Bispecific antibody against CEACAM5 and CD3

Abstract

The present invention relates to bispecific antibodies that bind to human carcinoembryonic antigen CEACAM5 (CEA) and human CD3ε. Additionally, the present invention relates to polynucleotides encoding such bispecific antibodies, as well as vectors and host cells containing such polynucleotides. The present invention further relates to methods for selecting and producing such antibodies, and to methods of using such antibodies in the treatment of disease. In one embodiment, the present invention relates to a bispecific antibody (also referred to as "bsAb CEAxCD3" or "CEAxCD3 bispecific antibody") comprising a first binding moiety that specifically binds to human CEACAM5 (also referred to as "CEA") and a second binding moiety that specifically binds to human CD3ε (also referred to as "CD3").

Description

Technical Field

[0001] Field of the Invention The present invention relates to a bispecific antibody (CEAxCD3 bispecific antibody) that binds to human cancer fetal antigen CEACAM5 (CEA) and human CD3ε. In addition, the present invention relates to a polynucleotide encoding such a bispecific antibody, as well as a vector and a host cell comprising such a polynucleotide. The present invention further relates to methods for selecting and generating such antibodies, as well as methods for using such antibodies in the treatment of diseases.

Background Art

[0002] Background of the Invention For example, it remains a challenge to successfully treat advanced / metastatic solid cancers such as pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, etc. The latest cancer immunotherapies have introduced methods / techniques to help the body's immune cells attack and kill cancer cells better. For example, several techniques / methods have been developed to increase the attack of tumor cells by T cells. Examples are, for example, immune checkpoint inhibitors such as monoclonal antibodies that inhibit PD-1 / PD-L1, T cell bispecific antibodies or CAR-T cells that bind to tumor-associated antigens (TAAs) and CD3 on T cells. CAR-T cells and bispecific antibodies are effective in hematological malignancies and are approved, for example, for the treatment of B cell malignancies or acute lymphoblastic leukemia ALL, but so far there has been no real breakthrough success of these methods in the treatment of advanced / metastatic solid cancers. Monoclonal antibodies and bispecific antibodies used in treatment can also cause various adverse effects. An important toxicity problem is cytokine release syndrome (CRS), which has been found, for example, in the treatment with alemtuzumab, muromonab-CD3, rituximab, tositumomab and the CD19xCD3 bispecific antibody blinatumomab.

[0003] Tabernero et al. (J Clin Oncol 35, 2017 (suppl. abstr. 3002)) presented phase 1 clinical data in patients with progressive / metastatic colorectal cancer with a CEAxCD3 bispecific antibody (RO6958688, sibisatamab, see below) in monotherapy and in combination with the anti-PD-L1 antibody atezolizumab at ASCO 2017. Sibisatamab has a so-called 2+1 format with one Fab fragment that binds to CD3 and two Fab fragments that bind to CEA. Such antibodies are described, for example, in US Patent Application Publication No. 20140242079 (International Publication No. 2014131712) and US Patent Application Publication No. 20140242080 (International Publication No. 2014131711).

[0004] As used herein, "TCB2014" refers to a bispecific antibody that binds to CEA and CD3 in a 2+1 format as described in US Patent Application Publication No. 20140242080 (which is incorporated by reference in its entirety), including the CDRs shown in SEQ ID NOs: 270-276 and 290-296 of US Patent Application Publication No. 20140242080 as CDRs (see also the CDRs of SEQ ID NOs: 4-10 and 24-30 of US Patent Application Publication No. 20140242079, which are incorporated by reference in their entirety). As used herein, "TCB2017" refers to molecule B in a "2+1 IgG CrossFab, inverted" format having charge modifications (VH / VL exchange in the CD3 binder, charge modifications in the CEA binder, humanized CEA binder) as described in International Publication No. 2017055389 (which is incorporated by reference in its entirety), including the CDRs shown in SEQ ID NOs: 4-6, 8-10 and 14-19 of International Publication No. 2017055389 as CDRs.

[0005] The 2+1 structure is quite different from native IgG antibodies. Its structure also contains two different heavy chains joined together by artificial amino acid (aa) crosslinking and a knobs-into-holes technique / aa sequence in the Fc portion (see, e.g., U.S. Patent No. 6,737,056 and International Publication No. WO 2013 / 055958). Such bispecific antibodies (e.g., RO6958688, sibrotuzumab) are immunogenic and thus cause the formation of anti-drug antibodies (ADA) and loss of drug exposure due to neutralization of the drug by ADA. Melero et al. reported loss of exposure in 50% or more of patients with ADA and in 45% of patients at 60–200 mg doses (Melero et al., ASCO 2017, Abstract 2549 and Poster No. 41; see Abstract in Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017) 2549–2549). Loss of exposure makes it difficult to manage the actual treatment and significantly reduces the probability of success. To minimize ADA formation, sibrotuzumab is being clinically tested in combination after pretreatment with the anti-CD20 antibody obinutuzumab in combinations of sibrotuzumab and atezolizumab, respectively (see ClinicalTrials.gov Trial NCT03866239). The pretreatment is performed to deplete B cells in patients with metastatic colorectal cancer. Depletion of B cells results in a decrease in the patient's immunoglobulins and thus potential ADA, but at the same time, this results in immunosuppression.

[0006] MEDI-565 (AMG211), a single-chain antibody of a further bispecific CEAxCD3 antibody, is in clinical development and results have been published (see, for example, M. Pishvaian et al., Clin Colorectal Cancer. 2016 DEC; 15(4) 345-351). Study NCT01284231 (ClinicalTrials.gov) has reported completion and no new trials have been initiated for several years. This single-chain bispecific antibody (two scFvs connected by an aa linker) has a very short excretion half-life of 2.2 - 6.5 hours (Pishvaian et al.; Clin. Colorectal Cancer, 2016 DEC; 15(4) 345-351) (incorporated herein by reference).

[0007] The present invention provides a CEAxCD3 bispecific antibody with high efficacy and lower immunogenicity. Such an antibody comprises a common heavy chain and, in one embodiment, a kappa light chain in the CEA-binding portion and a lambda light chain in the CD3-binding portion.

[0008] The concept of using a common heavy chain to obtain a bispecific antibody has been mentioned in Fischer et al., Nature Communications 6 (2015): 6113. https: / / doi.org / 10.1038 / ncomms7113 and Magistrelli G. et al., MABS 9 (2017) 231-239. Kappa-lambda bispecific antibodies are described, for example, in International Publication No. WO 2014 / 087248 (incorporated herein by reference in its entirety). Their structures are hardly distinguishable from the structure of native IgG, and as a result, there is no or minimal ADA formation, and thus less or minimal loss of exposure. The sequence of the common heavy chain variable region VH and the sequence of huCD3 VL 1A4 of the present invention are described in International Publication No. WO 2019 / 175658 (U.S. Patent Application Publication No. 2019 / 0284297) (incorporated herein by reference in its entirety).

[0009] As mentioned above, WO 2017 / 055389 describes bispecific CEAxCD3 antibodies that have a 2+1 format and bind to domains different from those of sibrotuzumab. One of these antibodies (RO7172508 or RG6123) has been tested in clinical trials in patients with locally advanced and / or metastatic CEA-positive solid tumors, even in combination with obinutuzumab pretreatment and atezolizumab (ClinicalTrials.gov; search for RO7172508). According to the description of the clinical trials in ClinicalTrials.gov for some cohorts, serum CEA (shed soluble CEACAM5, sCEA) levels below a certain threshold are required for patients to be eligible for treatment, suggesting that higher levels of shed soluble CEACAM5 can reduce the efficacy of this CEAxCD3 bispecific antibody. The antibodies of the present invention show minimal impact of shed soluble CEA on their tumor cell killing efficacy.

[0010] Shed soluble CEACAM5 is an established tumor marker. The level of sCEA in the plasma of cancer patients can exceed 1000 ng / ml, while the plasma concentration in healthy individuals is below 10 ng / ml (e.g., Sandler B et al. Anticancer Res 1999, 19(5B), 4229-33). Thus, shed soluble CEACAM5 can compete with membrane-bound CEA present on tumor cells for binding to therapeutic anti-CEA antibodies and anti-CEA bispecific antibodies, potentially causing a decrease in the efficacy of anti-CEA antibodies or CEAxCD3 antibodies. TCB2017 and TCB2014 (see above) have been tested by the inventors in vitro in assays for T cell-mediated lysis of CEA-positive tumor cells in the presence of soluble CEA. It has been found that the addition of sCEA to the assays shifts the lysis curves and thus the EC50 values of TCB2014 and TCB2017 to higher concentrations, suggesting that both TCB2014 and TCB2017 bind significantly to sCEA.

[0011] The human CEA family contains 29 genes, of which 7 belong to the CEA subgroup and 11 belong to the pregnancy-specific glycoprotein subgroup, and 18 are expressed. Members of some CEA subgroups are thought to have cell adhesion properties. CEACAM5 is expressed not only by colorectal cancer cells but also by pancreatic cancer, gastric cancer, lung cancer and other cancer types. CEACAM5 is thought to have a role in innate immunity (Hammarstroem S., Semin. Cancer Biol. 9(2):67-81 (1999)). Carcinoembryonic antigen 5 (CEA, CEACAM5 or CD66e; UniProtKB-P06731) is a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM family) and a tumor-associated antigen (Gold and Freedman, J Exp. Med., 121:439-462, 1965; Berinstein N. L., J Clin Oncol., 20:2197-2207, 2002). Multiple monoclonal antibodies have been produced against CEACAM5 for research purposes, as diagnostic tools, and for therapeutic purposes (see, for example, WO 2012117002). Members of the carcinoembryonic antigen family (CEACAM) are widely expressed and can regulate diverse functions including tumor promotion, tumor suppression, angiogenesis and neutrophil activation depending on the tissue. Four members of this family, CEACAM1, CEACAM3, CEACAM6 and CEACAM8, are expressed and enriched in human neutrophils (http: / / www.proteinatlas.org). Considering the mechanism of action of CEAxCD3 bispecific antibodies, cross-reactivity with other CEACAMs could lead to depletion of important circulating healthy cell populations. For example, cross-reactivity with CEACAM8 expressed by neutrophils or hematopoietic stem cells could lead to depletion of such cell populations.The present invention provides a CEAxCD3 bispecific antibody having low cross-reactivity with one or more members of the CEACAM family, namely CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20 and CEACAM21.

[0012] The murine monoclonal anti-CEACAM5 antibody PR1A3 was produced by fusing NS1 (P3 / NS1 / I-Ag-4-1) myeloma cells with spleen cells from a mouse immunized with normal colorectal epithelium. Richman P. I. and Bodmer W. F., Int. J. Cancer, 39:317-328, 1987 describe the murine monoclonal antibody PR1A3. Epitope mapping of PR1A3 indicates that this antibody targets the B3 domain and GPI anchor of the CEA molecule (Durbin H. et al., Proc. Natl. Acad. Sci. USA, 91:4313-4317, 1994). The epitope to which PR1A3 binds is a conformational epitope rather than a linear epitope (Stewart et al., Cancer Immunol. Immunother., 47 (1999) 299-06). The humanized PR1A3 (hPR1A3) antibody is described, for example, by Conaghhan P. J. et al., Br. J. Cancer, 98 (2008)1217-1225 and International Publication No. 2012117002. The CEA binder (referred to as CH1A1A) used in TCB2014 is a humanized, affinity matured and stability engineered version derived from the PR1A3 antibody. M. Bacac et al., Clin. Cancer Research 22(13);3286-97 (2016), Conaghan P, et al., Br J Cancer 2008;98:1217-25 and Durbin H, et al., Proc Natl Acad Sci U S A 1994;91:4313-7.

[0013] Methods for treating cancer by a combination of a human PD-1 axis antagonist and a bispecific anti-CEA×CD3 antibody are referred to in WO 2017 / 118657, and clinical results have been presented at the ASCO Conference 2017 (Tabernero et al., J Clin Oncol 35, 2017 (suppl. abstr. 3002)). Methods for treating tumors by administering an immune checkpoint antagonist that binds two or more different targets of the immune checkpoint pathway and a T cell redirecting agent that binds CEA and a T cell surface antigen are referred to in WO 2015 / 112534. A conjugate consisting of a single domain anti-CEACAM6 antibody and urease is currently in clinical trials (NCT02309892; WO 2016 / 116907). Class I antibodies that bind CEACAM5, CEACAM6, and granulocytes are referred to in US 2011 / 0064653. Bispecific antibodies comprising a first polypeptide chain and a second polypeptide chain covalently bound to each other are referred to in WO 2018 / 053328.

[0014] The anti-CD3ε antibodies described in the current state of the art are SP34 (Yang SJ, The Journal of Immunology (1986) 137; 1097-1100). SP34 reacts with both primate and human CD3. SP34 is available from BD Biosciences. A further anti-CD3 antibody described in the current state of the art is UCHT-1 (see International Publication No. WO 2000 / 041474). A further anti-CD3 antibody described in the current state of the art is BC-3 (Fred Hutchinson Cancer Research Institute; used in a Phase I / II trial of GvHD, Anasetti et al., Transplantation 54: 844 (1992)). SP-34 recognizes an epitope present exclusively on the ε chain of CD3 (see Salmeron et al., (1991) J. Immunol. 147: 3047), and is different from UCHT-1 and BC-3 in that UCHT-1 and BC-3 recognize epitopes contributed by both the ε and γ chains. Anti-CD3 antibodies are also described in International Publication Nos. WO 2007 / 042261, WO 2008 / 119565, WO 2008 / 119566, WO 2008 / 119567, WO 2010 / 037836, WO 2010 / 037837, WO 2010 / 037838 and U.S. Patent No. 8,236,308. Bispecific antibodies comprising a binding moiety specific for CEA and a binding moiety specific for CD3ε are described, for example, in U.S. Patent Application Publication No. 2014 / 0242079, International Publication Nos. WO 2007 / 071426, WO 2013 / 012414, WO 2015 / 112534, WO 2017 / 118675 and WO 2017 / 055389. Anti-CD3 antibodies comprising the sequence of the second binding moiety of the antibodies according to the invention are referred to in U.S. Patent Application No. 62 / 643,095 and International Application No. PCT / US2019 / 000232, which are hereby incorporated by reference in their entirety. U.S. Patent Application Publication No. 2012 / 321626 refers to multispecific Fab fusion proteins comprising Fab fragments that bind to the N-terminus of CD3 epsilon.WO 2018 / 199593 refers to bispecific antibodies that bind to HER3 and CD3.

[0015] As already mentioned above, the results of the first clinical trial with a T cell bispecific antibody TAAxCD3 (TAA = tumor-associated antigen) in patients with advanced solid tumors were disappointing. However, recent pre-phase 1 results show partial responses and stable disease in patients with advanced colorectal cancer, as monotherapy and in combination with PD-L1 inhibition (J. Tabernero et al., J. Clin. Oncol. 35, 2017 (suppl. Abstr. 3002)). Data have been published on the CEAxCD3 bispecific antibody sibrotuzumab (RO6958688, see for example Bacac et al., Clin. Cancer Res., 22(13), 3286-97 (2016); and US Patent Application Publication No. 2014 / 0242079). Another approach to obtaining better results is not only to add an inhibitor of the PD-1 checkpoint axis to the T cell bispecific antibody, but also to add additional checkpoint inhibitors or agonists. However, to date, there is no promising clinical data on such combinatorial approaches available.

[0016] The limited availability of T cells within advanced solid tumors is surely an important mechanism limiting the efficacy achievable with T cell bispecific antibodies and PD-1 axis inhibitors.

[0017] Instead of adding combinations of other therapeutic agents with T cell bispecific antibodies and PD-1 axis inhibitors, aiming to redirect T cells towards the tumor cells of advanced solid tumors, adding other immune cells, in particular, therapeutic agents that redirect macrophages or macrophages and natural killer (NK) cells towards tumor cells, may be more successful.

[0018] The present invention provides a novel CEAxCD3 bispecific antibody designed in a way that it can be administered in parallel with a CEAxCD47 bispecific antibody that redirects macrophages and NK cells against solid tumors expressing CEA. The combined attack of T cells, macrophages, and NK cells targeted to tumors expressing CEA provides a significant opportunity for excellent efficacy / killing and phagocytosis of tumor cells expressing CEA.

[0019] The hitherto unexpected results with CAR T cells in solid tumors may have a simple explanation that there were simply not enough CAR T cells infiltrating and distributing in solid tumors. This is certainly different in the majority of hematological malignancies where CAR T cells can approach tumor cells sufficiently, explaining the high efficacy difference in these malignancies compared to the unexpected efficacy in solid tumors. Additionally, CAR T cells can be severely suppressed by the tumor microenvironment (TME) of solid tumors, which is mostly strongly immunosuppressive.

[0020] The present invention provides a novel bispecific anti-CEAxCD3 antibody with high efficacy, low impact of sCEA on efficacy, low or no cross-reactivity with other CEACAMs other than CEACAM5 (=CEA), and thus reduced toxicity, low immunogenicity, the opportunity for parallel combination therapy with CEAxCD47 antibodies, and beneficial pharmacokinetic properties.

Prior Art Documents

Patent Documents

[0021]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0022]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0023] Summary of the Invention In one embodiment, the present invention relates to a bispecific antibody (also further referred to as "bsAb CEAxCD3" or "CEAxCD3 bispecific antibody") comprising a first binding portion (also further referred to as "CEA") that specifically binds to human CEACAM5 and a second binding portion (also further referred to as "CD3") that specifically binds to human CD3ε.

[0024] In one embodiment, the bispecific antibody is characterized in that the antibody is monovalent with respect to the first binding portion and monovalent with respect to the second binding portion.

[0025] In one embodiment, the bispecific antibody is characterized in that the sequences of the constant and variable framework regions are human.

[0026] In one embodiment, the bispecific antibody is characterized in that each of the first and second binding portions comprises a heavy chain of an immunoglobulin and a light chain of an immunoglobulin.

[0027] In one embodiment, the bispecific antibody has a first binding portion comprising a heavy chain and a second binding portion comprising a heavy chain, and the heavy chains in each binding portion are the same (i.e., a common heavy chain). In one embodiment, the common heavy chain variable region comprises, as CDRs, CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4. In one embodiment, the common heavy chain variable region is the one of SEQ ID NO: 1. In one embodiment, the common constant heavy chain is the one of SEQ ID NO: 30. In one embodiment, the common heavy chain is the one of SEQ ID NO: 43. In one embodiment, the common heavy chain is the one of SEQ ID NO: 44. In one embodiment, the common heavy chain is the one of SEQ ID NO: 45.

[0028] In one embodiment, the bispecific antibody is characterized in that it comprises, as heavy chains, a common heavy chain in the first binding portion and the second binding portion, a kappa light chain as a light chain in the first binding portion, and a lambda light chain as a light chain in the second binding portion. In one embodiment, the light chain of the second binding portion is the one of SEQ ID NO: 28, and the heavy chain of the second binding portion is the one of SEQ ID NO: 45 (for example, bispecific antibodies derived from AB-1L3-1 / N such as AB1 and AB13L3-1 / N, AB14L3-1 / N, AB15L3-1 / N, AB17L3-1 / N, AB20L3-1 / N, AB54L3-1 / N, AB60L3-1 / N, AB66L3-1N, AB71L3-1 / N, AB72L3-1 / N, and AB73L3-1 / N; see the sequence listing for CDR and VL sequences).

[0029] AB13, 14, 15, etc. represent the first binding portion (anti-CEACAM5 antibody arm) of the bispecific antibody of the present invention, and L3-1 represents the second binding portion (anti-CD3 antibody arm, also called 1A4) of the bispecific antibody of the present invention. Any ABXX anti-CEA arm can be combined with the L3-1 anti-CD3 arm to form a bispecific antibody. For example, ABXXL3-1 represents a CEAxCD3 bispecific antibody according to the present invention containing a WT hIgG1 Fc portion; ABXXL3-1 / D represents a CEAxCD3 bispecific antibody according to the present invention containing an hIgG1 Fc portion with L234A+L235A mutations; ABXXL3-1 / N represents a CEAxCD3 bispecific antibody according to the present invention containing an hIgG1 Fc portion with L234A+L235A+P329A mutations.

[0030] In one embodiment, the bispecific antibody is characterized in that it contains a common heavy chain as the heavy chain in the first binding portion and the second binding portion, a lambda-type region as the light chain variable region in the first binding portion, and a kappa-type region as the light chain constant region ( "hybrid format light chain"), and a lambda light chain as the light chain in the second binding portion (see, for example, L3-1AB8 H-CK5 / D, Figure 2 and the description of Figure 2).

[0031] In one embodiment, the bispecific antibody is characterized in that it contains a common heavy chain as the heavy chain in the first binding portion and the second binding portion, a lambda-type region as the light chain variable region in the first binding portion, and a lambda-type region as the light chain constant region, and a lambda-type region as the light chain variable region and a kappa-type region as the light chain constant region ( "hybrid format light chain") in the second binding portion; for example, AB8L3-1 H-CK5 / D.

[0032] The bispecific antibodies of the present invention exhibit low binding / cross-reactivity to CEACAM family members other than CEACAM5. In one embodiment, the bispecific antibody has an MFI value for binding to PEAKrapid cells (ATCC® CRL-2828™) expressing a CEACAM selected from the group consisting of CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20 and CEACAM21, which is 2-fold or less compared to the MFI value for binding to WT PEAK cells (i.e., untransfected PEAK cells) under the same experimental conditions. In one embodiment, the bispecific antibody has an MFI value for binding to PEAKrapid cells expressing a CEACAM selected from the group consisting of CEACAM1, CEACAM3, CEACAM4, CEACAM6 and CEACAM8, which is 2-fold or less compared to the MFI value for binding to WT PEAK cells under the same experimental conditions. In one embodiment, the bispecific antibody has an MFI value for binding to PEAKrapid cells expressing CEACAM8, which is 2-fold or less compared to the MFI value for binding to WT PEAK cells under the same experimental conditions. Experimental procedures for transfection of PEAK cells and for measuring the binding of antibodies to these PEAK cells are described in Examples 1 and 5.

[0033] In one embodiment, the bispecific antibody binds to MKN-45 cells (DSMZ number: ACC 409) with an EC50 value of 0.5 nM to 50 nM. In one embodiment, the bispecific antibody binds to MKN-45 cells with an EC50 value of 0.5 nM to 30 nM. In one embodiment, the bispecific antibody according to the present invention is characterized in that the MFI values for binding to MKN-45 cells at 200 nM, 1000 nM, and 5000 nM are at least twice the MFI values obtained with TCB2014. The binding assay is described in Example 7a. In one embodiment, the EC50 for the killing of the tumor cell line MKN-45 measured in an assay containing human PBMC is at least 40% lower for the bispecific antibody of the present invention than the EC50 measured for TCB2014. In one embodiment, the EC50 for the killing of the tumor cell line LS-174T measured in an assay containing human PBMC is at least 40% lower for the bispecific antibody of the present invention than the EC50 measured for TCB2014.

[0034] In one embodiment, the bispecific antibody kills LS174T cells in an assay containing human PBMC in a concentration-dependent manner with an EC50 value of 0.01 to 10 nM. In one embodiment, the bispecific antibody kills LS174T cells in an assay containing human PBMC in a concentration-dependent manner with an EC50 value of 0.01 to 1 nM.

[0035] The assay for measuring T cell retargeted lysis / killing of CEA-positive cells is described in Example 8.

[0036] In one embodiment, the bispecific antibody has an EC50 value in the same assay (killing of CEA-positive LS174T tumor cells) that does not increase by more than 20-fold, in one embodiment 15 or less, and in one embodiment 10 or less, in the presence of 5 μg / ml of soluble CEACAM5 compared to the EC50 value for lysis without soluble CEACAM5 under the same experimental conditions.

[0037] In another embodiment, the bispecific antibody is characterized in that the EC50 value in the same assay does not increase by more than 10-fold in the presence of 1 μg / ml of soluble CEACAM5 compared to the EC50 value for lysis without soluble CEACAM5 under the same experimental conditions, and in one embodiment is 5 or less.

[0038] In one embodiment, the bispecific antibody is characterized in that the bispecific antibody inhibits the growth of tumor volume in the HPAF-II model by 25% or more by day 18 compared to the growth of tumor volume in the vehicle group under the same experimental conditions. In one embodiment, the bispecific antibody is characterized in that the bispecific antibody inhibits the growth of tumor volume in the HPAF-II model in the same manner and does not differ in a statistically significant manner compared to TCB2014 under the same experimental conditions by day 18. The mouse tumor model is described in Example 9a.

[0039] In one embodiment, the bispecific antibody comprises amino acid substitutions in each subunit of the Fc domain that reduce binding to activating Fc receptors and / or reduce effector function, wherein the amino acid substitutions are substitutions of P329 selected from the group consisting of L234A and L235A, and / or P329A, P329G and P329R (Kabat EU indexing). In one embodiment, the bispecific antibody comprises the amino acid substitutions L234A and L235A and P329A (Kabat EU indexing) in each subunit of the Fc domain. L234A and L235A (LALA) mean that the leucine at positions 234 / 235 of the amino acid is replaced by alanine. P329A (PA) means that the proline at position 329 of the amino acid is replaced by alanine.

[0040] In one embodiment, the bispecific antibody comprises a common heavy chain. In one embodiment, the bispecific antibody comprises a common heavy chain that includes, as CDRs, CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4. In one embodiment, the bispecific antibody comprises, in a second binding portion, a light chain region that includes, as CDRs, CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20.

[0041] In one embodiment, the bispecific antibody comprises, in a first binding portion, a light chain constant region that includes the region of SEQ ID NO: 39. In one embodiment, the bispecific antibody comprises, in a first binding portion, a light chain constant region that includes the region of SEQ ID NO: 41. In one embodiment, the bispecific antibody comprises, in a first binding portion, a light chain constant region that includes the region of SEQ ID NO: 58. In one embodiment, the bispecific antibody comprises a common heavy chain of SEQ ID NO: 43, or a common heavy chain of SEQ ID NO: 44, or a common heavy chain of SEQ ID NO: 45. In one embodiment, the bispecific antibody comprises, in a first binding portion, a light chain constant region that includes the region of SEQ ID NO: 39 and a common heavy chain of SEQ ID NO: 45.

[0042] In one embodiment, the bispecific antibody comprises, in a second binding portion, a light chain selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, and 29, or a group of hybrid format light chains (LC) of SEQ ID NOs: 67, 68, 69, 70, and 71.

[0043] In one embodiment, the bispecific antibody comprises, in a first binding portion, a light chain constant region that includes the region of SEQ ID NO: 39, a common heavy chain of SEQ ID NO: 45, and, in a second binding portion, a light chain of SEQ ID NO: 28.

[0044] In one embodiment, the bispecific antibody competes with an anti-CEA antibody selected from the group consisting of an anti-CEA antibody (anti-CEA antibody MEDI) comprising VL and VH of the sequences of SEQ ID NOs: 48 and 49 as the VL and VH domains, an anti-CEA antibody (antibody SM3E) comprising VL and VH of the sequences of SEQ ID NOs: 46 and 47 as the VL and VH domains, an anti-CEA antibody (Lab) comprising VL and VH of the sequences of SEQ ID NOs: 56 and 57 as the VL and VH domains, an anti-CEA antibody (SAR) comprising VL and VH of the sequences of SEQ ID NOs: 50 and 51 as the VL and VH domains, an anti-CEA antibody (T86.66) comprising VL and VH of the sequences of SEQ ID NOs: 54 and 55 as the VL and VH domains, and an anti-CEA antibody (CH1A1A) comprising VL and VH of the sequences of SEQ ID NOs: 52 and 53 as the VL and VH domains. See also FIG. 1 and Example 5c).

[0045] Examples of antibodies useful as the CEA VL or CL region in the bispecific antibodies according to the present invention and competing with MEDI for binding to recombinant CEA are the anti-CEA antibody AB1 and antibodies obtained by lead optimization of AB1 (see Example 11 for experimental methods). AB13, 14, 15, 17, 20, 54, 60, 66, 71, 72, 73 of the anti-CEA antibodies, and AB13L3-1, AB14L3-1, AB15L3-1, AB17L3-1, AB20L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1 and AB73L3-1 of the respective bispecific anti-CEA x CD3 antibodies compete in the same manner as the respective AB1L3-1 of antibody AB1. Examples of antibodies useful as the CEA VL or CL region in the bispecific antibodies according to the present invention and competing with SM3E for binding to recombinant CEA are the anti-CEA antibody AB8 and antibodies obtained by oligonucleotide-specific mutagenesis of AB8 using degenerate oligonucleotides. Examples of antibodies useful as the CEA VL or CL region in the bispecific antibodies according to the present invention and competing with T84.66 for binding to recombinant CEA are the anti-CEA antibody 1B4 and antibodies obtained by oligonucleotide-specific mutagenesis of 1B4 using degenerate oligonucleotides.

[0046] Examples of antibodies useful as the CEA VL or CL region in the bispecific antibodies according to the present invention but not competing with any of the reference antibodies for binding to recombinant CEA are the anti-CEA antibody C11 and antibodies obtained by oligonucleotide-specific mutagenesis of C11 using degenerate oligonucleotides.

[0047] AB1 is an anti-CEA antibody having an HC of SEQ ID NO: 43 and a kappa LC of SEQ ID NO: 40, encoded by the nucleic acid sequences shown in SEQ ID NOs: 80 and 78, respectively.

[0048] AB8 is an anti-CEA antibody having an HC of SEQ ID NO: 43 and a lambda LC of SEQ ID NO: 42, which are encoded by the nucleic acid sequences shown in SEQ ID NOs: 80 and 79, respectively.

[0049] 1B4 is an anti-CEA antibody having an HC of SEQ ID NO: 43 and a lambda LC of SEQ ID NO: 74, which are encoded by the nucleic acid sequences shown in SEQ ID NOs: 80 and 77, respectively.

[0050] C11 is an anti-CEA antibody having an HC of SEQ ID NO: 43 and a kappa LC of SEQ ID NO: 73, which are encoded by the nucleic acid sequences shown in SEQ ID NOs: 80 and 76, respectively.

[0051] The CEA light chain useful as a kappa light chain is the one of SEQ ID NO: 40. The CEA light chain useful as a kappa light chain is the one of SEQ ID NO: 73. The CEA light chain useful as a lambda light chain is the one of SEQ ID NO: 74. The CEA light chain useful as a hybrid kappa light chain is the one of SEQ ID NO: 75.

[0052] In one embodiment, the bispecific antibody contains up to three amino acid substitutions in each subunit of the Fc domain that reduce binding to activating Fc receptors and / or effector functions, wherein the amino acid substitutions are substitutions of P329 selected from the group consisting of L234A, L235A, and P329A, P329G, and P329R (Kabat EU indexing). In one embodiment, the common heavy chain of the antibody according to the invention is the one of SEQ ID NO: 43, 44 or 45. In one embodiment, the common heavy chain of the antibody according to the invention is the one of SEQ ID NO: 45 (L234A, L235A and P329A).

[0053] In one embodiment, the bispecific antibody binds to MKN-45 cells with an EC50 value of 0.5 nM to 50 nM, competes with an anti-CEA antibody (MEDI) containing VL and VH of the sequences of SEQ ID NOs: 48 and 49 as VL and VH domains, or b2) competes with an anti-CEA antibody (SM3E) containing VL and VH of the sequences of SEQ ID NOs: 46 and 47 as VL and VH domains, or b3) competes with an anti-CEA antibody (T84.66) containing VL and CH of the sequences of SEQ ID NOs: 54 and 55 as VL and VH domains, or b4) does not compete with any of the tool antibodies (see Example 5c for tool antibodies), c) contains an amino acid substitution in each subunit of the Fc domain that reduces binding to and / or effector function of activating Fc receptors, wherein the amino acid substitution is a substitution of P329 selected from the group consisting of L234A and L235A, and P329A, P329G and P329R (Kabat EU indexing), d) exhibits one or more properties selected from the group of killing MKN-45, HPAF-II and / or LS174T cells in an assay containing human PBMCs in a concentration-dependent manner with an EC50 value of 0.01 to 10 nM.

[0054] In one embodiment, the bispecific antibody a) binds to MKN-45 cells with an EC50 value of 0.5 nM to 50 nM, b) kills MKN-45, HPAF-II or LS174T cells in an assay containing human PBMCs in a concentration-dependent manner with an EC50 value of 0.01 to 10 nM, c) binds to PEAK cells expressing CEACAM5 but does not cross-react with PEAK cells expressing CEACAM8, d) the killing EC50 in the TDCC assay (Example 8) does not increase by more than 5-fold in the presence of 1 μg / mL sCEA when LS174T tumor cells are used as target cells, e) inhibiting tumor growth in the HPAF-II model by 25% or more as compared to the control group (vehicle only); f) competing with the anti-CEA antibody (MEDI) comprising VL and VH of the sequences of SEQ ID NO: 48 and 49 as the VL and VH domains; and g) comprising the amino acid substitutions L234A, L235A and P329A (Kabat EU index numbering) in each subunit of the Fc domain and exhibiting one or more characteristics selected from the group of

[0055] In one embodiment, the bispecific antibody exhibits the characteristics of a) to d). In one embodiment, the bispecific antibody exhibits all the characteristics of a) to f). In one embodiment, the bispecific antibody exhibits the characteristics of a) to d) and g). In one embodiment, the bispecific antibody exhibits the characteristics of a) to d), f) and g). In one embodiment, the bispecific antibody exhibits all the characteristics of a) to g).

[0056] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, a) the first binding portion and the second binding portion each comprise, as a heavy chain, a common heavy chain (cHC), and as a variable region, a variable region comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3 and CDRH3 of SEQ ID NO: 4 as CDRH1, CDRH2 and CDRH3, respectively; b) the first binding portion i) a kappa light chain constant region (CL), and a light chain variable region (VL) comprising CDRL1 of SEQ ID NO: 32, CDRL2 of SEQ ID NO: 33 and CDRL3 of SEQ ID NO: 34 as CDRL1, CDRL2 and CDRL3, respectively, or a light chain variable region obtained from SEQ ID NO: 31 by oligonucleotide-specific mutagenesis using degenerate oligonucleotides and c) the second binding portion, as CDRL1, CDRL2 and CDRL3 I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8, II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12, III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16, IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24 comprising a variable light chain region comprising a group of CDRs selected from the group consisting of: d) the second binding moiety comprises a lambda light chain constant region characterized in that.

[0057] In one embodiment, the second binding moiety in c) comprises a light chain selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28 and 29.

[0058] In one embodiment, the bispecific antibody is characterized in that it comprises a first binding moiety that specifically binds to human CEACAM5 and a second binding moiety that specifically binds to human CD3ε, a) the first binding moiety and the second binding moiety each comprise, as a heavy chain, a heavy chain comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4 as CDRH1, CDRH2, and CDRH3, b) the first binding moiety comprises, as CDRs, a variable light chain region VL comprising CDRL1 obtained from SEQ ID NO: 32 by oligonucleotide-specific mutagenesis using degenerate oligonucleotides and containing a maximum of 4 amino acid substitutions, CDRL2 obtained from SEQ ID NO: 33 by oligonucleotide-specific mutagenesis using degenerate oligonucleotides and containing a maximum of 4 amino acid substitutions, and CDRL3 obtained from SEQ ID NO: 34 by oligonucleotide-specific mutagenesis using degenerate oligonucleotides and containing a maximum of 4 amino acid substitutions, c) The second binding portion includes a light chain variable region including CDRL1, CDRL2, and CDRL3 as CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20. It is characterized by this.

[0059] Such bispecific antibodies include, but are not limited to, AB13L3-1, AB14L3-1, AB15L3-1, AB17L3-1, AB20L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1, and AB73L3-1 of the bispecific anti-CEA×CD3 antibody.

[0060] In one embodiment, the bispecific antibody is characterized by including a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε. a) The first binding portion includes a heavy chain variable region VH including CDRH1, CDRH2, and CDRH3 as CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4. b) The first binding portion, as a light chain variable region, b1) CDRL1 of SEQ ID NO: 32, CDRL2 of SEQ ID NO: 33, and CDRL3 of SEQ ID NO: 34. b2) CDRL1 of SEQ ID NO: 81, CDRL2 of SEQ ID NO: 82, and CDRL3 of SEQ ID NO: 83. b3) CDRL1 of SEQ ID NO: 84, CDRL2 of SEQ ID NO: 85, and CDRL3 of SEQ ID NO: 86. b4) CDRL1 of SEQ ID NO: 87, CDRL2 of SEQ ID NO: 88, and CDRL3 of SEQ ID NO: 89. b5) CDRL1 of SEQ ID NO: 90, CDRL2 of SEQ ID NO: 91, and CDRL3 of SEQ ID NO: 92. b6) CDRL1 of SEQ ID NO: 93, CDRL2 of SEQ ID NO: 94, and CDRL3 of SEQ ID NO: 95. b7) CDRL1 of SEQ ID NO: 96, CDRL2 of SEQ ID NO: 97, and CDRL3 of SEQ ID NO: 98. b8) CDRL1 of SEQ ID NO: 99, CDRL2 of SEQ ID NO: 100, and CDRL3 of SEQ ID NO: 101. b9) a CDRL set selected from the group consisting of CDRL1 of SEQ ID NO: 102, CDRL2 of SEQ ID NO: 103, and CDRL3 of SEQ ID NO: 104, b10) a CDRL set selected from the group consisting of CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106, and CDRL3 of SEQ ID NO: 107, b11) a CDRL set selected from the group consisting of CDRL1 of SEQ ID NO: 108, CDRL2 of SEQ ID NO: 109, and CDRL3 of SEQ ID NO: 110, and b12) a CDRL set selected from the group consisting of CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, and CDRL3 of SEQ ID NO: 113 comprising a light chain variable region comprising a CDRL set selected from the group consisting of: c) the second binding portion comprises a heavy chain variable region VH 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 VL comprising CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20 characterized in that.

[0061] In one embodiment, the bispecific antibody is characterized in that it comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, a) the first binding portion comprises a heavy chain variable region VH comprising, as CDRH1, CDRH2, and CDRH3, CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4, b) the first binding portion, as a light chain variable region, b1) CDRL1 of SEQ ID NO: 90, CDRL2 of SEQ ID NO: 91, and CDRL3 of SEQ ID NO: 92, b2) CDRL1 of SEQ ID NO: 96, CDRL2 of SEQ ID NO: 97, and CDRL3 of SEQ ID NO: 98, b3) CDRL1 of SEQ ID NO: 99, CDRL2 of SEQ ID NO: 100, and CDRL3 of SEQ ID NO: 101, b4) CDRL1 of SEQ ID NO: 102, CDRL2 of SEQ ID NO: 103, and CDRL3 of SEQ ID NO: 104, b5) CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106, and CDRL3 of SEQ ID NO: 107, and b6) A light chain variable region comprising a set of CDRLs selected from the group consisting of CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, and CDRL3 of SEQ ID NO: 113 and comprising c) a second binding portion comprising a heavy chain variable region VH 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 VL comprising CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20 characterized in that

[0062] In one embodiment, the bispecific antibody is characterized in that it comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, a) the first binding portion comprises a heavy chain variable region VH comprising, as CDRH1, CDRH2, and CDRH3, CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4, b) the first binding portion comprises, as a light chain variable region, b1) CDRL1 of SEQ ID NO: 90, CDRL2 of SEQ ID NO: 91, and CDRL3 of SEQ ID NO: 92, b2) CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106, and CDRL3 of SEQ ID NO: 107, and b3) CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, and CDRL3 of SEQ ID NO: 113 and comprising a light chain variable region comprising a set of CDRLs selected from the group consisting of c) the second binding portion comprises a heavy chain variable region VH 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 VL comprising CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20 characterized in that

[0063] In one embodiment, the present invention a) In the first binding portion, a heavy chain variable region VH having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 1 and comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 3 and CDR3 of SEQ ID NO: 4, b) b1) A light chain variable region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 31 and comprising CDRL1 of SEQ ID NO: 32, CDRL2 of SEQ ID NO: 33 and CDRL3 of SEQ ID NO: 34, b2) A light chain variable region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 114 and comprising CDRL1 of SEQ ID NO: 81, CDRL2 of SEQ ID NO: 82 and CDRL3 of SEQ ID NO: 83, b3) A light chain variable region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 115 and having CDRL1 of SEQ ID NO: 84, CDRL2 of SEQ ID NO: 85 and CDRL3 of SEQ ID NO: 86, b4) A light chain variable region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 116 and having CDRL1 of SEQ ID NO: 87, CDRL2 of SEQ ID NO: 88 and CDRL3 of SEQ ID NO: 89, b5) A light chain variable region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 117 and having CDRL1 of SEQ ID NO: 90, CDRL2 of SEQ ID NO: 91 and CDRL3 of SEQ ID NO: 92, b6) A light chain variable region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 118 and having CDRL1 of SEQ ID NO: 93, CDRL2 of SEQ ID NO: 94 and CDRL3 of SEQ ID NO: 95, b7) A light chain variable region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 119 and having CDRL1 of SEQ ID NO: 96, CDRL2 of SEQ ID NO: 97 and CDRL3 of SEQ ID NO: 98, b8) A light chain variable region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 120 and having CDRL1 of SEQ ID NO: 99, CDRL2 of SEQ ID NO: 100 and CDRL3 of SEQ ID NO: 101, b9) A variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 121, and CDRL1 of SEQ ID NO: 102, CDRL2 of SEQ ID NO: 103 and CDRL3 of SEQ ID NO: 104 b10) A variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 122, and CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106 and CDRL3 of SEQ ID NO: 107 b11) A variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 123, and CDRL1 of SEQ ID NO: 108, CDRL2 of SEQ ID NO: 109 and CDRL3 of SEQ ID NO: 110, and b12) A variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 124, and CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112 and CDRL3 of SEQ ID NO: 113 A variable light chain region VL selected from the group consisting of c) In the second binding portion, a variable heavy chain region VH having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 1 and including CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 3 and CDR3 of SEQ ID NO: 4, and a variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 17 and including CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19 and CDRL3 of SEQ ID NO: 20 It relates to a bispecific antibody according to the present invention, characterized by comprising

[0064] In one embodiment, the present invention a) In the first binding portion, a variable heavy chain region VH of SEQ ID NO: 1, and b) b1) A variable light chain region VL of SEQ ID NO: 31 b2) A variable light chain region VL of SEQ ID NO: 114 b3) A variable light chain region VL of SEQ ID NO: 115 b4) A variable light chain region VL of SEQ ID NO: 116 b5) A variable light chain region VL of SEQ ID NO: 117 b6) The variable light chain region VL of SEQ ID NO: 118, b7) The variable light chain region VL of SEQ ID NO: 119, b8) The variable light chain region VL of SEQ ID NO: 120, b9) The variable light chain region VL of SEQ ID NO: 121, b10) The variable light chain region VL of SEQ ID NO: 122, b11) The variable light chain region VL of SEQ ID NO: 123, and b12) The variable light chain region VL of SEQ ID NO: 124 a variable light chain region VL selected from the group consisting of, and c) In the second binding portion, the heavy chain variable region VH of SEQ ID NO: 1 and the variable light chain region VL of SEQ ID NO: 17 characterized by comprising, relating to the bispecific antibody according to the present invention.

[0065] In one embodiment, the present invention a) In the first binding portion, the heavy chain variable region VH of SEQ ID NO: 1, and b) b1) The variable light chain region VL of SEQ ID NO: 117, b2) The variable light chain region VL of SEQ ID NO: 119, b3) The variable light chain region VL of SEQ ID NO: 120, b4) The variable light chain region VL of SEQ ID NO: 121, b5) The variable light chain region VL of SEQ ID NO: 122, and b6) The variable light chain region VL of SEQ ID NO: 124 a variable light chain region VL selected from the group consisting of, and c) In the second binding portion, the heavy chain variable region VH of SEQ ID NO: 1 and the variable light chain region VL of SEQ ID NO: 17 characterized by comprising, relating to the bispecific antibody according to the present invention.

[0066] In one embodiment, the present invention a) In the first binding portion, the heavy chain variable region VH of SEQ ID NO: 1, and b) b1) The variable light chain region VL of SEQ ID NO: 117, b2) The variable light chain region VL of SEQ ID NO: 122, and b3) The variable light chain region VL of SEQ ID NO: 124 a variable light chain VL selected from the group consisting of, and c) in the second binding portion, a heavy chain variable region VH of SEQ ID NO: 1 and a light chain variable region VL of SEQ ID NO: 17 relates to a bispecific antibody according to the invention, characterized in that it comprises

[0067] In one embodiment, the invention provides, in a first binding portion, a) a heavy chain variable region VH of SEQ ID NO: 1, and b) b1) a light chain of SEQ ID NO: 40, b2) a light chain of SEQ ID NO: 125, b3) a light chain of SEQ ID NO: 126, b4) a light chain of SEQ ID NO: 127, b5) a light chain of SEQ ID NO: 128, b6) a light chain of SEQ ID NO: 129, b7) a light chain of SEQ ID NO: 130, b8) a light chain of SEQ ID NO: 131, b9) a light chain of SEQ ID NO: 132, b10) a light chain of SEQ ID NO: 133, and b11) a light chain of SEQ ID NO: 134, and b12) a light chain of SEQ ID NO: 135 a light chain selected from the group consisting of, and, c) in the second binding portion, a heavy chain variable region VH of SEQ ID NO: 1 and a light chain of SEQ ID NO: 28 relates to a bispecific antibody according to the invention, characterized in that it comprises

[0068] In one embodiment, the invention provides, in a first binding portion, a) a1) a heavy chain of SEQ ID NO: 43, a2) a heavy chain of SEQ ID NO: 44, or a3) a heavy chain of SEQ ID NO: 45 a common heavy chain selected from the group consisting of, and b) b1) a light chain of SEQ ID NO: 40, b2) a light chain of SEQ ID NO: 125, b3) a light chain of SEQ ID NO: 126, b4) a light chain of SEQ ID NO: 127, b5) the light chain of SEQ ID NO: 128, b6) the light chain of SEQ ID NO: 129, b7) the light chain of SEQ ID NO: 130, b8) the light chain of SEQ ID NO: 131, b9) the light chain of SEQ ID NO: 132, b10) the light chain of SEQ ID NO: 133, b11) the light chain of SEQ ID NO: 134, or b12) the light chain of SEQ ID NO: 135 a light chain selected from the group consisting of, and c) in the second binding portion, the light chain of SEQ ID NO: 28 relates to a bispecific antibody according to the invention, characterized in that it comprises

[0069] In one embodiment, the bispecific antibody (AB17L3-1 / N) according to the invention comprises the common heavy chain ( / N) of SEQ ID NO: 45, and in the second binding portion, as the light chain, the light chain of SEQ ID NO: 28 (1A4 LC L3-1 respectively), and in the first binding portion, as the light chain, the light chain of SEQ ID NO: 128 (AB17). In one embodiment, the bispecific antibody (AB71L3-1 / N) according to the invention comprises the common heavy chain ( / N) of SEQ ID NO: 45, and in the second binding portion, as the light chain, the light chain of SEQ ID NO: 28 (L3-1), and in the first binding portion, as the light chain, the light chain of SEQ ID NO: 133 (AB71). In one embodiment, the bispecific antibody (AB73L3-1 / N) according to the invention comprises the common heavy chain ( / N) of SEQ ID NO: 45, and in the second binding portion, as the light chain, the light chain of SEQ ID NO: 28 (L3-1), and in the first binding portion, as the light chain, the light chain of SEQ ID NO: 135 (AB73).

[0070] In one embodiment, the bispecific antibody is characterized in that it comprises a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, a) the first binding portion and the second binding portion each comprise, as the heavy chain, a common heavy chain, and as the variable region, a variable region comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4 as CDRH1, CDRH2, and CDRH3, b) The first binding portion comprises i) a lambda light chain constant region (CL), and ii) a light chain variable region (VL) comprising, as CDRL1, CDRL2 and CDRL3, CDRL1 of SEQ ID NO: 36, CDRL2 of SEQ ID NO: 37 and CDRL3 of SEQ ID NO: 38, or a light chain variable region obtained from SEQ ID NO: 35 by oligonucleotide - specific mutagenesis using degenerate oligonucleotides and c) The second binding portion comprises, as CDRL1, CDRL2 and CDRL3, I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7 and CDRL3 of SEQ ID NO: 8, II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11 and CDRL3 of SEQ ID NO: 12, III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15 and CDRL3 of SEQ ID NO: 16, IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19 and CDRL3 of SEQ ID NO: 20, and V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23 and CDRL3 of SEQ ID NO: 24 and comprises a light chain variable region comprising a group of CDRs selected from the group consisting of d) The second binding portion comprises a hybrid kappa chain constant region characterized in that.

[0071] In one embodiment, the second binding portion in c) comprises a light chain selected from the group consisting of SEQ ID NOs: 67, 68, 69, 70 and 71.

[0072] In one embodiment, the second binding portion can comprise, as a light chain constant region, the lambda light chain constant region of SEQ ID NO: 41, in which case the first binding portion can comprise, in one embodiment, as a light chain constant region, the hybrid kappa light chain region of SEQ ID NO: 58. In one embodiment, the arm with the hybrid light chain constant region is based on the overall properties of the bsAb, including but not limited to stability and productivity.

[0073] In one embodiment, the light chain variable region of the first binding portion is obtained from SEQ ID NO: 35 by oligonucleotide-specific mutagenesis using degenerate oligonucleotides, the common heavy chain is that of SEQ ID NO: 45, and the variable region of the common heavy chain is that of SEQ ID NO: 1.

[0074] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε. a) The first binding portion and the second binding portion each comprise, as a heavy chain, a common heavy chain, and as variable regions, a variable region comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4 as CDRH1, CDRH2, and CDRH3. b) The first binding portion i) a kappa light chain constant region (CL), and ii) a light chain variable region (VL) comprising CDRL1 of SEQ ID NO: 64, CDRL2 of SEQ ID NO: 65, and CDRL3 of SEQ ID NO: 66 as CDRL1, CDRL2, and CDRL3, or a light chain variable region obtained from SEQ ID NO: 63 by oligonucleotide-specific mutagenesis and c) The second binding portion, as CDRL1, CDRL2, and CDRL3, I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8, II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12, III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16, IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24 and comprises a light chain variable region comprising a group of CDRs selected from the group consisting of d) The second binding portion comprises a lambda light chain constant region. It is characterized by the following.

[0075] In one embodiment, the second binding portion in c) comprises a light chain selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, and 29.

[0076] In one embodiment of the present invention, the light chain variable region of the first binding portion is obtained from SEQ ID NO: 63 by oligonucleotide-specific mutagenesis using degenerate oligonucleotides, and the common heavy chain is that of SEQ ID NO: 45.

[0077] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, a) The first binding portion and the second binding portion each include, as a heavy chain, a common heavy chain, and as variable regions, a variable region including CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4 as CDRH1, CDRH2, and CDRH3, b) The first binding portion includes a lambda light chain constant region (CL), and a light chain variable region (VL) including CDRL1 of SEQ ID NO: 60, CDRL2 of SEQ ID NO: 61, and CDRL3 of SEQ ID NO: 62 as CDRL1, CDRL2, and CDRL3, or a light chain variable region obtained from SEQ ID NO: 59 by oligonucleotide-specific mutagenesis using degenerate oligonucleotides, c) The second binding portion includes, as CDRL1, CDRL2, and CDRL3, I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8, II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12, III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16, IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24 comprising a light chain variable region comprising a group of CDRs selected from the group consisting of d) the second binding moiety comprises a hybrid kappa chain constant region characterized in that.

[0078] In a further embodiment of the invention, the second binding moiety comprises, as a light chain constant region, a lambda light constant chain region of SEQ ID NO: 41, in which case the first binding moiety, in one embodiment, comprises, as a light chain constant region, a hybrid kappa light chain constant region of SEQ ID NO: 58. The choice of the arm with the hybrid light chain constant region is based on the overall properties of the final bsAb, including but not limited to stability and productivity.

[0079] In one embodiment, the light chain variable region of the first binding moiety is obtained from SEQ ID NO: 59 by oligonucleotide site-directed mutagenesis using degenerate oligonucleotides.

[0080] In one embodiment, the bispecific antibody is characterized by comprising a first binding moiety that specifically binds to human CEACAM5 and a second binding moiety that specifically binds to human CD3ε, a) the first binding moiety and the second binding moiety each comprise, as a heavy chain, a common heavy chain and, as variable regions, a variable region comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3 and CDRH3 of SEQ ID NO: 4 as CDRH1, CDRH2 and CDRH3, b) the first binding moiety comprises a human kappa-type light chain constant region and a human kappa-type light chain variable region comprising CDRL1 of SEQ ID NO: 32 having 0, 1, 2, 3 or 4 amino acid substitutions as CDRL1, CDRL2 of SEQ ID NO: 33 having 0, 1, 2, 3 or 4 amino acid substitutions as CDRL2, and CDRL3 of SEQ ID NO: 34 having 0, 1, 2, 3, 4 or 5 amino acid substitutions as CDRL3, c) the second binding moiety comprises a human lambda-type light chain constant region and, as CDRL1, CDRL2 and CDRL3, I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8, II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12, III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16, IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24 comprising a group of CDRs selected from the group consisting of, characterized in that.

[0081] In one embodiment, the second binding moiety in c) comprises a light chain selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, and 29.

[0082] In one embodiment, the bispecific antibody is characterized in that it comprises a first binding moiety that specifically binds to human CEACAM5 and a second binding moiety that specifically binds to human CD3ε, a) the first binding moiety and the second binding moiety each comprise, as a heavy chain, a common heavy chain, and as a variable region, a variable region comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4 as CDRH1, CDRH2, and CDRH3, b) the first binding moiety comprises a human lambda light chain constant region, and as CDRL1, CDRL2, and CDRL3, a set of CDRLs selected from the group consisting of CDRL1 of SEQ ID NO: 36 having 0, 1, 2, 3, 4, or 5 amino acid substitutions, CDRL2 of SEQ ID NO: 37 having 0, 1, 2, 3, 4, or 5 amino acid substitutions, and CDRL3 of SEQ ID NO: 38 having 0, 1, 2, 3, 4, or 5 amino acid substitutions, c) the second binding moiety comprises a hybrid kappa light chain constant region, and as CDRL1, CDRL2, and CDRL3, I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8, II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12, III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16, IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24 comprising a group of CDRs selected from the group consisting of, characterized in that.

[0083] In one embodiment, the second binding moiety in c) comprises a light chain selected from the group consisting of SEQ ID NOs: 67, 68, 69, 70, and 71.

[0084] In one embodiment, the bispecific antibody is characterized in that it comprises a first binding moiety that specifically binds to human CEACAM5 and a second binding moiety that specifically binds to human CD3ε, a) the first binding moiety and the second binding moiety each comprise, as a heavy chain, a common heavy chain, and as a variable region, a variable region comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4 as CDRH1, CDRH2, and CDRH3, respectively, b) the first binding moiety comprises a human lambda light chain constant region, and as CDRL1, CDRL2, and CDRL3, a set of CDRLs selected from the group consisting of CDRL1 of SEQ ID NO: 60 having 0, 1, 2, 3, or 4 amino acid substitutions, CDRL2 of SEQ ID NO: 61 having 0, 1, 2, 3, or 4 amino acid substitutions, and CDRL3 of SEQ ID NO: 62 having 0, 1, 2, 3, 4, or 5 amino acid substitutions, c) the second binding moiety comprises a human kappa light chain constant region, and as CDRL1, CDRL2, and CDRL3, I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8, II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12, III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16, IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24 characterized by comprising a group of CDRs selected from the group consisting of and containing a human lambda - type light - chain variable region.

[0085] In one embodiment, the second binding moiety in c) comprises a light chain selected from the group consisting of SEQ ID NOs: 67, 68, 69, 70 and 71.

[0086] In one embodiment, the bispecific antibody is characterized by comprising a first binding moiety that specifically binds to human CEACAM5 and a second binding moiety that specifically binds to human CD3ε, a) The first binding moiety and the second binding moiety each comprise, as a heavy chain, a common heavy chain and, as variable regions, a variable region comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4 as CDRH1, CDRH2, and CDRH3, b) The first binding moiety comprises a human kappa - type light - chain constant region and, as CDRL1, CDRL2, and CDRL3, a set of CDRLs selected from the group consisting of CDRL1 of SEQ ID NO: 64 having 0, 1, 2, 3, 4, or 5 amino - acid substitutions, CDRL2 of SEQ ID NO: 65 having 0, 1, 2, 3, 4, or 5 amino - acid substitutions, and CDRL3 of SEQ ID NO: 66 having 0, 1, 2, 3, 4, or 5 amino - acid substitutions, and contains a human kappa - type light - chain variable region, c) The second binding moiety comprises a human lambda - type light - chain constant region and, as CDRL1, CDRL2, and CDRL3, I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8, II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12, III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16, IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24 characterized by comprising a group of CDRs selected from the group consisting of and comprising a human lambda - type light - chain variable region.

[0087] In one embodiment, the second binding moiety in c) comprises a light chain selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, and 29.

[0088] In one embodiment, the bispecific antibody comprises a first binding moiety that specifically binds to human CEACAM5 and a second binding moiety that specifically binds to human CD3ε, a) the first binding moiety comprises a heavy - chain variable region (VH) comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4, b) the first binding moiety comprises a light - chain variable region (VL) comprising a set of CDRLs where CDRL1 has the consensus sequence of SEQ ID NO: 136, CDRL2 has the consensus sequence of SEQ ID NO: 137, and CDRL3 has the consensus sequence of SEQ ID NO: 138, c) the second binding moiety comprises a VH comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4.

[0089] In one embodiment, the first binding portion includes, as a variable light chain framework array, the framework array of SEQ ID NO: 31. In one embodiment, the first binding portion includes, as a variable light chain framework array, the framework array of SEQ ID NO: 35. In one embodiment, the first binding portion includes, as a variable light chain framework array, the framework array of SEQ ID NO: 59. In one embodiment, the first binding portion includes, as a variable light chain framework array, the framework array of SEQ ID NO: 63.

[0090] In one embodiment, the first binding portion that specifically binds to CEA includes, as a heavy chain variable region, the heavy chain variable region of the amino acid sequence of SEQ ID NO: 1, and, as a light chain variable region, a light chain variable region having an amino acid sequence that is 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 31 or SEQ ID NO: 35, SEQ ID NO: 59 or SEQ ID NO: 63. The second binding portion that specifically binds to CD3 includes, as a heavy chain variable region, the heavy chain variable region of the amino acid sequence of SEQ ID NO: 1, and, as a light chain variable region, a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17 and SEQ ID NO: 21.

[0091] In one embodiment, the bispecific antibody according to the present invention is characterized by including a first binding portion specific for CEA, which includes a kappa light chain variable domain and a kappa light chain constant domain, and a second binding portion specific for CD3ε, which includes a lambda light chain variable domain and a lambda light chain constant domain.

[0092] In one embodiment, the bispecific antibody according to the present invention is characterized by including a first binding portion specific for CEA, which includes a lambda light chain variable domain and a kappa light chain constant domain, and a second binding portion specific for CD3ε, which includes a lambda light chain variable domain and a lambda light chain constant domain.

[0093] In one embodiment, the bispecific antibody according to the present invention is characterized by comprising a first binding portion specific for CEA, which comprises a lambda light chain variable domain and a lambda light chain constant domain, and a second binding portion specific for CD3ε, which comprises a lambda light chain variable domain and a kappa light chain constant domain.

[0094] In certain embodiments, the Fc domain exhibits a reduced binding affinity to Fc receptors and / or a reduced effector function as compared to the native / wild-type IgG1 Fc domain. In certain embodiments, the Fc domain is engineered to have a reduced binding affinity to Fc receptors and / or a reduced effector function as compared to the unengineered Fc domain. In one embodiment, the Fc domain comprises one or more amino acid substitutions that reduce binding to one or more Fc receptors and / or reduce effector function. In one embodiment, such one or more substitutions are selected from the group consisting of Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435 (Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al., FASEB J. 9 (1995) 115-119; Morgan, A., et al., Immunology 86 (1995) 319-324; European Patent Application No. 0307434). In one embodiment, the antibody has mutations in S228, L234, L235 and / or D265 that are involved in FcR binding of the IgG4 subclass or IgG1 or IgG2 subclasses, and / or contains the PVA236 mutation. In one embodiment, the mutations in the Fc domain are S228P, L234A, L235A, L235E and / or PVA236. In another embodiment, the mutations in the Fc domain are S228P in IgG4 and L234A and L235A in IgG1. In one embodiment, one or more amino acid substitutions in the Fc domain that reduce binding to one or more Fc receptors and / or reduce effector function are at one or more positions selected from the group of L234, L235 and P329 (Kabat EU indexing).In certain embodiments, each subunit of the Fc domain comprises two amino acid substitutions that reduce binding to Fc receptors and / or reduce effector function, wherein the amino acid substitutions are L234A and L235A (Kabat EU indexing). In certain embodiments, each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to Fc receptors and / or reduce effector function, wherein the amino acid substitutions are L234A, L235A and P329A (Kabat EU indexing). In such an embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain (Kabat EU indexing). In one embodiment, the Fc domain is of the IgG4 subclass, and in one embodiment, is of the IgG4 subclass having the S228P mutation.

[0095] In one embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is human FcγRIIIA, FcγRI and / or FcγRIIIA. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), but is not limited to ADCC only.

[0096] Embodiments of the invention are for use as a kappa CL region of SEQ ID NO: 58 as the CL region in the second binding portion in the construction of a bispecific antibody comprising the common heavy chain of SEQ ID NO: 43, 44 or 45 and the lambda CL region of SEQ ID NO: 41 as the CL region in the first binding portion.

[0097] Embodiments of the invention are for use as a kappa CL region of SEQ ID NO: 58 as the CL region in the second binding portion that specifically binds to CD3 in the construction of a bispecific antibody according to the invention, comprising the common heavy chain of SEQ ID NO: 43, 44 or 45 and the lambda CL region of SEQ ID NO: 41 as the CL region in the first binding portion that specifically binds to CEACAM5.

[0098] Embodiments of the present invention are for use as the CL region in the second binding portion specifically binding to CD3 in the construction of a bispecific antibody according to the present invention, including the common heavy chain of SEQ ID NO: 43, 44 or 45, the variable light chain of SEQ ID NO: 5, 9, 13, 17 or 21, and the lambda CL region of SEQ ID NO: 41 as the CL region in the first binding portion specifically binding to CEACAM5, which is the kappa CL region of SEQ ID NO: 58.

[0099] A further embodiment of the present invention is an oligonucleotide selected from the group consisting of SEQ ID NOs: 76, 77, 78 and 79 for use in affinity maturation of an antibody by oligonucleotide-specific mutagenesis using degenerate oligonucleotides of the light chain variable regions of SEQ ID NOs: 31, 35, 59 and 63 respectively.

[0100] In another aspect, there is provided a method for generating bispecificity of the present invention, comprising: a) culturing the host cell of the present invention under conditions suitable for the expression of a bispecific antibody; and b) recovering the bispecific antibody. The present invention also encompasses bispecific antibodies produced by the method of the present invention.

[0101] The present invention further provides a pharmaceutical composition comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier. Methods of using the bispecific antibody and pharmaceutical composition of the present invention are also encompassed by the present invention. In one aspect, the present invention provides the bispecific antibody or pharmaceutical composition of the present invention for use as a medicament. In one aspect, there is provided the bispecific antibody or pharmaceutical composition according to the present invention for use in the treatment of a disease in an individual in need thereof. In a specific embodiment, the disease is cancer.

[0102] The bispecific antibody of the present invention for use in the manufacture of a medicament for the treatment of a disease in an individual in need thereof, and a method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the bispecific antibody according to the present invention in a pharmaceutically acceptable form, are also provided. In a specific embodiment, the disease is cancer. In any of the above embodiments, the individual is preferably a mammal, particularly a human.

[0103] The present invention also provides a method for inducing lysis of target cells, particularly tumor cells, comprising contacting the target cells with the bispecific antibody of the present invention in the presence of T cells, particularly cytotoxic T cells.

[0104] A further embodiment of the present invention is the bispecific antibody according to the present invention for use in the manufacture of a medicament for treating a subject having a cancer that expresses CEA.

[0105] A further embodiment of the present invention is the bispecific antibody according to the present invention for use in the manufacture of a medicament according to the present invention, wherein the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer (NSCLC), esophageal cancer, gastric / esophageal junction cancer, pancreatic cancer and breast cancer.

[0106] A further embodiment of the present invention is the bispecific antibody according to the present invention for use in simultaneous, separate or sequential combination with an anti-CD47 antibody. In one embodiment, the anti-CD47 antibody is magrolimab, ALX148 or TTI-621 and / or TTI-622.

[0107] A further embodiment of the present invention is the bispecific antibody according to the present invention for use in simultaneous, separate or sequential combination with a second bispecific antibody comprising a third binding moiety that specifically binds to human CEACAM5 and a fourth binding moiety that specifically binds to human CD47 in the treatment of a subject having a cancer that expresses CEA.

[0108] Such second bispecific CEAxCD47 antibodies are described in International Application No. PCT / IB2019 / 054559 and US Application No. 16 / 428,359.

[0109] A further embodiment of the invention is a bispecific antibody according to the invention for use according to the invention, wherein the bispecific antibody according to the invention and the second bispecific CEAxCD47 antibody are administered to the subject alternately at intervals which are not limited to such intervals, but are between 6 and 15 days.

[0110] A further embodiment of the invention is a first bispecific antibody according to the invention comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3 for use in the treatment of cancer according to the invention, as well as a second bispecific antibody CEAxCD47 according to the invention, wherein the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, pancreatic cancer and breast cancer.

[0111] A further embodiment of the invention is a composition comprising a bispecific antibody according to the invention, which composition is characterized by not competing with the second CEAxCD47 bispecific antibody as defined above for use in the treatment of a subject having a cancer expressing CEA.

[0112] A further embodiment of the invention is a method for the treatment of a human patient diagnosed with a tumor (cancer), in particular a solid tumor, in particular a solid cancer expressing CEA, in particular colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, pancreatic cancer and breast cancer, the method comprising administering to the human patient an effective amount of a bispecific antibody according to the invention, as well as a second bispecific antibody described in International Application No. PCT / IB2019 / 054559 and US Application No. 16 / 428,359 against CEA and CD47, and the method then Administering to the patient the second anti-CEA x CD47 antibody at a dose of 0.1 to 30 mg / kg, in a further embodiment at a dose of 0.5 to 10 mg / kg, and in a further embodiment at a dose of 1 to 10 mg / kg, for example, weekly over a period of 4 to 12 weeks, Administering to the patient the second antibody at q1, q2w, q3w, or, if necessary, q4w, After these 4 to 12 weeks and after an additional 2 or 3 or 4 elimination half-lives of the anti-CEA x CD47 antibody, administering to the patient an antibody according to the invention at a dose of 0.1 to 10 mg / kg, Administering to the patient the antibody according to the invention at q1, q2w, q3w, or, if necessary, q4w, Waiting for 2 or 3 or 4 elimination half-lives of the antibody according to the invention, and then, if necessary, repeating the cycle of administration of the CEA x CD47 bispecific antibody followed by administration of the CEA x CD3 bispecific antibody, and, if necessary, repeating this cycle again A method comprising.

[0113] This "alternating" method is applicable when the antibody of the invention and the second bispecific antibody compete with respect to binding to CEA.

[0114] If the CEA x CD47 bispecific antibody and the CEA x CD3 bispecific antibody according to the invention are not competitive, the two bispecific antibodies can be administered, for example, to the patient in an approximately simultaneous manner at a dose of 0.1 to 30 mg / kg, in a further embodiment at a dose of 0.5 to 10 mg / kg, and in a further embodiment at a dose of 1 to 10 mg / kg of the CEA x CD47 bispecific antibody, and at a dose of 0.1 to 10 mg / kg of the CEA x CD3 bispecific antibody of the invention, followed by administration of one or more combinations of these, for example, at a frequency of q1w or q2w or q3w, or, if necessary, q4w, in a manner such that the patient experiences therapeutically effective plasma and tissue concentrations of both bispecific antibodies in parallel ("simultaneous manner").

[0115] The term "q1w" means once-weekly administration, and q2w means administration every two weeks, and so on.

[0116] A further embodiment of the present invention is a pharmaceutical composition comprising an antibody according to the present invention and a pharmaceutically acceptable excipient or carrier.

[0117] A further preferred embodiment of the present invention is a pharmaceutical composition comprising an antibody according to the present invention for use as a medicament.

[0118] A further preferred embodiment of the present invention is a pharmaceutical composition comprising an antibody according to the present invention for use as a medicament in the treatment of a solid tumor disorder expressing CEA.

[0119] A further preferred embodiment of the present invention is a pharmaceutical composition comprising an antibody according to the present invention for use as a medicament in the treatment of colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, esophageal cancer, pancreatic cancer or breast cancer.

[0120] A further embodiment of the present invention is a composition according to the present invention, characterized in that the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, pancreatic cancer or breast cancer.

[0121] A further embodiment of the present invention is an antibody according to the present invention for use in the manufacture of a pharmaceutical composition.

[0122] A further embodiment of the present invention is an antibody according to the present invention and a pharmaceutically acceptable excipient or carrier for use in the manufacture of a pharmaceutical composition.

[0123] A further embodiment of the present invention is an embodiment of an antibody according to the present invention for use in the manufacture of a medicament for the treatment of a solid tumor disorder.

[0124] A further embodiment of the present invention is an embodiment of an antibody according to the present invention for use in the treatment of colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, esophageal cancer, pancreatic cancer or breast cancer.

[0125] Another aspect of the present invention provides a method of inducing lysis of tumor cells, the method comprising contacting the tumor cells with a bispecific antibody of any of the embodiments described above. In some embodiments, the tumor cells are colorectal cancer cells, NSCLC (non-small cell lung cancer), gastric cancer cells, esophageal cancer cells, pancreatic cancer cells or breast cancer cells.

[0126] In one embodiment, the lysis is induced by T cell-dependent cytotoxicity (TDCC).

[0127] Another aspect of the present invention provides a method of treating a subject having a cancer that expresses CEA, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody of any of the embodiments described above.

[0128] Another aspect of the invention is a method of treating a subject having a cancer that expresses CEA, the method comprising administering to the subject a therapeutically effective amount of any of the bispecific antibodies of the embodiments described above in combination with a bispecific antibody that binds to human CEA and human CD47. If the CEAxCD47 antibody and the CEAxCD3 antibody are competing, they compete for the CEA receptor on the surface of tumor cells, and the receptor occupancy and efficacy for each combination partner depends on their binding affinity and their plasma concentration, and thus is difficult to predict, and also varies over time if the concentrations of the two drugs have different elimination half-lives of clearance from the body. Thus, competing CEAxCD3 and CEAxCD47 bispecific antibodies must be administered sequentially (alternately). If the CEAxCD3 and CEAxCD47 bispecific antibodies do not compete or minimally compete, they can be administered not only sequentially but also in parallel (simultaneously), which is sufficiently advantageous as tumor cell killing via the involvement of T cells by the CEAxCD3 bispecific antibody and via the involvement of macrophages by the simultaneous CEAxCD47 bispecific antibody is expected to be additive or even synergistic, meaning that the efficacy increases when both drugs are administered in parallel.

[0129] Another aspect of the invention is a method of increasing the progression-free survival and / or overall survival in a subject having a cancer that expresses CEA, the method comprising administering to the subject a therapeutically effective amount of any of the bispecific antibodies of the embodiments described above. In one embodiment, the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, head and neck cancer, uterine cancer, bladder cancer or another cancer that expresses CEA.

[0130] In certain embodiments of these methods, the bispecific antibody is administered in combination with chemotherapy or radiotherapy. In one embodiment, the subject is a patient suffering from colorectal cancer, or lung cancer, or gastric cancer, esophageal cancer, or pancreatic cancer, or breast cancer, or another cancer that expresses CEA.

[0131] In certain embodiments of these methods, the bispecific antibody of the present invention is administered to a patient at a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, either as a single dose or divided doses or by continuous infusion. In certain embodiments, the bispecific antibody of the present invention is administered to a patient at a dose in the range of 1 to 20 mg / kg.

[0132] Another aspect of the present invention is a method of treating a subject having a cancer that expresses CEA, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody of any of the embodiments described above in combination with a bispecific antibody against human CEA and human CD47. In certain embodiments of these methods, the bispecific antibody is administered in combination with the bispecific anti-CEA×CD47 antibody simultaneously, separately or in a sequential combination. In certain embodiments of these methods, the bispecific anti-CEA×CD47 antibody is administered at an interval of 6 to 15 days between the administration of the antibody of the present invention and the bispecific anti-CEA×CD47 antibody in an alternating pattern. In certain embodiments, the anti-CEA×CD47 antibody is administered to a patient at a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, either as a single dose or divided doses or by continuous infusion.

[0133] In certain embodiments of these methods, the bispecific antibody is administered in combination with a PD-1 axis antagonist, either simultaneously, separately, or in sequential combinations. In certain embodiments of these methods, the bispecific antibody is administered in combination with a bispecific anti-CEA x CD47 antibody and a PD-1 axis antagonist, either simultaneously, separately, or in sequential combinations. In certain embodiments, the PD-1 axis antagonist is administered to the patient at a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, either as a single dose, divided doses, or by continuous infusion.

[0134] Another aspect of the invention is a method of increasing the progression-free survival period and / or overall survival period in a subject having a cancer that abnormally expresses CEA, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody of any of the embodiments described above. In one embodiment, the cancer is colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, pancreatic cancer, or breast cancer.

[0135] In certain embodiments of these methods, the bispecific antibody is administered in combination with chemotherapy or radiation therapy. In one embodiment, the subject is a cancer patient having colorectal cancer, or lung cancer, or gastric cancer, esophageal cancer, or pancreatic cancer, or breast cancer, or another cancer that expresses CEA.

[0136] Another embodiment of the invention provides a bispecific antibody according to the invention for use in any of the methods of treatment described above. In one embodiment, the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, pancreatic cancer, and breast cancer.

[0137] Another embodiment of the present invention provides a polynucleotide encoding a bispecific antibody or a domain thereof (e.g., variable light chain region and / or variable heavy chain region) disclosed herein that immunospecifically binds to CEACAM5 or CD3ε. In certain embodiments, a polynucleotide is provided that comprises a nucleotide sequence encoding a light chain or a heavy chain of an antibody described herein. The polynucleotide can comprise a nucleotide sequence encoding a heavy chain that comprises a VH or a heavy chain CDR of an antibody described herein. The polynucleotide can comprise a nucleotide sequence encoding a light chain that comprises a VL or a light chain CDR of an antibody described herein.

[0138] Certain embodiments are vectors comprising an isolated polynucleotide disclosed herein. Certain other embodiments are cells comprising an isolated polynucleotide or vector encoding a bispecific antibody disclosed herein. In some embodiments, the cell is selected from the group consisting of Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

[0139] Certain embodiments are methods of making an antibody disclosed herein. In some embodiments, the method of making an antibody comprises expressing the antibody using a cell comprising an isolated polynucleotide or vector encoding a bispecific antibody disclosed herein. In some embodiments, the method of making an antibody comprises culturing a cell containing an isolated polynucleotide or vector encoding a bispecific antibody disclosed herein and isolating the antibody expressed therein. In an embodiment of the present invention, for example, the following items are provided. (Item 1) A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, a) the first binding portion comprises a heavy chain variable region (VH) comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4, b) the first binding portion is b1) CDRL1 of SEQ ID NO: 32, CDRL2 of SEQ ID NO: 33, and CDRL3 of SEQ ID NO: 34, b2) CDRL1 of SEQ ID NO: 81, CDRL2 of SEQ ID NO: 82, and CDRL3 of SEQ ID NO: 83, b3) CDRL1 of SEQ ID NO: 84, CDRL2 of SEQ ID NO: 85, and CDRL3 of SEQ ID NO: 86, b4) CDRL1 of SEQ ID NO: 87, CDRL2 of SEQ ID NO: 88, and CDRL3 of SEQ ID NO: 89, b5) CDRL1 of SEQ ID NO: 90, CDRL2 of SEQ ID NO: 91, and CDRL3 of SEQ ID NO: 92, b6) CDRL1 of SEQ ID NO: 93, CDRL2 of SEQ ID NO: 94, and CDRL3 of SEQ ID NO: 95, b7) CDRL1 of SEQ ID NO: 96, CDRL2 of SEQ ID NO: 97, and CDRL3 of SEQ ID NO: 98, b8) CDRL1 of SEQ ID NO: 99, CDRL2 of SEQ ID NO: 100, and CDRL3 of SEQ ID NO: 101, b9) CDRL1 of SEQ ID NO: 102, CDRL2 of SEQ ID NO: 103, and CDRL3 of SEQ ID NO: 104, b10) CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106, and CDRL3 of SEQ ID NO: 107, b11) CDRL1 of SEQ ID NO: 108, CDRL2 of SEQ ID NO: 109, and CDRL3 of SEQ ID NO: 110, and b12) CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, and CDRL3 of SEQ ID NO: 113 and comprises a light chain variable region (VL) comprising a set of CDRLs selected from the group consisting of c) the second binding portion comprises a VH comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4, d) the second binding portion comprises a VL comprising CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, bispecific antibody. (Item 2) The first binding portion is a1) CDRL1 of SEQ ID NO: 90, CDRL2 of SEQ ID NO: 91, and CDRL3 of SEQ ID NO: 92, a2) CDRL1 of SEQ ID NO: 96, CDRL2 of SEQ ID NO: 97, and CDRL3 of SEQ ID NO: 98, a3) CDRL1 of SEQ ID NO: 99, CDRL2 of SEQ ID NO: 100, and CDRL3 of SEQ ID NO: 101, a4) CDRL1 of SEQ ID NO: 102, CDRL2 of SEQ ID NO: 103, and CDRL3 of SEQ ID NO: 104, a5) CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106, and CDRL3 of SEQ ID NO: 107, and a6) CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, and CDRL3 of SEQ ID NO: 113 The bispecific antibody according to item 1, comprising a light chain variable region comprising a set of CDRLs selected from the group consisting of. (Item 3) The first binding portion is a1) CDRL1 of SEQ ID NO: 90, CDRL2 of SEQ ID NO: 91, and CDRL3 of SEQ ID NO: 92, a2) CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106, and CDRL3 of SEQ ID NO: 107, and a3) CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, and CDRL3 of SEQ ID NO: 113 The bispecific antibody according to item 1, comprising a light chain variable region comprising a set of CDRLs selected from the group consisting of. (Item 4) a) In the first binding portion, the heavy chain variable region VH of SEQ ID NO: 1, b) In the first binding portion, b1) The light chain variable region VL of SEQ ID NO: 31, b2) The light chain variable region VL of SEQ ID NO: 114, b3) The light chain variable region VL of SEQ ID NO: 115, b4) The light chain variable region VL of SEQ ID NO: 116, b5) The light chain variable region VL of SEQ ID NO: 117, b6) The light chain variable region VL of SEQ ID NO: 118, b7) The light chain variable region VL of SEQ ID NO: 119, b8) The light chain variable region VL of SEQ ID NO: 120, b9) The light chain variable region VL of SEQ ID NO: 121, b10) The light chain variable region VL of SEQ ID NO: 122, b11) The light chain variable region VL of SEQ ID NO: 123, and b12) The light chain variable region VL of SEQ ID NO: 124 A light chain variable region VL selected from the group consisting of, and c) In the second binding portion, the heavy chain variable region VH of SEQ ID NO: 1 and the light chain variable region VL of SEQ ID NO: 17 The bispecific antibody according to item 1, comprising. (Item 5) a) In the first binding portion, the heavy chain variable region VH of SEQ ID NO: 1, b) In the first binding portion, b1) The light chain variable region VL of SEQ ID NO: 117, b2) The light chain variable region VL of SEQ ID NO: 119, b3) The light chain variable region VL of SEQ ID NO: 120, b4) The light chain variable region VL of SEQ ID NO: 121, b5) The light chain variable region VL of SEQ ID NO: 122, and b6) The light chain variable region VL of SEQ ID NO: 124 a variable light chain VL selected from the group consisting of, and c) in the second binding portion, a heavy chain variable region VH of SEQ ID NO: 1 and a light chain variable region VL of SEQ ID NO: 17 The bispecific antibody according to item 2, comprising. (Item 6) a) in the first binding portion, a heavy chain variable region VH of SEQ ID NO: 1, b) in the first binding portion, b1) a light chain variable region VL of SEQ ID NO: 117, b1) a light chain variable region VL of SEQ ID NO: 122, and b4) a light chain variable region VL of SEQ ID NO: 124 a variable light chain VL selected from the group consisting of, and c) in the second binding portion, a heavy chain variable region VH of SEQ ID NO: 1 and a light chain variable region VL of SEQ ID NO: 17 The bispecific antibody according to item 3, comprising. (Item 7) A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, wherein the antibody is a) in the first binding portion, a heavy chain variable region VH of SEQ ID NO: 1, b) in the first binding portion, b1) a light chain of SEQ ID NO: 40, b2) a light chain of SEQ ID NO: 125, b3) a light chain of SEQ ID NO: 126, b4) a light chain of SEQ ID NO: 127, b5) a light chain of SEQ ID NO: 128, b6) a light chain of SEQ ID NO: 129, b7) a light chain of SEQ ID NO: 130, b8) a light chain of SEQ ID NO: 131, b9) a light chain of SEQ ID NO: 132, b10) a light chain of SEQ ID NO: 133, b11) a light chain of SEQ ID NO: 134, and b12) a light chain of SEQ ID NO: 135 a light chain selected from the group consisting of, and, c) in the second binding portion, a bispecific antibody comprising a heavy chain variable region VH of SEQ ID NO: 1 and a light chain of SEQ ID NO: 28. (Item 8) a) a heavy chain of SEQ ID NO: 43, b) a heavy chain of SEQ ID NO: 44, and c) a heavy chain of SEQ ID NO: 45 The bispecific antibody according to any one of items 1 to 7, characterized by comprising a common heavy chain selected from the group consisting of. (Item 9) A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, wherein the antibody comprises a common heavy chain of SEQ ID NO: 45, a light chain of SEQ ID NO: 28 in the second binding portion, and a light chain of SEQ ID NO: 128 in the first binding portion. (Item 10) A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, wherein the antibody comprises a common heavy chain of SEQ ID NO: 45, and in the second binding portion, a light chain of SEQ ID NO: 28, and in the first binding portion, a light chain of SEQ ID NO: 133. (Item 11) A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, wherein the antibody comprises a common heavy chain of SEQ ID NO: 45, and in the second binding portion, a light chain of SEQ ID NO: 28, and in the first binding portion, a light chain of SEQ ID NO: 135. (Item 12) A bispecific antibody for use in the treatment of cancer, 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 a) binds to MKN-45 cells with an EC50 value of 0.5 nM to 50 nM; b) kills MKN-45, HPAF-II or LS174T cells in an assay containing human PBMCs in a concentration-dependent manner with an EC50 value of 0.01 to 10 nM; c) binds to PEAK cells expressing CEACAM5 but does not cross-react with PEAK cells expressing CEACAM8; d) the EC50 value for killing in a TDCC assay using LS174T tumor cells as target cells does not increase by more than 5-fold in the presence of 1 μg / mL of sCEA. (Item 13) The antibody is a) inhibits tumor growth in the HPAF-II model by 25% or more compared to the vehicle control group; b) competes with an anti-CEA antibody comprising VL and VH domains of SEQ ID NOs: 48 and 49, respectively (MEDI); A bispecific antibody for use according to item 12. (Item 14) A bispecific antibody for use according to item 12 or 13, wherein each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329A (Kabat EU indexing). (Item 15) A bispecific antibody according to any one of items 1 to 14 for use in the treatment of colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer and / or breast cancer. (Item 16) The bispecific antibody according to any one of items 1 to 14 for use in treating cancer in vivo. (Item 17) The bispecific antibody according to any one of items 1 to 14 for use in administering a pharmaceutically effective amount of a composition containing the bispecific antibody of the present invention to a human subject suffering from cancer. (Item 18) The bispecific antibody according to any one of items 1 to 14 for use in the treatment of colorectal cancer, esophageal cancer, pancreatic adenocarcinoma, gastric cancer, non-small cell lung cancer, breast cancer, head and neck cancer, uterine cancer and bladder cancer. (Item 19) The bispecific antibody according to any one of items 1 to 14 for use in monotherapy for the treatment of solid tumors expressing CEA. (Item 20) The bispecific antibody according to any one of items 1 to 14 for use in the treatment of cancer in combination with a bispecific anti-CEA×CD47 antibody, either simultaneously, separately or sequentially. (Item 21) The bispecific antibody according to any one of items 1 to 14 for use in the treatment of cancer in combination with a bispecific anti-CEA×CD47 antibody and / or a PD-1 axis antagonist, either simultaneously, separately or sequentially. (Item 22) The bispecific antibody according to any one of items 1 to 14 for use in the treatment of cancer in combination with a PD-1 axis antagonist, either simultaneously, separately or sequentially. (Item 23) The bispecific antibody and the bispecific anti-CEA×CD47 antibody according to any one of items 1 to 14 for use in administration in an alternating administration with an interval of 6 to 15 days between the administration of the antibody of the present invention and the bispecific anti-CEA×CD47 antibody in the treatment of cancer. (Item 24) The bispecific antibody for use according to any one of items 15 to 23, wherein the antibody of the present invention is administered to a patient at a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, either as a single dose, divided doses or by continuous infusion. (Item 25) The bispecific antibody for use according to item 23, wherein the antibody of the present invention is administered to a patient at a dose in the range of 1 to 20 mg / kg. (Item 26) The bispecific antibody for use according to either item 20 or 21, wherein the anti-CEA×CD47 antibody is administered to a patient at a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, either as a single dose, divided doses or by continuous infusion. (Item 27) The bispecific antibody for use according to item 22, wherein the PD-1 axis antagonist is administered to a patient at a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, either as a single dose or in divided doses or by continuous infusion. (Item 28) A pharmaceutical composition comprising the bispecific antibody according to any one of items 1 to 14 and a pharmaceutically acceptable carrier. (Item 29) A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, a) wherein the first binding portion and the second binding portion each comprise a common heavy chain comprising, as a heavy chain, a variable region comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4, b) the first binding portion i) a lambda light chain constant region, and ii) a light chain variable region comprising CDRL1 obtained from SEQ ID NO: 32 by oligonucleotide-specific mutagenesis using degenerate oligonucleotides, CDRL2 obtained from SEQ ID NO: 33 by oligonucleotide-specific mutagenesis using degenerate oligonucleotides, and CDRL3 obtained from SEQ ID NO: 34 by oligonucleotide-specific mutagenesis using degenerate oligonucleotides comprising, c) the second binding portion comprises 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, d) the second binding portion comprises a light chain constant region of SEQ ID NO: 41, the bispecific antibody. (Item 30) The bispecific antibody according to item 29, wherein the second binding portion in c) comprises a light chain selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, and 29. (Item 31) A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, a) in the first binding portion, having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 1 and comprising a heavy chain variable region VH comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 3, and CDR3 of SEQ ID NO: 4, b) b1) having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 31 and comprising a light chain variable region VL comprising CDRL1 of SEQ ID NO: 32, CDRL2 of SEQ ID NO: 33, and CDRL3 of SEQ ID NO: 34, b2) a variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 114 and comprising CDRL1 of SEQ ID NO: 81, CDRL2 of SEQ ID NO: 82 and CDRL3 of SEQ ID NO: 83, b3) a variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 115 and having CDRL1 of SEQ ID NO: 84, CDRL2 of SEQ ID NO: 85 and CDRL3 of SEQ ID NO: 86, b4) a variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 116 and having CDRL1 of SEQ ID NO: 87, CDRL2 of SEQ ID NO: 88 and CDRL3 of SEQ ID NO: 89, b5) a variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 117 and having CDRL1 of SEQ ID NO: 90, CDRL2 of SEQ ID NO: 91 and CDRL3 of SEQ ID NO: 92, b6) a variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 118 and having CDRL1 of SEQ ID NO: 93, CDRL2 of SEQ ID NO: 94 and CDRL3 of SEQ ID NO: 95, b7) a variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 119 and having CDRL1 of SEQ ID NO: 96, CDRL2 of SEQ ID NO: 97 and CDRL3 of SEQ ID NO: 98, b8) a variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 120 and having CDRL1 of SEQ ID NO: 99, CDRL2 of SEQ ID NO: 100 and CDRL3 of SEQ ID NO: 101, b9) a variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 121 and having CDRL1 of SEQ ID NO: 102, CDRL2 of SEQ ID NO: 103 and CDRL3 of SEQ ID NO: 104, b10) a variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 122 and having CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106 and CDRL3 of SEQ ID NO: 107, b11) a variable light chain region VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 123 and having CDRL1 of SEQ ID NO: 108, CDRL2 of SEQ ID NO: 109 and CDRL3 of SEQ ID NO: 110, and b12) A variable light chain VL selected from the group consisting of having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 124, and having CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112 and CDRL3 of SEQ ID NO: 113 A variable light chain VL selected from the group consisting of c) In the second binding portion, a variable heavy chain VH having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 1 and containing CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 3 and CDR3 of SEQ ID NO: 4, and a variable light chain VL having 97%, 98%, 99% or 100% amino acid identity with SEQ ID NO: 17 and containing CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19 and CDRL3 of SEQ ID NO: 20 A bispecific antibody comprising (Item 32) The bispecific antibody according to any one of Items 29 to 31, wherein the common heavy chain is the one of SEQ ID NO: 43 (Item 33) The bispecific antibody according to any one of Items 29 to 31, wherein the common heavy chain is the one of SEQ ID NO: 44 (Item 34) The bispecific antibody according to any one of Items 29 to 31, wherein the common heavy chain is the one of SEQ ID NO: 45 (Item 35) A method for treating cancer, comprising administering to a subject in need thereof an effective amount of the bispecific antibody according to any one of Items 1 to 11 or 29 to 34, or the pharmaceutical composition according to Item 28 (Item 36) The method according to Item 35, wherein the bispecific antibody binds to MKN-45 cells with an EC50 value of 0.5 nM to 50 nM (Item 37) The method according to Item 35 or 36, wherein the bispecific antibody kills MKN-45, HPAF-II or LS174T cells in an assay containing human PBMCs in a concentration-dependent manner with an EC50 value of 0.01 to 10 nM (Item 38) The method according to any one of Items 35 to 37, wherein the bispecific antibody binds to PEAK cells expressing CEACAM5 but does not cross-react with PEAK cells expressing CEACAM8 (Item 39) The EC50 value of the bispecific antibody for killing in a TDCC assay using LS174T tumor cells as target cells does not increase by more than 5-fold in the presence of 1 μg / mL sCEA compared to the EC50 in the absence of sCEA. The method according to any one of Items 35 to 38 (Item 40) The method according to any one of items 35 to 39, wherein the bispecific antibody inhibits tumor growth in the HPAF-II model by 25% or more as compared to the vehicle control group. (Item 41) The method according to any one of items 35 to 40, wherein the bispecific antibody competes with an anti-CEA antibody comprising VL and VH of the sequences of SEQ ID NOs: 48 and 49 as VL and VH. (Item 42) The method according to any one of items 35 to 41, wherein the bispecific antibody is numbered according to the Kabat EU indexing and comprises amino acid substitutions L234A, L235A and P329A in each subunit of the Fc domain. (Item 43) The method according to any one of items 35 to 42, wherein the subject is human. (Item 44) The method according to any one of items 35 to 43, wherein the subject has colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer or breast cancer. (Item 45) The method according to any one of items 35 to 43, wherein the subject has cancer. (Item 46) The method according to any one of items 35 to 43, wherein the subject has colorectal cancer, esophageal cancer, pancreatic adenocarcinoma, gastric cancer, non-small cell lung cancer, breast cancer, head and neck cancer, uterine cancer or bladder cancer. (Item 47) The method according to any one of items 35 to 46, wherein the bispecific antibody is administered as a single agent therapy. (Item 48) The method according to item 47, wherein the bispecific antibody is administered for the treatment of solid tumors expressing CEA. (Item 49) The method according to any one of items 35 to 46, wherein the bispecific antibody is administered in combination with a bispecific anti-CEA x CD47 antibody simultaneously, separately or in a sequential combination. (Item 50) The method according to item 49, wherein the bispecific antibody is administered in an alternating manner with the bispecific anti-CEA x CD47 antibody, and the interval between the administrations of the bispecific antibody of the present invention and the bispecific anti-CEA x CD47 antibody is 6 to 15 days. (Item 51) The method according to any one of items 35 to 46, 49 or 50, wherein the bispecific antibody is administered in combination with a PD-1 axis antagonist simultaneously, separately or in a sequential combination. (Item 52) The method according to any one of items 35 to 51, wherein the bispecific antibody is administered at a dose of 0.1 to 100 mg / kg body weight per day or per week. (Item 53) The method according to any one of items 35 to 52, wherein the bispecific antibody is administered at a dose of 1 to 20 mg / kg. (Item 54) The method according to item 52 or 53, wherein the bispecific antibody is administered in a divided dose. (Item 55) The method according to item 52 or 53, wherein the bispecific antibody is administered by continuous infusion. (Item 56) The method according to any one of items 49 to 55, wherein the anti-CEA×CD47 bispecific antibody is administered to a patient at a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, either as a single dose, a divided dose, or by continuous infusion. (Item 57) The method according to any one of items 51 to 56, wherein the PD-1 axis antagonist is administered to a patient at a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, either as a single dose, a divided dose, or by continuous infusion. (Item 58) An isolated polynucleotide encoding the antibody or a binding portion thereof according to any one of items 1 to 34. (Item 59) A vector comprising the isolated polynucleotide according to item 58. (Item 60) A cell comprising the isolated polynucleotide according to item 58 or the vector according to item 59. (Item 61) The cell according to item 60, which is selected from the group consisting of Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture. (Item 62) A method for producing a bispecific antibody, comprising expressing the bispecific antibody in the cell according to item 60 or 61. (Item 63) A method for producing a bispecific antibody, comprising culturing the cell according to item 60 or 61 and isolating the antibody expressed therein.

Brief Description of the Drawings

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Figure 1

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Figure 7

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Figure 8

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Figure 9

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Figure 11-2

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Figure 12-1

Figure 12-2

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Figure 13-1

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Figure 14-1

Figure 14-2

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Figure 15-1

Figure 15-2

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Figure 16-1

Figure 16-2

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Figure 17-1

[0157] Despite statistically indistinguishable tumor cell lysis at 100 nM, the lower T cell activation of the antibodies of the present invention compared to TCB2014 suggests lower side effects at the same tumor lysis. Method described in Example 8c.

Figure 17-2

[0158]

Figure 18

Modes for Carrying Out the Invention

[0159] Definitions Unless otherwise defined, the terms are used herein as generally used in the art as follows.

[0160] As used herein, the term "antigen-binding portion, binding portion" refers, in its broadest sense, to the portion of an antibody that specifically binds to an antigenic determinant such as CEA, CD47, and CD3.

[0161] More specifically, as used herein, the binding moiety that binds to membrane-bound human carcinoembryonic antigen (CEA, same as CEACAM5) or to CD3 binds specifically to CEA or CD3, and more particularly, to cell surface or membrane-bound CEA or CD3. "Binds specifically to, 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, for example, about 10-fold, preferably >100-fold lower than the binding of the antibody to said target, as measured by surface plasmon resonance (SPR), for example, Biacore®, enzyme-linked immunosorbent assay (ELISA) or flow cytometry (FACS). The target is a protein discussed herein, for example, CEA, CD47 and CD3ε.

[0162] "Specifically binds to CEA, CD3, binds to CEA, CD3" refers, in one embodiment, to an antibody that can bind to target CEA and to CD3, respectively, with sufficient affinity such that the antibody is useful as a therapeutic agent in the retargeting of T cells to tumor cells via binding of CD3 and of CEA, respectively.

[0163] Preferably, the bispecific antibodies according to the invention bind to epitopes of CD3 that are conserved between different species, preferably between human and cynomolgus monkey.

[0164] As used herein, the term "antibody" refers to an antibody comprising two heavy chains and two light chains. In one embodiment, the antibody is a full-length antibody. As used herein, the term "antibody heavy chain" refers to an antibody heavy chain consisting of a variable region (variable domain) and a constant region (constant domain), as defined for a full-length antibody. As used herein, the term "antibody light chain" refers to an antibody light chain consisting of a variable region and a constant region, as defined for a full-length antibody. Constant light chains useful for the purposes of the present invention are included in the light chains disclosed in the present invention.

[0165] 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, in the direction from the N-terminus to the C-terminus, an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), and is abbreviated as VH-CH1-HR-CH2-CH3. A "full-length antibody light chain" is a polypeptide consisting of, in the direction from the N-terminus to the C-terminus, an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), and is abbreviated as VL-CL. The antibody light chain constant domain (CL) can be κ (kappa) or λ (lambda). The two full-length antibody domains are linked together via polypeptide interchain disulfide bonds between the CL domain and the CH1 domain, and between the hinge regions of the full-length antibody heavy chains. 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 antibodies according to the present invention are, in one embodiment, of the human IgG1 type, and in a further embodiment, contain one or more amino acid substitutions in the Fc portion defined below. The full-length antibodies according to the present invention contain two binding portions formed by pairs of VH and VL, one of which binds to CEA and the other to CD3.

[0166] As used herein, the term "Fc region; Fc domain" refers to the C-terminal region of the IgG heavy chain, and in the case of an IgG1 antibody, the C-terminal region includes -CH2-CH3 (see above). The boundaries of the Fc region of the IgG heavy chain can vary very slightly, but the human IgG heavy chain Fc region is usually defined as extending from the amino acid residue at the position of Cys226 to the carboxyl terminus.

[0167] The constant region is well-known in the current state of the art and is described, for example, by Kabat, E.A. (see, for example, Johnson, G., and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788).

[0168] The term "epitope" includes any polypeptide determinant that can bind specifically to an antibody. In certain embodiments, the epitope determinant includes chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features. An epitope is the region of a target that is bound by an antibody.

[0169] As used herein, the term "common heavy chain (cHC)" refers to a polypeptide consisting of, in the direction from the N-terminus to the C-terminus, 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), and is abbreviated as VH-CH1-HR-CH2-CH3. Suitable common heavy chains for bispecific antibodies according to the present invention are the heavy chains described in WO 2012023053, WO 2013088259, WO 2014087248, WO 2019175658, and WO 2016156537 (the entire contents of each of which are incorporated herein by reference). In one embodiment, the cHC of a bispecific antibody according to the present invention includes, as heavy chain CDRs, CDRL1 of SEQ ID NO: 2, CDRL2 of SEQ ID NO: 3, and CDRL3 of SEQ ID NO: 4. In one embodiment, the cHC of a bispecific antibody according to the present invention includes, as the heavy chain variable region, the VH region of SEQ ID NO: 1. In one embodiment, the cHC of a bispecific antibody according to the present invention is one of SEQ ID NO: 43, 44, or 45.

[0170] The format of the bispecific antibody comprising a common heavy chain according to the invention allows for affinity purification of the bispecific antibody (see, for example, WO 2013088259, WO 2012023053), which has characteristics that cannot be distinguished from a standard IgG molecule and cannot be distinguished from a standard monoclonal antibody, and is expected to have no or low immunogenic potential in patients.

[0171] As used herein, "AB1L3-1, AB17L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1, AB73L3-1, etc." refers to a bispecific CEAxCD3 antibody according to the invention, which comprises a common heavy chain comprising the CDRs of SEQ ID NOs: 2, 3 and 4 as heavy chain CDRs, and a light chain comprising the CDRs of SEQ ID NOs: 18, 19 and 20 as light chain CDRs in the second binding moiety. AB1, etc., thus represents the first binding moiety (anti-CEACAM5 binding moiety), and L3-1 represents the second binding moiety (anti-CD3 binding moiety).

[0172] In one embodiment, AB1L3-1, AB17L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1, AB73L3-1, etc. comprises the common heavy chain of SEQ ID NO: 43 (WT hIgG1), and as the light chain, the light chain of SEQ ID NO: 28 in the second binding moiety. Such bispecific antibodies of the invention are also designated as AB1L3-1, AB17L3-1, AB71L3-1, AB72L3-1, AB73L3-1 in the examples.

[0173] In one embodiment, AB1L3-1, AB17L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1, AB73L3-1, etc. include the common heavy chain of SEQ ID NO: 44 (hIgG1 having L234A+L235A mutations), and in the second binding portion, as the light chain, include the light chain of SEQ ID NO: 28. Such bispecific antibodies of the present invention are designated as AB1L3-1 / D, AB17L3-1 / D, AB71L3-1 / D, AB72L3-1 / D, AB73L3-1 / D, etc. in the examples.

[0174] In one embodiment, AB1L3-1, AB17L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1, AB73L3-1, etc. include the common heavy chain of SEQ ID NO: 45 (IgG1 having L234A+L235A+P329A mutations), and in the second binding portion, as the light chain, include the light chain of SEQ ID NO: 28. Such bispecific antibodies of the present invention are designated as AB1L3-1 / N, AB17L3-1 / N, AB54L3-1 / N, AB60L3-1 / N, AB66L3-1 / N, AB71L3-1 / N, AB72L3-1 / N, AB73L3-1 / N, etc. in the examples.

[0175] The bispecific antibodies of the present invention containing the common heavy chain can be prepared, for example, according to International Publication No. WO 2012 / 023053. The method described in International Publication No. WO 2012 / 023053 generates bispecific antibodies having the same structure as human immunoglobulins. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a kappa constant domain, and a second light chain variable region fused to a lambda constant domain.

[0176] In the bispecific antibody of the present invention, one binding site exhibits specificity for CEA, and the other site exhibits specificity for CD3, where for each, a heavy chain and each light chain contribute. The light chain variable region can be of the lambda or kappa family and is preferably fused to the lambda and kappa constant domains, respectively. This is preferred to avoid the occurrence of non-natural polypeptide binding. However, for either of the two specificities, an antibody arm that can be used for the production of the bispecific antibody of the present invention can also be obtained by fusing the kappa light chain variable domain to the lambda constant domain or, for either of the two specificities, by fusing the lambda light chain variable domain to the kappa constant domain. The bispecific antibody described in International Publication No. WO 2012 / 023053 is a "κλ-body". This κλ-body format enables affinity purification of bispecific antibodies that are indistinguishable from standard IgG molecules having characteristics indistinguishable from standard monoclonal antibodies and is thus preferred compared to previous formats that include, for example, amino acid cross-linking or other non-natural elements.

[0177] An essential step of the method is the identification of two antibody Fv regions having different antigen specificities that share the same heavy chain variable domain (each composed of a variable light domain and a variable heavy domain, respectively). Numerous methods for the production of monoclonal antibodies and their fragments have been described (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). A fully human antibody is an antibody molecule in which the sequences of both the light chain and the heavy chain, including CDR1 and 2, are derived from human genes. The CDR3 region can be of human origin or designed by synthetic means. Such antibodies are referred to as "human antibodies" or "fully human antibodies". Human monoclonal antibodies can be prepared by using the trioma technique; the human B cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique for producing human monoclonal antibodies (see Cole, et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be utilized and produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (see Cole, et al., supra).

[0178] The term "CD3ε or CD3", as used herein, relates to human CD3ε as described in UniProt P07766 (CD3E_HUMAN). The term "antibody to CD3, anti-CD3 antibody" relates to an antibody that binds to CD3ε.

[0179] As used herein, the term "CEA, CEACAM5" refers to the 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 "CEACAM6" refers to human CEACAM6 (CD66c; UniProtKB-P40199), which is also a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. As used herein, the term "CEACAM1" refers to human CEACAM1 (UniProtKB-P13688(CEAM1_HUMAN)), which is also a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. As used herein, the term "CEACAM8" refers to human CEACAM8 (UniProtKB-P31997(CEAM8_HUMAN)), which is also a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Further information and information on other members of the CEA family can be found at http: / / www.uniprot.org.

[0180] As used herein, the term "CEA-specific binding, CEA-binding, CEA-binding moiety" refers to specificity for CEACAM5 on the surface of cells in the context of the bispecific antibodies according to the invention. Binding to CEA in cells can be measured using gastric adenocarcinoma MKN-45 cells containing 100,000 to 400,000 copies of CEA per cell. The concentration of the antibodies according to the invention varies within an appropriate range with respect to the EC50 value obtained for binding to MKN-45 cells as defined above. The bispecific antibodies according to the invention specifically bind to such cell membrane-bound CEACAM5.

[0181] As used herein, the term "membrane-bound human CEA" refers to human carcinoembryonic antigen (CEA) that binds to the membrane portion of a cell or to the surface of a cell, particularly to the surface of a tumor cell. In certain circumstances, the term "membrane-bound human CEA" may refer to CEA that is constructed to preserve a membrane-bound CEA epitope that does not bind to the cell membrane but to which an antibody according to the invention binds.

[0182] As used herein, the term "no cross-reactivity with CEACAM8" refers, in the context of the bispecific antibodies according to the invention, to the binding of the bispecific antibodies according to the invention being tested in PEAK cells expressing CEACAM8 in comparison to binding to WT PEAK cells (see Examples 1 and 5 for details), and no cross-reactivity means that the MFI measured for PEAK cells expressing CEACAM8 is 2-fold or less than the MFI measured for WT PEAK cells. As used herein, the term "no cross-reactivity with a particular CEACAM" refers, in the context of the bispecific antibodies according to the invention, to said cross-reactivity under the same experimental procedures and definitions described for CEACAM8.

[0183] As used herein, the term "bispecific antibody that binds to human CEA and human CD3, CEAxCD3 bsAb" means a bispecific antibody that binds to human CEACAM5 and CD3ε.

[0184] As used herein, the term "complementary determining region" ("CDR") describes the non - contiguous antigen - binding (also known as antigen - binding region) sites found within the variable regions of both heavy and light chain polypeptides. CDRs are also referred to as "hypervariable regions", and the term is used interchangeably with the term "CDR" herein with respect to the portions of the variable regions that form the antigen - binding regions. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, ”Sequences of Proteins of Immunological Interest” (1983), and by Chothia et al., J. Mol. Biol. 196:901 - 917 (1987). Kabat et al. also defined a numbering system for variable domain sequences applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, ”Sequence of Proteins of Immunological Interest” (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in bispecific antibodies (e.g., CDR sequences) according to the present invention follow the Kabat numbering system.

[0185] As used herein, the term "oligonucleotide - specific mutagenesis" relates to such methods that use degenerate oligonucleotides. Combinations of various degenerate oligonucleotides are used for mutagenesis of each CDR. These include (but are not limited to) the degenerate codons NNS, HMT, DMT, NHT.

[0186] As used herein, the term "expression vector" refers to one or more vectors containing the heavy and light chains of an antibody according to the present invention, in a suitable manner known from the state of the art. As used herein, the term "host cell" encompasses any type of cell line that can be engineered to produce a bispecific antibody of the present invention. In one embodiment, the host cell is engineered to enable the production of antigen-binding molecules.

[0187] As used herein, the term "amino acid substitution" refers to the substitution of one amino acid by another amino acid from the group of 20 proteinogenic standard amino acids. Therapeutic applications and methods using the anti-CEAxCD3 antibodies according to the present invention

[0188] The CEACAMxCD3 bispecific antibodies according to the present invention are optimized for the treatment of solid tumors, either in monotherapy or in combination therapy, in particular in combination therapy with anti-CD47 antibodies, anti-CEAxCD47 antibodies and / or PD-1 axis antagonists. The antibodies according to the present invention and the CD47 antibodies or CEAxCD47 antibodies can be administered as described below.

[0189] In certain embodiments, each solid tumor of the disease is a cancer that expresses or further overexpresses CEA, including, but not limited to, the group of colorectal tumors, non-small cell lung tumors, gastric tumors, esophageal cancer, pancreatic tumors, and breast tumors. In certain embodiments, the tumor is a colorectal tumor. All therapeutic applications, methods of use, uses, combinations, etc. described herein are, in particular, embodiments for the treatment of these tumors / diseases.

[0190] The inventors recognize that the antibodies according to the present invention show that they have a low or no potential to form ADA that causes loss of exposure by neutralizing ADA that causes loss of efficacy, respectively.

[0191] In one embodiment, the present invention provides a method for treating cancer (carcinoma, tumor, e.g., human cancer), particularly tumors expressing CEA, in vivo. This method comprises administering to a subject a pharmaceutically effective amount of a composition containing the bispecific antibody of the present invention. "Subject" means a human subject, and in one embodiment, a patient suffering from cancer / tumor / carcinoma.

[0192] CEA expression in various tumor entities, particularly in colorectal cancer, esophageal cancer, pancreatic adenocarcinoma, gastric cancer, non-small cell lung cancer, breast cancer, head and neck cancer, uterine cancer, and bladder cancer, etc., is generally very high. In healthy normal glandular epithelium in the digestive tract, CEA is mainly expressed in a polarized pattern at the apical surface of cells. This polarized expression pattern limits the accessibility, and thus the potential toxicity, to systemically administered anti-CEA monospecific or bispecific antibodies. This polarized expression pattern is lost in the cells of malignant tumors of the digestive tract. CEA is expressed equally throughout the cell surface of cancer cells, which means that cancer cells are much more accessible to the antibodies of the present invention than normal healthy cells and can be selectively killed by the respective CEAxCD3 bispecific antibodies of the present invention by the combinations mentioned above.

[0193] In one embodiment, the bispecific antibody of the present invention can be used in monotherapy for the treatment of progressive solid tumors, in one embodiment, tumors expressing CEA. In one embodiment, the bispecific antibody according to the present invention is used in combination with CEAxCD47 bsAb, simultaneously, separately or sequentially. In one embodiment, the bispecific antibody according to the present invention is used in combination with CEAxCD47 bsAb and / or a PD-1 axis antagonist, simultaneously, separately or sequentially. In one embodiment, the bispecific antibody according to the present invention is used in combination with a PD-1 axis antagonist, simultaneously, separately or sequentially. Such PD-1 axis antagonists are described, for example, in WO 2017118675. Such combinations attack solid cancer by macrophages and T cells. CD47 antibodies are described, for example, in WO 2009091601, WO 2009091547, WO 2011143624, WO 2009131453, WO 2013119714, WO 2015105995, WO 2017181033, WO 2018026600, WO 2019157432 and WO 2013032948, and bispecific antibodies against CEA and CD47 are described in International Application No. PCT / IB2019 / 054559 and US Application No. 16 / 428,539.

[0194] As used herein, the term "combination, simultaneous, separate or sequential combination" of an antibody according to the invention, and a second antibody that binds to human CD47 or human CEA and human CD47, refers to any administration, either separately or together, of two antibodies (or, in the case of a combination of an antibody of the invention, a CD47 mAb or a CEAxCD47 bsAb and a PD-1 axis antagonist, three antibodies), where the two or three antibodies are administered as part of a suitable dosing regimen designed to obtain the benefits of combination therapy, for example, in separate, sequential, simultaneous, parallel, time-shifted or alternating administrations. Thus, the two or three antibodies can be administered either as part of the same pharmaceutical composition or in separate pharmaceutical compositions. The antibody according to the invention can be administered before, simultaneously with, or after the administration of the second bispecific antibody, or in a combination of some of them. If the antibody according to the invention is administered to a patient, for example, at repeated intervals during the standard course of treatment, the second bispecific antibody can be administered before, simultaneously with, or after each administration of the antibody of the invention, or in a combination of some of them, or at different intervals in relation to the treatment with the antibody of the invention, or as a single administration before, at any time during, or after the course of treatment with the antibody of the invention. In one embodiment, the antibody according to the invention and the second bispecific antibody are administered in alternating administrations at intervals of 6 to 15 days between the administrations of the antibody of the invention and the second antibody. In such alternating administrations, the first administration can be the antibody of the invention or the second antibody.

[0195] The term "PD-1 axis antagonist" refers to an anti-PD-1 antibody or an anti-PD-L1 antibody. Anti-PD-1 antibodies include, for example, pembrolizumab (Keytruda®, MK-3475), nivolumab, pidilizumab, lambrolizumab, MEDI-0680, PDR001, and REGN2810. Anti-PD-1 antibodies are described, for example, in WO 2008 / 15671, WO 2013 / 173223, WO 2015 / 026634, US Patent No. 7,521,051, US Patent No. 8,008,449, US Patent No. 8,354,509, WO 2009 / 14335, WO 2015 / 026634, WO 2008 / 156712, WO 2015 / 026634, WO 2003 / 099196, WO 2009 / 101611, WO 2010 / 027423, WO 2010 / 027827, WO 2010 / 027828, WO 2008 / 156712, and WO 2008 / 156712. Anti-PD-L1 antibodies include, for example, atezolizumab, MDX-1105, durvalumab, and avelumab. Anti-PD-L1 antibodies are described, for example, in WO 2015 / 026634, WO 2013 / 019906, WO 2010 / 077634, US Patent No. 8,383,796, WO 2010 / 077634, WO 2007 / 005874, and WO 2016 / 007235.

[0196] Regarding the combined administration of the antibody according to the present invention and the second bispecific antibody, both compounds can be present in one single dosage form or in separate dosage forms, for example, in two different or identical dosage forms.

[0197] If the antibody of the present invention and the second antibody do not compete with respect to CEACAM5, in one embodiment, both antibodies are administered simultaneously. If the antibody of the present invention and the second antibody compete with respect to CEACAM5, in one embodiment, the antibodies are administered in an alternating fashion.

[0198] The antibodies of the present invention are typically administered to a patient in a dosing regimen that provides the most effective treatment (from both the perspective of efficacy and safety) of the cancer for which the patient is being treated, as is known in the art. To achieve the full therapeutic potential of this approach, preferably, the tumor cells are simultaneously attacked by T cells and macrophages, and the CEAxCD3 and CEAxCD47 bispecific antibodies must be non-competing with respect to binding to CEA on the cell surface.

[0199] As discussed above, the amount of antibody administered and the timing of administration of the antibodies of the present invention can depend on the type of patient being treated (e.g., gender, age, weight) and condition, the severity of the disease or condition being treated, and the route of administration. For example, the antibodies of the present invention and the second antibody can be administered to a patient in a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, in a single or divided dose or by continuous infusion. In one embodiment, each of the antibodies of the present invention and the second antibody is administered to a patient in a dose in the range of 1 to 20 mg / kg. In some cases, dosage levels below the lower limit of the foregoing range may be appropriate, while in other cases, even higher doses can be used without causing any adverse side effects.

[0200] As used herein, the term "antibody half-life" refers to the half-life of the antibody as measured in a conventional pharmacokinetic assay. The antibodies according to the present invention and the second bispecific antibody against CEA and CD47 have an elimination half-life of 3 to 14 days.

[0201] In another aspect, the invention also encompasses the use of the bispecific antibodies according to the invention in the treatment of a disease, in particular a cell proliferation disorder in which CEA is expressed, in particular abnormally expressed (e.g., overexpressed or expressed in a different pattern on the cell surface) compared to normal tissue of the same cell type, such as, but not limited to, colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, esophageal cancer, pancreatic cancer, and breast cancer. The expression level of CEA can be determined by methods known in the art (e.g., via immunohistochemical assays, immunofluorescence assays, immunoenzymatic assays, ELISA, flow cytometry, radioimmunoassays, etc.).

[0202] In one aspect, the bispecific antibodies of the invention can be used to target cells in vivo or in vitro that express CEA. The bispecific antibodies of the invention are particularly useful in the prevention of tumor formation, eradication of tumors, and inhibition of tumor growth or metastasis through the induction of TDCC of tumor cells. The bispecific antibodies of the invention can be used to treat any tumor that expresses CEA. Specific malignant tumors that can be treated with the bispecific antibodies of the invention include, but are not limited to, colorectal cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, pancreatic cancer, and breast cancer.

[0203] The bispecific antibodies of the invention can be administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form including, but not limited to, those discussed below, such as by intravenous bolus, or by sustained infusion over a period of time, intramuscularly, intraperitoneally, intrathecal, subcutaneously, intra-articularly, intrasynovially, intramedullary, orally, topically, or by inhalation route. The bispecific antibodies of the invention are also preferably administered by routes within the tumor, around the tumor, within the lesion, or around the lesion to exert local and systemic therapeutic effects.

[0204] Regarding the treatment of diseases, the appropriate dosage of the bispecific antibodies of the present invention depends on the type of disease being treated, the severity and course of the disease, previous treatments, the patient's medical history and response to the antibody, as well as the discretion of the attending physician. The bispecific antibodies of the present invention are preferably administered to a patient once or over a series of treatments. The present invention provides a method for selectively killing tumor cells expressing CEA.

[0205] This method involves the interaction between the bispecific antibody of the present invention and the tumor cells. These tumor cells can be derived from human cancers including colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, pancreatic cancer, and breast cancer.

[0206] In another aspect, the present invention is directed to the bispecific antibodies of the present invention for use in the manufacture of a medicament for treating diseases associated with abnormal CEA expression. In certain embodiments, the disease is a cancer that expresses or further overexpresses CEA, including but not limited to colorectal tumors, non-small cell lung tumors, gastric tumors, esophageal cancer, pancreatic tumors, and breast tumors. In certain embodiments, the tumor is a colorectal tumor. Compositions, formulations, dosages, and routes of administration

[0207] In one aspect, the present invention is directed to a pharmaceutical composition comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier. The present invention is further directed to such a pharmaceutical composition for use in a method of treating a disease such as cancer or for use in the manufacture of a medicament for treating a disease such as cancer. Specifically, the present invention is directed to a method for treating a disease, more particularly for treating cancer, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention.

[0208] In one aspect, the present invention encompasses pharmaceutical compositions, combinations, and methods for treating human cancers and tumors as defined above. For example, the present invention includes a pharmaceutical composition comprising a pharmaceutically effective amount of the antibody of the present invention and a pharmaceutically acceptable carrier for use in the treatment of human cancers.

[0209] The bispecific antibody composition of the present invention can be administered using conventional modes of administration, including, but not limited to, intravenous, intraperitoneal, oral, intralymphatic, or direct intratumoral administration. Intravenous administration or subcutaneous administration is preferred.

[0210] In one aspect of the present invention, a therapeutic formulation containing the bispecific antibody of the present invention is prepared for storage in the form of a lyophilized formulation or a liquid formulation by mixing the antibody having the desired purity with a pharmaceutically acceptable carrier, excipient, or stabilizer as required (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dosages and concentrations used. The formulations used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane. The most effective mode of administration and dosage regimen for the pharmaceutical composition of the present invention depend on the severity and course of the disease, the condition of the patient and the response to the treatment, as well as the judgment of the treating physician. Therefore, the dosage of the composition can be a uniform dosage or can be adapted to the individual patient, for example, body weight. Nevertheless, the effective dosage of the composition of the present invention is generally in the range of 0.1 to 20 mg / kg.

[0211] The bispecific antibodies of the present invention have a molecular weight of 150 kD per mole. In one embodiment, they have an Fc portion. The elimination half-life in patients ranges from 3 to 14 days. This half-life allows for administration once a day, once a week, or once every two weeks, among other possibilities.

[0212] The bispecific antibodies of the present invention and their respective compositions can be in various dosage forms, including, but not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions. The preferred form depends on the mode of administration and the therapeutic application.

[0213] The composition comprising the bispecific antibody of the present invention is formulated, dosed, and administered in a manner compatible with good medical practice. Factors for consideration in this context include the particular disease or disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disease or disorder, the site of drug delivery, the method of administration, the dosing schedule, and other factors known to medical practitioners.

[0214] Product In another aspect of the invention, there is provided a product containing a material useful for the treatment, prevention and / or diagnosis of the disorders described above. The product includes a container, and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container holds the composition, either by itself or in combination with another composition effective for treating, preventing and / or diagnosing the condition, and may have a sterile access port (for example, the container can be an infusion bag or vial with a stopper pierceable by a hypodermic needle). One of the active agents in the composition is the bispecific antibody of the present invention. The label or package insert indicates that the composition is used for treating the selected condition. Also, the product can include (a) a first container in which the composition is contained, the composition comprising the bispecific antibody of the present invention; and (b) a second container in which the composition is contained, the composition comprising a further cytotoxic agent or other therapeutic agent. The product in this embodiment of the invention may further include a package insert indicating that the composition can be used for treating a particular condition. Alternatively, or in addition, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. Table 1 Sequence Listing

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Examples

[0215] (Example 1) Cloning, Expression, and Purification of Human CEACAM Family Members

[0216] Cloning Sequences corresponding to the full extracellular domain (ECD) and A3-B3 domain of CEACAM5 were synthesized and subcloned into the pEAK8 mammalian expression vector (Edge Biosystems, Gaithersburg, Md.). The vector was modified to introduce either an AviTag™ (Avidity, Denver, Colo.) and a hexahistidine tag, a human FC region, or a mouse FC region at the C-terminus. The construct was verified by DNA sequencing. Recombinant soluble proteins were purified by IMAC (immobilized metal ion affinity chromatography), FcXL, or CaptureSelect™ IgG-Fc(ms) Affinity Matrix (Thermo Fisher Scientific).

[0217] Vectors encoding the full-length versions of human CEACAM1, 3, 4, 5, 6, 7, 8, 18, 19, 20, 21, and cynomolgus monkey CEACAM5 and CEACAM6 were also generated for expression on the cell surface of PEAK and / or CHO cells. Soluble full-length human CEACAM16 was similarly cloned.

[0218] In addition, vectors encoding the following truncated versions of human CEACAM5 were also generated for expression on the cell surface of PEAK and / or CHO cells: A1-B1-A2-B2-A3-B3; B1-A2-B2-A3-B3; A2-B2-A3-B3; B2-A3-B3; A3-B3. The B3 subdomain is expressed as a fusion protein to the first 140 aa of the human CD86 protein.

[0219] Expression The plasmids mentioned above were then transfected into mammalian cells using a liposomal transfection reagent such as Lipofectamine 2000 (Thermo Fisher Scientific). The transfection step requires very small amounts of DNA and cells, typically 4×10 5 cells per well and 2 μg of plasmid DNA, and the transfection was performed in 6-well plates. Different mammalian cell lines can be used, but in the examples shown below, transformed human fetal kidney monolayer epithelial cells (PEAK cells) are transfected. These cells stably express the EBNA-1 gene, which further supports the episomal replication process, are semi-adherent, and can grow under standard cell culture conditions (5% CO2; DMEM medium supplemented with 10% fetal bovine serum, 37 °C). After 24 hours, the cells are placed under selective conditions by adding medium containing 0.5 - 2 μg / mL puromycin: cells carrying the episomal vector are resistant to this antibiotic.

[0220] Two to three weeks after transfection, the amplified and selected cells were seeded into disposable CELLine™ bioreactors (Sigma Aldrich) for the production step. CELLine™ is a two-compartment bioreactor that can be used in a standard cell culture incubator. The smaller compartment (15 ml) contains the cells and is separated by a semi-permeable membrane with a 10 kDa cut-off size from the larger (1 liter) compartment containing the medium (Bruce et al 2002, McDonald et al 2005). This system allows diffusion of nutrients, gazes and metabolic waste while retaining the cells and secreted proteins in the smaller compartment. The cultures were maintained for 7–10 days before harvest of the supernatant. Since the medium contains serum, the cells maintain a good viability and several production runs can be performed using the same cells and vessel.

[0221] Purification After recovery, the cell culture supernatant is clarified by centrifugation. Next, 100 mM imidazole is supplemented to the supernatant and loaded onto Ni-NTA affinity chromatography resin (Qiagen). A relatively high concentration of imidazole minimizes the binding of contaminants to the resin. After washing the column, the protein is eluted at a flow rate of 2 mL / min using a 30 mL imidazole gradient (20 - 400 mM imidazole) in an AKTA Prime chromatography system (Cytiva). The elution gradient further improves the purity of the recombinant protein, but can be replaced by a stepwise elution method if a chromatography system is not available. The eluted fractions can be analyzed by SDS-PAGE or ELISA to determine their content in the recombinant protein. The fractions of interest are pooled and desalted in an Amicon® 10 kDa column (Millipore) equilibrated with phosphate-buffered saline or another appropriate buffer. The desalted protein can then be quantified using various techniques, and its purity can be analyzed by SDS-PAGE. The recombinant protein is biotinylated in vitro using biotin ligase (Avidity, Denver Colo.) according to the manufacturer's instructions. After desalting, the biotinylation level is evaluated by a pull-down assay using streptavidin magnetic beads and SDS-PAGE analysis. (Example 2) Phage display selection of CEACAM5 Fv using a human scFv library containing immobilized variable heavy domains

[0222] General procedures for the construction and handling of a human scFv library displayed on M13 bacteriophage are described in Vaughan et al. (Nat. Biotech. 1996, 14:309-314), which is hereby incorporated by reference in its entirety. Libraries for selection and screening encode scFvs that all share the same VH domain and are diversified only in the VL domain. Methods for the generation of a fixed VH library, and their use for the identification and assembly of bispecific antibodies are described in U.S. Patent Application Publication No. 2012 / 0184716 and International Publication No. 2012 / 023053, each of which is hereby incorporated by reference in its entirety. Procedures for identifying scFvs that bind to human CEACAM5 are described below. Protein Selection

[0223] An aliquot of the scFv phage library (10 12Block the Pfu) with PBS containing 3% (w / v) skim milk in a rotary mixer at room temperature for 1 hour. Release the blocked phage on streptavidin magnetic beads (Dynabeads™ M-280) in a rotary mixer at room temperature for 1 hour. Incubate the deselected phage with either 100 nM biotinylated human CEACAM5 or the A3-B3 domain captured on streptavidin magnetic beads in a rotary mixer at room temperature for 2 hours. Capture the beads using a magnetic stand, then wash 5 times with PBS / 0.1% Tween® 20 and 2 times with PBS. Elute the phage with 100 nM TEA in a rotary mixer at room temperature for 30 minutes. Neutralize the eluted phage and beads with 1 M Tris-HCl pH 7.4 and add directly to 10 ml of exponentially growing TG1 cells (an E. coli strain commonly used in phage display), and incubate at 37 °C for 1 hour with gentle shaking (90 rpm). Aliquots of the infected TG1 are serially diluted and the selective output is titered. Spin the remaining infected TG1 at 3800 rpm for 10 minutes, resuspend in 2 ml of 2xTY, and spread on 2xTYAG (2xTY medium containing 100 μg / ml ampicillin and 2% glucose) agar bioassay plates. After an overnight incubation at 30 °C, add 10 ml of 2xTY to the plates, scrape the cells from the surface, and transfer to a 50 ml polypropylene tube. Add a 50% glycerol solution to the cell suspension to obtain a final concentration of 17% glycerol. Aliquots of the selection rounds are stored at -80 °C. Phage rescue

[0224] Add 50 μl of the cell suspension obtained from the previous selection round to 50 ml of 2xTYAG and grow with stirring (240 rpm) at 37 °C until an OD 600 of 0.3 - 0.5 is reached. Then, the culture is 11Superinfect with individual M13K07 helper phage and incubate at 37 °C (90 rpm) for 1 hour. Exchange the medium by centrifuging the cells at 3800 rpm for 10 minutes, removing the medium, and resuspending the pellet in 50 ml of 2xTYAK (2xTY medium containing 100 μg / ml ampicillin; 50 μg / ml kanamycin). Then grow the culture overnight at 30 °C (240 rpm). The next day, use the supernatant containing the phage for the next round of selection. Cell surface selection

[0225] Block the supernatant containing the phage with PBS containing 3% (w / v) skim milk on a rotary mixer at room temperature for 1 hour. Then unselect the blocked phage on MKN-45 CEACAM5 cells that do not express human CEACAM5 KO for 1 hour. Unselected phage was incubated with 2×10 7 MKN-45 cells expressing CEACAM5 (blocked with PBS, 3% BSA, 0.1% NaN3) with gentle stirring at room temperature for 2 hours. Pellet the cells and wash 6 times with PBS. Elute the bound phage with 76 mM citric acid and shake for 10 minutes. After neutralization with 1 M Tris-HCl pH 8, add the cells directly to 10 ml of exponentially growing TG1 and incubate at 37 °C for 1 hour with gentle shaking. Aliquots of infected TG1 are serially diluted and the selection output is titered. Spin the infected TG1 at 3800 rpm for 10 minutes, resuspend in 2 ml of 2xTY medium, and spread on a 2xTYAG agar bioassay plate. After overnight incubation at 30 °C, add 10 ml of 2xTY to the plate, scrape the cells from the surface, and transfer to a 50 ml polypropylene tube. Add 50% glycerol solution to the cell suspension to obtain a final glycerol concentration of 17%. Aliquots of the selection round are stored at -80 °C. (Example 3) Screening for scFv binding / unbinding to soluble CEACAM5, CEACAM6, and CEACAM1 scFv periplasm preparation for binding and functional assays

[0226] Inoculate individual transformed TG1 clones from the selection output into deep-well microtiter plates containing 0.9 ml of 2xTYAG medium (2xTY medium containing 100 μg / ml ampicillin, 0.1% glucose) per well and grow at 37 °C for 5 - 6 hours (240 rpm). Then add 100 μl of 0.2 mM IPTG per well in 2xTY medium to obtain a final concentration of 0.02 mM IPTG. Incubate the plates overnight at 30 °C with shaking at 240 rpm. Centrifuge the deep-well plates at 3200 rpm for 10 minutes at 4 °C and carefully remove the supernatant. Resuspend the pellet in 150 μl of TES buffer (50 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8), 20% sucrose, supplemented with complete protease inhibitor, Roche). Induce a hypotonic shock by adding 150 μl of diluted TES buffer (1:5 TES:water dilution) and incubating for 30 minutes on ice. Centrifuge the plates at 4000 rpm for 10 minutes at 4 °C to pellet the cells and debris. Carefully transfer the supernatant to another microtiter plate and keep it on ice for immediate testing in a functional assay or a binding assay. Binding

[0227] Screening of scFvs for binding to CEACAM5 is tested in a homogeneous assay using CellInsight™ technology. Mix the following reagents in each well of a 384 clear-bottom well plate (Corning): 30 μl of streptavidin polystyrene bead suspension (Polysciences; 3000 beads / well) coated with either biotinylated CEACAM5, biotinylated domain A3-B3, or biotinylated NusA for the control protein; 60 μl of blocked scFv periplasm preparation; 10 μl of detection buffer (PBS containing 5 μg / ml mouse anti-c-myc antibody; anti-mouse Fc AlexaFluor® 647 diluted 1:200). After mixing at 600 rpm for 5 minutes, incubate the 384-well plate at room temperature and read it after 2 hours on a CellInsight™ CX5 High-Content Screening platform (ThermoFisher Scientific). Select clones expressing scFvs that give a signal specific to CEACAM5 rather than NusA for further analysis or sequencing.

[0228] Binding to CEACAM1, CEACAM6, and other CEACAMs can be measured in the same manner. Clone sequencing

[0229] Inoculate single clones into a 96-deep well microtiter plate containing 1 ml of LBAG medium (LB medium with 100 μg / ml ampicillin and 2% glucose) per well and grow overnight at 37 °C at 300 rpm. Extract DNA using the Zyppy-96 Plasmid Miniprep kit (Zymo Research) and sequence it. (Example 4) Reformatting of immobilized VH candidates to IgG and transient expression in mammalian cells

[0230] After screening and sequencing, scFv candidates with desired binding properties are reformatted into IgG and expressed by transient transfection into PEAK cells. The VH and VL sequences of the selected scFv are amplified using specific oligonucleotides, cloned into an expression vector containing the heavy and light chain constant regions, and the construct is verified by sequencing. The expression vector is transfected into mammalian cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, 4×10 6 cells of PEAK are cultured in a 25 ml culture medium containing fetal bovine serum in a T75 flask. The transfected cells are cultured at 37 °C for 5 - 6 days, and IgG production is quantified using an Octet RED96 instrument. The supernatant is recovered for IgG purification in FcXL affinity resin (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, the supernatant from the transfected cells is incubated overnight at 4 °C with an appropriate amount of FcXL resin. After washing the resin with PBS, the sample is loaded onto an Amicon Pro column, and then IgG is eluted with 50 mM glycine pH 3.5. The eluted IgG fraction is then dialyzed against histidine NaCl pH 6.0 buffer using Amicon 50 kDa, and the IgG content is quantified by absorption at 280 nm. Purity and IgG integrity are verified by electrophoresis using an Agilent Bioanalyzer 2100 according to the manufacturer's instructions (Agilent Technologies, Santa Clara, Calif., USA). (Example 5) Characterization of CEACAM5 Monoclonal Antibody a) Binding to cells transfected with different members of the CEACAM family of the anti-CEACAM5 arm

[0231] The specificity of the anti-CEACAM5 antibody arm, tested as a bivalent mAb or a monovalent bsAb, is demonstrated by flow cytometry using PEAK and / or CHO cells transfected with different members of the CEACAM family.

[0232] Using vectors encoding the full-length versions of human CEACAM1, 3, 4, 5, 6, 7, 8, 18, 19, 20 and 21 and 20, these proteins are expressed on the surface of PEAK and / or CHO cells as described in Example 1. Similarly, vectors encoding the full-length versions of cynomolgus CEACAM5 and 6 are also used to express these proteins on the surface of PEAK and / or CHO cells. Untransfected PEAK and / or CHO cells are used as negative controls. The cells are harvested, counted, checked for viability and resuspended in FACS buffer (2% BSA in PBS, 0.1% NaN3) at 3×10 6 cells / ml. 100 μl of the cell suspension is dispensed into a V-bottom 96-well plate (3×10 5 cells / well). The supernatant is removed by centrifugation at 1300 rpm for 3 minutes at 4°C and the cells are incubated with increasing concentrations of the antibody according to the invention for 15 minutes at 4°C. The antibody with the anti-CEACAM5 arm to be tested is diluted in FACS buffer and the concentration range is from 30 pM to 500 nM. The cells are washed twice with cold FACS buffer and reincubated with a compatible anti-human IgG secondary antibody for a further 15 minutes at 4°C. The cells are washed twice with cold FACS buffer and resuspended in 300 μl of FACS buffer with TOPRO-3 (Invitrogen) diluted 1:1500. Fluorescence is measured using a FACSCalibur™ (BD Biosciences) or Cytoflex Platform (Beckman Coulter). The dose-response binding curve is fitted using GraphPad Prism8 software. In the same manner, CEACAM1, CEACAM6 and other CEACAMs can be characterized.

[0233] The results obtained by using the experimental procedures described in Examples 1 and 5a are shown in Tables 2 and 3 (tested with full-sized antibodies at 10 mcg / ml; BiTE MEDI-565 was tested at equimolar concentration). For the bispecific antibodies AB17L3-1 / N, AB71L3-1 / N, AB72L3-1 / N, AB73L3-1 / N, the MFIs measured for binding to CEACAM5-transfected cells were found to be between 29,000 and 41,000 (Table 2). In contrast, the MFIs found by using PEAK cells transfected with CEACAM1, 3, 4, 6, 8 were found to be below 1000, except for the strong signal for AB72L3-1 / N in CEACAM8-transfected cells. When the MFI values obtained in transfected cells expressing any given CEACAM are divided by the values obtained in WT PEAK cells, a "fold over PEAK WT" can be calculated (Table 3). Except for AB72L3-1 / N, the antibodies of the present invention are all specific for CEACAM5 since all "fold over PEAK WT" values are below 2.0. In contrast, MEDI-565 BiTE shows a "fold over PEAK WT" higher than 2 for CEACAM8, suggesting cross-reactivity to such CEACAM family members. This can, for example, cause neutrophil killing since, as already mentioned above, human neutrophils express CEACAM8 on their surface. Table 2 Binding to transiently expressed CEACAMx in PEAK cells [MFI] [Table 2] Table 3 Binding to transiently expressed CEACAMx in PEAK cells [fold over PEAK WT] [Table 3] b) Binding of CEACAM5 monoclonal antibodies to recombinant proteins in enzyme-linked immunosorbent assay (ELISA)

[0234] Capture biotinylated recombinant human CEACAM5 protein at 0.5 μg / mL in a 96-well microplate coated with streptavidin. Wash the plate and add the monoclonal anti-TAA bivalent antibody of the present invention at a wide concentration range (e.g., 5×10 -4 ~1 μg / mL) and incubate for 1 hour. Wash the plate and detect the bound antibody with anti-human IgG (Fc)-HRP (Jackson ImmunoResearch). After washing, reveal the plate with Amplex Red® reagent (Molecular Probes). Measure the fluorescence signal in a Synergy HT plate reader (Biotek).

[0235] Binding to other recombinant CEACAM family members such as CEACAM1 and CEACAM6 can be evaluated similarly. The binding results are shown in Figures 8 and 9 for 1B4 mAb and C11 mAb, respectively. c) Epitope binning of the antibody of the present invention by competition with a reference antibody

[0236] Epitope binning is a competitive immunoassay used, for example, to characterize the binding of a new monoclonal antibody to a target protein. Generate a competitive blocking profile for the new antibody that binds to the target protein with an antibody that also binds to this target protein and whose binding epitope has already been established / published. Competition with one of these reference antibodies indicates that the new antibody has an epitope in the same or a nearby location and that they are "binned" together.

[0237] The ability of the CEACAM5 mAb of the present invention to compete with CEACAM5 reference antibodies is tested by ELISA on recombinant human CEACAM5 using the following reference antibodies with mouse Fc regions: SM3E, an mAb derived from sm3E described in US Patent Application Publication No. 20050147614A1; MEDI, an mAb derived from MEDI-565 described in International Publication No. 2016036678A1; SAR, an mAb derived from Mab2_VLg5VHg2 described in European Patent Application Publication No. 3199552A1; CH1A1A, an mAb derived from CH1A1A-2F1 described in US Patent Application Publication No. 20120251529 and Klein et al in Oncoimmunology, 2017 Jan 11;6(3); humanized T84.66, an mAb derived from variant 1 described in International Publication No. 2017055389; LAB, an mAb derived from hMN14 described in US Patent Application Publication No. 2002 / 0165360A1.

[0238] SM3E binds more to the distal part of the cell membrane at the N-terminus of CEA, for example, MEDI binds to the central part, and CH1A1A binds near the membrane.

[0239] Biotinylated human CEACAM5 is coated at 0.5 μg / ml on a 96-well plate coated with streptavidin and incubated for 1 hour with 10 μg / ml of the reference mAb or an irrelevant mAb with a mouse Fc region. The CEACAM5 mAb of the present invention (meaning a bivalent monoclonal anti-CEA antibody) is added at 0.2 μg / ml for 1 hour at room temperature. The plate is washed and the bound CEACAM5 mAb is detected with anti-human IgG (Fc)-HRP (Jackson ImmunoResearch). After washing, the plate is developed with Amplex® Red reagent. The fluorescence signal is measured in a Synergy HT plate reader (Biotek).

[0240] Based on the results found with the CEACAM5 mAb, the induced CEAxCD3 according to the invention is considered to compete with the reference antibody if the binding to CEACAM5 is reduced by more than 80% when comparing the results with and without the addition of the tool antibody. The CEAxCD3 antibody is identified as non-competing with the tool antibody if the binding to CEACAM5 is reduced by less than 20% when comparing the results with and without the addition of the tool antibody. Figure 1 shows schematically the binding region of the reference antibody used in Example 5c. d) Determination of the CEACAM5 domain to which the antibody of the invention binds using truncated CEACAM5

[0241] Using truncated CEACAM5 lacking one or more of its extracellular subdomains, the subdomain to which the antibody of the invention binds can be determined.

[0242] Using a vector encoding the full-length version of human CEACAM5 (containing all of its extracellular domains, i.e., N-A1-B1-A2-B2-A3-B3), and vectors encoding subsets of the extracellular domain of CEACAM5 (A1-B1-A2-B2-A3-B3; B1-A2-B2-A3-B3; A2-B2-A3-B3; B2-A3-B3; A3-B3 and B3) only, these proteins are expressed on the surface of PEAK and / or CHO cells as described in Example 1. Un-transfected PEAK and / or CHO cells are used as negative controls. Flow cytometry staining and acquisition are performed as described in subsection a) of Example 5.

[0243] The antibody according to the invention is found to bind to a given truncated CEACAM5 protein when the bound antibody is detected by a PE-conjugated anti-human IgG Fc secondary antibody. (Example 6) Expression and purification of bispecific antibodies with lambda and kappa light chains

[0244] Co-expression of one heavy chain and two light chains in the same cell can result in the assembly of three different antibodies. Co-expression can be achieved in different ways, such as transfection of multiple vectors expressing one of the co-expressed chains, or by using a vector that drives the expression of multiple genes. Vectors encoding different anti-CEACAM5 antibodies are co-transfected with another vector expressing the heavy and light chains of the anti-CD3 antibody. Alternatively, each of them is cloned into the vector pNovi κHλ previously generated to enable co-expression of two light chains, one heavy chain, one kappa light chain and one lambda light chain, as described in US Patent Application Publication No. 2012 / 0184716 and International Publication No. 2012 / 023053, which are hereby incorporated by reference in their entirety. The expression of the three genes is driven by the human cytomegalovirus promoter (hCMV), and the vector also contains the glutamine synthetase gene (GS) that enables the selection and establishment of a stable cell line. The common VH and VL genes of anti-CEACAM5 IgG and anti-CD3 IgG are cloned into the vector pNovi κHλ for transient expression in mammalian cells. Expi293 cells are cultured in suspension in an appropriate Erlenmeyer flask with an appropriate number of cells and culture medium volume. Plasmid DNA is finally transfected into Expi293 cells using PEI. The antibody concentration in the supernatant of the transfected cells is measured using Octet RED96 during production. According to the antibody concentration, the supernatant is collected 5-7 days after transfection and clarified by centrifugation at 1300 g for 10 minutes. Purification is based on a three-step purification process. First, the CaptureSelect™ FcXL affinity matrix (Thermo Fisher Scientific) is washed with PBS and then added to the clarified supernatant. After overnight incubation at +4 °C and 20 rpm, the supernatant is centrifuged at 2000 g for 10 minutes, the flow-through is saved, and the resin is washed twice with PBS. The resin is then transferred to an Amicon Pro column and a solution containing 50 mM glycine at pH 3.0 is used for elution.Generate several elution fractions, neutralize with Tris-HCl pH 7.4, and pool. Pool containing total human IgG (bispecific and two monospecific antibodies) is quantified using a Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, Del.), and then incubated with an appropriate volume of CaptureSelect™ KappaXL affinity matrix (Thermo Fisher Scientific, GE Healthcare) at 20 rpm for 30 minutes at room temperature. The resin recovery, washing, elution, and neutralization steps are performed as described previously. The final affinity purification step is performed using the CaptureSelect™ lambda Fab affinity matrix (Thermo Fisher Scientific) applying the same process as for the kappa purification step. All elution fractions are pooled and desalted against His-NaCl pH 6 formulation buffer using a 50 kDa Amicon ultrafiltration filter unit (Merck Millipore). The final product is quantified using a Nanodrop.

[0245] The purified bispecific antibody is analyzed by electrophoresis under denaturing and reducing conditions using an Agilent 2100 Bioanalyzer and Protein 80 kit as described by the manufacturer (Agilent Technologies, Santa Clara, Calif., USA). Mix 4 μL of the purified sample with sample buffer supplemented with dithiothreitol (DTT; Sigma Aldrich, St. Louis, Mo.). Heat the sample at 95 °C for 5 minutes and then load onto the chip. All samples are tested for endotoxin contamination using the Limulus Amebocyte Lysate test (LAL; Charles River Laboratories, Wilmington, Mass.). (Example 7) In vitro characterization of monovalent and bispecific antibodies a) Binding of monovalent and bispecific antibodies to cells expressing CEACAM5 and cells not expressing CEACAM5

[0246] To demonstrate the binding of the CD3xCEACAM5κλ antibody to target cells, a series of flow cytometry-based experiments can be performed comparing the binding of the CD3xCEACAM5κλ antibody to its monovalent counterparts. Examples of cells that can be used include CEACAM5-positive cell lines such as the gastric adenocarcinoma cell line MKN45 (expressing 155,000 CEACAM5 molecules per cell), or the pancreatic adenocarcinoma cell line HPAF-II (expressing 108,000 CEACAM5 molecules per cell), or the colorectal adenocarcinoma cell line LS174T (expressing 26,000 CEACAM5 molecules per cell), as well as CEACAM5-negative cell lines such as the lung cancer cell line A549 and the MKN45 CEACAM5 knockout cell line generated by CRISPR-CAS9 methodology. Cell staining and binding evaluation can be performed as described above. The resulting binding curves are shown in Figures 4, 5, 11, and 14. The EC50 values can be calculated for binding to MKN45 cells using GraphPad Prism8, and the data are shown in Table 4. The binding of the bsAb of the present invention at 200 nM, 1000 nM, and 5000 nM is 40% and higher compared to the binding of TCB2014 (see Table 4 and Figures 11 and 14). Table 4: Binding EC50 to MKN-45 cells. N / A: Not applicable

Table 4

[0247] To demonstrate the binding of the CD3xCEACAM5κλ antibody to effector T cells, a series of flow cytometry-based experiments can be performed to compare the binding of the CD3xCEACAM5κλ antibody with its monovalent counterparts. Examples of cells that can be used include human primary T cells and CD3-positive (Jurkat and / or HuT 78) or CD3-negative (TIB-153 and / or JKT-beta-del) cell lines. Cell staining and binding evaluation can be performed as described above. The results are shown in Figures 3 and 10. c) Epitope binding of CEACAM5 antibody by competition with reference antibodies

[0248] Epitope binding is a competitive immunoassay used to characterize the binding of the antibodies according to the invention or, for example, the binding of a related anti-CEA (target protein) antibody of the first binding moiety. The competitive blocking profile of an antibody that binds to the target protein is generated against an antibody that also binds to this target protein and whose binding epitope has already been established / disclosed. Competition with one of these reference antibodies indicates that the antibodies have epitopes in the same or nearby locations and that they are "binned" together. The ability of the anti-CEACAM5 arm, which is part of the bispecific antibody of the invention that competes with the anti-CEACAM5 reference antibody, is tested by ELISA using the following reference antibodies with recombinant human CEACAM5 and mouse Fc regions: SM3E, the sequence of the mAb derived from SM3E described in US Patent Application Publication No. 20050147614A1, the mAb produced using standard methods; MEDI, the mAb derived from MEDI-565 described in International Publication No. 2016036678A1; CH1A1A, the mAb derived from CH1A1A-2F1 described in US Patent Application Publication No. 20120251529 and Klein et al in Oncoimmunology, 2017 Jan 11;6(3). SM3E binds more to the distal part of the cell membrane at the N-terminus of CEA, MEDI binds to the central part, and CH1A1A binds near the membrane.

[0249] The κλ body used at 1 μg / ml is captured by goat anti-human IgG (Fcγ) (Jackson ImmunoResearch) coated at 10 μg / ml on a 96-well black microplate and blocked with blocking buffer (2% BSA in PBS, 0.05% Tween® 20). The competitor IgG (0.03 - 20 μg / ml) is pre-incubated for 1 hour with 0.1 μg / ml biotinylated human CEACAM5 in the blocking buffer. The plate of κλ body is washed and incubated for 1 hour with the pre-incubated competitor IgG / CEACAM5 mixture. After washing, CEACAM5 is detected using streptavidin-HRP (Jackson ImmunoResearch). The plate is developed with Amplex®Red reagent (Molecular Probes) and the fluorescence signal is measured in a Synergy HT plate reader (Biotek).

[0250] If the binding of CEACAM5 to the κλ body is reduced by 80% or more by each of the tool antibodies, it can be concluded that the CEAxCD3 bispecific antibody is classified as binding competitively with the tool antibody. Thus, the CEAxCD3 antibody is identified as non-competitive with the tool antibody if the binding of CEACAM5 to each κλ body is reduced by 20% or less when comparing the results with and without the addition of the tool antibody. d) Binding of bispecific antibody to primary human blood cells

[0251] To demonstrate the binding of the CD3xCEACAM5 κλ antibody to primary T cells and the lack of binding to primary B cells and monocytes (CEA-negative population), a series of experiments based on flow cytometry can be performed. Cell staining and binding evaluation can be carried out as described in Example 7a. The data are shown in Figure 13. (Example 8) T cell-dependent cytotoxicity (TDCC) mediated by bispecific antibody a) TDCC of CEACAM5-positive and CEACAM5-negative cell lines

[0252] The T cell-dependent cytotoxicity (TDCC) of different CEACAM5-positive and CEACAM5-negative tumor cell lines induced by the CEAxCD3 bispecific antibody of the present invention is evaluated using either human PBMC or purified primary T cells as effector cells.

[0253] Target cells are detached with trypsin or cell dissociation solution after two washes with PBS. After the centrifugation step, the cells are resuspended in assay medium, adjusted to the required concentration, and seeded in 96-well plates.

[0254] The effector cells can be either human peripheral blood mononuclear cells (PBMC) or purified T cells. PBMC are isolated from buffy coats obtained from healthy human donors using SepMate™ tubes (Stemcell Technologies) and Lymphoprep™ buffer (Stemcell Technologies). When using purified T cells as effector cells, an additional purification step is performed, where the T cells are negatively isolated from PBMC by using a T cell immunomagnetic negative selection kit (STEMCELL Technologies).

[0255] For the TDCC assay, when using PBMC as effector cells, add these to target cells at a final E:T ratio of 10:1, and when using purified T cells, use a final E:T ratio of 5:1. Next, add the CEAxCD3 antibodies and related control antibodies of the present invention at concentrations in a dose range (up to 100 nM, in duplicate) to pre-seeded target cells and effector cells. Target cell killing is evaluated by quantifying the LDH released into the medium by apoptotic / necrotic cells (Cytotoxicity Detection Kit PLUS (LDH), Roche) after incubation at 37 °C, 5% CO2 for either 24, 48, or 72 hours. Maximum LDH release (= 100% lysis) was obtained by incubating target cells with 1% Triton® X-100. Spontaneous LDH release (= 0% lysis) refers to target cells co-cultured with effector cells without the addition of any antibody. TDCC curves (Figures 6, 7, 12, and 15) and EC50 values (Table 5) can be calculated for the MKN-45 and LS174T cell lines using GraphPad Prism 8, and the EC50 found for the bsAbs of the present invention shown in Table 5 is significantly lower than the EC50 measured with TCB2014, demonstrating higher potency for in vitro tumor cell killing by these bsAbs of the present invention. Table 5 Killing EC50 of three CEA+ cell lines

Table 5

[0256] The combination of the bispecific antibody of the present invention and an anti-CD47 mAb (e.g., described in US Patent Application Publication No. 20140140989 and International Publication No. 2017196793), or a CEAxCD47 bispecific antibody (described in International Application No. PCT / IB2019 / 054559, incorporated herein by reference), can be tested in the models described above. Additional test conditions can be added to the experimental design, where such CD47-targeting antibodies (monospecific or bispecific) are used at different doses, either alone or in combination with the CEAxCD3 antibody of the present invention. c) Upregulation of T cell activation markers in the killing of CEA-expressing tumor cells induced by CEAxCD3 bsAb

[0257] The killing of CEA-positive tumor cells induced by CEAxCD3 bsAb requires T cell activation, which can be quantified by flow cytometry using antibodies that recognize specific T cell activation markers such as CD69 (early activation marker) or CD25 (late activation marker).

[0258] To evaluate the activation state of T cells at the end of the killing assay (described in Example 8a above), continue with the following procedure: Transfer the floating cells (including both CD4+ and CD8+ T cells) into a new V-bottom 96-well plate. Remove the supernatant by centrifugation (3 minutes at 1300 rpm at 4°C), wash the cells twice with cold FACS buffer (2% BSA in PBS, 0.1% NaN3), and then incubate with the Fc-blocking reagent (BD Biosciences) for 15 minutes at 4°C. After washing twice with FACS buffer, incubate the cells with the following antibodies for 15 minutes at 4°C (used according to the manufacturer's recommendations): anti-CD45 (V500 conjugated, BD Biosciences), CD69 (FITC conjugated, Biolegend), CD8 (PerCP-Cy5.5 conjugated, Biolegend), CD25 (PE conjugated, Biolegend), CD4 (APC conjugated, ThermoFisher), CD3 (APC-R700 conjugated, BD Biosciences).

[0259] Wash the cells twice with cold FACS buffer and resuspend in 200 μl of FACS buffer. Measure the fluorescence using the Cytoflex Platform (Beckman Coulter) and analyze the data using FlowJo™ v10 software (BD Life Sciences). The results are shown in Figure 17. d) T cell proliferation induced by the CEAxCD3 bsAb molecule

[0260] The CEAxCD3 bsAbs are analyzed for their ability to induce T cell proliferation upon crosslinking in the presence of CEA-positive tumor target cells. As negative controls, CEA-negative malignant cells are used similarly. Freshly isolated human PBMCs are adjusted to 1 million cells per mL with warm PBS and stained with 0.2 μM carboxyfluorescein diacetate succinimidyl ester (CFSE, ThermoFisher Scientific) in PBS for 15 minutes at 37 °C, washed several times with complete RPMI medium (containing 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 50 μM 2-mercaptoethanol and 25 μg / mL gentamicin), and transferred to 96-well plates at 2×10 6 cells per mL. 0.02×10 6 target cells are seeded per well of a flat-bottom 96-well plate, and different CEAxCD3 bsAbs are added at the indicated concentrations. CFSE-labeled PBMCs are added to obtain a final E:T ratio of 10:1, and the assay plates are incubated at 37 °C for 5 days in a humidified incubator. On day 5, the effector cells are harvested, washed twice with FACS buffer (PBS, 2% BSA, 0.1% NaN3), and then the cells are stained with BD Horizon 620 (BD Biosciences, 564996) to exclude dead cells, as well as anti-CD45 (V500 conjugated, BD Biosciences), anti-CD4-APC (ThermoFischer, 17-0049-41) and anti-CD8-PerCP-Cy5.5 (Biolegend, 301032). CFSE staining is analyzed by flow cytometry using CytoFLEX (Beckman Coulter) in live CD4 + or CD8 + cells, and the results are evaluated by FlowJo software. e) Cytokines released into the supernatant upon killing of tumor cells expressing CEA induced by CEAxCD3 bsAbs

[0261] Killing of CEA-positive tumor cells induced by the CEAxCD3 bsAb requires T cell activation. Upon activation, T cells can release multiple cytokines that can further act as immunomodulators. The ability of the bispecific antibodies of the invention to induce cytokine release by T cells during the killing of tumor cells expressing CEA was evaluated by quantifying selected cytokines in the supernatant at the end of the TDCC assay described in Example 8a. Two days after co-culture of CEA-positive target cells and CD3-positive effector T cells, the culture supernatant was collected by centrifugation and stored frozen at -80 °C until further analysis. Cytokines / enzymes such as Granzyme B, IL2, IL6, IL10, TNFα and IFNγ were quantified using the Mesoscale Discovery Platform by using a multiplex kit, and the results are shown in Figure 16. f) TDCC of CEACAM5-positive cells in the presence of shed CEA

[0262] CEA-positive tumors are known to shed CEA. Such shed CEA can negatively impact the antitumor efficacy of CEA-targeting antibodies that do not preferentially bind to membrane-bound CEA. To evaluate whether the bispecific antibodies of the invention are affected by shed CEA (sCEA), the T cell-dependent cytotoxicity (TDCC) assay described in Example 8a is performed in the presence of various concentrations of spiked sCEA (BioRad# PHP282). The EC50 value in the presence of sCEA is then compared to the EC50 obtained in the absence of sCEA (Table 6). The EC50 calculated for a given sCEA concentration (0.2, 1 or 1 μg / mL) is then compared to that obtained in the absence of sCEA (0 μg / mL) and expressed as the fold change in EC50 compared to the condition without shed CEA. Such values are reported in Table 7. Table 6 EC50 for killing of LS174T cells in the presence of sCEA [Table 6-1] [Table 6-2] *Without top plateau Table 7 Fold change in EC50 compared to the condition without shedding CEA (0 μg / mL)

Table 7

[0263] At 1 and 5 μg / mL of sCEA, a significantly higher shift in EC50 for tumor cell killing was found in TCB2014 and TCB2017 when sCEA was added, compared to the bispecific antibody of the present invention. Concentrations of sCEA of 1 μg / mL and above are found in patients with CEA-positive tumors. The lower shift in the killing curve of the bsAb of the present invention due to sCEA suggests that high sCEA levels have less inhibitory effect on the efficacy of the bsAb of the present invention compared to TCB2014 and TCB2017. g) TDCC of CEACAM5-negative primary blood cell population

[0264] Considering the mechanism of action of the CEAxCD3 bispecific antibody, cross-reactivity with other CEACAMs can lead to depletion of important circulating healthy cell populations. For example, cross-reactivity with CEACAM8 expressed by neutrophils can lead to depletion of such cell populations. To confirm the absence of binding and thus the absence of killing of such CEA-negative circulating healthy cell populations, purified primary cells such as neutrophils are used as "target cells" instead of the CEA-positive cell line in the experimental procedure described in Example 8a. (Example 9) Evaluation of the antitumor activity of the CEAxCD3 T cell retargeting molecule as a single agent or in combination therapy with a CD47-targeting antibody in a humanized mouse tumor model a) Antitumor activity of the CEAxCD3 molecule in a PBMC humanized mouse tumor model

[0265] 8 - 10-week-old NOG mice (NOD / Shi-scid / OL-2Rγ nullmice (Taconic Biosciences), 1–5 × 10 6 CEA-positive tumor cells (cell line-derived or patient-derived) are implanted subcutaneously (sc) and randomized into treatment groups. Four to seven days later, all mice are inoculated with 10 × 10 mAbs, either i.p. or iv., for the humanization process. 6 pcs or 20 x 10 6 Human PBMCs (peripheral blood mononuclear cells) are injected into the mice. CD3xCEA molecule or control is then administered iv at different doses once or twice a week starting 3-6 days after PBMC injection. Mice are monitored three times a week for tumor development and tumors are grown at the experimental endpoint (tumor volume = 1500 mm). 3 Tumor volumes are measured by digital calipers according to the formula (length x width). 2 )×0.5. Statistical analysis is performed at the end of the study using one-way comparative analysis of variance. Results from an experiment in which 1 million HPAF-II cells were subcutaneously engrafted in NOG mice followed by injection of 10 million human PBMCs are shown in FIG. 19. b) CD34 + Antitumor activity of CEAxCD3 molecules in humanized mouse tumor models

[0266] >25% human CD45 in blood at 14 weeks of age + Fully humanized CD34 bearing cells + -huNOG mice (CD34 + engraftedNOD / Shi-scid / OL-2Rγ null mice (Taconic Biosciences), 1–5 × 10 6 CEA-positive tumor cells (cell line-derived or patient-derived) are implanted subcutaneously (sc) and randomized into treatment groups. The mean tumor volume is adjusted to a predefined value (100-200 mm). 3 When tumor volume reaches 1000 mg / kg / day (range 1000 mg / kg / day), CD3xCEA molecule or control is administered iv once or twice weekly at different doses. Mice are monitored for tumor growth three times a week and tumors are grown to reach the experimental endpoint (tumor volume = 1500 mm3).3 ) up to, measured by a digital caliper. The tumor volume is calculated using the formula (length × width 2 ) × 0.5. Statistical analysis is performed at the end of the study using a comparative analysis of one-way analysis of variance. c) Anti-tumor activity of the CEAxCD3 molecule in combination with a CD47-targeted antibody (monospecific or bispecific) in a humanized mouse tumor model

[0267] The combination of the bispecific antibody of the present invention with an anti-CD47 mAb (e.g., described in US Patent Application Publication No. 20140140989 and International Publication No. 2017196793) or a CEAxCD47 bispecific antibody (described in International Application No. PCT / IB2019 / 054559, incorporated herein by reference) can be tested in the models described above. An additional group is added to the experimental design that includes treatment groups in which the CD47-targeted antibody (monospecific or bispecific) is administered i.v. once or twice a week, either alone or in combination with the CEAxCD3 antibody of the present invention, at various doses. d) Anti-tumor activity of the CEAxCD3 molecule in combination with a CD47-targeted antibody (monospecific or bispecific) in a transgenic mouse tumor model

[0268] The combination of the bispecific antibody of the present invention with an anti-CD47 mAb (e.g., described in US Patent Application Publication No. 20140140989 and International Publication No. 2017196793) or a CEAxCD47 bispecific antibody (described in International Application No. PCT / IB2019 / 054559, incorporated herein by reference) can be tested in transgenic mice engineered to express human CEA and human CD47 and subcutaneously (s.c.) implanted with 0.5 - 5 × 10 6 mouse tumor cells and engineered to express human CD3, human CD47, and human SIRPα. The average tumor volume is at a predetermined value (100 - 200 mm 3When the range is reached, randomize the mice. Administer the treatment i.v. once or twice a week at different doses. Monitor the mice for tumor growth three times a week and measure the tumors with digital calipers until the end point of the experiment (tumor volume = 1500 mm 3 ). Measure the tumor volume using the formula (length × width 2 ) × 0.5. Perform statistical analysis at the end of the study using a comparative analysis of one-way analysis of variance. (Example 10) Cytokine release tested in whole blood and PBMCs derived from human blood of healthy human donors

[0269] Perform the in vitro cytokine release assay using whole blood at the minimum dilution (WB CRA) with the test antibody (95% v / v blood) in an aqueous presentation. This assay format is thought to closely mimic the in vivo environment, containing factors at physiological concentrations that can affect the mechanism of cytokine release. However, this format is thought to be insufficient for predicting T cell-mediated cytokine release (e.g., anti-CD28).

[0270] Alternatively, the cytokine release assay can be performed using peripheral blood mononuclear cells (PBMCs) from healthy human donors with an antibody in an aqueous phase (AP) to evaluate T cell-mediated cytokine release (PBMC AP CRA). This format limits the cross-linking of mAbs to avoid the high cytokine release observed with anti-CD3 antibodies during cross-linking.

[0271] For each assay format, negative controls (anti-EGFR mAb and PBS) and specific positive controls (anti-CD52 mAb, CEAxCD3 BiTE and / or anti-CD28 mAb) are tested in parallel with the CEAxCD3 bispecific antibody. After 24 hours for WB CRA and 48 hours for PBMC AP CRA, the supernatants are tested for cytokines in a multiplex assay (Mesoscale Discovery, Sector 600) using electrochemiluminescence as the readout. IFNγ, TNFα and IL-6 are measured for WB CRA, and IFNγ, IL-2, IL-10 and TNFα are measured for PBMC AP CRA. The results are plotted for each cytokine and each donor is represented as a single data point. (Example 11) Affinity maturation of CEA antibodies by oligonucleotide-specific mutagenesis (lead optimization; LO) using degenerate oligonucleotides

[0272] The antibodies identified during the screening process described in Example 3 are selected for affinity maturation in order to increase their affinity and potency. All of these antibodies share the same variable heavy chain but have different variable light chains. AB1 and C11 contain kappa light chains (IGKV3-11 and IGKV1-5, respectively, according to the IMGT nomenclature), while AB8 and 1B4 contain lambda light chains (IGLV2-14 and IGLV3-21, respectively). Several phage libraries presenting scFv variants are generated by introducing diversity into CDR1, CDR2, and CDR3 of the variable light chain region while keeping the heavy chain variable region unmodified. Different diversification strategies are used to generate libraries for each candidate, where CDRL1+CDRL2; or CDRL3 only, or all three CDRLs, are diversified by oligonucleotide-specific mutagenesis of the parental sequence using degenerate oligonucleotides (CDRL1+CDRL2+CDRL3). CDRL1 is diversified at amino acid positions 1 to 5, CDRL2 is diversified at amino acid positions 1 to 4, and CDRL3 is diversified at amino acid positions 1 to 5. A total of up to 5×10 14 transformants, covering up to 10 9 theoretical diversities, are generated for each candidate.

[0273] For candidates AB1 and 1B4, additional libraries diversified at up to 17 amino acid positions across all CDRLs are generated with up to 5×10 21 transformants, covering up to 10 9 theoretical diversities.

[0274] These libraries are used for phage display selection as described in Example 2, except that the selection stringency is increased between rounds by gradually reducing the concentration of recombinant hCEACAM5 from 100 nM to 0.01 nM between different selection rounds, or by using cells that express lower levels of hCEACAM5 such as the SNUC-1 cell line. The selected variants are screened for their ability to bind to CEACAM5 using the assays described in Example 3. Positive clones are reformatted as IgG and characterized as described in Examples 4 and 5, respectively.

[0275] The anti-CEA arm AB1 (SEQ ID NOs: 31 - 34) was optimized in two consecutive lead optimization waves. Wave 1 resulted in anti-CEA arms AB13, AB14, AB15, AB17 and AB20. Wave 2 resulted in anti-CEA arms AB54, AB60, AB66, AB71, AB72 and AB73. (Example 12) TDCC (T cell-dependent cytotoxicity) and / or TDCC and ADCP of tumor-derived organoids

[0276] Organoids derived from tumor cells are an advanced translational model for testing T cell retargeting compounds and / or macrophage and NK cell retargeting compounds.

[0277] Organoids are prepared according to standard procedures (Schuette et al., Nature Communications 2017; DOI: 10.1038 / ncomms14262), incubated with compounds for up to 8 days in co-culture with PBMC to generate macrophages in vitro. The medium is changed every 4 days and replaced with fresh medium.

[0278] Collect the organoids and enzymatically dissociate them into single cells using Accutase at 37 °C for 5 minutes. Pellet the cells, resuspend them in FACS buffer (PBS, 2% FBS, 2 mM EDTA), and filter them through a 400 μm cell strainer. Incubate the cell suspension of equal cell numbers with antibodies against CD45, CD4, CD8, CEA, and CD14 (all from Thermo Fisher Scientific, Dreieich, Germany) for 30 minutes on ice. Use propidium iodide for live cell gating, measure it, and analyze it using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

[0279] Freeze the supernatant from each well at -80 °C for analysis of T cell activity by using ELISA. (Example 13) TDCC and / or TDCC and ADCP of tumor tissue slices derived from patients

[0280] Fresh tumor tissue slices derived from patients are another advanced translational model for testing T cell and / or macrophage and / or NK cell retargeting compounds.

[0281] Fresh tumor tissue samples are sectioned according to previously published standard procedures (Soennichsen et al., Clinical Colorectal Cancer, 17 (2018) e189 - e199). Briefly, immediately after surgical resection and initial gross pathological evaluation, tumor samples are sectioned into 350 - μm slices using a tissue chopper (McIlwain TC752; Campden Instruments, Leicestershire, England). The diameter of the tissue slices is then standardized by using a 3 - mm coring tool (kai Europe, Solingen, Germany). Three tissue slices are randomly pooled, placed on membrane inserts, and cultured in 6 - well plates. The slices are incubated under standardized conditions of 37 °C and 5% CO₂. The medium is changed 2 hours and 24 hours after preparation, prior to treatment.

[0282] After 24 hours of pre - incubation in standard cell culture medium, triplicate slices can be exposed to the bispecific antibodies according to the invention, either individually or in combination, for up to 120 hours. If necessary, the incubation time can be reduced to 72 hours. The medium is changed after 72 hours.

[0283] After compound exposure, tumor slices are fixed overnight using 4% paraformaldehyde. The supernatant from each well is frozen at - 80 °C for analysis of T - cell activity using ELISA.

[0284] Paraformaldehyde - fixed slices are embedded in paraffin and processed into 5 - μm sections. Hematoxylin and eosin (HE) staining is performed to evaluate histopathological aspects and the proportion of tumor cells. Total cell count, tumor cell count, and proliferation are analyzed by immunofluorescence staining. Briefly, paraffin sections are de - paraffinized. After antigen retrieval, the sections are washed with 0.3% PBS / Triton® X and blocked with 5% normal goat serum (Jackson ImmunoResearch, Suffolk, UK) for 30 minutes. Each, cytokeratin [Chem.] Primary antibodies against CEA, Ki67, and cleaved PARP are diluted in 0.5% bovine serum albumin and incubated overnight at 4°C. The sections are rinsed with 0.3% phosphate-buffered saline / Triton® X and labeled with secondary antibodies. The nuclei are stained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO). Antibodies against CEA (tumor cells), CD163 (macrophages), and CD3, CD4, CD8, PD-L1, and FoxP3 (all T cells) are included depending on the availability of tumor slices for further analysis.

[0285] Regions containing tumor cells are analyzed in HE sections using a slide scanner (Pannoramic SCAN and Pannoramic Viewer, 3D Histech, Budapest, Hungary) to examine various tumor cell fractions. Slices containing more benign epithelial cells than neoplastic epithelial cells are excluded from the analysis. Slices without tumor cells are excluded from the analysis of the proliferating tumor cell fraction but included in the analysis of tumor cells for each condition. For further analysis, five pictures (20×) per tissue slice are obtained from the fluorescently stained sections using an Olympus BX51 fluorescence microscope (Olympus Deutschland, Hamburg, Germany). The positive pixel count is determined for Hoechst 33342, cytokeratin, Ki67, and cleaved PARP staining using a staining-specific segmentation algorithm for Image J. The proliferation / apoptosis tumor area is calculated by analyzing the pixels of Ki67 / cleaved PARP-positive nuclei around the cytokeratin-positive pixels.

[0286] For all photographs, calculate the total cell count (Hoechst positive), the tumor cell count (Hoechst and cytokeratin positive), and the proliferating tumor cell count (Hoechst, cytokeratin, and Ki67 positive / cleaved PARP). Normalize the tumor cell count relative to the total cell count, and normalize the proliferating tumor cell count relative to the tumor cell count, taking into account the different tumor cell fractions for each photograph. Then, calculate the average slice value from the individual image values. Calculate the average value for the condition using the average slice values.

[0287] All publications, patents, patent applications, Internet sites, and accession numbers / database sequences, including both polynucleotide and polypeptide sequences, cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, Internet site, or accession number / database sequence were specifically and individually indicated to be incorporated by reference in that manner.

Claims

1. A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, a) the first binding portion comprises a heavy chain variable region (VH) comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4, b) the first binding portion, b1) CDRL1 of SEQ ID NO: 90, CDRL2 of SEQ ID NO: 91, and CDRL3 of SEQ ID NO: 92, b2) CDRL1 of SEQ ID NO: 96, CDRL2 of SEQ ID NO: 97, and CDRL3 of SEQ ID NO: 98, b3) CDRL1 of SEQ ID NO: 99, CDRL2 of SEQ ID NO: 100, and CDRL3 of SEQ ID NO: 101, b4) CDRL1 of SEQ ID NO: 102, CDRL2 of SEQ ID NO: 103, and CDRL3 of SEQ ID NO: 104, b5) CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106, and CDRL3 of SEQ ID NO: 107, b6) CDRL1 of SEQ ID NO: 108, CDRL2 of SEQ ID NO: 109, and CDRL3 of SEQ ID NO: 110, and b7) CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, and CDRL3 of SEQ ID NO: 113 comprising a light chain variable region (VL) comprising a set of CDRLs selected from the group consisting of, c) the second binding portion comprises a VH comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4, d) the second binding portion comprises a VL comprising CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, the bispecific antibody.

2. a) in the first binding portion, the heavy chain variable region VH of SEQ ID NO: 1, b) in the first binding portion, b1) the light chain variable region VL of SEQ ID NO: 117, b2) the light chain variable region VL of SEQ ID NO: 119, b3) the light chain variable region VL of SEQ ID NO: 120, b4) the light chain variable region VL of SEQ ID NO: 121, b5) the light chain variable region VL of SEQ ID NO: 122, b6) the light chain variable region VL of SEQ ID NO: 123, and b7) the light chain variable region VL of SEQ ID NO: 124 a light chain variable region VL selected from the group consisting of, and c) in the second binding portion, the heavy chain variable region VH of SEQ ID NO: 1 and the light chain variable region VL of SEQ ID NO: 17 The bispecific antibody according to claim 1.

3. a) in the first binding portion, the heavy chain variable region VH of SEQ ID NO: 1, b) in the first binding portion, b1) the light chain variable region VL of SEQ ID NO: 117, b2) a variable light chain region VL of SEQ ID NO: 122, and b3) a variable light chain region VL of SEQ ID NO: 124 A variable light chain region VL selected from the group consisting of, and c) In the second binding portion, a variable heavy chain region VH of SEQ ID NO: 1 and a variable light chain region VL of SEQ ID NO: 17 The bispecific antibody according to claim 2, comprising

4. A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, wherein the antibody is a) In the first binding portion, a variable heavy chain region VH of SEQ ID NO: 1, b) In the first binding portion, b1) a light chain of SEQ ID NO: 128, b2) a light chain of SEQ ID NO: 130, b3) a light chain of SEQ ID NO: 131, b4) a light chain of SEQ ID NO: 132, b5) a light chain of SEQ ID NO: 133, b6) a light chain of SEQ ID NO: 134, and b7) a light chain of SEQ ID NO: 135 A light chain selected from the group consisting of, and c) In the second binding portion, a bispecific antibody comprising a variable heavy chain region VH of SEQ ID NO: 1 and a light chain of SEQ ID NO:

28.

5. a) a heavy chain of SEQ ID NO: 43, b) a heavy chain of SEQ ID NO: 44, and c) a heavy chain of SEQ ID NO: 45 The bispecific antibody according to any one of claims 1 to 4, comprising a common heavy chain selected from the group consisting of

6. A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, wherein the antibody comprises a common heavy chain of SEQ ID NO: 45, and in the second binding portion, a light chain of SEQ ID NO: 28, and in the first binding portion, a light chain of SEQ ID NO:

128.

7. A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, wherein the antibody comprises a common heavy chain of SEQ ID NO: 45, and in the second binding portion, a light chain of SEQ ID NO: 28, and in the first binding portion, a light chain of SEQ ID NO:

133.

8. A bispecific antibody comprising a first binding portion that specifically binds to human CEACAM5 and a second binding portion that specifically binds to human CD3ε, wherein the antibody comprises a common heavy chain of SEQ ID NO: 45, and in the second binding portion, a light chain of SEQ ID NO: 28, and in the first binding portion, a light chain of SEQ ID NO:

135.

9. The bispecific antibody according to any one of claims 1 to 8, characterized in that each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329A (Kabat EU numbering).

10. A composition comprising the bispecific antibody according to any one of claims 1 to 9 for use in treating cancer in vivo.

11. A composition comprising the bispecific antibody according to any one of claims 1 to 9 for use in administration to a human subject suffering from cancer.

12. A composition comprising the bispecific antibody according to any one of claims 1 to 9 for use in the treatment of colorectal cancer, esophageal cancer, pancreatic adenocarcinoma, gastric cancer, non-small cell lung cancer, breast cancer, head and neck cancer, uterine cancer and bladder cancer.

13. A composition comprising the bispecific antibody according to any one of claims 1 to 9 for use in a monotherapy for the treatment of solid tumors expressing CEA.

14. A composition comprising the bispecific antibody according to any one of claims 1 to 9 for use in the treatment of cancer, characterized in that it is administered in combination with a bispecific anti-CEA x CD47 antibody simultaneously, separately or in a sequential combination.

15. A composition comprising the bispecific antibody according to any one of claims 1 to 9 for use in the treatment of cancer, characterized in that it is administered in combination with a bispecific anti-CEA x CD47 antibody and / or a PD-1 axis antagonist simultaneously, separately or in a sequential combination.

16. A composition comprising the bispecific antibody according to any one of claims 1 to 9 for use in the treatment of cancer, characterized in that it is administered in combination with a PD-1 axis antagonist simultaneously, separately or in a sequential combination.

17. The composition according to claim 16, wherein the PD-1 axis antagonist is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, and ramucirumab.

18. A combination comprising a bispecific antibody according to any one of claims 1 to 9 and a bispecific anti-CEAxCd47 antibody for use in the treatment of cancer, wherein the antibody of the present invention and the bispecific anti-CEAxCd47 antibody are administered in an alternating administration with an interval of 6 to 15 days between the administration of the antibody of the present invention and the bispecific anti-CEAxCd47 antibody.

19. The composition according to any one of claims 10 to 17 or the combination according to claim 18, wherein the antibody is administered to a patient at a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, in a single dose or divided doses or by continuous infusion.

20. The composition or combination according to claim 19, wherein the antibody of the present invention is administered to a patient at a dose in the range of 1 to 20 mg / kg.

21. The composition according to any one of claims 14, 15 or 18 or the combination according to claim 18, wherein the anti-CEAxCd47 antibody is administered to a patient at a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, in a single dose or divided doses or by continuous infusion.

22. The composition according to claim 19, wherein the PD-1 axis antagonist is administered to a patient at a dose in the range of 0.1 to 100 mg / kg body weight per day or per week, in a single dose or divided doses or by continuous infusion.

23. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

24. An isolated polynucleotide encoding an antibody according to any one of claims 1 to 9 or an antigen-binding fragment thereof.

25. A vector comprising the isolated polynucleotide according to claim 24.

26. A cell comprising the isolated polynucleotide according to claim 24 or the vector according to claim 25.

27. A composition for use in the treatment of cancer, comprising a bispecific antibody according to any one of claims 1 to 9, wherein the composition is administered in an alternating administration with the bispecific anti-CEAxCd47 antibody with an interval of 6 to 15 days between the administration of the composition and the bispecific anti-CEAxCd47 antibody.

28. A composition comprising a bispecific anti-CEA x CD47 antibody for use in the treatment of cancer, wherein the composition is administered in an alternating administration with the bispecific antibody according to any one of claims 1 to 9, the interval between administrations of the composition being between 6 and 15 days, characterized in that it is administered in an alternating administration with the bispecific antibody according to any one of claims 1 to 9.

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