 30% killing at the highest concentration (saturation of the concentration response curve), and combination with 1 or 10 μg / ml K2AC100 increased the effect to 80% killing. Figure 2 shows the effect achieved by 1 μg / ml and 10 μg / ml K2AC100 in monotherapy in the same study, with % killing significantly lower than that achieved by the combination shown in Figure 2.

[0338] Figure 3 The results of setup No. 1 are shown again, but with different donors and different tumor cells (LS-174T instead of MKN-45). Monotherapy with AB73 already achieved a maximum of approximately 70% killing (saturation of the concentration response curve), which increased to approximately 80% killing in combination with K2AC100. If K2AC100 is added, approximately 80% killing can also be achieved at lower concentrations of AB73 - see Figure 4. At the concentrations tested (i.e. 1 μg / ml and 10 μg / ml), K2AC100 as a monotherapy did not achieve more than 40% killing.

[0339] Figure 5 and Figure 6 Results from studies conducted in setting 2 are shown. PBMCs and macrophages from the donors used in this experiment caused nearly 100% killing in K2AC100 monotherapy ( Figure 5 ). The combination resulted in the same maximum killing. But again, the combination of the bispecific antibodies at lower concentrations achieved higher killing than the killing achieved with the same concentrations of the two bispecific antibodies in monotherapy.

[0340] Example 12: Antitumor activity: Tissue slice culture.

[0341] according to The anti-tumor activity of the combination therapy of K2AC100 and AB73L3-1 / N was measured in tumor tissue slice cultures from patients diagnosed with CEA-expressing tumors using the method described by R. et al. (Clinical Colorectal Cancer, Vol. 17, No. 2, e189-99, 2018).

[0342] 1. Tissue Slice Culture and Processing

[0343] according to R. et al., fresh tumor tissue samples were cut and processed. Briefly, tumor samples were cut into 350 μm slices using a tissue chopper immediately after surgical resection and the first macroscopic pathological assessment. The diameter of the tissue slices was then standardized using a 3 mm coring tool. Three tissue slices were randomly pooled, placed on a membrane insert, and cultured in a 6-well plate. The slices were incubated under standardized conditions of 37 ° C and 5% CO2. After pre-culture in standard cell culture medium, the slices were exposed to the bispecific antibodies in triplicate, either alone or in combination, and in combination with PD-L1 inhibitors for up to 120 hours. After compound exposure, the tumor slices were fixed overnight using 4% paraformaldehyde.

[0344] 2. Staining

[0345] Paraformaldehyde-fixed sections were embedded in paraffin and processed into 5 μm sections. Hematoxylin and eosin (HE) staining was performed to evaluate histopathological aspects and tumor cell proportions. Total cell counts, tumor cell counts, and proliferation were analyzed by immunofluorescence staining. Briefly, paraffin sections were dewaxed. After antigen retrieval, sections were washed with 0.3% PBS / TritonX and blocked with 5% normal goat serum for 30 minutes. Primary antibodies against cytokeratin (AE1t3), Ki67, and cleaved PARP were diluted in 0.5% bovine serum albumin and incubated overnight at 4°C. Sections were rinsed with 0.3% phosphate-buffered saline / TritonX and labeled with secondary antibodies. Nuclei were stained with Hoechst 33342. Additional staining (e.g., for CEA expression) may be included.

[0346] 3. Data Analysis

[0347] Five images (20x) were taken from each tissue section on fluorescently stained sections using a fluorescence microscope. Positive pixel counts for Hoechst 33342, cytokeratin, Ki67, and cleaved PARP staining were determined using staining-specific segmentation algorithms. Proliferation / apoptosis tumor areas were calculated by analyzing the pixels of Ki67 / cleaved PARP-positive nuclei surrounded by cytokeratin-positive pixels. For each picture, total cell counts (Hoechst positive), tumor cell counts (Hoechst positive and cytokeratin positive), and proliferating tumor cell counts (Hoechst positive, cytokeratin positive, and Ki67 positive / cleaved PARP) were calculated. Tumor cell counts were normalized to total cell counts, and proliferating tumor cell counts were normalized to tumor cell counts to account for different tumor cell fractions for each picture. Then, average slice values ​​were calculated from individual image values. The average values ​​for each condition were calculated using the average slice values. Preliminary data support the beneficial effects of the combination of K2AC100 and AB73L3-1 / N on patients with solid tumors expressing CEA.

[0348] Example 13: In vivo anti-tumor activity of the CEAxCD47 and CEAxCD3 bispecific antibody combination in a mouse tumor model co-transplanted with human PBMCs and macrophages.

[0349] According to the knowledge of the present inventors, the anti-tumor activity of the combination of the present invention can be evaluated in the following mouse models:

[0350] Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats (healthy donors). A portion of the PBMCs was cryopreserved in a medium containing 90% FCS and 10% cryoprotectant and used on the day of implantation. A major portion of the PBMCs was used to prepare monocyte-derived macrophages.

[0351] In short, 10 7 PBMCs were seeded in T175cm 2 In a culture flask, in medium containing 10% decomplemented FCS supplemented with 10% AB+ human serum and 20 ng / mL human M-CSF. After incubation for 24 hours at 37°C, 5% CO2, the medium was removed to remove floating cells and replaced with medium supplemented with 20 ng / mL-1 human M-CSF alone. After incubation for an additional 2 days, half of the medium was replaced with fresh medium supplemented with human M-CSF. After 6 days of differentiation, the macrophages were detached and resuspended in PBS.

[0352] Frozen PBMCs were thawed and resuspended in sterile PBS.

[0353] At the same time, the target cells expressing CEA (LS174T or HPAF-II or other CEA-positive tumor cells) in the exponential growth phase were detached and the cell suspension was adjusted in PBS. The cell suspension was counted using an automatic cell counter based on trypan blue (ViCell XR#Beckman).

[0354] Finally, the three different cell suspensions were mixed for implantation. The final mixed cell suspension contained 10 6 Target cells expressing CEA, 10 6 NSG mice (or NOG mice) were subcutaneously implanted with 100 μL of cell suspension containing LS174T or other tumor cells, human PBMCs and human macrophages.

[0355] On day +1 after implantation or later, mice in a dedicated group are injected with CEAxCD3 alone or in combination with CEAxCD47 molecules for the first time. In the following weeks, the mice are injected with the combination of the present invention, for example, intravenously twice or once a week. In parallel, a control group is injected with only each single molecule. Finally, a group is injected with the vector to obtain a tumor growth reference curve. Mice are monitored for tumor growth at least twice a week, and tumors are measured by digital calipers until the end of the experiment (tumor volume = 1500mm 3 or GvHD symptoms). Tumor volume was calculated using the formula (length x width) x 0.5. Statistical analysis was performed using one-way ANOVA comparative analysis at the time of study.

[0356] Example 14: In vivo anti-tumor activity in a transgenic mouse model

[0357] According to the knowledge of the inventors, the anti-tumor activity of the combination according to the invention can be evaluated in transgenic mice as single agents and in combination therapy, respectively.

[0358] 1. Cell Line Generation and Growth Testing

[0359] hCEACAM5(Tg)hCD47(Tg)mCD47(ko) cell lines will be generated, for example based on the murine colon cancer cell lines CT26 or MC38. Knockout (KO) of the endogenous mouse CD47 gene is performed using CRISPR / Cas9, followed by isolation of KO clones by cell sorting. The KO clones are transfected with a cassette that drives expression of both hCD47 and hCEACAM5 using an internal ribosome entry site (IRES), and the engineered clones are then isolated based on, for example, total expression levels and ratios. Three validated clones are selected for subsequent testing of their engraftment / tumorigenicity in vivo for final clone selection.

[0360] 2. In vivo antitumor activity

[0361] A mouse strain of a BALB / cJGpt background expressing human CD3e (T001550 heterozygous BALB / c-hCD3ET / Wt mouse) and human CD47 / human SIRPα (T037264 homozygous BALB / c-hCD47 / hSIRPα mouse) can be obtained from GemPharmatech. Alternatively, a mouse strain of a C57BL / 6 / Bcgen background expressing human CD3e (homozygous B-hCD3E mouse) and human CD47 / human SIRPα (homozygous B-hSIRPα / hCD47 mouse) can be obtained from Biocytogen. In each case, the two mouse strains were crossed to obtain triple humanized hCD3e / hSIRPa / hCD47 mice, and the offspring were used in subsequent experiments to test the bispecific antibodies according to the present invention as single agents or combination therapies.

[0362] Triple humanized hCD3e / hSIRPa / hCD47 mice were inoculated with 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) on day 0. Once the median tumor size in the cohort reached, e.g., 200 mm 3 , start treatment with the bispecific antibody according to the invention as a single agent and in combination, intravenous bolus, with an interval of, for example, 2 treatments per week, until one mouse shows a tumor volume, for example, exceeding 3000 mm 3 or the occurrence of any one or more pre-specified animal protection and care endpoints. Tumor volume and body weight were measured 3 times per week. The following formula was used to express the tumor volume in mm 3 Tumor volume is given as: TV = 0.5a × b 2 , where a and b are the long and short diameters of the tumor, respectively.

Claims

1. A first bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, characterized in that: a) the first binding moiety comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises CDRH1 of SEQ ID NO: 23, CDRH2 of SEQ ID NO: 24, and CDRH3 of SEQ ID NO: 25, and the light chain variable region comprises CDRL1 of SEQ ID NO: 35, CDRL2 of SEQ ID NO: 36, and CDRL3 of SEQ ID NO: 37, b) the second binding moiety comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises CDRH1 of SEQ ID NO: 23, CDRH2 of SEQ ID NO: 24, and CDRH3 of SEQ ID NO: 25, and the light chain variable region comprises CDRL1 of SEQ ID NO: 29, CDRL2 of SEQ ID NO: 30, and CDRL3 of SEQ ID NO: 31, It is used for treating cancer in combination with a second bispecific antibody, The second bispecific antibody comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, characterized in that: a) the first binding moiety comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4, the light chain variable region comprises CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and b) the second binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3 and CDRH3 of SEQ ID NO: 4, and the light chain variable region comprises CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7 and CDRL3 of SEQ ID NO:

8.

2. A first bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, characterized in that: a) the first binding moiety comprises a heavy chain variable region of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 38, and b) the second binding moiety comprises a heavy chain variable region of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 32, It is used for treating cancer in combination with a second bispecific antibody, The second bispecific antibody comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, characterized in that: a) the first binding moiety comprises a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 21, and b) the second binding portion comprises a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO:

5.

3. A first bispecific antibody, comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, characterized in that: a) the first binding moiety comprises a heavy chain region of SEQ ID NO: 27 and a light chain of SEQ ID NO: 39, and b) the second binding moiety comprises a heavy chain region of SEQ ID NO: 27 and a light chain of SEQ ID NO: 33, It is used for treating cancer in combination with a second bispecific antibody, The second bispecific antibody comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, characterized in that: a) the first binding moiety comprises a heavy chain region of SEQ ID NO: 15 and a light chain of SEQ ID NO: 22, and b) the second binding portion comprises a heavy chain region of SEQ ID NO: 15 and a light chain of SEQ ID NO:

9.

4. The bispecific antibody for use according to any one of claims 1 to 3, characterized in that The cancer is a solid cancer.

5. The bispecific antibody for use according to claim 4, characterized in that The cancer is colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer, breast cancer or other cancer expressing CEACAM5.

6. The first bispecific antibody for use according to any one of claims 1 to 3, characterized in that The ratio of the KD values ​​for binding to recombinant CEACAM3 to binding to recombinant CEACAM5 is 100 or higher.

7. The first bispecific antibody for use according to any one of claims 1 to 3, characterized in that The ratio of the KD values ​​for binding to recombinant CEACAM3 to binding to recombinant CEACAM5 was 100-200.

8. The bispecific antibody for use according to any one of claims 1 to 7, characterized in that The first bispecific antibody and the second bispecific antibody are administered to the subject simultaneously at an interval of 2-15 days.

9. A combination of a first bispecific antibody and a second bispecific antibody, wherein: The first bispecific antibody comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, characterized in that: a) the first binding moiety comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises CDRH1 of SEQ ID NO: 23, CDRH2 of SEQ ID NO: 24, and CDRH3 of SEQ ID NO: 25, and the light chain variable region comprises CDRL1 of SEQ ID NO: 35, CDRL2 of SEQ ID NO: 36, and CDRL3 of SEQ ID NO: 37, b) the second binding moiety comprises a heavy chain variable region comprising CDRH1 of SEQ ID NO: 23, CDRH2 of SEQ ID NO: 24, and CDRH3 of SEQ ID NO: 25, and a light chain variable region comprising CDRL1 of SEQ ID NO: 29, CDRL2 of SEQ ID NO: 30, and CDRL3 of SEQ ID NO: 31; and The second bispecific antibody comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, characterized in that: a) the first binding moiety comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4, the light chain variable region comprises CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and b) the second binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3 and CDRH3 of SEQ ID NO: 4, and the light chain variable region comprises CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7 and CDRL3 of SEQ ID NO:

8.

10. A combination of a first bispecific antibody and a second bispecific antibody, wherein: The first bispecific antibody comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, characterized in that: a) the first binding moiety comprises a heavy chain variable region of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 38, and b) the second binding moiety comprises a heavy chain variable region of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 32; and The second bispecific antibody comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, characterized in that: a) the first binding moiety comprises a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 21, and b) the second binding portion comprises a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO:

5.

11. A combination of a first bispecific antibody and a second bispecific antibody, wherein: The first bispecific antibody comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD47, characterized in that: a) the first binding moiety comprises a heavy chain region of SEQ ID NO: 27 and a light chain of SEQ ID NO: 39, and b) the second binding moiety comprises a heavy chain region of SEQ ID NO: 27 and a light chain of SEQ ID NO: 33; and The second bispecific antibody comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, characterized in that: a) the first binding portion comprises a heavy chain region of SEQ ID NO: 15 and a light chain of SEQ ID NO: 22, and b) the second binding portion comprises a heavy chain region of SEQ ID NO: 15 and a light chain of SEQ ID NO:

9.

12. A pharmaceutical composition comprising the combination of any one of claims 9 to 11 and a pharmaceutically acceptable excipient or carrier.

13. The pharmaceutical composition according to claim 12, for use as a medicine.

14. The pharmaceutical composition according to claim 12 or 13, for use as a drug for treating solid cancer.

15. The pharmaceutical composition according to any one of claims 12 to 14, for use as a medicament for treating colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, pancreatic cancer or breast cancer.

16. A method of treating a patient suffering from a cancer expressing CEACAM5, the method comprising administering to the patient a therapeutically effective amount of the combination according to any one of claims 9 to 11 or the pharmaceutical composition according to any one of claims 12 to 15.

17. The method of claim 16, wherein the antibodies are administered simultaneously.

18. The method of claim 16 or 17, wherein the patient is administered one or more doses of 0.01 to 10 mg / kg of the combination or composition of any one of claims 9 to 15.

19. The method of claim 16 or 17, wherein the patient is administered one or more doses of 0.01 to 10 mg / kg of the second bispecific antibody and one or more doses of 1 to 20 mg / kg of the first bispecific antibody.

20. A method of increasing the survival time of a patient suffering from a cancer expressing CEACAM5, the method comprising administering to the patient a therapeutically effective amount of the combination or composition of any one of claims 7 to 11.

21. A combination as claimed in any one of claims 9 to 11 for use in the manufacture of a medicament for treating a patient suffering from a cancer expressing CEACAM5.

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