Bispecific antibodies against ceacam5 and cd3

Abstract

The present invention relates to bispecific antibodies that bind to human carcinoembryonic antigen CEACAM5 (CEA) and human CD3s. Furthermore, the present invention relates to polynucleotides encoding such bispecific antibodies and vectors and host cells comprising such polynucleotides. The present invention also relates to methods of selecting and producing such antibodies as well as methods of using such antibodies for the treatment of disease.

Description

Technical Field

[0001] This invention relates to bispecific antibodies (CEAxCD3 bispecific antibodies) that bind to human carcinoembryonic antigen CEACAM5 (CEA) and human CD3ε. Furthermore, this invention relates to polynucleotides encoding such bispecific antibodies, and to vectors and host cells containing such polynucleotides. The invention also relates to methods for selecting and producing such antibodies, and methods for treating diseases using such antibodies. Background Technology

[0002] Successfully treating advanced / metastatic solid tumors (such as pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, etc.) remains a challenge. Modern cancer immunotherapy introduces methods / technologies to help the body's immune cells better attack and kill cancer cells. For example, several technologies / methods have been developed to increase the attack of T cells on tumor cells. Examples include immune checkpoint inhibitors, such as monoclonal antibodies that inhibit PD-1 / PD-L1, and T cell bispecific antibodies that bind tumor-associated antigens (TAAs) to CD3 on T cells or CAR-T cells. CAR-T cells and bispecific antibodies are effective against hematologic malignancies and are approved for the treatment of diseases such as B-cell malignancies or acute lymphoblastic leukemia (ALL), but to date, these methods have not yet achieved a real breakthrough in the treatment of advanced / metastatic solid tumors. Monoclonal antibodies and bispecific antibodies used for treatment can cause a variety of adverse reactions. An important toxicity issue is cytokine release syndrome (CRS), which has been observed, for example, in treatment with alemtuzumab, muromonab-CD3, rituximab, tosituzumab, and the CD19xCD3 bispecific antibody blinatumomab.

[0003] Tabernero et al. (J Clin Oncol 35, 2017 (suppl.abstr.3002)) presented phase 1 clinical data at ASCO 2017 on the use of a CEAxCD3 bispecific antibody (RO 6958688, cibisatamab, see below) in monotherapy and in combination with the anti-PD-L1 antibody atezolizumab for the treatment of patients with advanced / metastatic colorectal cancer. Cibisatamab has a so-called 2+1 form, with one Fab fragment binding to CD3 and two Fab fragments binding to CEA. Such antibodies have been described, for example, in US20140242079 (WO2014131712) and US20140242080 (WO2014131711).

[0004] As used herein, “TCB2014” refers to a bispecific antibody that binds CEA and CD3 in a 2+1 configuration, as described in US20140242080 (incorporated in its entirety by reference), which contains the CDRs shown as shown in SEQ ID NOs 270-276 and 290-296 of US20140242080 as the CDR (see also the CDRs of SEQ ID NOs 4-10 and 24-30 of US20140242079, in their entirety by reference). As used herein, “TCB2017” refers to molecule B (VH / VL exchange in CD3 binder, charge modification in CEA binder, humanized CEA binder) in the “2+1IgG CrossFab, inverted” form with charge modification, as described in WO2017055389 (incorporated in its entirety by reference), which contains as a CDR the CDR shown in SEQ ID NO: 4-6, 8-10 and 14-19 of WO2017055389.

[0005] The 2+1 structure differs significantly from natural IgG antibodies. This structure also includes an artificial amino acid (aa) bridge and two distinct heavy chains linked by an aa sequence in the knob-to-hole / Fc region (see, for example, US6737056, WO2013055958). Such bispecific antibodies (e.g., RO6958688, cyproheptadine) are immunogenic, thus leading to the formation of anti-drug antibodies (ADA) and loss of drug exposure due to ADA neutralization. Melero et al. reported 50% or more of ADA patients and 45% of patients with lost exposure at doses of 60–200 mg (Melero et al., ASCO 2017, Abstract 2549 and Poster No. 41, Abstract; see Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017) 2549-2549). The loss of exposure makes actual treatment difficult to control and significantly reduces the likelihood of success. To minimize ADA formation, cybituximab and combinations of cybituximab and atezolizumab were clinically tested after pretreatment with the anti-CD20 antibody obinutuzumab (see ClinicalTrials.gov Trial NCT03866239). Pretreatment was given to deplete B cells in patients with metastatic colorectal cancer. B cell depletion leads to a decrease in patient immunoglobulins, resulting in a potential decrease in ADA, but also weakens the immune system.

[0006] MEDI-565 (AMG211) (another bispecific CEAxCD3 antibody, a single-chain antibody) has entered clinical development, and results have been published (see, for example, M. Pishvaian et al., Clin. Colorectal Cancer. 2016 DEC; 15(4) 345-351). The NCT01284231 study (ClinicalTrials.gov) has been reported completed, and no new trials have been initiated in the past few years. This single-chain bispecific antibody (two scFvs linked by an aa linker) has an extremely low elimination half-life between 2.2 and 6.5 hours (Pishvaian et al.; Clin. Colorectal Cancer, 2016 DEC; 15(4) 345-351) (incorporated hereby by reference).

[0007] This invention provides a CEAxCD3 bispecific antibody with low immunogenicity and high efficacy. This antibody comprises a common heavy chain and, in one embodiment, a κ light chain in the CEA-binding moiety and a λ light chain in the CD3-binding moiety.

[0008] Fischer et al., Nature Communications 6 (2015):6113. https: / / doi.org / 10.1038 / ncomms7113 and Magistrelli G. et al., MABS 9 (2017)231-239 mention the concept of using a common heavy chain to obtain bispecific antibodies. κλ bispecific antibodies are described, for example, in WO2014087248 (incorporated herein by reference in its entirety). Their structure is almost indistinguishable from that of natural IgG, resulting in little or no formation of ADA and thus low or minimal exposure loss. The sequence of the variable region VH of the inventive common heavy chain and the sequence huCD3 VL 1A4 are described in WO2019175658 (US2019 / 0284297) (incorporated herein by reference in its entirety).

[0009] As described above, WO2017055389 describes a bispecific CEAxCD3 antibody with a 2+1 form but binding a domain different from that of cytoxumab. One of these antibodies (RO7172508 or RG 6123) has been tested in clinical trials in patients with locally advanced and / or metastatic CEA-positive solid tumors (ClinicalTrials.gov; search RO7172508), and has also been tested with olibutuzumab pretreatment and in combination with atezolibutumab. Based on descriptions of some cohorts of clinical trials in ClinicalTrials.gov, serum CEA (exfoliated soluble CEACAM5, sCEA) levels below a certain threshold are required in treatment-seeking patients to qualify them for treatment, suggesting that higher levels of exfoliated soluble CEACAM5 may reduce the efficacy of this CEAxCD3 bispecific antibody. The antibody of the present invention exhibits minimal impact of exfoliated soluble CEA on its tumor cell-killing efficacy.

[0010] Exfoliated soluble CEACAM5 is an established tumor marker. Plasma sCEA levels in cancer patients can exceed 1000 ng / ml, while in healthy individuals plasma concentrations are below 10 ng / ml (e.g., Sandler B. et al., Anticancer Res 1999, 19(5B), 4229-33). Therefore, exfoliated 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 leading to reduced efficacy of anti-CEA antibodies or CEAxCD3 antibodies. TCB2017 and TCB2014 (see above) have been tested in vitro by the inventors in the presence of soluble CEA in an assay targeting T cell-mediated lysis of CEA-positive tumor cells. The addition of sCEA to the test was found to shift the fragmentation curve, thus shifting the EC50 values ​​of TCB2014 and TCB2017 to higher concentrations, indicating that both TCB2014 and TCB2017 are significantly bound to sCEA.

[0011] The human CEA family comprises 29 genes, 18 of which are expressed: 7 belong to the CEA subgroup and 11 to the pregnancy-specific glycoprotein subgroup. Several CEA subgroup members are thought to possess cell adhesion properties. CEACAM5 is expressed not only in colorectal cancer cells but also in pancreatic cancer, gastric cancer, lung cancer, and other cancer types. CEACAM5 is believed to play a role in innate immunity. 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-associated 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). Various monoclonal antibodies against CEACAM5 have been developed for research purposes (as a diagnostic tool) and for therapeutic purposes (see, for example, WO2012117002). Members of the carcinoembryonic antigen family (CEACAM) are widely expressed and, depending on the tissue, can regulate a variety of functions, including tumor promotion, tumor suppression, angiogenesis, and neutrophil activation. Four members of this family, CEACAM1, CEACAM3, CEACAM6, and CEACAM8, are expressed and enriched on human neutrophils (http: / / www.proteinatlas.org). Given the mechanism of action of CEAxCD3 bispecific antibodies, cross-reactivity with other CEACAMs may lead to the depletion of important circulating healthy cell populations. For example, cross-reactivity with CEACAM8, expressed by neutrophils or hematopoietic stem cells, may lead to the depletion of such cell populations. This invention provides a CEAxCD3 bispecific antibody with low cross-reactivity to one or more members of the CEACAM family: CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, and CEACAM21.

[0012] The mouse monoclonal anti-CEACAM5 antibody PR1A3 was generated by fusing NS1 (P3 / NS1 / I-Ag-4-1) myeloma cells with spleen cells from mice immunized with normal colorectal epithelium. The mouse monoclonal antibody PR1A3 was described by Richman PI and Bodmer WF, Int. J. Cancer, 39:317-328, 1987. Epitope mapping of PR1A3 showed that the 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 that PR1A3 binds to is a conformational epitope, not a linear epitope (Stewart et al., Cancer Immunol. Immunother., 47(1999)299-06). Humanized PR1A3 (hPR1A3) antibodies are described, for example, in Conaghhan PJ et al., Br. J. Cancer, 98(2008) 1217-1225 and WO2012117002. The CEA binder used in TCB2014 (named CH1A1A) is a humanized, affinity-matured, and stability-engineered form derived from PR1A3 antibodies. 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 USA 1994; 91:4313–7).

[0013] WO2017118657 describes a combination therapy for cancer using a human PD-1 axis antagonist and a bispecific anti-CEAxCD3 antibody; clinical results were presented at the 2017 ASCO meeting (Tabernero et al., J Clin Oncol 35, 2017 (suppl.abstr. 3002)). WO2015112534 describes a method for treating tumors by administering immune checkpoint antagonists that bind to two or more different targets of the immune checkpoint pathway and T-cell retargeting agents that bind to CEA and T-cell surface antigens. A conjugate consisting of a single-domain anti-CEACAM6 antibody and urease is currently in clinical trials (NCT02309892; WO2016116907). US20110064653 mentions a class I antibody that binds to CEACAM5, CEACAM6, and granulocytes. WO2018053328 mentions a bispecific antibody containing a first polypeptide chain and a second polypeptide chain that are covalently bound to each other.

[0014] The anti-CD3ε antibody described in the prior art is SP34 (Yang SJ, The Journal of Immunology (1986) 137; 1097-1100). SP34 reacts with CD3 in both primates and humans. SP34 is available from BDBiosciences. Another anti-CD3 antibody described in the prior art is UCHT-1 (see WO2000041474). Another anti-CD3 antibody described in the prior art is BC-3 (Fred Hutchinson Cancer Research Institute; for a phase I / II trial of GvHD, Anasetti et al., Transplantation 54:844 (1992)). SP34 differs from UCHT-1 and BC-3 in that SP-34 recognizes epitopes present only on the ε chain of CD3 (see Salmeron et al., (1991) J. Immunol. 147:3047), while UCHT-1 and BC-3 recognize epitopes facilitated by both the ε and γ chains. Anti-CD3 antibodies are also described in WO2007042261, WO2008119565, WO2008119566, WO2008119567, WO2010037836, WO2010037837, WO2010037838, and US8236308. Bispecific antibodies containing both a CEA-specific binding moiety and a CD3ε-specific binding moiety are described, for example, in US20140242079, WO2007071426, WO2013012414, WO2015112534, WO2017118675, and WO2017055389. Anti-CD3 antibodies containing the second binding portion of the antibody according to the invention are mentioned in US62 / 643,095 and PCT / US2019 / 000232, which are incorporated herein by reference in their entirety. US2012321626 mentions a multispecific Fab fusion protein comprising a Fab fragment that binds to the N-terminus of CD3ε. WO2018199593 mentions a bispecific antibody that binds to HER3 and CD3.

[0015] As mentioned above, the initial clinical trial results of the T-cell bispecific antibody TAA x CD3 (TAA = tumor-associated antigen) in patients with advanced solid tumors were disappointing. However, preliminary Phase 1 results for the CEAxCD3 bispecific antibody cyproxetine (RO6958688, see, for example, Bacac et al., Clin. Cancer Res., 22(13), 3286-97 (2016); and US20140242079) have recently been published, showing partial response and stable disease in patients with advanced colorectal cancer in both monotherapy and in combination with PD-L1 inhibitors (J. Tabernero et al., J. Clin. Oncol. 35, 2017 (suppl. Abstr. 3002)). Another approach to achieving better results may be to add not only inhibitors of the PD-1 checkpoint axis to the T-cell bispecific antibody, but also additional checkpoint inhibitors or agonists. However, to date, there are no promising clinical data believed for this combination approach.

[0016] The limited availability of T cells in advanced solid tumors is undoubtedly an important mechanism that limits the efficacy achievable with T cell bispecific antibodies plus PD-1 axis inhibitors.

[0017] Instead of adding another therapeutic agent designed to redirect T cells to fight tumor cells in advanced solid tumors to the combination of T cell bispecific antibodies and PD-1 axis inhibitors, it may be more successful to add the therapeutic agent redirected to tumor cells to other immune cells, especially macrophages or macrophages and natural killer (NK) cells.

[0018] This invention provides novel CEAxCD3 bispecific antibodies designed to be administered in parallel with CEAxCD47 bispecific antibodies targeting macrophages and NK cells to combat CEA-expressing solid tumors. The combined attack by T cells, macrophages, and NK cells targeting CEA-expressing tumors offers a significant opportunity for superior efficacy in killing and phagocytizing CEA-expressing tumor cells.

[0019] The disappointing results of CAR T cell therapy in solid tumors to date may have a simple explanation—the number of CAR T cells penetrating and distributing within the solid tumor is insufficient. This is certainly different in most hematologic malignancies; CAR T cells can access tumor cells very well, which explains the difference between the high efficacy in these malignancies and the disappointing efficacy in solid tumors. Furthermore, the tumor microenvironment (TME) of solid tumors (which is predominantly immunosuppressive) may severely suppress CAR T cell activity.

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

[0021] In one embodiment, the present invention relates to a bispecific antibody (also known as “bsAb CEAxCD3” or “CEAxCD3 bispecific antibody”) comprising a first binding portion that specifically binds to human CEACAM5 (also known as “CEA”) and a second binding portion that specifically binds to human CD3ε (also known as “CD3”).

[0022] In one embodiment, the bispecific antibody is characterized in that the antibody is monovalent for both the first binding portion and the second binding portion.

[0023] In one implementation, the bispecific antibody is characterized by constant and variable frame region sequences being human.

[0024] In one embodiment, the bispecific antibody is characterized in that the first and second binding portions each comprise an immunoglobulin heavy chain and an immunoglobulin light chain.

[0025] In one embodiment, the bispecific antibody has a first binding portion comprising a heavy chain and a second binding portion comprising a heavy chain, wherein the heavy chain in each binding portion is the same (i.e., a common heavy chain). In one embodiment, the common heavy chain variable region comprises CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4 as a CDR. In one embodiment, the common heavy chain variable region is SEQ ID NO: 1. In one embodiment, the common constant heavy chain is SEQ ID NO: 30. In one embodiment, the common heavy chain is SEQ ID NO: 43. In one embodiment, the common heavy chain is SEQ ID NO: 44. In one embodiment, the common heavy chain is SEQ ID NO: 45.

[0026] In one embodiment, the bispecific antibody is characterized by including a common heavy chain as a heavy chain in the first binding portion and the second binding portion, including a κ light chain as a light chain in the first binding portion, and including a λ light chain as a light chain in the second binding portion. In one embodiment, the light chain of the second binding portion is SEQ ID NO: 28, and the heavy chain of the second binding portion is SEQ ID NO: 45 (e.g., bispecific antibodies derived from AB1 and AB-1L3-1 / N, such as 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; CDR and VL sequences are listed in the sequence listing).

[0027] 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 known as 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 the CEAxCD3 bispecific antibody according to the present invention, which contains the WT hIgG1 Fc portion; ABXXL3-1 / D represents the CEAxCD3 bispecific antibody according to the present invention, which contains the hIgG1 Fc portion carrying the L234A+L235A mutation; ABXXL3-1 / N represents the CEAxCD3 bispecific antibody according to the present invention, which contains the hIgG1 Fc portion carrying the L234A+L235A+P329A mutation.

[0028] In one embodiment, the bispecific antibody is characterized by comprising a common heavy chain as a heavy chain in both the first and second binding portions, the first binding portion comprising a λ-type region as a variable region of the light chain and a κ-type region as a constant region of the light chain (“hybrid light chain”), and the second binding portion comprising a λ-light chain as a light chain (e.g., L3-1AB8 H-CK5 / D, see [link]). Figure 2 and Figure 2 (Description).

[0029] In one embodiment, the bispecific antibody is characterized by including a common heavy chain as a heavy chain in the first binding portion and the second binding portion, including a λ-type region as a variable region of the light chain and a λ-type region as a constant region of the light chain in the first binding portion, and including a λ-type region as a variable region of the light chain and a κ-type region as a constant region of the light chain (“hybrid light chain”) in the second binding portion; for example, AB8L3-1 H-CK5 / D.

[0030] The bispecific antibody of the present invention exhibits low binding / cross-reactivity against CEACAM family members other than CEACAM5. In one embodiment, the bispecific antibody is characterized by binding PEAKrapid cells expressing CEACAMs selected from CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, and CEACAM21 to WT PEAK cells (i.e., untransfected PEAK cells) compared to the MFI value of PEAKrapid cells (i.e., untransfected PEAK cells) bound under the same experimental conditions. CRL-2828 TM The MFI value of the bispecific antibody is not more than twice that of WT PEAK cells. In one embodiment, the bispecific antibody is characterized in that the MFI value of PEAKrapid cells expressing CEACAMs selected from CEACAM1, CEACAM3, CEACAM4, CEACAM6, and CEACAM8 is not more than twice that of WT PEAK cells under the same experimental conditions. In one embodiment, the bispecific antibody is characterized in that the MFI value of PEAKrapid cells expressing CEACAM8 is not more than twice that of WT PEAK cells under the same experimental conditions. The experimental procedures for transfecting PEAK cells and measuring the binding of the antibody to these PEAK cells are described in Examples 1 and 5.

[0031] In one embodiment, the bispecific antibody binds to MKN-45 cells (DSMZ No: 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 invention is characterized in that the MFI value of binding to MKN-45 cells at 200 nM, 1000 nM, and 5000 nM is at least twice the MFI value obtained with TCB2014. Binding assays are described in Example 7a. In one embodiment, the EC50 of the bispecific antibody of the invention for the tumor-killing cell line MKN-45, measured in an assay containing human PBMCs, is 40% or more lower than the EC50 measured against TCB2014. In one embodiment, the EC50 of the bispecific antibody of the invention for the tumor-killing cell line LS-174T, measured in an assay containing human PBMCs, is 40% or more lower than the EC50 measured against TCB2014.

[0032] In one embodiment, in an assay containing human PBMCs, the bispecific antibody kills LS174T cells in a concentration-dependent manner, with an EC50 value of 0.01 to 10 nM. In another embodiment, in an assay containing human PBMCs, the bispecific antibody kills LS174T cells in a concentration-dependent manner, with an EC50 value of 0.01 to 1 nM.

[0033] The assay for measuring the lysis / killing of T cells that retarget CEA-positive cells is described in Example 8.

[0034] In one embodiment, the bispecific antibody is characterized in that, compared with the EC50 value in the absence of soluble CEACAM5 cleavage under the same experimental conditions, the EC50 value in the presence of 5 μg / ml soluble CEACAM5 in the same assay (killing CEA-positive LS174T tumor cells) is increased by no more than 20-fold in one embodiment, no more than 15-fold in another embodiment, and no more than 10-fold in yet another embodiment.

[0035] In another embodiment, the bispecific antibody is characterized in that, compared with the EC50 value in the absence of soluble CEACAM5 cleavage under the same experimental conditions, the EC50 value in the presence of 1 μg / ml soluble CEACAM5 in the same assay increases by no more than 10-fold, and in one embodiment by no more than 5-fold.

[0036] In one embodiment, the bispecific antibody is characterized in that, compared with tumor volume growth in the mediator group under the same experimental conditions, the bispecific antibody inhibits tumor volume growth by 25% or more up to day 18 in the HPAF-II model. In one embodiment, the bispecific antibody is characterized in that, compared with TCB2014 under the same experimental conditions, the bispecific antibody shows similar and statistically insignificant inhibition of tumor volume growth up to day 18 in the HPAF-II model. A mouse tumor model is described in Example 9a.

[0037] In one embodiment, the bispecific antibody contains amino acid substitutions in each subunit of the Fc domain that reduce binding to the activated Fc receptor and / or reduce effector function, wherein said amino acid substitutions are L234A and L235A and / or P329A selected from P329A, P329G, and P329R (Kabat EU index numbers). In one embodiment, the bispecific antibody contains amino acid substitutions L234A and L235A and P329A (Kabat EU index numbers) in each subunit of the Fc domain. L234A and L235A (LALA) refer to the substitution of leucine at position 234 / 235 with alanine. P329A (PA) refers to the substitution of proline at position 329 with alanine.

[0038] In one embodiment, the bispecific antibody comprises a common heavy chain. In one embodiment, the bispecific antibody comprises a common heavy chain containing CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4 as a CDR. In one embodiment, the bispecific antibody comprises a light chain region containing CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20 as a CDR in the second binding portion.

[0039] In one embodiment, the bispecific antibody includes the region of SEQ ID NO: 39 as a light chain constant region in the first binding region. In one embodiment, the bispecific antibody includes the region of SEQ ID NO: 41 as a light chain constant region in the first binding region. In one embodiment, the bispecific antibody includes the region of SEQ ID NO: 58 as a light chain constant region in the first binding region. In one embodiment, the bispecific antibody includes the common heavy chain of SEQ ID NO: 43, or the common heavy chain of SEQ ID NO: 44, or the common heavy chain of SEQ ID NO: 45. In one embodiment, the bispecific antibody includes the region of SEQ ID NO: 39 and the common heavy chain of SEQ ID NO: 45 as light chain constant regions in the first binding region.

[0040] In one embodiment, the bispecific antibody comprises, as a light chain, a light chain selected from the group consisting of SEQ ID NO: 25, 26, 27, 28 and 29 or a group consisting of hybrid light chains (LC) selected from the group consisting of SEQ ID NO: 67, 68, 69, 70 and 71, as a light chain in the second binding portion.

[0041] In one embodiment, the bispecific antibody comprises a region of SEQ ID NO: 39 as a light chain constant region in the first binding portion, a common heavy chain of SEQ ID NO: 45, and a light chain of SEQ ID NO: 28 in the second binding portion.

[0042] In one embodiment, the bispecific antibody competes with an anti-CEA antibody selected from: anti-CEA antibodies comprising VL and VH as VL and VH domains (anti-CEA antibody MEDI), comprising VL and VH as VL and VH domains (antibody SM3E), comprising VL and VH as VL and VH domains (SEQ ID NO: 48 and 49), comprising VL and VH as VL and VH domains (Labetuzumab (Lab)), comprising VL and VH as VL and VH domains (SEQ ID NO: 50 and 51), comprising VL and VH as VL and VH domains (T86.66), and comprising VL and VH as VL and VH domains (SEQ ID NO: 52 and 53) (CH1A1A). See also Figure 1 (and Example 5c).

[0043] Examples of antibodies that can be used as the CEA VL or CL region in the bispecific antibodies according to the present invention and compete with MEDI for binding to recombinant CEA are anti-CEA antibody AB1 and antibodies obtained through leader optimization of AB1 (experimental methods see Example 11). Anti-CEA antibodies AB13, 14, 15, 17, 20, 54, 60, 66, 71, 72, 73 and the corresponding bispecific anti-CEAxCD3 antibodies AB13L3-1, AB14L3-1, AB15L3-1, AB17L3-1, AB20L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1 and AB73L3-1 compete with antibody AB1 rsp.AB1L3-1 in the same manner. Examples of antibodies that can be used as the CEA VL or CL region in the bispecific antibody according to the present invention and compete with SM3E for binding to recombinant CEA are anti-CEA antibody AB8 and antibodies obtained by oligonucleotide-directed mutagenesis of AB8 using degenerate oligonucleotides. Examples of antibodies that can be used as the CEA VL or CL region in the bispecific antibody according to the present invention and compete with T84.66 for binding to recombinant CEA are anti-CEA antibody 1B4 and antibodies obtained by oligonucleotide-directed mutagenesis of 1B4 using degenerate oligonucleotides.

[0044] Examples of antibodies that can be used as the CEA VL or CL region in the bispecific antibodies according to the present invention but do not compete with any reference antibody for binding recombinant CEA are anti-CEA antibody C11 and antibodies obtained by oligonucleotide-directed mutagenesis of C11 using degenerate oligonucleotides.

[0045] AB1 is an anti-CEA antibody having HC SEQ ID NO: 43 and κLC SEQ ID NO: 40, encoded by the nucleic acid sequences shown in SEQ ID NO: 80 and 78, respectively.

[0046] AB8 is an anti-CEA antibody having HC SEQ ID NO: 43 and λLC SEQ ID NO: 42, encoded by the nucleic acid sequences shown in SEQ ID NO: 80 and 79, respectively.

[0047] 1B4 is an anti-CEA antibody having HC SEQ ID NO: 43 and λLC SEQ ID NO: 74, encoded by the nucleic acid sequences shown in SEQ ID NO: 80 and 77, respectively.

[0048] C11 is an anti-CEA antibody having HC SEQ ID NO: 43 and κLC SEQ ID NO: 73, encoded by the nucleic acid sequences shown in SEQ ID NO: 80 and 76, respectively.

[0049] The CEA light chain that can be used as the κ light chain is SEQ ID NO: 40. The CEA light chain that can be used as the κ light chain is SEQ ID NO: 73. The CEA light chain that can be used as the λ light chain is SEQ ID NO: 74, and the CEA light chain that can be used as the hybrid κ light chain is SEQ ID NO: 75.

[0050] In one embodiment, the bispecific antibody contains up to three amino acid substitutions in each subunit of the Fc domain, which reduce binding and / or effector function with the activated Fc receptor, wherein the amino acid substitutions are substitutions for L234A, L235A, and P329 selected from P329A, P329G, and P329R (Kabat EU index number). In one embodiment, the common heavy chain of the antibody according to the invention is SEQ ID NO: 43, 44, or 45. In one embodiment, the common heavy chain of the antibody according to the invention is SEQ ID NO: 45 (L234A, L235A, and P329A).

[0051] In one embodiment, the bispecific antibody exhibits one or more properties selected from: a) binding to MKN-45 cells at an EC50 value of 0.5 nM to 50 nM, b1) competing with an anti-CEA antibody comprising the VL and VH (MEDI) sequences SEQ ID NO: 48 and 49 as VL and VH domains, or b2) competing with an anti-CEA antibody comprising the VL and VH (SM3E) sequences SEQ ID NO: 46 and 47 as VL and VH domains, or b3) competing with an anti-CEA antibody comprising the sequence SEQ ID NO: 48 and 49 as VL and VH domains. NO: 54 and 55 of VL and CH (T84.66), or b4) not competing with any tool antibody (see Example 5c for tool antibodies), c) containing amino acid substitutions in each subunit of the Fc domain that reduce binding and / or effector function with the activated Fc receptor, wherein the amino acid substitutions are substitutions of L234A and L235A and P329 selected from P329A, P329G and P329R (Kabat EU index number), d) killing MKN-45, HPAF-II and / or LS174T cells in a concentration-dependent manner in assays containing human PBMCs, with EC50 values ​​from 0.01 to 10 nM.

[0052] In one embodiment, the bispecific antibody exhibits one or more properties selected from the following:

[0053] a) Binding to MKN-45 cells, with EC50 values ​​ranging from 0.5 nM to 50 nM.

[0054] b) In assays containing human PBMCs, MKN-45, HPAF-II, or LS174T cells were killed in a concentration-dependent manner, with EC50 values ​​ranging from 0.01 to 10 nM.

[0055] c) It binds to PEAK cells expressing CEACAM5, but does not cross-react with PEAK cells expressing CEACAM8.

[0056] d) When LS174T tumor cells were used as target cells, the cytotoxic EC50 in the TDCC assay (Example 8) increased by no more than 5-fold in the presence of 1 μg / mL sCEA.

[0057] e) Compared with the control group (mediator only), tumor growth in the HPAF-II model was inhibited by 25% or more.

[0058] f) Competing with anti-CEA antibodies containing VL and VH (MEDI) as VL and VH domains (SEQ ID NO: 48 and 49), and

[0059] g) Each subunit of the Fc domain contains amino acid substitutions for L234A, L235A, and P329A (Kabat EU index number).

[0060] In one embodiment, the bispecific antibody exhibits properties a) through d). In one embodiment, the bispecific antibody exhibits all properties a) through f). In one embodiment, the bispecific antibody exhibits properties a) through d) and g). In one embodiment, the bispecific antibody exhibits properties a) through d) and f) and g). In one embodiment, the bispecific antibody exhibits all properties a) through g).

[0061] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that...

[0062] a) The first and second binding portions each contain a common heavy chain (cHC) as a heavy chain and a variable region as a variable region, which contains CDRH1 (SEQ ID NO: 2), CDRH2 (SEQ ID NO: 3), and CDRH3 (SEQ ID NO: 4) as CDRH1, CDRH2, and CDRH3, respectively.

[0063] b) The first connecting part includes

[0064] i) The light chain constant region (CL) and light chain variable region (VL), including CDRL1 of SEQ ID NO: 32, CDRL2 of SEQ ID NO: 33, and CDRL3 of SEQ ID NO: 34 as CDRL1, CDRL2, and CDRL3, or derived from the light chain variable region of SEQ ID NO: 31 by oligonucleotide directed mutagenesis using degenerate oligonucleotides.

[0065] c) The second binding portion includes a light chain variable region containing a set of CDRs as CDRL1, CDRL2, and CDRL3, selected from:

[0066] I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8

[0067] II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12,

[0068] III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16,

[0069] IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and

[0070] V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24.

[0071] d) The second binding part contains the constant region of the λ light chain.

[0072] In one embodiment, the second binding portion in c) comprises a light chain selected from SEQ ID NO: 25, 26, 27, 28 and 29.

[0073] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that...

[0074] a) The first and second binding portions each contain a heavy chain as a heavy chain, which includes CDRH1 as SEQ ID NO: 2, CDRH2 as SEQ ID NO: 3, and CDRH3 as SEQ ID NO: 4, respectively.

[0075] b) The first binding portion comprises a light chain variable region (VL) containing CDRL1, derived from SEQ ID NO: 32 by directed mutagenesis using degenerate oligonucleotides and containing up to four amino acid substitutions, CDRL2, derived from SEQ ID NO: 33 by directed mutagenesis using degenerate oligonucleotides and containing up to four amino acid substitutions, and CDRL3, derived from SEQ ID NO: 34 by directed mutagenesis using degenerate oligonucleotides and containing up to four amino acid substitutions.

[0076] c) The second binding portion includes 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 as CDRL1, CDRL2, and CDRL3.

[0077] Such bispecific antibodies are, but are not limited to, bispecific anti-CEAxCD3 antibodies AB13L3-1, AB14L3-1, AB15L3-1, AB17L3-1, AB20L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1, and AB73L3-1.

[0078] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that:

[0079] a) The first binding portion includes the heavy chain variable region VH, which includes CDRH1 as SEQ ID NO: 2, CDRH2 as SEQ ID NO: 3, and CDRH3 as SEQ ID NO: 4.

[0080] b) The first binding portion includes a light chain variable region as a light chain variable region, which contains a selection from the following CDRL group.

[0081] b1) CDRL1 (SEQ ID NO: 32), CDRL2 (SEQ ID NO: 33), and CDRL3 (SEQ ID NO: 34),

[0082] b2) CDRL1 of SEQ ID NO: 81, CDRL2 of SEQ ID NO: 82, and CDRL3 of SEQ ID NO: 83,

[0083] b3) CDRL1 of SEQ ID NO: 84, CDRL2 of SEQ ID NO: 85, and CDRL3 of SEQ ID NO: 86,

[0084] b4) CDRL1 of SEQ ID NO: 87, CDRL2 of SEQ ID NO: 88, and CDRL3 of SEQ ID NO: 89,

[0085] b5) CDRL1 (SEQ ID NO: 90), CDRL2 (SEQ ID NO: 91), and CDRL3 (SEQ ID NO: 92),

[0086] b6) CDRL1 (SEQ ID NO: 93), CDRL2 (SEQ ID NO: 94), and CDRL3 (SEQ ID NO: 95),

[0087] b7) CDRL1 (SEQ ID NO: 96), CDRL2 (SEQ ID NO: 97), and CDRL3 (SEQ ID NO: 98),

[0088] b8) CDRL1 (SEQ ID NO: 99), CDRL2 (SEQ ID NO: 100), and CDRL3 (SEQ ID NO: 101)

[0089] b9) CDRL1 of SEQ ID NO: 102, CDRL2 of SEQ ID NO: 103, and CDRL3 of SEQ ID NO: 104,

[0090] b10) CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106, and CDRL3 of SEQ ID NO: 107,

[0091] b11) CDRL1 of SEQ ID NO: 108, CDRL2 of SEQ ID NO: 109, and CDRL3 of SEQ ID NO: 110, and

[0092] b12) CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, and CDRL3 of SEQ ID NO: 113, and

[0093] c) The second binding portion includes the heavy chain variable region VH, which includes CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3 and CDRH3 of SEQ ID NO: 4, and the light chain variable region VL, which includes CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19 and CDRL3 of SEQ ID NO: 20.

[0094] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that:

[0095] a) The first binding portion includes the heavy chain variable region VH, which includes CDRH1 as SEQ ID NO: 2, CDRH2 as SEQ ID NO: 3, and CDRH3 as SEQ ID NO: 4.

[0096] b) The first binding portion includes a light chain variable region as a light chain variable region, which contains a selection from the following CDRL group.

[0097] b1) CDRL1 (SEQ ID NO: 90), CDRL2 (SEQ ID NO: 91), and CDRL3 (SEQ ID NO: 92),

[0098] b2) CDRL1 (SEQ ID NO: 96), CDRL2 (SEQ ID NO: 97), and CDRL3 (SEQ ID NO: 98)

[0099] b3) CDRL1 (SEQ ID NO: 99), CDRL2 (SEQ ID NO: 100), and CDRL3 (SEQ ID NO: 101)

[0100] b4) CDRL1 of SEQ ID NO: 102, CDRL2 of SEQ ID NO: 103, and CDRL3 of SEQ ID NO: 104,

[0101] b5) CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106, and CDRL3 of SEQ ID NO: 107, and

[0102] b6) CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, and CDRL3 of SEQ ID NO: 113, and

[0103] c) The second binding portion includes the heavy chain variable region VH, which includes CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3 and CDRH3 of SEQ ID NO: 4, and the light chain variable region VL, which includes CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19 and CDRL3 of SEQ ID NO: 20.

[0104] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that:

[0105] a) The first binding portion includes the heavy chain variable region VH, which includes CDRH1 as SEQ ID NO: 2, CDRH2 as SEQ ID NO: 3, and CDRH3 as SEQ ID NO: 4.

[0106] b) The first binding portion includes a light chain variable region as a light chain variable region, which contains a selection from the following CDRL group.

[0107] b1) CDRL1 (SEQ ID NO: 90), CDRL2 (SEQ ID NO: 91), and CDRL3 (SEQ ID NO: 92),

[0108] b2) CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106, and CDRL3 of SEQ ID NO: 107, and

[0109] b3) CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, and CDRL3 of SEQ ID NO: 113, and

[0110] c) The second binding portion includes the heavy chain variable region VH, which includes CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3 and CDRH3 of SEQ ID NO: 4, and the light chain variable region VL, which includes CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19 and CDRL3 of SEQ ID NO: 20.

[0111] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that it comprises...

[0112] a) The heavy chain variable region VH in the first binding portion having 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 1, comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 3, and CDR3 of SEQ ID NO: 4, and

[0113] b) The variable region VL of the light chain, which is selected from...

[0114] b1) Having 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 31 and containing the light chain variable region VL of CDRL1 of SEQ ID NO: 32, CDRL2 of SEQ ID NO: 33, and CDRL3 of SEQ ID NO: 34.

[0115] b2) Having 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 114 and containing the light chain variable region VL of CDRL1 of SEQ ID NO: 81, CDRL2 of SEQ ID NO: 82, and CDRL3 of SEQ ID NO: 83.

[0116] b3) has 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 115 and contains the light chain variable region VL of CDRL1 of SEQ ID NO: 84, CDRL2 of SEQ ID NO: 85, and CDRL3 of SEQ ID NO: 86.

[0117] b4) has 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 116 and contains the light chain variable region VL of CDRL1 of SEQ ID NO: 87, CDRL2 of SEQ ID NO: 88, and CDRL3 of SEQ ID NO: 89.

[0118] b5) has 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 117 and contains the light chain variable region VL of CDRL1 of SEQ ID NO: 90, CDRL2 of SEQ ID NO: 91, and CDRL3 of SEQ ID NO: 92.

[0119] b6) has 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 118 and contains the light chain variable region VL of CDRL1 of SEQ ID NO: 93, CDRL2 of SEQ ID NO: 94, and CDRL3 of SEQ ID NO: 95.

[0120] b7) has 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 119 and contains the light chain variable region VL of CDRL1 of SEQ ID NO: 96, CDRL2 of SEQ ID NO: 97, and CDRL3 of SEQ ID NO: 98.

[0121] b8) has 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 120 and contains the light chain variable region VL of CDRL1 of SEQ ID NO: 99, CDRL2 of SEQ ID NO: 100, and CDRL3 of SEQ ID NO: 101.

[0122] b9) has 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 121 and contains the light chain variable region VL of CDRL1 of SEQ ID NO: 102, CDRL2 of SEQ ID NO: 103, and CDRL3 of SEQ ID NO: 104.

[0123] b10) has 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 122 and contains the light chain variable region VL of CDRL1 of SEQ ID NO: 105, CDRL2 of SEQ ID NO: 106, and CDRL3 of SEQ ID NO: 107.

[0124] b11) has 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 123 and contains the light chain variable region VL of CDRL1 of SEQ ID NO: 108, CDRL2 of SEQ ID NO: 109, and CDRL3 of SEQ ID NO: 110.

[0125] b12) has 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 124 and contains the light chain variable region VL of CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, and CDRL3 of SEQ ID NO: 113.

[0126] c) The heavy chain variable region VH of the second binding portion 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, and the light chain variable region VL having 97%, 98%, 99%, or 100% amino acid identity with SEQ ID NO: 17 and comprising CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20.

[0127] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that it comprises...

[0128] a) The heavy chain variable region VH SEQ ID NO: 1 in the first binding portion, and

[0129] b) The variable region VL of the light chain, which is selected from...

[0130] b1) The light chain variable region VL of SEQ ID NO: 31,

[0131] b2) The light chain variable region VL of SEQ ID NO: 114,

[0132] b3) The light chain variable region VL of SEQ ID NO: 115,

[0133] b4) The light chain variable region VL of SEQ ID NO: 116

[0134] b5) The light chain variable region VL of SEQ ID NO: 117,

[0135] b6) The light chain variable region VL of SEQ ID NO: 118,

[0136] b7) The light chain variable region VL of SEQ ID NO: 119

[0137] b8) The light chain variable region VL of SEQ ID NO: 120

[0138] b9) The light chain variable region VL of SEQ ID NO: 121,

[0139] b10) SEQ ID NO: 122 light chain variable region VL,

[0140] b11) SEQ ID NO: 123 light chain variable region VL, and

[0141] b12) SEQ ID NO: 124 light chain variable region VL, and

[0142] c) The heavy chain variable region VH of SEQ ID NO: 1 and the light chain variable region VL of SEQ ID NO: 17 in the second binding portion.

[0143] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that it comprises...

[0144] a) The heavy chain variable region VH SEQ ID NO: 1 in the first binding portion, and

[0145] b) The variable region VL of the light chain, which is selected from...

[0146] b1) The light chain variable region VL of SEQ ID NO: 117,

[0147] b2) The light chain variable region VL of SEQ ID NO: 119,

[0148] b3) The light chain variable region VL of SEQ ID NO: 120,

[0149] b4) The light chain variable region VL of SEQ ID NO: 121,

[0150] b5) The light chain variable region VL of SEQ ID NO: 122, and

[0151] b6) The light chain variable region VL of SEQ ID NO: 124, and

[0152] c) The heavy chain variable region VH of SEQ ID NO: 1 and the light chain variable region VL of SEQ ID NO: 17 in the second binding portion.

[0153] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that it comprises...

[0154] a) The heavy chain variable region VH SEQ ID NO: 1 in the first binding portion, and

[0155] b) The variable region VL of the light chain, which is selected from...

[0156] b1) The light chain variable region VL of SEQ ID NO: 117,

[0157] b2) The light chain variable region VL of SEQ ID NO: 122, and

[0158] b3) The light chain variable region VL of SEQ ID NO: 124, and

[0159] c) The heavy chain variable region VH of SEQ ID NO: 1 and the light chain variable region VL of SEQ ID NO: 17 in the second binding portion.

[0160] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that the first binding portion contains

[0161] a) Heavy chain variable region VH SEQ ID NO: 1, and

[0162] b) Light chains, selected from:

[0163] b1) Light chain of SEQ ID NO: 40

[0164] b2) The light chain of SEQ ID NO: 125,

[0165] b3) The light chain of SEQ ID NO: 126

[0166] b4) Light chain of SEQ ID NO: 127

[0167] b5) Light chain of SEQ ID NO: 128,

[0168] b6) Light chain of SEQ ID NO: 129

[0169] b7) Light chain of SEQ ID NO: 130

[0170] b8) Light chain of SEQ ID NO: 131,

[0171] b9) Light chain of SEQ ID NO: 132

[0172] b10) SEQ ID NO: 133 light chain, and

[0173] b11) The light chain of SEQ ID NO: 134, and

[0174] b12) SEQ ID NO: 135 light chain, and

[0175] c) The heavy chain variable region VH of SEQ ID NO: 1 and the light chain of SEQ ID NO: 28 in the second binding portion.

[0176] In one embodiment, the present invention relates to a bispecific antibody according to the present invention, characterized in that the first binding portion contains

[0177] a) Common heavy chains, selected from:

[0178] a1) Heavy chain of SEQ ID NO: 43

[0179] a2) The heavy chain of SEQ ID NO: 44, or

[0180] a3) The heavy chain of SEQ ID NO: 45, and

[0181] b) Light chains, selected from:

[0182] b1) The light chain of SEQ ID NO: 40,

[0183] b2) The light chain of SEQ ID NO: 125,

[0184] b3) The light chain of SEQ ID NO: 126

[0185] b4) Light chain of SEQ ID NO: 127

[0186] b5) Light chain of SEQ ID NO: 128,

[0187] b6) Light chain of SEQ ID NO: 129

[0188] b7) Light chain of SEQ ID NO: 130

[0189] b8) Light chain of SEQ ID NO: 131,

[0190] b9) Light chain of SEQ ID NO: 132

[0191] b10) light chain of SEQ ID NO: 133,

[0192] b11) light chain of SEQ ID NO: 134, or

[0193] b12) SEQ ID NO: 135 light chain, and

[0194] c) The light chain of SEQ ID NO: 28 in the second binding portion.

[0195] 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 a light chain (1A4 LC, L3-1) of SEQ ID NO: 28 as a light chain in the second binding portion, and a light chain (AB17) of SEQ ID NO: 128 as a light chain in the first binding portion. 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 a light chain (L3-1) of SEQ ID NO: 28 as a light chain in the second binding portion, and a light chain (AB71) of SEQ ID NO: 133 as a light chain in the first binding portion. In one embodiment, the bispecific antibody (AB73L3-1 / N) according to the invention comprises a common heavy chain ( / N) of SEQ ID NO: 45 and a light chain (L3-1) of SEQ ID NO: 28 as a light chain in the second binding portion and a light chain (AB73) of SEQ ID NO: 135 as a light chain in the first binding portion.

[0196] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that...

[0197] a) The first and second binding portions each contain a common heavy chain as a heavy chain and a variable region as a variable region, which contains CDRH1 (SEQ ID NO: 2), CDRH2 (SEQ ID NO: 3), and CDRH3 (SEQ ID NO: 4) as CDRH1, CDRH2, and CDRH3, respectively.

[0198] b) The first connecting part includes

[0199] i) λ light chain constant region (CL) and

[0200] ii) Light chain variable region (VL), comprising CDRL1 as CDRL1 of SEQ ID NO: 36, CDRL2 of SEQ ID NO: 37, and CDRL3 of SEQ ID NO: 38, or derived from the light chain variable region of SEQ ID NO: 35 by oligonucleotide directed mutagenesis using degenerate oligonucleotides.

[0201] c) The second binding portion includes a light chain variable region containing a set of CDRs as CDRL1, CDRL2, and CDRL3, selected from:

[0202] I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8

[0203] II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12,

[0204] III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16,

[0205] IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and

[0206] V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24.

[0207] d) The second binding region contains the constant region of the heterozygous κ chain.

[0208] In one embodiment, the second binding portion in c) comprises a light chain selected from SEQ ID NO: 67, 68, 69, 70, and 71.

[0209] In one embodiment, the second binding portion may include the λ light chain constant region of SEQ ID NO: 41 as the light chain constant region; in this case, the first binding portion in one embodiment includes the heterozygous κ light chain region of SEQ ID NO: 58 as the light chain constant region. In one embodiment, the arm carrying the heterozygous light chain constant region is based on the overall properties of bsAb, including but not limited to stability and productivity.

[0210] In one embodiment, the light chain variable region of the first binding portion is derived from SEQ ID NO: 35 by oligonucleotide-directed mutagenesis using degenerate oligonucleotides, the common heavy chain is SEQ ID NO: 45, and the variable region of the common heavy chain is SEQ ID NO: 1.

[0211] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that...

[0212] a) The first and second binding portions each contain a common heavy chain as a heavy chain and a variable region as a variable region, which contains CDRH1 (SEQ ID NO: 2), CDRH2 (SEQ ID NO: 3), and CDRH3 (SEQ ID NO: 4) as CDRH1, CDRH2, and CDRH3, respectively.

[0213] b) The first connecting part includes

[0214] i) The constant region (CL) of the κ light chain, and

[0215] ii) Light chain variable region (VL), comprising CDRL1 as 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 derived from SEQ ID NO: 63 via oligonucleotide directed mutagenesis.

[0216] c) The second binding portion includes a light chain variable region containing a set of CDRs as CDRL1, CDRL2, and CDRL3, selected from:

[0217] I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8

[0218] II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12,

[0219] III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16,

[0220] IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and

[0221] V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24.

[0222] d) The second binding part contains the constant region of the λ light chain.

[0223] In one embodiment, the second binding portion in c) comprises a light chain selected from SEQ ID NO: 25, 26, 27, 28 and 29.

[0224] In one embodiment of the invention, the light chain variable region of the first binding portion is derived from SEQ ID NO: 63 by oligonucleotide-directed mutagenesis using degenerate oligonucleotides, and the common heavy chain is SEQ ID NO: 45.

[0225] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that...

[0226] a) The first and second binding portions each contain a common heavy chain as a heavy chain and a variable region as a variable region, which contains CDRH1 (SEQ ID NO: 2), CDRH2 (SEQ ID NO: 3), and CDRH3 (SEQ ID NO: 4) as CDRH1, CDRH2, and CDRH3, respectively.

[0227] b) The first binding region comprises a λ light chain constant region (CL) and a light chain variable region (VL), including CDRL1 as SEQ ID NO: 60, CDRL2 as SEQ ID NO: 61, and CDRL3 as SEQ ID NO: 62, or derived from the light chain variable region of SEQ ID NO: 59 by oligonucleotide directed mutagenesis using degenerate oligonucleotides.

[0228] c) The second binding portion includes a light chain variable region containing a set of CDRs as CDRL1, CDRL2, and CDRL3, selected from:

[0229] I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8

[0230] II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12,

[0231] III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16,

[0232] IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and

[0233] V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24.

[0234] d) The second binding region contains the constant region of the heterozygous κ chain.

[0235] In another embodiment of the invention, the second binding portion comprises the λ light chain constant region of SEQ ID NO: 41 as the light chain constant region; in this case, the first binding portion in one embodiment comprises the hybrid κ light chain constant region of SEQ ID NO: 58 as the light chain constant region. The selection of the arm carrying the hybrid light chain constant region is based on the overall properties of the final bsAb, including but not limited to stability and productivity.

[0236] In one embodiment, the light chain variable region of the first binding portion is derived from SEQ ID NO: 59 by oligonucleotide-directed mutagenesis using degenerate oligonucleotides.

[0237] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that...

[0238] a) The first and second binding portions each contain a common heavy chain as a heavy chain and a variable region as a variable region, which contains CDRH1 as SEQ ID NO: 2, CDRH2 as SEQ ID NO: 3, and CDRH3 as SEQ ID NO: 4.

[0239] b) The first binding region comprises a constant region of the human κ-type light chain and a variable region of the human κ-type light chain, including CDRL1 (SEQ ID NO: 32) having substitutions of 0, 1, 2, 3, or 4 amino acids as CDRL1, CDRL2 (SEQ ID NO: 33) having substitutions of 0, 1, 2, 3, or 4 amino acids as CDRL1, CDRL2, and CDRL3 (SEQ ID NO: 34) having substitutions of 0, 1, 2, 3, 4, or 5 amino acids as CDRL3.

[0240] c) The second binding portion comprises a constant region of the human-laminar light chain and a variable region of the human-laminar light chain, and includes a set of CDRs as CDRL1, CDRL2, and CDRL3, selected from:

[0241] I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8

[0242] II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12,

[0243] III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16,

[0244] IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and

[0245] V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24.

[0246] In one embodiment, the second binding portion in c) comprises a light chain selected from SEQ ID NO: 25, 26, 27, 28 and 29.

[0247] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that...

[0248] a) The first and second binding portions each contain a common heavy chain as a heavy chain and a variable region as a variable region, which contains CDRH1 (SEQ ID NO: 2), CDRH2 (SEQ ID NO: 3), and CDRH3 (SEQ ID NO: 4) as CDRH1, CDRH2, and CDRH3, respectively.

[0249] b) The first binding portion comprises a constant region of the human λ-type light chain and a variable region of the human λ-type light chain, comprising the CDRL group as CDRL1, CDRL2, and CDRL3, selected from CDRL1 of SEQ ID NO: 36 having substitutions of 0, 1, 2, 3, 4, or 5 amino acids, CDRL2 of SEQ ID NO: 37 having substitutions of 0, 1, 2, 3, 4, or 5 amino acids, and CDRL3 of SEQ ID NO: 38 having substitutions of 0, 1, 2, 3, 4, or 5 amino acids.

[0250] c) The second binding region comprises a constant region of the heterozygous κ light chain and a variable region of the human λ-type light chain, containing a set of CDRs as CDRL1, CDRL2, and CDRL3, selected from:

[0251] I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8

[0252] II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12,

[0253] III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16,

[0254] IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and

[0255] V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24.

[0256] In one embodiment, the second binding portion in c) comprises a light chain selected from SEQ ID NO: 67, 68, 69, 70 and 71.

[0257] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that...

[0258] a) The first and second binding portions each contain a common heavy chain as a heavy chain and a variable region as a variable region, which contains CDRH1 (SEQ ID NO: 2), CDRH2 (SEQ ID NO: 3), and CDRH3 (SEQ ID NO: 4) as CDRH1, CDRH2, and CDRH3, respectively.

[0259] b) The first binding portion comprises a constant region of the human λ-type light chain and a variable region of the human λ-type light chain, comprising the CDRL group as CDRL1, CDRL2, and CDRL3, selected from CDRL1 of SEQ ID NO: 60 having substitutions of 0, 1, 2, 3, or 4 amino acids, CDRL2 of SEQ ID NO: 61 having substitutions of 0, 1, 2, 3, or 4 amino acids, and CDRL3 of SEQ ID NO: 62 having substitutions of 0, 1, 2, 3, 4, or 5 amino acids.

[0260] c) The second binding region comprises a constant region of the human κ-type light chain and a variable region of the human λ-type light chain, and includes a set of CDRs as CDRL1, CDRL2, and CDRL3, selected from:

[0261] I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8

[0262] II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12,

[0263] III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16,

[0264] IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and

[0265] V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24.

[0266] In one embodiment, the second binding portion in c) comprises a light chain selected from SEQ ID NO: 67, 68, 69, 70 and 71.

[0267] In one embodiment, the bispecific antibody is characterized by comprising a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3ε, characterized in that...

[0268] a) The first and second binding portions each contain a common heavy chain as a heavy chain and a variable region as a variable region, which contains CDRH1 (SEQ ID NO: 2), CDRH2 (SEQ ID NO: 3), and CDRH3 (SEQ ID NO: 4) as CDRH1, CDRH2, and CDRH3, respectively.

[0269] b) The first binding portion comprises a constant region of the human κ-type light chain and a variable region of the human κ-type light chain, comprising the CDRL group as CDRL1, CDRL2, and CDRL3, selected from CDRL1 of SEQ ID NO: 64 having substitutions of 0, 1, 2, 3, 4, or 5 amino acids, CDRL2 of SEQ ID NO: 65 having substitutions of 0, 1, 2, 3, 4, or 5 amino acids, and CDRL3 of SEQ ID NO: 66 having substitutions of 0, 1, 2, 3, 4, or 5 amino acids.

[0270] c) The second binding portion comprises a constant region of the human-laminar light chain and a variable region of the human-laminar light chain, and includes a set of CDRs as CDRL1, CDRL2 and CDRL3, selected from I) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7 and CDRL3 of SEQ ID NO: 8.

[0271] II) CDRL1 of SEQ ID NO: 10, CDRL2 of SEQ ID NO: 11, and CDRL3 of SEQ ID NO: 12,

[0272] III) CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16,

[0273] IV) CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19, and CDRL3 of SEQ ID NO: 20, and

[0274] V) CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24.

[0275] In one embodiment, the second binding portion in c) comprises a light chain selected from SEQ ID NO: 25, 26, 27, 28 and 29.

[0276] 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, wherein:

[0277] a) The first binding portion includes a heavy chain variable region (VH), which includes CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4.

[0278] b) The first binding portion includes a light chain variable region (VL) containing the CDRL group, wherein CDRL1 has the shared sequence of SEQ ID NO: 136, CDRL2 has the shared sequence of SEQ ID NO: 137, and CDRL3 has the shared sequence of SEQ ID NO: 138, and

[0279] c) The second binding portion includes VH, which includes CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4.

[0280] In one embodiment, the first binding portion comprises the frame sequence of SEQ ID NO: 31, which is a variable light chain frame sequence. In one embodiment, the first binding portion comprises the frame sequence of SEQ ID NO: 35, which is a variable light chain frame sequence. In one embodiment, the first binding portion comprises the frame sequence of SEQ ID NO: 59, which is a variable light chain frame sequence. In one embodiment, the first binding portion comprises the frame sequence of SEQ ID NO: 63, which is a variable light chain frame sequence.

[0281] In one embodiment, the first binding portion specifically binding to CEA comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 1 as the heavy chain variable region and the light chain variable region of the amino acid sequence having 98%, 99%, or 100% identity with the amino acid sequence selected from SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 59, or SEQ ID NO: 63 as the light chain variable region, and the second binding portion specifically binding to CD3 comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 1 as the heavy chain variable region and the light chain variable region of the amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, and SEQ ID NO: 21 as the light chain variable region.

[0282] In one embodiment, the bispecific antibody according to the invention is characterized by comprising a first binding portion specific to CEA, which includes a κ light chain variable domain and a κ light chain constant domain, and a second binding portion specific to CD3ε, which includes a λ light chain variable domain and a λ light chain constant domain.

[0283] In one embodiment, the bispecific antibody according to the invention is characterized by comprising a first binding portion specific to CEA, which includes a λ light chain variable domain and a κ light chain constant domain, and a second binding portion specific to CD3ε, which includes a λ light chain variable domain and a λ light chain constant domain.

[0284] In one embodiment, the bispecific antibody according to the invention is characterized by comprising a first binding portion specific to CEA, which includes a λ light chain variable domain and a λ light chain constant domain, and a second binding portion specific to CD3ε, which includes a λ light chain variable domain and a κ light chain constant domain.

[0285] In one specific embodiment, the Fc domain exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to the natural / wild-type IgG1 Fc domain. In some embodiments, the Fc domain is engineered to have reduced binding affinity to Fc receptors and / or reduced effector function 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, one or more such substitutions are selected from Pro238, Asp265, Asp270, Asn297 (without Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 (Shields, RL 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; EP 0 307 434). In one embodiment, regarding FcR binding, the antibody is an IgG4 subclass or an IgG1 or IgG2 subclass having mutations in S228, L234, L235, and / or D265, and / or containing a PVA236 mutation. In one embodiment, the mutation in the Fc domain is S228P, L234A, L235A, L235E, and / or PVA236. In another embodiment, the mutation in the Fc domain is in IgG4 S228P and in IgG1 L234A and L235A. 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 located at one or more positions selected from L234, L235, and P329 (Kabat EU index number). In a particular embodiment, each subunit of the Fc domain contains two amino acid substitutions that reduce binding to Fc receptors and / or reduce effector function, wherein said amino acid substitutions are L234A and L235A (Kabat EU index number). In a particular embodiment, each subunit of the Fc domain contains three amino acid substitutions that reduce binding to the Fc receptor and / or reduce effector function, wherein the amino acid substitutions are L234A, L235A, and P329A (Kabat EU index number). In one such embodiment, the Fc domain is the IgG1 Fc domain, particularly the human IgG1 Fc domain (Kabat EU index number).In one embodiment, the Fc domain is an IgG4 subclass, and in another embodiment, it is an IgG4 subclass with the mutation S228P.

[0286] 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 activated 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.

[0287] One embodiment of the present invention is that the κCL region of SEQ ID NO: 58 used for the second binding portion is used as the CL region in constructing a bispecific antibody containing a common heavy chain of SEQ ID NO: 43, 44 or 45, and the λCL region of SEQ ID NO: 41 is used as the CL region in the first binding portion.

[0288] One embodiment of the present invention is that the κCL region of SEQ ID NO: 58, used for the second binding portion specifically binding to CD3, serves as the CL region in constructing a bispecific antibody according to the present invention comprising a common heavy chain of SEQ ID NO: 43, 44, or 45, and the λCL region of SEQ ID NO: 41 serves as the CL region in the first binding portion specifically binding to CEACAM5.

[0289] One embodiment of the invention is the κCL region of SEQ ID NO: 58, used for the second binding portion specifically binding to CD3, as the CL region in constructing a bispecific antibody according to the invention comprising a common heavy chain of SEQ ID NO: 43, 44 or 45 and a variable light chain of SEQ ID NO: 5, 9, 13, 17 or 21, and the λCL region of SEQ ID NO: 41, as the CL region in the first binding portion specifically binding to CEACAM5.

[0290] Another embodiment of the invention is an oligonucleotide selected from SEQ ID NO: 76, 77, 78 and 79, used for antibody affinity maturation by oligonucleotide-directed mutagenesis of degenerate oligonucleotides with corresponding light chain variable regions of SEQ ID NO: 31, 35, 59 and 63.

[0291] In another aspect, a method for generating the bispecific antibody of the present invention is provided, comprising the steps of: a) culturing the host cells of the present invention under conditions suitable for expressing the bispecific antibody and b) recovering the bispecific antibody. The present invention also includes bispecific antibodies generated by the method of the present invention.

[0292] The present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier. The present invention also includes methods of using the bispecific antibody and pharmaceutical composition of 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 another aspect, a bispecific antibody or pharmaceutical composition according to the present invention is provided for treating a disease in an individual in need. In one specific embodiment, the disease is cancer.

[0293] Also provided is the bispecific antibody of the present invention for preparing a medicament for treating a disease in an individual in need; and a method of treating an individual's disease comprising administering to the individual a therapeutically effective amount of a composition comprising a pharmaceutically acceptable form of the bispecific antibody according to the present invention. In one specific embodiment, the disease is cancer. In any of the above embodiments, the individual is preferably a mammal, particularly a human.

[0294] The present invention also provides a method for inducing the 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.

[0295] Another embodiment of the present invention is the use of the bispecific antibody according to the present invention to prepare a medicament for treating subjects with cancer expressing CEA.

[0296] Another embodiment of the present invention is a bispecific antibody according to the present invention, used to prepare a drug according to the present invention, characterized in that the cancer is selected from colorectal cancer, non-small cell lung cancer (NSCLC), esophageal cancer, gastric / esophageal junction cancer, pancreatic cancer and breast cancer.

[0297] Another embodiment of the invention is a bispecific antibody according to the invention, which is used in combination with an anti-CD47 antibody simultaneously, alone, or sequentially. In one embodiment, the anti-CD47 antibody is magrolimab, ALX148, or TTI-621 and / or TTI-622.

[0298] Another embodiment of the present invention is a bispecific antibody according to the present invention, for use in treating a subject with CEA-expressing cancer in a simultaneous, individual or sequential combination with a second bispecific antibody comprising a third binding portion specifically binding to human CEACAM5 and a fourth binding portion specifically binding to human CD47.

[0299] This type of second bispecific CEAxCD47 antibody is described in PCT / IB2019 / 054559 and US16 / 428,359.

[0300] Another embodiment of the present invention is a bispecific antibody according to the present invention, characterized in that the bispecific antibody according to the present invention and the second bispecific CEAxCD47 antibody are alternately administered to the subject at an interval of 6 to 15 days, but not limited to such an interval.

[0301] Another embodiment of the present invention is a first bispecific antibody according to the present invention (which comprises a first binding portion specifically binding to human CEACAM5 and a second binding portion specifically binding to human CD3 according to the present invention) and a second bispecific antibody CEAxCD47, which are used to treat cancer 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 and breast cancer.

[0302] Another embodiment of the present invention is a composition comprising a bispecific antibody according to the present invention, characterized in that it does not compete with the second CEAxCD47 bispecific antibody as defined above for the treatment of subjects with cancers expressing CEA.

[0303] Another embodiment of the present invention is a method for treating a human patient diagnosed with tumors (cancer), particularly solid tumors, especially solid cancers expressing CEA, particularly colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, pancreatic cancer, and breast cancer, comprising administering to the human patient an effective amount of the bispecific antibody according to the present invention and a second bispecific antibody against CEA and CD47 as described in PCT / IB2019 / 054559 and US16 / 428,359, the method subsequently comprising:

[0304] The second anti-CEAxCD47 antibody is administered to the patient at a dose of 0.1 to 30 mg / kg, in a further embodiment 0.5 to 10 mg / kg, and in a further embodiment 1 to 10 mg / kg, for example once weekly for 4 to 12 weeks.

[0305] The second antibody was administered to the patient q1, q2w, q3w, or optionally q4w.

[0306] Four to twelve weeks later, and after another two, three, or four elimination half-lives of the anti-CEAxCD47 antibody, the patient is administered a dose of 0.1 to 10 mg / kg of the antibody according to the invention.

[0307] The antibody according to the invention is administered to the patient at q1, q2w, q3w, or optionally q4w.

[0308] Wait for 2, 3, or 4 elimination half-lives of the antibody according to the invention, and then

[0309] Optionally, repeat the cycle of CEA x CD47 bispecific antibody administration followed by CEA x CD3 bispecific antibody administration and optionally repeat the cycle again.

[0310] This "alternating" approach is applied if the antibody of the present invention and the second bispecific antibody are competitive in binding to CEA.

[0311] Where the CEA x CD47 bispecific antibody and the CEA x CD3 bispecific antibody according to the invention are not competitive, the two bispecific antibodies may also be administered in parallel in a manner in which the patient experiences therapeutically effective plasma and tissue concentrations (“simultaneous administration”), for example by administering to the patient at approximately the same dose of 0.1 to 30 mg / kg of the CEA x CD47 bispecific antibody and 0.1 to 10 mg / kg of the CEA x CD3 bispecific antibody according to the invention, followed by one or more of these combined administrations at a frequency of q1w, q2w, q3w, or optionally q4w.

[0312] The term "q1w" means to be applied once a week; "q2w" means to be applied once every two weeks, and so on.

[0313] Another embodiment of the present invention is a pharmaceutical composition comprising an antibody according to the invention and a pharmaceutically acceptable excipient or carrier.

[0314] Another preferred embodiment of the present invention is a pharmaceutical composition comprising an antibody according to the present invention, which is used as a medicament.

[0315] Another preferred embodiment of the present invention is a pharmaceutical composition comprising an antibody according to the present invention, which is used as a medicament for treating solid tumors expressing CEA.

[0316] Another preferred embodiment of the present invention is a pharmaceutical composition comprising the antibody according to the present invention, which is used as a medicament for treating colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, esophageal cancer, pancreatic cancer, or breast cancer.

[0317] Another 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.

[0318] Another embodiment of the present invention is the use of the antibody according to the present invention in the preparation of a pharmaceutical composition.

[0319] Another embodiment of the present invention is the use of antibodies and pharmaceutically acceptable excipients or carriers according to the present invention to prepare pharmaceutical compositions.

[0320] Another embodiment of the present invention is the use of the antibody according to the present invention in the preparation of a medicament for treating solid tumors.

[0321] Another embodiment of the present invention is the use of the antibody according to the present invention to treat colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, esophageal cancer, pancreatic cancer, or breast cancer.

[0322] Another aspect of the invention provides a method for inducing cell lysis of tumor cells, comprising contacting the tumor cells with a bispecific antibody according to any of the above embodiments. 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.

[0323] In one implementation, cell lysis is induced by T cell-directed cytotoxicity (TDCC).

[0324] Another aspect of the present invention provides a method for treating a subject with cancer expressing CEA, the method comprising administering to the subject a therapeutically effective amount of any of the above-described embodiments of a bispecific antibody.

[0325] Another aspect of the invention provides a method for treating a subject with cancer expressing CEA, the method comprising administering to the subject a therapeutically effective amount of any of the above-described embodiments of a bispecific antibody in combination with a bispecific antibody binding to human CEA and human CD47. If the CEAxCD47 antibody and the CEAxCD3 antibody compete, they will compete for CEA receptors on the surface of tumor cells. The receptor occupancy and efficacy of each combination partner depend on their binding affinity and plasma concentration, and are therefore difficult to predict and will also vary over time if the concentrations of the two drugs have different elimination half-lives and clearance rates from the body. Therefore, the competitive CEAxCD3 and CEAxCD47 bispecific antibodies should be administered sequentially (alternatingly). If there is no competition or very little competition between the CEAxCD3 and CEAxCD47 bispecific antibodies, they can not only be administered sequentially but also in parallel (simultaneously). This is likely to be an advantage because killing tumor cells through the binding of the CEAxCD3 bispecific antibody to T cells and simultaneously through the binding of the CEAxCD47 bispecific antibody to macrophages is expected to produce an additive effect, or even a synergistic effect, meaning that efficacy is enhanced if the two drugs are administered in parallel.

[0326] Another aspect of the invention provides a method for increasing progression-free survival and / or overall survival in subjects suffering from CEA-expressing cancers, the method comprising administering to the subject a therapeutically effective amount of any of the bispecific antibodies 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 CEA-expressing cancer.

[0327] In some implementations of these methods, the bispecific antibody is administered in combination with chemotherapy or radiotherapy. In one implementation, the subject is a patient with colorectal cancer or lung cancer or stomach cancer, esophageal cancer or pancreatic cancer or breast cancer or another cancer that expresses CEA.

[0328] In some embodiments of these methods, the bispecific antibody of the present invention is administered to the patient in a single dose or divided doses or by continuous infusion at a dose of 0.1 to 100 mg / kg body weight daily or weekly. In some embodiments, the bispecific antibody of the present invention is administered to the patient at a dose of 1 to 20 mg / kg.

[0329] Another aspect of the invention provides a method of treating a subject with cancer expressing CEA, the method comprising administering to the subject a therapeutically effective amount of any of the above embodiments of a bispecific antibody in combination with a bispecific antibody targeting human CEA and human CD47. In some embodiments of these methods, the bispecific antibody is administered in combination with a bispecific anti-CEAxCD47 antibody in a simultaneous, single, or sequential manner. In some embodiments of these methods, the bispecific anti-CEAxCD47 antibody is administered in an alternating pattern with the antibody of the present invention, with an interval of 6 to 15 days between administrations of the antibody of the present invention and the bispecific anti-CEAxCD47 antibody. In some embodiments, the anti-CEAxCD47 antibody is administered to the patient in a single dose or divided doses or by continuous infusion at a dose of 0.1 to 100 mg / kg body weight daily or weekly.

[0330] In some embodiments of these methods, the bispecific antibody is administered in combination with the PD-1 axis antagonist, either simultaneously, alone, or sequentially. In some embodiments of these methods, the bispecific antibody is administered in combination with a bispecific anti-CEAxCD47 antibody and a PD-1 axis antagonist, either simultaneously, alone, or sequentially. In some embodiments, the PD-1 axis antagonist is administered to the patient at a dose of 0.1 to 100 mg / kg body weight daily or weekly, either as a single or divided dose or via continuous infusion.

[0331] Another aspect of the invention provides a method for increasing progression-free survival and / or overall survival in subjects with cancers that abnormally express CEA, the method comprising administering to the subject a therapeutically effective amount of any of the bispecific antibodies 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.

[0332] In some implementations of these methods, the bispecific antibody is administered in combination with chemotherapy or radiotherapy. In one implementation, the subject is a patient with colorectal cancer or lung cancer or stomach cancer, esophageal cancer, or pancreatic cancer or breast cancer or another cancer that expresses CEA.

[0333] Another embodiment of the invention provides a bispecific antibody according to the invention for use in any of the above-described treatment methods. In one embodiment, the cancer is selected from colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, pancreatic cancer, and breast cancer.

[0334] Another embodiment of the invention provides a polynucleotide encoding a bispecific antibody disclosed herein or an immune-specific binding domain (e.g., a variable light chain region and / or a variable heavy chain region) of CEACAM5 or CD3ε. In some aspects, this invention provides a polynucleotide comprising a nucleotide sequence encoding a light chain or heavy chain of the antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding a heavy chain comprising a VH or CDR of the antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding a light chain comprising a VL or CDR of the antibody described herein.

[0335] Some embodiments are vectors containing isolated polynucleotides disclosed herein. Other embodiments are cells containing isolated polynucleotides or vectors encoding bispecific antibodies disclosed herein. In some embodiments, the cells are selected from Streptomyces, yeast, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS 1, COS 7, BSC1, BSC40, BMT10 cells in tissue culture, plant cells, insect cells, and human cells.

[0336] Some embodiments are methods for preparing the antibodies disclosed herein. In some embodiments, the method of preparing antibodies includes expressing antibodies using cells containing isolated polynucleotides or vectors encoding bispecific antibodies disclosed herein. In some embodiments, the method of preparing antibodies includes culturing cells containing isolated polynucleotides or vectors encoding bispecific antibodies disclosed herein and isolating the antibodies expressed therein. Attached Figure Description

[0337] Figure 1 Epitope binning of the novel CEA binder used in the κ / λ(KL)CEAxCD3 bispecific antibody (details in Example 5c)

[0338] To characterize the novel CEA-binding antibodies of the present invention, a competitive immunoassay was used. Competitive blocking profiles were created against antibodies that all bind to CEACAM5 (CEA) and whose binding epitopes are publicly known. SM3E, MEDI (=MEDI-565), SAR, T84.66, labectocilizumab, and CH1A1A are such antibodies; details of these antibodies and the assay are given in Example 5c.

[0339] Figure 2 Agilent signature profile of purified bispecific antibodies

[0340] The expression, purification, and analysis of the novel κλ bispecific CEAxCD3 antibody are described in Example 6. The purified bispecific antibody was analyzed by electrophoresis under denaturing and reducing conditions. Using an Agilent 2100 bioanalyzer, this figure shows typical results obtained for the κλ antibodies of the present invention: AB1L3-1 / D (KL CEAxCD3 bispecific antibody with λCD3 LC SEQ ID NO: 28 and κCEA LC SEQ ID NO: 40 and common HC SEQ ID NO: 44) and L3-1AB8 H-CK5 / D (hybrid KLCEAxCD3 bispecific antibody with hybrid -κCD3 LC SEQ ID NO: 70 and λCEA LC SEQ ID NO: 42 and common HC SEQ ID NO: 44). Y4 L3-1 / D is a bispecific antibody with the same CD3 arm as AB1L3-1 / D and L3-1AB8 H-CK5 / D, but the second arm does not bind to CEA. This antibody is often used as a control antibody in pharmacological assays.

[0341] Figure 3 With CD3 POS Jurkat cells (and CD3) NEG The binding of TIB-153 cells

[0342] The KL bispecific antibodies AB1L3-1 / D and L3-1AB8 H-CK5 / D of this invention bind in a concentration-dependent manner to CD3-expressing Jurkat cells. Both KL bispecific antibodies possess the same CD3 arm. Y4L3-1 / D is a KL bispecific antibody with the same CD3 arm, but the second arm does not bind to CEA. The right figure shows that even at a concentration of 100 nM, the bispecific antibodies do not bind to the CD3-negative cell line TIB-153.

[0343] Figure 4 With CEA POS MKN-45 cells (and CEA) NEG MKN-45_hCEA KO (cell) binding

[0344] The KL bispecific antibodies AB1L3-1 / D and L3-1AB8 H-CK5 / D of this invention bind to CEA-expressing MKN-45 cells in a concentration-dependent manner. Both KL bispecific antibodies possess the same CD3 arm. Y4L3-1 / D is a KL bispecific antibody with the same CD3 arm, but the second arm does not bind to CEA and therefore does not bind to MKN-45 cells. The right figure shows that even at a concentration of 100 nM, it does not bind to the MKN-45 cell line after CEACAM5 knockout.

[0345] Figure 5 With CEA POS LS174T cell binding

[0346] The KL bispecific antibodies AB1L3-1 / D and L3-1AB8 H-CK5 / D of this invention bind in a concentration-dependent manner to CEA-expressing LS174T cells. Both KL bispecific antibodies possess the same CD3 arm. Y4L3-1 / D is a KL bispecific antibody with the same CD3 arm, but the second arm does not bind to CEA and therefore does not bind to LS174T cells.

[0347] Figure 6 CEA POS MKN-45 cells (and CEA) NEG MKN-45_hCEA KO Cell killing

[0348] The KL bispecific antibodies AB1L3-1 / D and L3-1AB8 H-CK5 / D of the present invention demonstrate concentration-dependent T cell retargeting killing / lysis of MKN-45 cells (as determined in Example 8a). Both KL bispecific antibodies possess the same CD3 arm. Both KL bispecific antibodies exhibit the same killing efficacy. This is surprising / unexpected because the binding of AB1L3-1 / D to MKN-45 is much weaker than that of L3-1AB8 H-CK5 / D (see Example 8a). Figure 4 ).

[0349] Y4L3-1 / D is a KL bispecific antibody with the same CD3 arm, but the second arm does not bind to CEA, and it shows very low killing / cleavage efficacy at concentrations approximately 100-fold higher.

[0350] Right figure: All three bispecific antibodies showed only very low nonspecific killing / lysis of CEA knockout MKN-45 cells.

[0351] Figure 7 CEA POS LS-174T's lethality

[0352] The KL bispecific antibodies AB1L3-1 / D and L3-1AB8 H-CK5 / D of the present invention demonstrated concentration-dependent T cell retargeting killing / lysis of LS-174T cells (as described in Example 8a). Both KL bispecific antibodies possess the same CD3 arm. L3-1AB8 H-CK5 / D exhibited stronger killing potency than AB1L3-1 / D. L3-1AB8 H-CK5 / D bound to LS-174T cells more strongly than AB1L3-1 / D (see Example 8a). Figure 5 ).

[0353] Y4L3-1 / D is a KL bispecific antibody with the same CD3 arm, but the second arm does not bind to CEA, and it shows very low killing / cleavage efficacy at concentrations up to 100-fold higher.

[0354] Figure 8 The binding of monoclonal antibody CEA mAb 1B4 to different recombinant proteins (ELISA).

[0355] CEA ECD = extracellular domain of CEA; CEA A3B3 is the A3B3 domain of CEA. Recombinant mesothelin MSLN was used as a control to measure nonspecific binding (non-specific binding to the ECD and A3B3 domains of CEA).

[0356] Figure 9The binding of monoclonal antibody CEA mAb C11 to different recombinant proteins (ELISA).

[0357] CEA ECD = extracellular domain of CEA; CEA A3B3 is the A3B3 domain of CEA. Recombinant mesothelin MSLN was used as a control to measure nonspecific binding (non-specific binding to the ECD and A3B3 domains of CEA).

[0358] Figure 10 Lead optimization of wave 1 antibody and CD3 POS Jurkat cells (and CD3) NEG The binding of TIB-153 cells

[0359] The KL bispecific antibodies AB13L3-1 / N, AB14L3-1 / N, AB15L3-1 / N, AB17L3-1 / N, and AB20L3-1 / N of this invention bind in a concentration-dependent manner to CD3-expressing HUT-78 cells. All KL bispecific antibodies possess the same CD3 arm. AB1L3-1 / N is a parental KL bispecific antibody derived from the aforementioned bsAb through leader optimization. Y4L3-1 / N is a KL bispecific antibody with the same CD3 arm, but the second arm does not bind to CEA. TCB 2014 corresponds to another CEAxCD3 T cell bispecific antibody described in US20140242079, which is included as a reference antibody. hIgG1 corresponds to a non-binding human IgG1 antibody used as an isotype control. Figure 11 B showed that even at a concentration of 100 nM bispecific antibody, it did not bind to the CD3-negative cell line JKTβ-del. The method is described in Example 7b.

[0360] Figure 11 Lead optimization of wave 1 antibody and CEA POS MKN-45 cells, HPAF-II cells and LS174T ( CL-188 TM ) cells (and CEA) NEG MKN-45_hCEA KO (cell) binding

[0361] The KL bispecific antibodies AB13L3-1 / N, AB14L3-1 / N, AB15L3-1 / N, AB17L3-1 / N, and AB20L3-1 / N of the present invention are effective against CEA-positive MKN-45(A); HPAF-II(C) and LS174T(D) or CEA-negative MKN-45_hCEA. KO(B) Cell concentration-dependent binding. All KL bispecific antibodies have the same CD3 arm. AB1L3-1 / N is a parental KL bispecific antibody derived from the above bsAb through affinity maturation. Y4L3-1 / N is a KL bispecific antibody with the same CD3 arm, but the second arm does not bind to CEA. TCB 2014 corresponds to another CEAxCD3T cell bispecific antibody described in US20140242079, which is included as a reference antibody. hIgG1 corresponds to a non-binding human IgG1 antibody used as an isotype control. Figure 10 B showed that even at a concentration of 100 nM bispecific antibody, it did not interact with the CEA-negative cell line MKN-45_hCEA. KO Cell binding. At a concentration of 100 nM, the antibody of the present invention binds 40% or more more to MKN-45 and / or HPAF-II cells than to TCB2014. The method is described in Example 7a.

[0362] Figure 12 Lead optimization of wave 1 antibody against CEA POS MKN-45, HPAF-II and LS174T cells (and CEA) NEG MKN-45_hCEA KO Cell killing

[0363] The KL bispecific antibodies AB13L3-1 / N, AB14L3-1 / N, AB15L3-1 / N, AB17L3-1 / N, and AB20L3-1 / N of the present invention are effective against CEA-positive MKN-45(A); HPAF-II(C) and LS174T(D) or CEA-negative MKN-45_hCEA. KO (B) Concentration-dependent T cell retargeting killing / lysis (assay described in Example 8a). All KL bispecific antibodies have the same CD3 arm. When using CEA-positive cells, all KL bispecific antibodies showed improved killing compared to their parental AB1L3-1 / N antibodies derived from them via leader optimization (A, C, D), but only CEA-knockout MKN-45 cells showed very low nonspecific killing / lysis (B). Y4L3-1 / N is a KL bispecific antibody with the same CD3 arm, but the second arm does not bind to CEA, and it showed only very low killing / lysis potency at the highest tested concentration. TCB2014 corresponds to another CEAxCD3 T cell bispecific antibody described in US20140242079, which is included as a reference antibody. The EC50 of the bispecific antibodies of the present invention is lower than that of TCB2014, demonstrating improved potency against tumor cell killing. The method is described in Example 8a.

[0364] Figure 13 Lead optimization of wave 2 antibody and CD3 POS Primary T cells (CD4+ and CD8+) and CD3 NEG The combination of B cells and monocytes

[0365] The KL bispecific antibodies AB54L3-1 / N, AB60L3-1 / N, AB66L3-1 / N, AB71L3-1 / N, AB72L3-1 / N, and AB73L3-1 / N of the present invention bind in a concentration-dependent manner to CD3-positive primary CD4+ T cells (A) and primary CD8+ T cells (B) or CD3-negative B cells (C) and monocytes (D). All KL bispecific antibodies have the same CD3 arm and bind similarly to the CD3-positive T cell population. Y4L3-1 / N is a KL bispecific antibody with the same CD3 arm, but the second arm does not bind to CEA; it binds equally well to CD3-positive T cells. TCB 2014 corresponds to another CEAxCD3 T cell bispecific antibody described in US20140242079, which is included as a reference antibody. Anti-human CD47 antibody B6H12 (aCD47 mAb) was used as a positive control (T cells, B cells, and monocytes expressing CD47). The second Ab corresponds only to the condition where only the detection antibody is added to the cells and used to determine the background signal (negative control). Even at a concentration of 200 nM, none of the antibodies tested showed binding to the CD3-negative cell population.

[0366] Figure 14 Lead optimization of wave 2 antibody and CEA POS MKN-45 cells, HPAF-II cells, and LS174T cells (and CEA) NEG MKN-45_hCEA KO (cell) binding

[0367] The KL bispecific antibodies AB54L3-1 / N, AB60L3-1 / N, AB66L3-1 / N, AB71L3-1 / N, AB72L3-1 / N, and AB73L3-1 / N of the present invention are effective against CEA-positive MKN-45(A); HPAF-II(C) and LS174T(D) or CEA-negative MKN-45_hCEA. KO (B) Concentration-dependent binding of cells. All KL bispecific antibodies have the same CD3 arm. Y4L3-1 / N is a KL bispecific antibody with the same CD3 arm, but the second arm does not bind to CEA. TCB 2014 corresponds to another CEAxCD3 T cell bispecific antibody described in US20140242079, which is included as a reference antibody. Figure 14 B showed that even at a concentration of 200 nM bispecific antibody, it did not interact with the CEA-negative cell line MKN-45_hCEA. KO Cell binding. For Figure 14 A. EC50 values ​​are reported in Table 4. The bispecific antibody of this invention binds 40% or more more than TCB2014 at a concentration of 200 nM.

[0368] Figure 15 Lead optimization of wave 2 antibody against CEA POS MKN-45, HPAF-II and LS174T cells (and CEA) NEG MKN-45_hCEA KO Cell killing

[0369] The KL bispecific antibodies AB54L3-1 / N, AB60L3-1 / N, AB66L3-1 / N, AB71L3-1 / N, AB72L3-1 / N, and AB73L3-1 / N of the present invention are effective against CEA-positive MKN-45(A); HPAF-II(C) and LS174T(D) or CEA-negative MKN-45_hCEA. KO (B) Concentration-dependent T cell retargeting killing / lysis (assay described in Example 8a). All KL bispecific antibodies have the same CD3 arm. Y4L3-1 / N is a KL bispecific antibody with the same CD3 arm, but the second arm does not bind to CEA, and it showed very low killing / lysis potency at the highest tested concentration. TCB2014 corresponds to another CEAxCD3 T cell bispecific antibody described in US20140242079, which is included as a reference antibody. Compared to the reference antibody TCB2014, the KL bispecific antibodies AB54L3-1 / N, AB60L3-1 / N, AB66L3-1 / N, AB71L3-1 / N, AB72L3-1 / N, and AB73L3-1 / N of the present invention all showed lower EC50 values ​​and stronger killing of CEA-positive target cells (A, C, D), but significantly lower nonspecific killing / lysis in CEA-knockout MKN-45 cells (B). Figure 15 A, C, and D, the EC50 values ​​are reported in Table 5.

[0370] Figure 16 T-cell-mediated cytokine secretion in MKN45 tumor cells

[0371] Following T-cell-mediated killing of MKN45 tumor cells, the KL bispecific antibodies AB17L3-1 / N, AB72L3-1 / N, and AB73L3-1 / N of the present invention mediated the secretion of perforin (B); granzyme B (C); IFN-γ (D); TNF-α (E); IL-2 (F); IL-6 (G); and IL-10 (H) (E:T(human PBMC:tumor cells) = 10:1), after incubation for 48 hours. Specific lysis is shown in (A). The method is described in Example 8e.

[0372] Figure 17 T cell activation following cytotoxicity in MKN-45 tumor cells

[0373] Two days after T-cell-mediated killing of CEA-positive MKN-45 tumor cells, the KL bispecific antibodies AB17L3-1 / N, AB72L3-1 / N, and AB73L3-1 / N of this invention upregulated human CD4+ and CD8+ T cells mediated by CD25 (A and B) and CD69 (C and D). Figure 16 A shows the killing / lysis of tumor cells.

[0374] Although there was no statistically significant difference in tumor cell lysis at 100 nM, the lower T cell activation of the antibody of the present invention compared to TCB2014 indicates lower side effects at the same tumor lysis, the method described in Example 8c.

[0375] Figure 18 In vivo antitumor efficacy in the HPAF-II model of NOG mice with huPBMC metastases

[0376] The in vivo antitumor efficacy of the KL bispecific antibodies AB17L3-1 / N, AB72L3-1 / N, and AB73L3-1 / N, and TCB2014 (all administered as a single 10 mg / kg injection) in an HPAF-II tumor cell line model in NOG mice with huPBMC metastases was demonstrated. Mice were randomly assigned to groups on day 11, at which point the average tumor volume was approximately 150 mmHg. 3 The results show the mean tumor volume and SEM values ​​of eight mice measured by calipers in different study groups. No statistically significant difference was found between AB73L3-1 / N and TCB2014. The methods are described in Example 9a.

[0377] definition

[0378] Unless otherwise defined below, the terms used herein are the same as those commonly used in the art.

[0379] As used herein, the term "antigen-binding portion" in its broadest sense refers to the portion of an antibody that specifically binds to antigenic determinants such as CEA, CD47, and CD3.

[0380] More specifically, as used herein, the binding site specifically binds to membrane-bound human carcinoembryonic antigen (CEA, identical to CEACAM5) or CD3-bound CEA or CD3, more specifically binding to cell surface or membrane-bound CEA or CD3. "Specific binding," "specific to," and "binding" mean that the binding is selective to the antigen and can be distinguished from unwanted or non-specific interactions. In some embodiments, the anti-target antibody binds to unrelated non-target proteins to a degree about 10-fold less, preferably >100-fold less, than the antibody binds to the target, such as, for example, through surface plasmon resonance (SPR). Measured by enzyme-linked immunosorbent assay (ELISA) or flow cytometry (FACS). Targets are proteins discussed in this article—such as CEA, CD47, and CD3ε.

[0381] "Specifically binding to CEA and CD3" in one implementation refers to an antibody that can bind to target CEA and CD3, having sufficient affinity to make the antibody usable as a therapeutic agent for retargeting T cells to tumor cells through binding to CD3 and CEA.

[0382] Preferably, the bispecific antibody according to the invention binds to a conserved CD3 epitope from different species, preferably between humans and cynomolgus monkeys.

[0383] 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 variable regions (variable domains) and constant regions (constant domains) defined for a full-length antibody. As used herein, the term "antibody light chain" refers to an antibody light chain consisting of variable regions and constant regions defined for a full-length antibody. The constant light chain that can be used in this invention is included in the light chains disclosed herein.

[0384] The term "full-length antibody" refers to an antibody composed of two "full-length antibody heavy chains" and two "full-length antibody light chains." The "full-length antibody heavy chain" is a polypeptide composed of the antibody heavy chain variable domain (VH), antibody constant heavy chain domain 1 (CH1), antibody hinge region (HR), antibody heavy chain constant domain 2 (CH2), and antibody heavy chain constant domain 3 (CH3) in the N-terminal to C-terminal direction, abbreviated as VH-CH1-HR-CH2-CH3. The "full-length antibody light chain" is a polypeptide composed of the antibody light chain variable domain (VL) and antibody light chain constant domain (CL) in the N-terminal to C-terminal direction, abbreviated as VL-CL. The antibody light chain constant domain (CL) can be κ (kappa) or λ (lambda). The two full-length antibody domains are linked together by interpeptide disulfide bonds between the CL and CH1 domains and between the hinge regions of the full-length antibody heavy chain. Typical examples of full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE. In one embodiment, the full-length antibody according to the invention is of the human IgG1 type; in another embodiment, it contains one or more amino acid substitutions in the Fc portion as defined below. The full-length antibody according to the invention comprises two binding moieties, each formed by a pair of VH and VL, one binding CEA and the other binding CD3.

[0385] As used herein, the term "Fc region; Fc domain" refers to the C-terminal region of the IgG heavy chain; in the case of IgG1 antibodies, the C-terminal region contains –CH2-CH3 (see above). Although the boundaries of the Fc region of the IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at Cys226 to the carboxyl terminus.

[0386] The constant region is well known in the prior art, for example as described in Kabat, EA, (see, for example, Johnson, G. and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218; Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788).

[0387] The term "epitaph" includes any polypeptide determinant capable of specifically binding to an antibody. In some embodiments, the epitope determinant includes chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. An epitope is a target region that is bound by an antibody.

[0388] As used herein, the term "common heavy chain (cHC)" refers to a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3) in the N-terminal to C-terminal direction, abbreviated as VH-CH1-HR-CH2-CH3. The common heavy chain suitable for the bispecific antibody according to the invention is the heavy chain described in WO2012023053, WO2013088259, WO2014087248, WO2019175658, and WO2016156537 (the entire contents of each of these are incorporated herein by reference). In one embodiment, the cHC of the bispecific antibody according to the invention comprises CDRL1 of SEQ ID NO: 2, CDRL2 of SEQ ID NO: 3, and CDRL3 of SEQ ID NO: 4 as heavy chain CDRs. In one embodiment, the cHC of the bispecific antibody according to the present invention includes the VH region of SEQ ID NO: 1 as a heavy chain variable region. In one embodiment, the cHC of the bispecific antibody according to the present invention is SEQ ID NO: 43, 44 or 45.

[0389] The present invention, in the form of a bispecific antibody comprising a common heavy chain, allows for affinity purification of bispecific antibodies that are indistinguishable from standard IgG molecules and have characteristics indistinguishable from standard monoclonal antibodies (see, for example, WO2013088259, WO2012023053), promising no or low immunogenicity potential in patients.

[0390] As used herein, “AB1L3-1, AB17L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1, AB73L3-1, etc.” refer to the bispecific CEAxCD3 antibody according to the present invention, which comprises a common heavy chain (which contains the CDRs of SEQ ID NO: 2, 3 and 4 as the heavy chain CDR) and a light chain in the second binding moiety (which contains the CDRs of SEQ ID NO: 18, 19 and 20 as the light chain CDR). Therefore, AB1 et al. represent the first binding moiety (anti-CEACAM5 binding moiety), and L3-1 represents the second binding moiety (anti-CD3 binding moiety).

[0391] In one embodiment, AB1L3-1, AB17L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1, AB73L3-1, etc., comprise a common heavy chain of SEQ ID NO: 43 (WT hIgG1) and a light chain of SEQ ID NO: 28 as the light chain in the second binding moiety. Such bispecific antibodies of the present invention are also designated as AB1L3-1, AB17L3-1, AB71L3-1, AB72L3-1, and AB73L3-1 in the examples.

[0392] In one embodiment, AB1L3-1, AB17L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1, AB73L3-1, etc., comprise a common heavy chain of SEQ ID NO: 44 (hIgG1 with L234A+L235A mutation) and a light chain of SEQ ID NO: 28 as the light chain in the second binding moiety. Such bispecific antibodies of the present invention are designated in the examples as AB1L3-1 / D, AB17L3-1 / D, AB71L3-1 / D, AB72L3-1 / D, AB73L3-1 / D, etc.

[0393] In one embodiment, AB1L3-1, AB17L3-1, AB54L3-1, AB60L3-1, AB66L3-1, AB71L3-1, AB72L3-1, AB73L3-1, etc., comprise a common heavy chain (IgG1 with the L234A+L235A+P329A mutation) of SEQ ID NO: 45 and a light chain of SEQ ID NO: 28 as a light chain in the second binding moiety. Such bispecific antibodies of the present invention are designated in the examples 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.

[0394] The bispecific antibody comprising a common heavy chain of the present invention can be prepared, for example, according to WO2012023053. The method described in WO2012023053 produces a bispecific antibody that is structurally identical to human immunoglobulin. This type of molecule consists of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a κ constant domain, and a second light chain variable region fused to a λ constant domain.

[0395] In the bispecific antibody of the present invention, one binding site exhibits specificity for CEA, while the other binding site exhibits specificity for CD3, with both the heavy chain and the corresponding light chain contributing to each. The light chain variable region can be from the λ or κ family and is preferably fused to the λ and κ constant domains, respectively. This is preferred to avoid the generation of non-natural peptide linkages. However, it is also possible to obtain antibody arms suitable for generating the bispecific antibody of the present invention by fusing the κ light chain variable domain to the λ constant domain for either of the two specificities or by fusing the λ light chain variable domain to the κ constant domain for either of the two specificities. The bispecific antibody described in WO2012023053 is a “κλ body”. This κλ body form allows for affinity purification of bispecific antibodies that are indistinguishable from standard IgG molecules and have characteristics indistinguishable from standard monoclonal antibodies, and is therefore advantageous compared to previous forms that included, for example, amino acid bridges or other non-natural elements.

[0396] A key step in this method is identifying two antibody Fv regions (each consisting of a variable light domain and a variable heavy domain) with different antigen specificities that share the same heavy chain variable domain. Many methods for generating 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). Completely human antibodies are antibody molecules in which both the light and heavy chain sequences (including CDRs 1 and 2) are derived from human genes. The CDR3 region can be of human origin or synthesized. Such antibodies are called "human antibodies" or "completely human antibodies." Human monoclonal antibodies can be prepared using trioma technology; human B-cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4:72); and EBV hybridoma technology 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 Cole et al., 1983 Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see Cole et al., ibid.).

[0397] As used herein, the term "CD3ε or CD3" refers to human CD3ε as described under UniProt P07766 (CD3E_HUMAN). The terms "antibody against CD3" or "anti-CD3 antibody" refer to antibodies that bind to CD3ε.

[0398] As used herein, the terms “CEA, CEACAM5” refer to human carcinoembryonic antigen (CEA, CEACAM-5, or CD66e; UniProtKB-P06731), 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-associated 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-associated 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-associated cell adhesion molecule (CEACAM) family. Further information and information about other members of the CEA family are available at http: / / www.uniprot.org.

[0399] As used herein, the terms "specifically binding to CEA," "bound to CEA," and "CEA-binding moiety" refer to specificity for CEACAM5 on the cell surface in the context of the bispecific antibody according to the invention. Binding to CEA on the cell surface can be measured using gastric adenocarcinoma cells MKN-45 containing 100,000 to 400,000 copies of CEA per cell. The concentration of the antibody according to the invention varies within a suitable range related to the resulting EC50 value for binding to MKN-45 cells as defined above. The bispecific antibody according to the invention specifically binds to this cell membrane-bound CEACAM5.

[0400] As used herein, the term "membrane-bound human CEA" refers to human carcinoembryonic antigen (CEA) that is bound to the membrane portion or cell surface of a cell, particularly the surface of tumor cells. In some cases, the term "membrane-bound human CEA" may refer to CEA that is not bound to the cell membrane, but it has been constructed to preserve the membrane-bound CEA epitopes bound by the antibodies according to the invention.

[0401] As used herein, the term "no cross-reactivity to CEACAM8" in the context of bispecific antibodies according to the invention means testing the binding of the bispecific antibody according to the invention on PEAK cells expressing CEACAM8 compared to binding on WT PEAK cells (see Examples 1 and 5 for details), and no cross-reactivity means that the MFI measured on PEAK cells expressing CEACAM8 is no more than twice the MFI measured on WT PEAK cells. As used herein, the term "no cross-reactivity to a certain CEACAM" in the context of bispecific antibodies according to the invention means the cross-reactivity under the same experimental procedures and definitions described for CEACAM8.

[0402] As used herein, the terms "bispecific antibody binding human CEA and human CD3, CEAxCD3 bsAb" refer to a bispecific antibody binding human CEACAM5 and CD3ε.

[0403] As used herein, the term “complementarity-determining region” (“CDR”) describes a discontinuous antigen-binding site (also known as an antigen-binding region) found within the variable region of heavy and light chain polypeptides. The CDR is also referred to as a “hypervariable region,” and this term is used interchangeably with “CDR” herein, referring to the variable region portion that forms the antigen-binding region. This specific region is described in Kabat et al., USDept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987). Kabat et al. also defined a numbering system applicable to the variable domain sequences of any antibody. Those skilled in the art can readily assign this “Kabat numbering” system to any variable domain sequence without relying on any experimental data outside the sequence itself. As used herein, “Kabat numbering” refers to the numbering system proposed by Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). Unless otherwise stated, references to the numbering (e.g., CDR sequence) of specific amino acid residue positions in the bispecific antibodies according to the invention are based on the Kabat numbering system.

[0404] As used herein, the term "oligonucleotide directed mutagenesis" refers to such methods that use degenerate oligonucleotides. For the mutagenesis of each CDR, a combination of various degenerate oligonucleotides is used. These include (but are not limited to) the degenerate codons NNS, HMT, DMT, and NHT.

[0405] As used herein, the term "expression vector" refers to one or more vectors that contain the heavy and light chains of the antibody according to the invention in a manner known in the art. As used herein, the term "host cell" encompasses any kind of cell system that can be engineered to produce the bispecific antibody of the invention. In one embodiment, the host cell is engineered to allow the production of antigen-binding molecules.

[0406] As used in this article, the term "amino acid substitution" refers to the replacement of one amino acid with another amino acid from the 20 standard amino acid groups for proteins.

[0407] Therapeutic applications and methods of use of the anti-CEAxCD3 antibody according to the present invention

[0408] The CEACAM x CD3 bispecific antibody according to the present invention is optimized for the treatment of solid tumors, whether in monotherapy or combination therapy, especially in combination therapy with anti-CD47 antibodies, anti-CEAxCD47 antibodies, and / or PD-1 axis antagonists. The antibody and CD47 antibody or CEAxCD47 antibody according to the present invention can be administered as described below.

[0409] In one specific implementation, the disease or solid tumor is a cancer that expresses or even overexpresses CEA, including but not limited to colorectal tumors, non-small cell lung tumors, gastric tumors, esophageal cancer, pancreatic tumors, and breast tumors. In one specific implementation, the tumor is a colorectal tumor. All therapeutic applications, uses, combinations, etc., described herein are particularly implementations for treating these tumors / diseases.

[0410] The inventors recognize that the antibodies according to the invention exhibit low or no ADA formation potential or exposure loss due to neutralization of ADA or loss of potency.

[0411] In one embodiment, the present invention provides a method for treating cancer (cancer, tumor, such as human cancer), particularly tumors expressing CEA, in vivo. The method includes administering to a subject a pharmaceutically effective amount of a composition containing the bispecific antibody of the present invention. "Subject" refers to a human subject, and in one embodiment, a patient suffering from cancer / tumor / carcinoma.

[0412] CEA expression is typically very high in various tumor solids, 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. In healthy, normal glandular epithelial cells of the gastrointestinal tract, CEA is primarily expressed in a polarized pattern on the apical surface of the cell. This polarized expression pattern limits the accessibility of systemically administered anti-CEA monospecific or bispecific antibodies, thus posing a potential toxicity. This polarized expression pattern is lost in cells of gastrointestinal malignancies. CEA is expressed uniformly across the entire cell surface of cancer cells, meaning that cancer cells are more easily approached by the antibodies of this invention than normal healthy cells and can be selectively killed by the CEAxCD3 bispecific antibody of this invention or a combination thereof.

[0413] In one embodiment, the bispecific antibody of the present invention can be used as a monotherapy to treat advanced solid tumors, in one embodiment, tumors expressing CEA. In one embodiment, the bispecific antibody of the present invention is used in combination with CEAxCD47 bsAb, either simultaneously, alone, or in a sequential combination. In one embodiment, the bispecific antibody of the present invention is used in combination with CEAxCD47 bsAb and / or a PD-1 axis antagonist, either simultaneously, alone, or in a sequential combination. In one embodiment, the bispecific antibody of the present invention is used in combination with a PD-1 axis antagonist, either simultaneously, alone, or in a sequential combination. Such PD-1 axis antagonists are described, for example, in WO2017118675. This combination attacks solid tumors via macrophages and T cells. CD47 antibodies are described, for example, in WO2009091601, WO2009091547, WO2011143624, WO2009131453, WO2013119714, WO2015105995, WO2017181033, WO2018026600, WO2019157432 and WO2013032948, and bispecific antibodies against CEA and CD47 are described in PCT / IB2019 / 054559 and US16 / 428,539.

[0414] As used herein, the term "combination, simultaneous, individual, or sequential combination" of the antibody according to the invention and a second antibody binding human CD47 or human CEA and human CD47 refers to any administration of two antibodies (or, in the case of a combination of the antibody of the invention, CD47 mAb or CEAxCD47bsAb, and a PD-1 axis antagonist) individually or together, wherein the two or three antibodies are administered as part of an appropriate dosing regimen intended to obtain the benefit of combination therapy, for example, in a manner that is administered individually, sequentially, simultaneously, concurrently, chronologically staggered, or alternately. Thus, two or three antibodies may be administered as part of the same pharmaceutical composition or in separate pharmaceutical compositions. The antibody according to the invention may be administered before, simultaneously, or after the administration of the second bispecific antibody, or in some combination thereof. When the antibody according to the invention is administered to a patient at repeated intervals (e.g., during standard treatment), the second bispecific antibody may be administered before, simultaneously, or after each administration of the antibody of the invention, or in some combination thereof, or at different intervals related to treatment using the antibody of the invention, or as a single dose at any time before, during, or after the treatment process using the antibody of the invention. In one embodiment, the antibody and the second bispecific antibody according to the invention are administered alternately, with an interval of 6 to 15 days between administrations of the antibody and the second antibody. In such alternating administration, the first dose may be either the antibody or the second antibody according to the invention.

[0415] The term "PD-1 axis antagonist" refers to anti-PD-1 antibodies or anti-PD-L1 antibodies. Anti-PD-1 antibodies are, for example, pembrolizumab (PD-1 antibody). MK-3475), nivolumab, pidilizumab, lambolizumab, MEDI-0680, PDR001, and REGN2810. Anti-PD-1 antibodies are described in, for example, WO200815671, WO2013173223, WO2015026634, US7521051, US8008449, US8354509, WO20091 / 14335, WO2015026634, WO2008156712, WO2015026634, WO2003099196, WO2009101611, WO2010 / 027423, WO2010 / 027827, WO2010 / 027828, WO2008 / 156712, and WO2008 / 156712. Anti-PD-L1 antibodies are, for example, atezolizumab, MDX-1 105, durvalumab, and avermectin. Anti-PD-L1 antibodies are described in, for example, WO2015026634, WO2013 / 019906, WO2010077634, US8383796, WO2010077634, WO2007005874 and WO2016007235.

[0416] Regarding the combined administration of the antibody and the second bispecific antibody according to the present invention, the two compounds may be present in a single dosage form or in separate dosage forms, for example, in two different or the same dosage forms.

[0417] If the antibody and the second antibody of the present invention do not compete for CEACAM5, in one embodiment, the two antibodies are administered simultaneously. If the antibody and the second antibody of the present invention compete for CEACAM5, in one embodiment, the antibodies are administered alternately.

[0418] As is known in the art, the antibodies of the present invention are typically administered to patients in a dosage regimen that provides the most effective treatment for the cancer the patient is being treated (from an efficacy and safety perspective). Preferably, the tumor cells are attacked by both T cells and macrophages simultaneously, and to realize the full therapeutic potential of this approach, the CEAxCD3 and CEAxCD47 bispecific antibodies should be non-competitive in binding to CEA on the cell surface.

[0419] As described above, the timing and dosage of the antibodies of the present invention may depend on the type (e.g., sex, age, weight) and condition of the patient being treated, the severity of the disease or condition being treated, and the route of administration. For example, the antibodies and second antibodies of the present invention may be administered to the patient in a single or divided dose or by continuous infusion at a dose of 0.1 to 100 mg / kg body weight daily or weekly. In one embodiment, each of the antibodies and second antibodies of the present invention is administered to the patient at a dose of 1 to 20 mg / kg. In some cases, dose levels below the lower limit of the above range may be sufficient, while in other cases, larger doses may be used without causing any harmful side effects.

[0420] As used herein, the term "antibody half-life" refers to the half-life of the antibody as measured in a typical pharmacokinetic assay. The antibodies according to the invention and the second bispecific antibody against CEA and CD47 have elimination half-lives of 3-14 days.

[0421] In another aspect, the present invention also relates to the use of the bispecific antibody according to the invention in the treatment of diseases, particularly cell proliferation disorders in which CEA is expressed, especially in which CEA is abnormally expressed compared to normal tissues of the same cell type (e.g., expressed or overexpressed in a different pattern on the cell surface). Such disorders include, but are not limited to, colorectal cancer, NSCLC (non-small cell lung cancer), gastric cancer, esophageal cancer, pancreatic cancer, and breast cancer. CEA expression levels can be determined by methods known in the art (e.g., by immunohistochemistry, immunofluorescence assay, immunoenzyme assay, ELISA, flow cytometry, radioimmunoassay, etc.).

[0422] On the one hand, the bispecific antibody of the present invention can be used to target cells expressing CEA in vivo or in vitro. The bispecific antibody of the present invention is particularly suitable for preventing tumor formation, eradicating tumors, and inhibiting tumor growth or metastasis by inducing TDCC in tumor cells. The bispecific antibody of the present invention can be used to treat any tumor expressing CEA. Specific malignancies that can be treated with the bispecific antibody of the present invention include, but are not limited to, colorectal cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, pancreatic cancer, and breast cancer.

[0423] The bispecific antibodies of the present invention, in pharmaceutically acceptable dosage forms (such as those discussed below, including those that can be administered to humans by bolus injection or by intravenous infusion over a period of time), can be administered to mammals, preferably humans, via intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local, or inhalation routes. The bispecific antibodies of the present invention are also suitable for administration via intratumoral, peritumoral, intralesional, or perilesional routes to exert local and systemic therapeutic effects.

[0424] For the treatment of diseases, the appropriate dosage of the bispecific antibody of the present invention will depend on the type of disease to be treated, the severity and progression of the disease, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The bispecific antibody of the present invention is suitable for administration to the patient in a single treatment or in a series of treatments. The present invention provides a method for selectively killing tumor cells expressing CEA.

[0425] This method involves the interaction of the bispecific antibody of the present invention with the tumor cells. These tumor cells can originate from human cancers, including colorectal cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, pancreatic cancer, and breast cancer.

[0426] In another aspect, the present invention relates to a bispecific antibody for preparing a medicament for treating diseases associated with abnormal CEA expression. In one specific embodiment, the disease is a cancer that expresses or even overexpresses CEA, including but not limited to colorectal tumors, non-small cell lung tumors, gastric tumors, esophageal cancer, pancreatic tumors, and breast tumors. In one specific embodiment, the tumor is a colorectal tumor.

[0427] Composition, formulation, dosage and route of administration

[0428] On one hand, the present invention relates to pharmaceutical compositions comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier. The present invention further relates to methods for using such pharmaceutical compositions to treat diseases such as cancer, or to the preparation of medicaments for treating diseases such as cancer. Specifically, the present invention relates to a method for treating a disease, and more specifically, a method for treating cancer, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention.

[0429] On one hand, the present invention includes pharmaceutical compositions, combinations, and methods for treating human cancers and tumors as defined above. For example, the present invention includes pharmaceutical compositions for treating human cancers comprising a pharmaceutically effective amount of the antibody of the present invention and a pharmaceutically acceptable carrier.

[0430] The bispecific antibody composition of the present invention can be administered using conventional methods, including but not limited to intravenous, intraperitoneal, oral, intralymphatic, or direct intratumoral administration. Intravenous or subcutaneous administration is preferred.

[0431] In one aspect of the invention, therapeutic formulations containing the bispecific antibodies of the invention are prepared for storage in lyophilized or liquid form by mixing antibodies of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used. Formulations intended for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filter membranes. The most effective administration method and dosage regimen of the pharmaceutical compositions of the invention depends on the severity and progression of the disease, the patient's condition and response to treatment, and the judgment of the treating physician. Therefore, the dosage of the composition may be a fixed dose or may be adapted to individual patients, such as body weight. However, the effective dose of the compositions of the invention is generally in the range of 0.1 to 20 mg / kg.

[0432] The bispecific antibodies of this invention have a molecular weight of 150 kDa / mol. In one embodiment, they carry an Fc moiety. The elimination half-life for patients is 3 to 14 days. This half-life allows for, but is not limited to, administration once daily, once weekly, or once every two weeks.

[0433] The bispecific antibodies and their respective compositions of the present invention can be in various dosage forms, including but not limited to liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microbubbles, liposomes, and injections or insoluble solutions. Preferred forms depend on the method of administration and the therapeutic application.

[0434] Compositions containing the bispecific antibodies of the present invention will be formulated, administered, and applied in accordance with good medical practice. Factors considered herein include the specific disease or condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease or condition, the site of delivery of the drug, the method of administration, the timing of administration, and other factors known to the physician.

[0435] Products

[0436] In another aspect of the invention, articles comprising materials for treating, preventing, and / or diagnosing the aforementioned conditions are provided. The articles include a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from various materials such as glass or plastic. The container contains the composition alone or in combination with another effective composition for treating, preventing, and / or diagnosing the condition, and may have a sterile access port (e.g., a container with a stopper that can be punctured by a hypodermic needle may be an intravenous solution bag or vial). One active agent in the composition is the bispecific antibody of the present invention. The label or packaging insert indicates that the composition is for treating the selected condition. Furthermore, the articles may comprise (a) a first container containing the composition, wherein the composition contains the bispecific antibody of the present invention; and (b) a second container containing the composition, wherein the composition contains additional cytotoxic agents or other therapeutic agents. The articles in this embodiment of the invention may also include a packaging insert indicating that the composition is suitable for treating a specific condition. Alternatively or additionally, the article may also include a second (or third) container containing pharmaceutically acceptable buffer solutions, such as bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may also include other materials required from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes.

[0437] Table 1

[0438] sequence list

[0439]

[0440]

[0441]

[0442]

[0443]

[0444] Example

[0445] Example 1: Cloning, expression, and purification of human CEACAM family members

[0446] clone

[0447] Sequences corresponding to the complete 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 Avitag at the C-terminus. TM (Avidity, Denver Colo.) and a hexahistine tag, human FC region, or mouse FC region. The construct was validated by DNA sequencing. It was then analyzed using IMAC (immobilized metal ion affinity chromatography), FcXL, or CaptureSelect. TM Purification of recombinant soluble proteins using IgG-Fc(ms) Affinity Matrix (Thermo Fisher Scientific).

[0448] Vectors encoding the full-length forms of human CEACAM 1, 3, 4, 5, 6, 7, 8, 18, 19, 20, 21 and cynomolgus monkey CEACAM 5 and CEACAM 6 were also generated for expression on the cell surface of PEAK and / or CHO cells. A soluble full-length human CEACAM 16 was similarly cloned.

[0449] In addition, vectors encoding the following truncated forms of human CEACAM5 were 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 was expressed as a fusion protein with the first 140 amino acids of the human CD86 protein.

[0450] Express

[0451] The plasmids were then transfected into mammalian cells using a liposome-based transfection reagent such as Lipofectamine 2000 (Thermo Fisher Scientific). The transfection step requires only a small amount of DNA and cells, typically 4 x 10⁴ cells per well. 5 Transfection was performed in 6-well plates with 1 cell and 2 μg of plasmid DNA. Although different mammalian cell lines can be used, in the examples given below, transformed human embryonic kidney monolayer epithelial cells (PEAK cells) were transfected. These cells stably express the EBNA-1 gene (further supporting the appendage replication process), are semi-adherent, and can grow under standard cell culture conditions (5% CO2; 37°C in DMEM medium supplemented with 10% fetal bovine serum). After 24 hours, the cells were placed under selective conditions by adding medium containing 0.5–2 μg / mL puromycin: cells containing the appendage vector are resistant to this antibiotic.

[0452] Two to three weeks after transfection, the expanded and selected cells were injected into a single-use celline. TM Used in the production steps within a bioreactor (Sigma-Aldrich). CELLine TM This is a two-chamber bioreactor that can be used with a standard cell culture incubator. The smaller compartment (15 ml) contains cells and is separated from the larger (1 L) compartment containing culture medium by a semi-permeable membrane with a cutoff size of 10 kDa (Bruce et al. 2002, McDonald et al. 2005). This system allows the diffusion of nutrients, gases, and metabolic waste while retaining cells and secreted proteins in the smaller compartment. The culture is maintained for 7–10 days before harvesting the supernatant. Because the culture medium contains serum, the cells maintain good viability, and multiple production runs can be performed using the same cells and containers.

[0453] purification

[0454] After harvesting, the cell culture supernatant was clarified by centrifugation. The supernatant was then replenished with 100 mM imidazole and loaded onto Ni-NTA affinity chromatography resin (Qiagen). The relatively high concentration of imidazole minimized contaminant binding to the resin. After washing the column, the protein was eluted using a 30 mL imidazole gradient (20–400 mM imidazole) at a flow rate of 2 mL / min on an AKTA Prime chromatography system (Cytiva). The elution gradient further improved the purity of the recombinant protein, but if the chromatography system is unavailable, a stepwise elution method can be used instead. The eluted fractions can be analyzed by SDS-PAGE or ELISA to determine their content in the recombinant protein. The fractions were equilibrated with phosphate-buffered saline or another suitable buffer. Fractions of interest were pooled and desalted on a 10 kDa column (Millipore). The desalted proteins were then quantified using various techniques, and their purity was analyzed by SDS-PAGE. Recombinant proteins were biotinylated in vitro using biotin ligase (Avidity, Denver Colo.) according to the manufacturer's instructions. Following desalting, biotinylation levels were assessed using a streptavidin magnetic bead pull-down assay and SDS-PAGE analysis.

[0455] Example 2: Phage display selection of CEACAM5 Fvs using a human scFv library containing fixed variable heavy chain domains

[0456] Vaughan et al. (Nat. Biotech. 1996, 14:309-314) described a general procedure for constructing and processing human scFv libraries displayed on M13 phage, which is incorporated herein by reference in its entirety. The libraries used for selection and screening encode all scFvs that share the same VH domain and are individually diverse in the VL domain. Methods for generating immobilized VH libraries and for identifying and assembling bispecific antibodies are described in US2012 / 0184716 and WO2012 / 023053, each of which is incorporated herein by reference in its entirety. The procedure for identifying scFvs that bind to human CEACAM5 is described below.

[0457] Protein selection

[0458] scFv phage library (10 12 Aliquots of Pfu were blocked for one hour at room temperature on a rotary mixer with PBS containing 3% (w / v) skim milk. The blocked phages were then encapsulated in streptavidin-coated magnetic beads (Dynabeads). TM Deselected phages were placed on a rotary mixer at room temperature for one hour. The deselected phages were then incubated with 100 nM biotinylated human CEACAM5 or streptavidin-captured A3-B3 domains on magnetic beads at room temperature for two hours. The beads were then captured using a magnetic scaffold and incubated with PBS / 0.1%... Wash five times with PBS, then twice with PBS. Elute the phage with 100 nM TEA for 30 min at room temperature on a rotary mixer. 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 (a commonly used E. coli strain for phage display) and incubate at 37°C with gentle shaking (90 rpm) for 1 h. Serially dilute an aliquot of infected TG1 to titrate the selected output. Spin the remaining infected TG1 at 3800 rpm for 10 min, then resuspend in 2 ml of 2xTY and spread on a 2xTYAG agar bioassay plate (containing 100 μg / ml ampicillin and 2% glucose). After incubation overnight at 30°C, add 10 ml of 2xTY to the plate, scrape 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. Hold the selected aliquots at -80°C.

[0459] bacteriophage rescue

[0460] Add 50 μl of cell suspension obtained from previous selections to 50 ml of 2xTYAG and grow at 37°C with stirring (240 rpm) until OD.600 Reach 0.3 to 0.5. Then use 1.2 x 10 11 M13K07 helper phage was used to superinfect the culture, and the cells were incubated at 37°C (90 rpm) for one hour. The culture medium was replaced by centrifuging the cells at 3800 rpm for 10 minutes, removing the medium and resuspending the pellet in 50 ml of 2xTYAK medium (2xTY medium containing 100 μg / ml ampicillin and 50 μg / ml kanamycin). The culture was then incubated overnight at 30°C (240 rpm). The next day, the supernatant containing the phage was used for the next round of selection.

[0461] Cell surface selection

[0462] At room temperature, the phage-containing supernatant was blocked for one hour in PBS containing 3% (w / v) skim milk on a rotary mixer. Then, MKN-45 CEACAM5, which does not express human CEACAM5, was used as a buffer. KO Cells were selected to block phages one hour later. The selected phages were then compared with 2 x 10⁻⁶ cells expressing CEACAM5. 7 MKN-45 cells (blocked in PBS, 3% BSA, 0.1% NaN3) were incubated at room temperature with gentle shaking for 2 hours. The cell pellet was collected and washed six times with PBS. Bacteriophages were eluted with 76 mM citrate and shaken for 10 min. After neutralization with 1 M pH 8 Tris-HCl, the cells were added directly to 10 ml of exponentially growing TG1 and incubated at 37°C with gentle shaking for 1 hour. An aliquot of infected TG1 was serially diluted to titrate the selection output. The infected TG1 was spun at 3800 rpm for 10 min, then resuspended in 2 ml of 2xTY medium and spread onto a 2xTY AG agar bioassay plate. After incubation overnight at 30°C, 10 ml of 2xTY was added to the plate, cells were scraped from the surface and transferred to 50 ml polypropylene tubes. 50% glycerol solution was added to the cell suspension to obtain a final concentration of 17% glycerol. The aliquots of the selection round were held at -80°C.

[0463] Example 3: Screening for scFvs that bind / do not bind to soluble CEACAM5, CEACAM6, and CEACAM1

[0464] scFv periplate formulation for binding and functional testing

[0465] Transformed TG1 clones from selected individuals were inoculated into deep-well microtiter plates containing 0.9 ml of 2xTYAG medium (2xTY medium containing 100 μg / ml ampicillin and 0.1% glucose) per well and grown at 37°C for 5–6 h (240 rpm). Then, 100 μl of 0.2 mM IPTG in 2xTY medium was added to each well to obtain a final concentration of 0.02 mM IPTG. The plates were incubated overnight at 30°C with shaking at 240 rpm. The deep-well plates were centrifuged at 3200 rpm for 10 min at 4°C, and the supernatant was carefully removed. The precipitate was resuspended in 150 μl of TES buffer (50 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8), 20% sucrose, supplemented with a complete protease inhibitor, Roche). Hypotonic shock was induced by adding 150 μl of diluted TES buffer (1:5 TES:water dilution) and incubating on ice for 30 min. Centrifuge the plate at 4000 rpm for 10 minutes at 4°C to precipitate cells and debris. Carefully transfer the supernatant to another microtiter plate and keep it on ice for immediate testing in functional or binding assays.

[0466] Combination

[0467] Using CellInsight TM The technique was used to test scFv screening for binding to CEACAM5 in a homogeneous assay. The following reagents were mixed in each well of a 384-well clear plate (Corning): 30 μl of streptavidin polystyrene bead suspension (Polysciences; 3000 beads / well) coated with biotinylated CEACAM5, biotinylated domains A3-B3, or biotinylated NusA (control protein); 60 μl of blocking scFv periplasmic preparation; 10 μl of detection buffer (PBS containing 5 μg / ml mouse anti-c-myc antibody; anti-mouse Fc diluted 1:200). 647). After mixing at 600 rpm for 5 minutes, the 384-well plate was incubated at room temperature for 2 hours, and then analyzed in CellInsight. TM Readings were taken on the CX5 high-content screening platform (ThermoFisher Scientific). Clones expressing scFvs that give a specific signal to CEACAM5 rather than NusA were selected for further analysis or sequencing.

[0468] The combination with CEACAM1, CEACAM6 and other CEACAMs can be measured in the same way.

[0469] Cloning sequencing

[0470] Single clones were inoculated into 96-well deep-well microtiter plates containing 1 ml of LBAG medium (LB medium containing 100 μg / ml ampicillin and 2% glucose) per well and grown overnight at 37°C and 300 rpm. DNA was extracted and sequenced using the Zyppy-96Plasmid Miniprep kit (Zymo Research).

[0471] Example 4: Reformatting immobilized VH candidates into IgG and transient expression in mammalian cells

[0472] Following screening and sequencing, candidate scFvs with the desired binding properties were reformulated as IgG and expressed via transient transfection into PEAK cells. The VH and VL sequences of the selected scFvs were amplified with specific oligonucleotides and cloned into expression vectors containing heavy and light chain constant regions, with the construction validated by sequencing. The expression vectors were transfected into mammalian cells using a Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. In short, 4x10 6 PEAK cells were cultured in 25 ml of culture medium containing fetal bovine serum in T75 culture flasks. Transfected cells were cultured at 37°C for 5–6 days, and IgG production was quantified using an Octet RED96 instrument. Following the manufacturer's instructions, the supernatant was collected for IgG purification on FcXL affinity resin (Thermo Fisher Scientific). Briefly, the supernatant of transfected cells was incubated with an appropriate amount of FcXL resin overnight at 4°C. After washing the resin with PBS, the sample was loaded onto an Amicon Pro column, and IgG was eluted in 50 mM Glycine pH 3.5. The eluted IgG fraction was then dialyzed against Amicon 50 kDa histidine-targeted NaCl pH 6.0 buffer, and the IgG concentration was quantified by absorbance at 280 nm. Purity and IgG integrity were verified by electrophoresis using an Agilent Bioanalyzer 2100, following the manufacturer's instructions (Agilent Technologies, Santa Clara, Calif., USA).

[0473] Example 5: Characterization of CEACAM5 monoclonal antibody

[0474] a) Binding of the anti-CEACAM 5-arm to cells transfected with different members of the CEACAM family

[0475] The specificity of the anti-CEACAM5 antibody arm (tested as a bivalent mAb or a monovalent bsAb) was demonstrated by flow cytometry using PEAK and / or CHO cells transfected with different members of the CEACAM family.

[0476] As described in Example 1, vectors encoding the full-length forms of human CEACAM 1, 3, 4, 5, 6, 7, 8, 18, 19, 20, and 21 and 20 were used to express these proteins on the surface of PEAK and / or CHO cells. Similarly, vectors encoding the full-length forms of cynomolgus monkey CEACAM 5 and 6 were also used to express these proteins on the surface of PEAK and / or CHO cells. Untransfected PEAK and / or CHO cells were used as negative controls. Cells were harvested, counted, viability checked, and expressed at 3 × 10⁻⁶ cells. 6 Cells / ml were resuspended in FACS buffer (PBS, 2% BSA, 0.1% NaN3). 100 μl of the cell suspension was distributed into 3 × 10⁶ wells of a V-bottom 96-well plate. 5 Cells / well. The supernatant was removed by centrifugation at 4°C, 1300 rpm for 3 min, and the cells were incubated at 4°C with an increased concentration of the antibody according to the invention for 15 min. The antibody carrying the anti-CEACAM5 arm to be tested was diluted in FACS buffer to a concentration ranging from 30 pM to 500 nM. Cells were washed twice with cold FACS buffer and then incubated again at 4°C with a compatible anti-human IgG secondary antibody for 15 min. Cells were washed twice with cold FACS buffer and resuspended in 300 μl of FACS buffer with a 1:1500 dilution of TOPRO-3 (Invitrogen). Using FACSCalibur... TM Fluorescence was measured using either BD Biosciences or the Cytoflex Platform (Beckman Coulter). Dose-response binding curves were fitted using GraphPad Prism8 software. CEACAM1, CEACAM6, and other CEACAMs were characterized in the same manner.

[0477] Results obtained using the experimental procedures described in Examples 1 and 5a are shown in Tables 2 and 3 (full-size antibody tested at 10 mcg / ml; BiTE MEDI-565 tested at equimolar concentrations). For the bispecific antibodies AB17L3-1 / N, AB71L3-1 / N, AB72L3-1 / N, and AB73L3-1 / N, the measured MFIs for binding to CEACAM5-transfected cells ranged between 29,000 and 41,000 (Table 2). In contrast, MFIs found using PEAK cells transfected with CEACAM1, 3, 4, 6, and 8 were below 1000, with the sole exception of a strong signal for AB72L3-1 / N on CEACAM8-transfected cells. The “relative PEAKWT factor” can be calculated by dividing the MFI value obtained on transfected cells expressing any given CEACAM by the value obtained on WT PEAK cells (Table 3). Except for AB72L3-1 / N, all antibodies of this invention are specific for CEACAM5 because their "relative PEAK WT factor" values ​​are all below 2.0. In contrast, MEDI-565BiTE showed a "relative PEAK WT factor" for CEACAM8 above 2, indicating cross-reactivity among this type of CEACAM family member. This could lead to killing, for example, neutrophils, since, as mentioned above, human neutrophils express CEACAM8 on their surface.

[0478] Table 2. Binding of CEACAMx to transiently expressed CEACAMx on PEAK cells [MFI]

[0479]

[0480] Table 3. Binding of CEACAMx transiently expressed on PEAK cells [factor relative to PEAK WT]

[0481]

[0482] b) Binding of CEACAM5 monoclonal antibody to recombinant protein in enzyme-linked immunosorbent assay (ELISA)

[0483] Biotinylated recombinant human CEACAM5 protein was captured at 0.5 μg / mL in streptavidin-coated 96-well microplates. The plates were washed and the monoclonal anti-TAA bivalent antibody of the present invention was added at a wide concentration range (e.g., from 5 x 10⁻⁶). -4 Add to 1 μg / mL and incubate for 1 hour. Wash the plate and detect the bound antibody using anti-human IgG (Fc)-HRP (Jackson ImmunoResearch). After washing, use Amplex... Molecular Probes display plate. Fluorescence signal was measured on a Synergy HT microplate reader (Biotek).

[0484] Binding with other recombinant CEACAM family members, such as CEACAM1 and CEACAM6, can be evaluated similarly. Figure 8 and Figure 9 The binding results for 1B4 mAb and C11 mAb are shown respectively.

[0485] c) Epitope compartmentation of the antibody of the present invention by competing with a reference antibody.

[0486] Epitope compartmentalization is a competitive immunoassay used, for example, to characterize the binding of novel monoclonal antibodies to target proteins. A competitive blocking profile of novel antibodies binding to the target protein is created against antibodies that also bind to the target protein and whose binding epitopes have been established / disclosed. Competition with one of these reference antibodies indicates that the novel antibody has the same or closely located epitopes and that they are "compartmentalized" together.

[0487] The ability of the CEACAM5 mAb of the present invention to compete with a CEACAM5 reference antibody was tested by ELISA using recombinant human CEACAM5 and the following reference antibodies carrying the mouse Fc region: SM3E, mAb derived from sm3E described in patent US20050147614A1; MEDI, mAb derived from MEDI-565 described in patent WO2016036678A1; SAR, mAb derived from Mab2_VLg5VHg2 described in patent EP3199552A1; CH1A1A, derived from patent US20120251529 and Klein et al., Oncoimmunology, 2017 Jan. The mAb of CH1A1A-2F1 described in 11; 6(3); the mAb of humanized T84.66, derived from variant 1 described in patent WO2017055389; and the mAb of hMN14, derived from patent US2002 / 0165360A1.

[0488] SM3E, for example, binds more to the N-terminus of CEA, the distal portion of the cell membrane; MEDI binds to the middle portion; and CH1A1A binds to the position closer to the membrane.

[0489] Biotinylated human CEACAM5 was coated at 0.5 μg / ml in streptavidin-coated 96-well plates and incubated for 1 hour with a 10 μg / ml reference mAb or an unrelated mAb carrying the mouse Fc region. The CEACAM5 mAb of this invention (i.e., a bivalent monoclonal anti-CEA antibody) was added at 0.2 μg / ml at room temperature for 1 hour. The plate was washed and the bound CEACAM5 mAb was detected using anti-human IgG (Fc)-HRP (Jackson Immuno Research). After washing, [the plate was then used...]. Red reagent display plate. Fluorescence signal was measured on a Synergy HT microplate reader (Biotek).

[0490] Based on the results obtained using CEACAM5 mAb, if the binding to CEACAM5 is reduced by more than 80% compared to the result without the tool antibody, the CEAxCD3 bsAb derived according to the present invention is considered to compete with the reference antibody. If the binding to CEACAM5 is reduced by less than 20% compared to the result without the tool antibody, the CEAxCD3 antibody is identified as not competing with the tool antibody. Figure 1 The binding region of the reference antibody used in Example 5c is shown schematically.

[0491] d) Determining the CEACAM5 domain bound by the antibody of the present invention using a truncated form of CEACAM5.

[0492] The antibody-binding subdomains of the present invention can be determined using a truncated form of CEACAM5 that lacks one or more extracellular subdomains.

[0493] Vectors encoding the full-length form of human CEACAM5 (containing all its extracellular domains, i.e., N-A1-B1-A2-B2-A3-B3) and vectors encoding only subsets of the extracellular domains of CEACAM5 (A1-B1-A2-B2-A3-B3; B1-A2-B2-A3-B3; A2-B2-A3-B3; B2-A3-B3; A3-B3 and B3) were used to express these proteins on the surface of PEAK and / or CHO cells, as described in Example 1. Untransfected PEAK and / or CHO cells were used as negative controls. Flow cytometry staining and collection were performed as described in Section 5,a).

[0494] If the bound antibody is detected by the PE-conjugated anti-human IgG Fc secondary antibody, it is found that the antibody according to the invention binds to the given truncated CEACAM5 protein.

[0495] Example 6: Expression and purification of bispecific antibodies carrying λ and κ light chains

[0496] Simultaneous expression of one heavy chain and two light chains in the same cell can lead to the assembly of three different antibodies. Simultaneous expression can be achieved in various ways, such as by transfecting multiple vectors expressing one of the co-expressed chains or by using a vector that drives the expression of multiple genes. A vector encoding different anti-CEACAM5 antibodies was co-transfected with another vector expressing both the heavy and light chains of an anti-CD3 antibody. Alternatively, as described in US2012 / 0184716 and WO2012 / 023053 (each incorporated herein by reference in its entirety), two light chains were cloned into a previously generated vector pNoviκHλ to allow co-expression of one heavy chain, one κ light chain, and one λ light chain. The expression of these three genes is driven by a human cytomegalovirus promoter (hCMV), and the vector also contains a glutamine synthase gene (GS), which enables the selection and establishment of stable cell lines. The common VH and VL genes for anti-CEACAM5 IgG and anti-CD3 IgG were cloned into the vector pNoviκHλ for transient expression in mammalian cells. Expi293 cells were suspended in suitable Erlenmeyer flasks with appropriate cell numbers and culture medium volumes. Plasmid DNA was then transfected into Expi293 cells using a PEI (polydioxanone) solution. During production, antibody concentration in the transfected cell supernatant was measured using an Octet RED96. Based on antibody concentration, the supernatant was collected 5–7 days post-transfection and clarified by centrifugation at 1300g for 10 minutes. Purification was based on a three-step process. First, CaptureSelect… TM FcXL affinity matrix (Thermo Fisher Scientific) was washed with PBS and then added to the clear supernatant. After incubation overnight at +4°C and 20 rpm, the supernatant was centrifuged at 2000 g for 10 min, the eluent was stored, and the resin was washed twice with PBS. The resin was then transferred to an Amicon Pro column and eluted with a solution containing 50 mM glycine at pH 3.0. Several elution fractions were generated, neutralized with Tris-HCl at pH 7.4, and combined. The library containing total human IgG (bispecific and two monospecific antibodies) was quantified using a Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, Del.), and then precipitated with an appropriate volume of Capture Select at RT and 20 rpm. TM Incubate with KappaXL affinity matrix (Thermo Fisher Scientific, GE Healthcare) for 30 minutes. Perform resin recovery and washing, elution, and neutralization steps as previously described. The final affinity purification step is performed using CaptureSelect. TMThe λFab affinity matrix (Thermo Fisher Scientific) was used, employing the same process as the κ purification step. All elution fractions were collected and desalted using a 50 kDa Amicon Ultra centrifuge filter unit (MerckMillipore) prepared with a buffer formulated for His-NaCl pH 6. The final product was quantified using Nanodrop.

[0497] As described by the manufacturer (Agilent Technologies, Santa Clara, Calif., USA), the purified bispecific antibody was analyzed by electrophoresis under denaturing and reducing conditions using an Agilent 2100 Bioanalyzer with a Protein 80 kit. 4 μL of purified sample was mixed with sample buffer supplemented with dithiothreitol (DTT; Sigma Aldrich, St. Louis, MO.). The sample was heated at 95°C for 5 minutes and then loaded onto the chip. Endotoxin contamination of all samples was tested using the horseshoe crab lysate assay (LAL; Charles River Laboratories, Wilmington, Mass.).

[0498] Example 7: In vitro characterization of monovalent and bispecific antibodies

[0499] a) Binding of monovalent and bispecific antibodies to cells expressing CEACAM5 and cells not expressing CEACAM5.

[0500] To demonstrate the binding of the CD3 x CEACAM5κλ antibody to target cells, a series of flow cytometry-based experiments can be performed to compare the binding of the CD3 x CEACAM5κλ antibody to its monovalent counterpart. Examples of cell lines 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), and CEACAM5-negative cell lines, such as the lung cancer cell lines A549 and MKN45 CEACAM5 knockout cell lines generated via the CRISPR-CAS9 method. Cell staining and binding assessment can be performed as described above. The obtained binding curves are shown in the figure. Figure 4 , 5As shown in 11 and 14. EC50 values ​​for binding to MKN45 cells were calculated using GraphPad Prism8; the data are shown in Table 4. Compared to binding to TCB2014, the bsAbs of this invention showed 40% or more higher binding at 200 nM, 1000 nM, and 5000 nM (see Table 4 and...). Figure 11 and 14 ).

[0501] Table 4: EC50 binding to MKN-45 cells. N / A: Not applicable.

[0502] EC50(nM) Highest MFI value AB17L3-1 / N 59.2 862’218 AB54L3-1 / N 66.4 759’782 AB60L3-1 / N 64.1 989’186 AB66L3-1 / N 24.3 759’391 AB71L3-1 / N 34.1 833’116 AB72L3-1 / N 24.5 962’960 AB73L3-1 / N 27.8 920’523 Y4L3-1 / N N / A N / A TCB2014 11.6 327’315

[0503] b) Binding of monovalent and bispecific antibodies to cells expressing CD3 and cells not expressing CD3.

[0504] To demonstrate the binding of the CD3 x CEACAM5 κλ antibody to effector T cells, a series of flow cytometry-based experiments can be performed to compare the binding of the CD3 x CEACAM5 κλ antibody to its monovalent counterpart. Examples of cell lines that can be used include human primary T cells and CD3-positive (Jurkat and / or HuT78) or CD3-negative (TIB-153 and / or JKT-beta-del) cell lines. Cell staining and binding assessment can be performed as described above. Results are as follows: Figure 3 and Figure 10 As shown.

[0505] c) Epitope fragmentation of CEACAM5 antibody through competition with reference antibody.

[0506] Epitope compartmentalization is a competitive immunoassay used to characterize the binding of antibodies according to the invention, or the binding of related anti-CEA (target protein) antibodies, such as those with a first binding site. A competitive blocking profile of antibodies binding to the target protein is created against antibodies that also bind to the target protein and whose binding epitopes have been established / disclosed. Competition with one of these reference antibodies indicates that the antibodies have the same or closely spaced epitopes and that they are "compartmentalized" together. The ability of the anti-CEACAM5 arm, which is part of the bispecific antibody of this invention, to compete with the anti-CEACAM5 reference antibody was tested by ELISA using recombinant human CEACAM5 and the following reference antibodies carrying the mouse Fc region: SM3E, a mAb derived from the sequence of the SM3E mAb described in patent US20050147614A1, generated using a standard method; MEDI, a mAb derived from MEDI-565 described in patent WO2016036678A1; and CH1A1A, a mAb derived from CH1A1A-2F1 described in patent US20120251529 and Klein et al., Oncoimmunology, 2017 Jan 11; 6(3). SM3E binds more to the N-terminus of CEA, the distal portion of the cell membrane, MEDI binds to the middle portion, and CH1A1A binds to the position closer to the membrane.

[0507] κλ bodies used at 1 μg / ml were captured by goat anti-human IgG (Fcγ) (Jackson ImmunoResearch) at 10 μg / ml coated on 96-well black microplates and blocked with blocking buffer (PBS 2% BSA, 0.05% Tween 20). Competitive IgG (0.03 to 20 μg / ml) was pre-incubated with 0.1 μg / ml biotinylated human CEACAM5 in blocking buffer for 1 hour. The κλ body plate was washed and incubated with the pre-incubated competitive IgG / CEACAM5 mixture for 1 hour. After washing, CEACAM5 was detected using streptavidin-HRP (Jackson ImmunoResearch). Amplex was used to detect CEACAM5. TM The Red reagent (Molecular Probes) was used to expose the plate, and the fluorescence signal was measured on a Synergy HT microplate reader (Biotek).

[0508] If the corresponding tool antibody reduces the binding of CEACAM5 to the κλ body by 80% or more, it can be concluded that the CEAxCD3 bispecific antibody is classified as competing for binding with the tool antibody. Therefore, if the results of adding and not adding the tool antibody result in a reduction of 20% or less in the binding of CEACAM5 to the corresponding κλ body, the CEAxCD3 antibody is identified as not competing with the tool antibody.

[0509] d) Binding of bispecific antibodies to human primary blood cells

[0510] To demonstrate the binding of the CD3 x CEACAM5κλ antibody to primary T cells, and its lack of binding to primary B cells and monocytes (CEA-negative population), a series of flow cytometry-based experiments can be performed. Cell staining and binding assessment can be performed as described in Example 7a. Data are as follows: Figure 13 As shown.

[0511] Example 8: Bispecific antibody-mediated T cell-dependent cytotoxicity (TDCC)

[0512] a) TDCC in CEACAM5-positive and CEACAM5-negative cell lines

[0513] 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 was evaluated using human PBMCs or purified primary T cells as effector cells.

[0514] After washing twice with PBS, target cells were separated using trypsin or cell dissociation solution. Following centrifugation, the cells were resuspended in assay medium, adjusted to the desired concentration, and plated in 96-well plates.

[0515] Effector cells can be human peripheral blood mononuclear cells (PBMCs) or purified T cells. Use Lymphoprep... TM SepMate (Stemcell Technologies) TM Stemcell Technologies isolates PBMCs from the erythrocyte sedimentation rate (ESR) layer derived from healthy human donors. If the purified T cells are intended to be used as effector cells, an additional purification step is performed, in which T cells are negatively isolated from the PBMCs using a T cell immunomagnetic negative selection kit (STEMCELL Technologies).

[0516] For the TDCC assay, when PBMCs were used as effector cells, these cells were added to target cells at a final E:T ratio of 10:1; when purified T cells were used, a final E:T ratio of 5:1 was used. The CEAxCD3 antibody of the present invention and related control antibodies were then added to pre-coated target and effector cells at dose-range concentrations (up to 100 nM, in duplicate). Target cell killing was assessed by quantifying the release of LDH (Cytotoxicity Detection Kit PLUS (LDH)) from apoptotic / necrotic cells into the culture medium after incubation at 37°C and 5% CO2 for 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-incubated with effector cells without the addition of any antibody. TDCC curve ( Figure 6 , 7 The EC50 values ​​(12 and 15) and EC50 values ​​(Table 5) can be calculated using GraphPad Prism8. For the MKN-45 and LS174T cell lines, the EC50 values ​​found using the bsAbs of the present invention shown in Table 5 were significantly lower than those measured using TCB2014, indicating that these bsAbs of the present invention have higher in vitro tumor cell killing efficacy.

[0517] Table 5. Killing EC50 of three CEA+ cell lines

[0518] EC50(nM) MKN45 HPAFII LS174T AB17L3-1 / N 0.11 0.16 0.25 AB54L3-1 / N 0.11 0.10 0.13 AB60L3-1 / N 0.13 0.28 0.21 AB66L3-1 / N 0.05 0.20 0.22 AB71L3-1 / N 0.09 0.16 0.16 AB72L3-1 / N 0.02 0.12 0.11 AB73L3-1 / N 0.03 0.13 0.06 TCB2014 0.85 0.45 1.87 Y4L3-1 / N N / A N / A N / A

[0519] b) Kill assay using a combination of CEAxCD3 and CEAxCD47 bispecific antibodies

[0520] The bispecific antibody of the present invention, in combination with an anti-CD47 mAb (e.g., described in US20140140989 and WO2017196793) or with a CEAxCD47 bispecific antibody (described in PCT / IB2019 / 054559, incorporated herein by reference), can be tested in the models described above. Additional testing conditions can be added to the experimental design, wherein such CD47-targeting antibodies (monospecific or bispecific) are used alone or in combination with the CEAxCD3 antibody of the present invention at different doses.

[0521] c) Upregulation of T cell activation markers after killing CEA-expressing tumor cells induced by CEAxCD3 bsAb.

[0522] 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 (an early activation marker) or CD25 (a late activation marker).

[0523] To assess the activation status of T cells at the end of the kill assay (as described above, Example 8a), the following procedure was followed: Floating cells (including CD4+ and CD8+ T cells) were transferred to new V-bottom 96-well plates. After removing the supernatant by centrifugation (3 min at 4°C, 1300 rpm), the cells were washed twice with cold FACS buffer (PBS 2% BSA, 0.1% NaN3) and then incubated at 4°C with Fc blocking reagent (BD Biosciences) for 15 min. After washing twice with FACS buffer, cells were incubated at 4°C for 15 minutes with the following antibodies (used according to the manufacturer’s recommendations): anti-CD45 (V500 conjugated, BDBiosciences), CD69 (FITC conjugated, Biolegend), CD8 (PerCP-Cy5.5-conjugated, Biolegend), CD25 (PE-conjugated, Biolegend), CD4 (APC-conjugated, ThermoFisher), and CD3 (APC-R700-conjugated, BDBiosciences).

[0524] Cells were washed twice with cold FACS buffer and resuspended in 200 μl of FACS buffer. Fluorescence was measured using the Cytoflex Platform (Beckman Coulter) and analyzed using FlowJo. TM Data was analyzed using the v10 software (BD LifeSciences). Results are as follows: Figure 17 As shown.

[0525] d) T cell proliferation induced by CEAxCD3 bsAb molecules

[0526] The ability of CEAxCD3 bsAb to induce T cell proliferation after cross-linking in the presence of CEA-positive tumor target cells was analyzed. CEA-negative malignant cells were also used as a negative control. Freshly isolated human PBMCs were adjusted to 1 million cells / mL in warm PBS and stained with 0.2 μM carboxyfluorescein diacetate succinimide (CFSE, ThermoFisher Scientific) in PBS at 37°C for 15 min. The cells were 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 stained with 2 x 10⁻⁶ mol / L solution.6 Transfer cells / mL to 96-well plates. Seed 0.02 x 10⁻⁶ cells / mL in each well of a flat-bottomed 96-well plate. 6 Target cells were selected, and different concentrations of CEAxCD3 bsAb were added. CFSE-labeled PBMCs were added to obtain a final E:T ratio of 10:1, and the assay plates were incubated in a humidified incubator at 37°C for 5 days. On day 5, effector cells were collected, washed twice with FACS buffer (PBS, 2% BSA, 0.1% NaN3), stained with BD Horizon 620 (BD Biosciences, 564996) to exclude dead cells, and stained with anti-CD45 (V500-conjugated, BD Biosciences), anti-CD4-APC (ThermoFischer, 17-0049-41), and anti-CD8-PerCP-Cy5.5 (Biolegend, 301032). CFSE staining of live CD4+ or CD8+ cells was analyzed by flow cytometry using CytoFLEX (Beckman Coulter), and the results were evaluated using FlowJo software.

[0527] e) Cytokines released in the supernatant following CEAxCD3 bsAb-induced killing of CEA-expressing tumor cells.

[0528] The killing of CEA-positive tumor cells induced by CEAxCD3 bsAb requires T cell activation. Upon activation, T cells can release various cytokines, which can further act as immunomodulators. The ability of the bispecific antibody of this invention to induce T cell cytokine release during the killing of CEA-expressing tumor cells was assessed by quantifying selected cytokines in the supernatant at the end of the TDCC assay described in Example 8a. After co-culturing CEA-positive target cells and CD3-positive effector T cells for 2 days, the culture supernatant was collected by centrifugation and frozen at -80°C until further analysis. Cytokines / enzymes (e.g., granzyme B, IL2, IL6, IL10, TNFα, and IFNγ) were quantified using a multiplex kit on the Mesoscale Discovery Platform, and the results are as follows: Figure 16 As shown.

[0529] f) TDCC in CEACAM5 positive cells in the presence of shed CEA

[0530] It is known that CEA-positive tumors shed CEA. This shed CEA may negatively impact the antitumor efficacy of CEA-targeting antibodies that do not preferentially bind to membrane-bound CEA. To assess whether the bispecific antibody of the present invention is affected by shed CEA (sCEA), the T-cell-dependent cytotoxicity (TDCC) assay described in Example 8a was performed in the presence of different concentrations of incorporated sCEA (BioRad#PHP282). The EC50 values ​​in the presence of sCEA were then compared with those obtained in the absence of sCEA (Table 6). The EC50 calculated for a given sCEA concentration (0.2, 1, or 1 μg / mL) was then compared with the EC50 obtained in the absence of sCEA (0 μg / mL), and expressed as a fold change in EC50 compared to the condition without shed CEA. These values ​​are reported in Table 7.

[0531] Table 6. EC50 of LS174T cell killing in the presence of sCEA

[0532]

[0533]

[0534] *No highest platform

[0535] Expect

[0536] Table 7. Changes in EC50 compared to the condition without CEA shedding (0 μg / mL)

[0537]

[0538] *No highest platform

[0539] Expect

[0540] At 1 and 5 μg / mL sCEA levels, compared to the bispecific antibodies of the present invention, the addition of sCEA to TCB2014 and TCB2017 resulted in a significantly higher shift in the EC50 for tumor cell killing. Concentrations of sCEA at 1 μg / mL and above were found in CEA-positive tumor patients. The lower shift in the killing curve of the bsAbs of the present invention due to sCEA indicates that high sCEA levels have a smaller inhibitory effect on the efficacy of the bsAbs of the present invention compared to TCB2014 and TCB2017.

[0541] g) TDCC of CEACAM5 negative primary blood cell population.

[0542] Given the mechanism of action of the CEAxCD3 bispecific antibody, cross-reactions with other CEACAMs could lead to the depletion of an important circulating healthy cell population. For example, cross-reactions with CEACAM8 expressed by neutrophils could lead to the depletion of such cell populations. To confirm the absence of binding and therefore the absence of killing of this CEA-negative circulating healthy cell population, purified primary cells such as neutrophils were used instead of CEA-positive cell lines as “target cells” in the experimental procedure described in Example 8a.

[0543] Example 9: Evaluation of the antitumor activity of CEAxCD3 T cell retargeting molecules as a single agent or in combination with CD47-targeting antibodies in a humanized mouse tumor model.

[0544] a) Antitumor activity of CEAxCD3 molecules in the PBMC humanized mouse tumor model

[0545] 8-10 week old NOG mice (NOD / Shi-scid / OL-2Rγ) null Mice (Taconic Biosciences) subcutaneous (sc) implantation of 1 to 5 x 10 6 Mice were randomly assigned to several treatment groups and subjected to CEA-positive tumor cells (cell line or patient-derived) via intraperitoneal or intravenous injection of 10 or 20 x 10⁻⁶ cells. Four to seven days later, all mice were injected intraperitoneally or intravenously with 10 or 20 x 10⁻⁶ cells. 6 Personal PBMCs (peripheral blood mononuclear cells) were used in the humanization process. Then, 3–6 days after PBMC injection, CD3xCEA molecules or controls were administered intravenously once or twice weekly at different doses. Tumor development in mice was monitored three times weekly, and tumors were measured using a digital caliper until the end of the experiment (tumor volume = 1500 mm²). 3 Or GvHD symptoms may occur. Use the formula (length x width) 2 The tumor volume was calculated by multiplying the result by 0.5. Statistical analysis was performed using univariate ANOVA at the end of the study. Figure 18 The results show the effects of subcutaneous transplantation of 1 million HPAF-II cells into NOG mice followed by injection of 10 million human PBMCs.

[0546] b) Antitumor activity of CEAxCD3 molecules in a CD34+-humanized mouse tumor model

[0547] Fully humanized CD34+-huNOG mice (CD34+ transplanted NOD / Shi-scid / OL-2Rγ) null Mice (Taconic Biosciences), 14 weeks old, with >25% human CD45 in their blood. +Cells, subcutaneous (sc) implantation 1 to 5 x 10 6 CEA-positive tumor cells (cell line-derived or patient-derived) were randomly assigned to several treatment groups. Treatment was initiated when the mean tumor volume reached a predetermined value (range from 100 to 200 mm). 3 During this period, CD3xCEA molecules or controls were administered intravenously once or twice a week at different doses. Tumor growth in mice was monitored three times a week, and tumor volume was measured using a digital caliper until the end of the experiment (tumor volume = 1500 mm²). 3 Use the formula (length x width) 2 The tumor volume was calculated by multiplying the result by 0.5. Statistical analysis was performed using univariate ANOVA at the end of the study.

[0548] c) Antitumor activity of CEAxCD3 molecules in combination with CD47-targeting antibodies (monospecific or bispecific) in humanized mouse tumor models.

[0549] The bispecific antibody of the present invention, in combination with an anti-CD47 mAb (e.g., described in US20140140989 and WO2017196793) or with a CEAxCD47 bispecific antibody (described in PCT / IB2019 / 054559, incorporated herein by reference), can be tested in the models described above. Additional groups were added to the experimental design, including treatment groups receiving intravenous administration of CD47-targeting antibodies (monospecific or bispecific) alone or in combination with the CEAxCD3 antibody of the present invention at different doses once or twice weekly.

[0550] d) Antitumor activity of CEAxCD3 molecules in combination with CD47-targeting antibodies (monospecific or bispecific) in transgenic mouse tumor models.

[0551] The bispecific antibody of the present invention, in combination with anti-CD47 mAb (e.g., described in US20140140989 and WO2017196793) or with CEAxCD47 bispecific antibody (described in PCT / IB2019 / 054559, incorporated herein by reference), can be tested in transgenic mice engineered to express human CD3, human CD47, and human SIRPα, which are subcutaneously (sc) implanted with 0.5 to 5 x 10 6 A number of engineered mouse tumor cells expressing human CEA and human CD47 were selected. The tumor volume was measured when the mean tumor volume reached a predetermined value (range from 100 to 200 mm). 3 Mice were randomly assigned to receive intravenous treatment once or twice a week at varying doses. Tumor growth was monitored three times a week, and tumor volume was measured using a digital caliper until the end of the experiment (tumor volume = 1500 mm²). 3Use the formula (length x width) 2 The tumor volume was calculated by multiplying the result by 0.5. Statistical analysis was performed using univariate ANOVA at the end of the study.

[0552] Example 10: Cytokine release tested in whole blood and PBMCs from healthy human donors

[0553] In vitro cytokine release assays were performed using whole blood (WB CRA) in an aqueous presentation with minimal dilution of the test antibody (95% v / v blood). This assay format is considered to closely mimic the in vivo environment, containing factors at physiological concentrations that may influence the mechanisms of cytokine release. However, this format is not considered to be a good predictor of T cell-mediated cytokine release (e.g., anti-CD28).

[0554] Alternatively, cytokine release assays can be performed using antibodies from peripheral blood mononuclear cells (PBMCs) and aqueous presentation (aqueous phase, AP) derived from healthy human donors to assess T cell-mediated cytokine release (PBMC APCRA). This format restricts mAb cross-linking to avoid the high cytokine release observed with anti-CD3 antibodies during cross-linking.

[0555] Negative controls (anti-EGFR mAb and PBS) and specific positive controls (anti-CD52 mAb, CEA x CD3 BiTE, and / or anti-CD28 mAb) were tested in parallel with the CEA x CD3 bispecific antibody for each assay. Cytokines in the supernatant were tested in multiplex assays using electrochemiluminescence as readout (Mesoscale Discovery, Sector 600) 24 hours after Western blotting (WB) CRA and 48 hours after PBMC aPCRA CRA. IFNγ, TNFα, and IL-6 were measured for WB CRA, and IFNγ, IL-2, IL-10, and TNFα were measured for PBMC aPCRA. Results for each cytokine were plotted, with each donor shown as a single data point.

[0556] Example 11: CEA antibody affinity maturation using degenerate oligonucleotides via oligonucleotide-directed mutagenesis (leader optimization; LO)

[0557] Antibodies identified during the screening process described in Example 3 were selected for affinity maturation to increase their affinity and potency. All these antibodies shared the same variable heavy chain but had different variable light chains. AB1 and C11 contained a κ light chain (IGKV3-11 and IGKV1-5, respectively, according to IMGT nomenclature), while AB8 and 1B4 contained a λ light chain (IGLV2-14 and IGLV3-21, respectively). Several phage libraries displaying scFv variants were generated by introducing diversity in the CDR1, CDR2, and CDR3 regions of the light chain variable regions while keeping the heavy chain variable regions unmodified. Different diversification strategies were used to generate libraries for each candidate, where CDRL1+CDRL2; or only CDRL3; or all three CDRLs were diversified by oligonucleotide-directed mutagenesis of the parental sequences using degenerate oligonucleotides (CDRL1+CDRL2+CDRL3). CDRL1 diversifies at 1 to 5 amino acid positions; CDRL2 diversifies at 1 to 4 amino acid positions, and CDRL3 diversifies at 1 to 5 amino acid positions. A total of up to 5 x 10⁻⁶ samples were generated for each candidate. 9 There are 10 transformants, partially covering up to 10 14 Theoretical diversity.

[0558] For candidates AB1 and 1B4, additional libraries with diversity at up to 17 amino acid positions across all CDRLs were generated, with a maximum of 5 x 10^6 amino acids. 9 There are 10 transformants, partially covering up to 10 21 Theoretical diversity.

[0559] These libraries were used for phage display selection as described in Example 2, except that selection stringency could be increased by gradually decreasing the concentration of recombinant hCEACAM5 from 100 nM to 0.01 nM between different selection rounds or by using cells expressing lower levels of hCEACAM5, similar to the SNUC-1 cell line. The ability of selected variants to bind to CEACAM5 was screened using the assays described in Example 3. Positive clones were reformatted as IgG and characterized as described in Examples 4 and 5, respectively.

[0560] Anti-CEA arms AB1 (SEQ ID NO: 31 to 34) were optimized in two consecutive leading 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.

[0561] Example 12: TDCC (T cell-dependent cytotoxicity) and / or TDCC plus ADCP in tumor-derived organoids

[0562] Tumor cell-derived organoids are an advanced translational model for testing T cell retargeting compounds and / or macrophage and NK cell retargeting compounds.

[0563] Organoids were prepared according to standard procedures (Schütte et al., Nature Communications 2017; DOI:10.1038 / ncomms14262) and incubated with the compound for up to 8 days in a co-culture of PBMCs and in vitro-generated macrophages. The culture medium was changed and replaced with fresh medium every 4 days.

[0564] Organoids were collected and enzymatically dissociated into single cells using Accutase at 37°C for 5 minutes. Cells were pelleted, resuspended in FACS buffer (PBS, 2% FBS, 2 mM EDTA), and filtered through a 400 μm cell filter. An equal volume of the cell suspension was incubated on ice for 30 minutes with antibodies against CD45, CD4, CD8, CEA, and CD14 (all from Thermo Fisher Scientific, Dreieich, Germany). For live-cell gating, measurements and analyses were performed using propidium iodide and FlowJo software (FlowJo, LLC, Ashland, OR, USA).

[0565] The supernatant from each well was frozen at -80°C for analysis of T cell activity using ELISA.

[0566] Example 13: TDCC and / or TDCC plus ADCP in patient-derived tumor tissue sections

[0567] Fresh tumor tissue sections from patients are another advanced translational model for testing compounds that retarget T cells and / or macrophages and / or NK cells.

[0568] Fresh tumor tissue samples will be processed according to previously published standard procedures ( Cutting was performed immediately after surgical resection and initial macroscopic pathological evaluation using a tissue mincer (McIlwain TC752; Campden Instruments, Leicestershire, England). The tissue section diameter was then normalized using a 3-mm corer (Kai Europe, Solingen, Germany). Three tissue sections were randomly pooled, placed on membrane inserts, and cultured in 6-well plates. Sections were incubated at 37°C and 5% CO2 under standard conditions. The culture medium was changed before treatment at 2 hours and 24 hours after preparation.

[0569] After pre-culturing in standard cell culture medium for 24 hours, the sliced ​​triplet can be exposed individually or in combination to the bispecific antibody according to the invention for up to 120 hours. If necessary, the incubation time can be shortened to 72 hours. The culture medium will be replaced after 72 hours.

[0570] Following compound exposure, tumor sections were fixed overnight with 4% paraformaldehyde. The supernatant from each well was frozen at -80°C for T cell viability analysis using ELISA.

[0571] Paraformaldehyde-fixed sections were embedded in paraffin and processed into 5 μm sections. Hematoxylin and eosin (HE) staining was performed to assess histopathological aspects and the proportion of tumor cells. Total cell count, tumor cell count, and proliferation were analyzed by immunofluorescence staining. Briefly, the paraffin sections were dewaxed. After antigen retrieval, sections were washed with 0.3% PBS / Triton X and blocked for 30 minutes with 5% normal goat serum (Jackson Immuno Research, Suffolk, UK). Sections targeting cytokeratin... Primary antibodies against Ki67 and cleaved PARP were diluted in 0.5% bovine serum albumin and incubated overnight at 4°C. Sections were washed with 0.3% phosphate-buffered saline / Triton X and labeled with secondary antibodies. Cell nuclei were stained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO). For further analysis, antibodies against CEA (tumor cells), CD163 (macrophages), and CD3, CD4, CD8, PD-L1, and FoxP3 (all T cells) were used, depending on the availability of tumor sections.

[0572] Regions containing tumor cells were analyzed in HE sections using slide scanning (Pannoramic SCAN and Pannoramic Viewer, 3D Histech, Budapest, Hungary) to investigate different tumor cell fractions. Sections containing more benign epithelial cells than tumor epithelial cells were excluded from the analysis. Sections without tumor cells were excluded from the analysis of proliferating tumor cell fractions but included in the tumor cell analysis for each condition. For further analysis, five images (20x) of each tissue section were taken from the fluorescently stained sections using an Olympus BX51 fluorescence microscope (Olympus Deutschland, Hamburg, Germany). The staining-specific segmentation algorithm of Image J was used to determine the positive pixel counts for Hoechst 33342, cytokeratin, Ki67, and cleaved PARP staining. Proliferating / apoptotic tumor regions were calculated by analyzing pixels surrounded by Ki67 / cleaved PARP positive cell nuclei.

[0573] For each image, the total cell count (Hoechst positive), tumor cell count (Hoechst and cytokeratin positive), and proliferating tumor cell count (Hoechst, cytokeratin, and Ki67 positive / cut PARP) were calculated. The tumor cell count was normalized to the total cell count, and the proliferating tumor cell count was normalized to the total tumor cell count to account for the different tumor cell fractions in each image. Then, a mean slice value was calculated from the individual image values. The mean slice value was used to calculate the average condition.

[0574] All publications, patents, patent applications, websites, and accession numbers / database sequences, including the polynucleotide and polypeptide sequences cited herein, are incorporated herein in their entirety by reference for all purposes, to the extent that each individual publication, patent, patent application, website, or accession number / database sequence is specifically and individually identified and incorporated by reference. sequence list <110> Ramkap BioAlpha Inc. <120> Bispecific antibodies against CEACAM5 and CD3 <130> 4130.003PC02 <150> EP19198124 <151> 2019-09-18 <150> US62 / 902150 <151> 2019-09-18 <160> 138 <170> PatentIn version 3.5 <210> 1 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> huCD3 VH <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> huCD3 CDRH1 <400> 2 Thr Tyr Ala Met Asn 1 5 <210> 3 <211> 19 <212> PRT <213> Artificial sequence <220> <223> huCD3 CDRH2 <400> 3 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 4 <211> 14 <212> PRT <213> Artificial sequence <220> <223> huCD3 CDRH3 <400> 4 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 5 <211> 109 <212> PRT <213> Artificial sequence <220> <223> huCD3 VL 1B6 <400> 5 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ala Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 6 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> huCD3 1B6 CDRL1 <400> 6 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> huCD3 1B6 CDRL2 <400> 7 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> huCD3 1B6 CDRL3 <400> 8 Ala Leu Trp Tyr Ala Asn Arg Trp Val 1 5 <210> 9 <211> 109 <212> PRT <213> Artificial sequence <220> <223> huCD3 VL 1A10 <400> 9 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Lys Gly 85 90 95 Tyr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 10 <211> 14 <212> PRT <213> Artificial sequence <220> <223> huCD3 1A10 CDRL1 <400> 10 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial sequence <220> <223> huCD3 1A10 CDRL2 <400> 11 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <220> <223> huCD3 1A10 CDRL3 <400> 12 Ala Leu Trp Tyr Lys Gly Tyr Trp Val 1 5 <210> 13 <211> 109 <212> PRT <213> Artificial sequence <220> <223> huCD3 VL 1F8 <400> 13 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Asp Gly 85 90 95 Lys Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> huCD3 1F8 CDRL1 <400> 14 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> huCD3 1F8 CDRL2 <400> 15 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 16 <211> 9 <212> PRT <213> Synthetic sequence <220> <223> huCD3 1F8 CDRL3 <400> 16 Ala Leu Trp Tyr Asp Gly Lys Trp Val 1 5 <210> 17 <211> 109 <212> PRT <213> Synthetic sequence <220> <223> huCD3 VL 1A4 <400> 17 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Lys Gln 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 18 <211> 14 <212> PRT <213> Artificial sequence <220> <223> huCD3 1A4 CDRL1 <400> 18 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 19 <211> 7 <212> PRT <213> Artificial sequence <220> <223> huCD3 1A4 CDRL2 <400> 19 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial sequence <220> <223> huCD3 1A4 CDRL3 <400> 20 Ala Leu Trp Tyr Lys Gln Arg Trp Val 1 5 <210> twenty one <211> 109 <212> PRT <213> Artificial sequence <220> <223> huCD3 VL 1H4 <400> twenty one Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Asn Gln 85 90 95 His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 22 <211> 14 <212> PRT <213> Synthetic Sequence <220> <223> huCD3 1H4 CDRL1 <400> 22 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 23 <211> 7 <212> PRT <213> Synthetic Sequence <220> <223> huCD3 1H4 CDRL2 <400> 23 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 24 <211> 9 <212> PRT <213> Synthetic Sequence <220> <223> huCD3 1H4 CDRL3 <400> 24 Ala Leu Trp Tyr Asn Gln His Trp Val 1 5 <210> 25 <211> 215 <212> PRT <213> Synthetic Sequence <220> <223> huCD3 1B6 LC <400> 25 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe<000168�>50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ala Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 <210> 26 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> huCD3 1A10 LC <400> 26 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Lys Gly 85 90 95 Tyr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 <210> 27 <211> 215 <212> PRT <213> Synthetic Sequence <220> <223> huCD3 1F8 LC <400> 27 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15<000175!>Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Asp Gly 85 90 95 Lys Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 <210> 28 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> huCD3 1A4 LC <400> 28 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Lys Gln 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 <210> 29 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> huCD3 1H4 LC <400> 29 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Asn Gln 85 90 95 His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 <210> 30 <211> 329 <212> PRT <213> Artificial sequence <220> <223> Common constant heavy chain (WT IgG1) <400> 30 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 31 <211> 109 <212> PRT <213> Synthetic Sequence <220> <223> CEA VL AB1 (2F2) <400> 31 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Leu Arg His Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 32 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB1 CDRL1 <400> 32 Arg Ala Ser Gln Ser Val Asn Ser Asn Leu Asn 1 5 10 <210> 33 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB1 CDRL2 <400> 33 His Gly Ser Asn Arg Ala Thr 1 5 <210> 34 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB1 CDRL3 <400> 34 Gln Gln Phe Asp Leu Arg His Glu His Asn Thr 1 5 10 <210> 35 <211> 112 <212> PRT <213> Artificial sequence <220> <223> CEA VL AB8 (2A3) <400> 35 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Ile Glu Asn 20 25 30 Ala Ile Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Pro 35 40 45 Met Ile Tyr Thr Leu Ser Asp Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Trp Asp Thr Phe 85 90 95 Ala Ile Gly Pro Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 36 <211> 14 <212> PRT <213> Artificial sequence <220> <223> CEA AB8 CDRL1 <400> 36 Thr Gly Thr Ser Ser Asp Val Ile Glu Asn Ala Ile Val Ser 1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB8 CDRL2 <400> 37 Thr Leu Ser Asp Arg Pro Ser 1 5 <210> 38 <211> 12 <212> PRT <213> Synthetic sequence <220> <223> CEA AB8 CDRL3 <400> 38 Ser Ser Trp Asp Thr Phe Ala Ile Gly Pro Ala Val 1 5 10 <210> 39 <211> 117 <212> PRT <213> Synthetic sequence <220> <223> Constant κ light chain (CK) <400> 39 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 1 5 10 15 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 20 25 30 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 35 40 45 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 50 55 60 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 65 70 75 80 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 85 90 95 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 100 105 110 Asn Arg Gly Glu Cys 115 <210> 40 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> CEA AB1 Light Chain (VKCK_2F2) <400> 40 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 [[ID=The original text to be translated is as below which wraped by : Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 85 90 95 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 100 105 110 Asn Arg Gly Glu Cys 115 [[ID=The original text to be translated is as below which wraped by : Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 85 90 95 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 100 105 110 Asn Arg Gly Glu Cys 115 <210> 40 <211> 216 <212> PRT <00]29]] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Leu Arg His Glu 85 90 95 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Leu Arg His Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 41 <211> 116 <212> PRT <213> Synthetic Sequence <220> <223> Constant lambda light chain (CL) <400> 41 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala 1 5 10 15 Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn 20 25 30 Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val 35 40 45 Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu 50 55 60 Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser 65 70 75 80 Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser 85 90 95 Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro 100 105 110 Thr Glu Cys Ser 115 <210> 42 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> CEA AB8 Light Chain (VLCL_2A3) <400> 42 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Ile Glu Asn 20 25 30 Ala Ile Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Pro 35 40 45 Met Ile Tyr Thr Leu Ser Asp Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Trp Asp Thr Phe 85 90 95 Ala Ile Gly Pro Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 165 170 175 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 180 185 190 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 195 200 205 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 43 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> Common Heavy Chain (Wild Type) <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly 450 <210> 44 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> Common Heavy Chain (LALA Mutation) <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly 450 <210> 46 <211> 106 <212> PRT <213> artificial sequence <220> <223> VK_SM3E <400> 46 Glu Asn Val Leu Thr Gln Ser Pro Ser Ser Met Ser Val Ser Val Gly 1 5 10 15 Asp Arg Val Asn Ile Ala Cys Ser Ala Ser Ser Ser Val Pro Tyr Met 20 25 30 His Trp Leu Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Val Gln Pro Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 47 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH_SM3E <400> 47 Gln Val Lys Leu Glu Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser 20 25 30 Tyr Met His Trp Leu Arg Gln Gly Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Leu Gly Leu Ser Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 48 <211> 116 <212> PRT <213> artificial sequence <220> <223> VL_MEDI <400> 48 Gln Ala Val Leu Thr Gln Pro Ala Ser Leu Ser Ala Ser Pro Gly Ala 1 5 10 15 Ser Ala Ser Leu Thr Cys Thr Leu Arg Arg Gly Ile Asn Val Gly Ala 20 25 30 Tyr Ser Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr 35 40 45 Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val 50 55 60 Ser Ser Arg Phe Ser Ala Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile 65 70 75 80 Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90 95 Met Ile Trp His Ser Gly Ala Ser Ala Val Phe Gly Gly Gly Thr Lys 100 105 110 Leu Thr Val Leu 115 <210> 49 <211> 121 <212> PRT <213> Artificial sequence <220> <223> VH_MEDI <400> 49 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Gly Thr Thr Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly 100​​​​​​​​​​​​​​​<223> VK_SAR <400> 50 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Phe Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Thr Arg Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro<00***2409>65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 51 <211> 120 <212> PRT <213> Artificial sequence <220> <223> VH_SAR <400> 51 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Tyr Phe Gly Ser Ser Gly Pro Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 52 <211> 108 <212> PRT <213> artificial sequence <220> <223> VK_CH1A1A <400> 52 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu 85 90 95 Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 53 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH_ CH1A1A <400> 53 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60 Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 54 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VK_T84.66 <400> 54 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe 20 25 30 Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 55 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH_T84.66 <400> 55 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 56 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> VK_Rablatuzumab <400> 56 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 57 It should be noted that in the above translation, for the numbers in Chinese characters in line 28, it is assumed that they are still numbers and just presented in Chinese characters in the original text. If there is a misunderstanding, please correct according to the actual situation.<211> 119 <212> PRT <213> Artificial sequence <220> <223> VH_Labetuzumab <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr Thr Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gly Gly 100 105 110 Thr Pro Val Thr Val Ser Ser 115 <210> 58 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Constant heterozygous - κ light chain (H - CK 5) <400> 58 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Arg Thr Val Ala Ala 1 5 10 15 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 20 25 30 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 35 40 45 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 50 55 60 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 65 70 75 80 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 85 90 95 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 100 105 110 Phe Asn Arg Gly Glu Cys 115 <210> 59 <211> 110 <212> PRT <213> Artificial sequence <220> <223> CEA VL 1B4 <400> 59 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Leu Ile Gly Lys Asn Ala Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Asn Tyr Gly Ile Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Leu Lys Leu Glu 85 90 95 Pro Asp Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 60 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA 1B4 CDRL1 <400> 60 Gly Gly Asn Leu Ile Gly Lys Asn Ala Val His 1 5 10 <210> 61 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA 1B4 CDRL2 <400> 61 Asn Tyr Gly Ile Arg Pro Ser 1 5 <210> 62 <211> 13 <212> PRT <213> artificial sequence <220> <223> CEA 1B4 CDRL3 <400> 62 Gln Val Trp Asp Thr Leu Lys Leu Glu Pro Asp Ala Val 1 5 10 <210> 63 <211> 107 <212> PRT <213> artificial sequence <220> <223> CEA VL C11 <400> 63 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Leu Ile Ser Asn Gly 20 25 30 Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ile Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Leu Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 64 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA C11 CDRL1 <400> 64 Arg Ala Ser Gln Leu Ile Ser Asn Gly Leu Tyr 1 5 10 <210> 65 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA C11 CDRL2 <400> 65 Asp Ala Ser Ile Leu Glu Ser 1 5 <210> 66 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CEA C11 CDRL3 <400> 66 Gln Gln Arg Leu Ser Trp Pro Leu Thr 1 5 <210> 67 <211> 217 <212> PRT <213> Artificial sequence <220> <223> huCD3 1B6 LC-heterozygous κ <400> 67 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ala Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 68 <211> 217 <212> PRT <213> Artificial sequence <220> <223> huCD3 1A10 LC - Hybrid κ <400> 68 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Lys Gly 85 90 95 Tyr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 69 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> huCD3 1F8 LC-Hybrid κ <400> 69 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Asp Gly 85 90 95 Lys Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 70 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> huCD3 1A4 LC-Hybrid κ <400> 70 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Lys Gln 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 71 <211> 217 <212> PRT <213> Artificial sequence <220> <223> huCD3 1H4 LC - Hybrid κ <400> 71 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Asn Gln​​​​​​​​​​​​ 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 72 <211> 220 <212> PRT <213> Artificial sequence <220> <223> CEA 2A3 LC - Hybrid κ <400> 72 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Ile Glu Asn 20 25 30 Ala Ile Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Pro 35 40 45 Met Ile Tyr Thr Leu Ser Asp Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Trp Asp Thr Phe 85 90 95 Ala Ile Gly Pro Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 73 <211> 214 <212> PRT <213> Artificial sequence <220> <223> CEA C11 LC (VKCK_C11) <400> 73 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Leu Ile Ser Asn Gly 20 25 30 Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ile Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Leu Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 74 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> CEA 1B4 LCEA 1B4 LC (VLCL_1B4) <400> 74 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Leu Ile Gly Lys Asn Ala Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Asn Tyr Gly Ile Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Leu Lys Leu Glu 85 90 95 Pro Asp Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 75 <211> 218 <212> PRT <213> Synthetic Sequence <220> <223> CEA 1B4 LC - Hybrid κ (VLCK_1B4) <400> 75 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Leu Ile Gly Lys Asn Ala Val 20 25 30 [[ID=NO]]His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Asn Tyr Gly Ile Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Leu Lys Leu Glu 85 90 95 Pro Asp Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 76 <211> 645 <212> DNA <213> Artificial sequence <220> <223> VKCK_C11 (DNA) <400> 76 gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gcttattagt aatggtttgt attggtatca gcagaaacca gggaaagccc ctaagctcct gatctatgat gcttccattt tggaaagtgg ggtcccatca aggttcagcg gcagtggatc tgggacagag ttcactctca ccatcagcag cctgcagcct gatgattttg caacttatta ctgtcagcag cgtctcagct ggcctctcac tttcggccaa gggaccaagg tggaaatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg ctgagcaaag cagactcga gaaacacaaa gtctacgcct gcgagtcac ccatcagggc ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttaa <210> 77 <211> 651 <212> DNA <213> The snowstorm <220> <223> VLCL_1B4 (DNA) <400> 77 tcctatgtgc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatt 60 acctgtgggg gaaaccttat tggaaagaat gctgtgcact ggtaccagca gaagccaggc 120 caggcccctg tgctggtcat ctataattat ggtattcggc cctcagggat tcctgagcga 180 ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatactctta agcttgagcc tgatgctgtg 300 ttcggcggag ggaccaagct gaccgtccta ggtcagccca aggctgcccc ctcggtcact 360 ctgttcccgc cctcctctga ggagcttcaa gccaacaagg ccacactggt gtgtctcata 420 agtgacttct acccgggagc cgtgacagtg gcttggaaag cagatagcag ccccgtcaag 480 gcgggagtgg agaccaccac accctccaaa caaagcaaca acaagtacgc ggccagcagc 540 tatctgagcc tgacgcctga gcagtggaag tcccacagaa gctacagctg ccaggtcacg 600 catgaaggga gcaccgtgga gaagacagtg gcccctacag aatgttcata a 651 <210> 78 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> VKCK_2F2 (AB1) (DNA) <400> 78 gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gtctgttaat tctaatttaa attggtacca acagaaacct 120 ggccaggctc ccaggctcct catctatcat gggtccaata gggccactgg catcccagcc 180 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240 gaagattttg cagtttatta ctgtcagcag tttgatctta ggcatgagca taataccttc 300 ggccaaggga ccaaggtgga aatcaaacgt acggtggctg caccatctgt cttcatcttc 360 ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 420 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 480 tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 540 ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 600 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta a 651 <210> 79 <211> 657 <212> DNA <213> artificial sequence <220> <223> VLCL_2A3 (AB8) (DNA) <400> 79 cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60 tcctgcactg gaaccagcag tgacgttat gagaatgcta ttgtctcctg gtaccaacag 120 cacccaggca aagcccccaa acccatgatt tatactctta gtgatcggcc ctcaggggtt 180 tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240 caggctgagg acgaggctga ttattactgc agctcatggg atacttttgc gattggtcct 300 gctgtgttcg gcggagggac caagctgacc gtcctaggtc agcccaaggc tgccccctcg 360 gtcactctgt tcccgccctc ctctgaggag cttcaagcca acaaggccac actggtgtgt 420 ctcataagtg acttctaccc gggagccgtg acagtggctt ggaaagcaga tagcagcccc 480 gtcaaggcgg gagtggagac caccacaccc tccaaacaaa gcaacaacaa gtacgcggcc 540 agcagctatc tgagcctgac gcctgagcag tggaagtccc acagaagcta cagctgccag 600 gtcacgcatg aagggagcac cgtggagaag acagtggccc ctacagaatg ttcataa 657 <210> 80 <211> 1365 <212> DNA <213> Artificial Sequence <220> <223> Common Heavy Chain VHCH (Wild - type; DNA) <400> 80 gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgaaactc 60 tcctgtgcag cctctgggtt caccttcaac acctatgcta tgaactgggt ccgccaggct 120 cccgggaaag ggctggagtg ggttggccgt attagaagca aatataacaa ttacgcgaca 180 tactatgctg actcggtgaa agacaggttc accatctcca gagatgattc aaagaacacg 240 gcgtatctgc aaatgaacag cctgaaaacc gaggacacgg ccgtgtatta ctgtgtgaga 300 cacgggaatt tcggcaattc ttatgtctcg tggttcgctt actggggcca agggactctg 360 gtcacagtct cgagcgcctc caccaagggc ccatcggtct tccccctggc accctcctcc 420 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480 ccggtgacag tctcgtggaa ctcaggagcc ctgaccagcg gcgtgcacac cttcccggct 540 gtcctacagt cctcaggact ctactccctc agcagcgtgg tgactgtgcc ctccagcagc 600 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacc caaggtggac aagagagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720 gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 780 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 840 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg gaggagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1080. gagaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta taccctgccc ccatctcggg aggagatgac caagaccag gtcagcctga cttgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtggggag agcaacgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctatagcaa gctcaccgtg 1260 gacaagtcca ggtggcagca ggggacgtc ttctcatgct ccgtgatgca tgaggctctg 1320 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gttaa 1365 <210> 81 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB13 CDRL1 <400> 81 Arg Ala Ser Gln Ser Val Asn Ser Asn Leu Asn 1 5 10 <210> 82 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB13 CDRL2 <400> 82 His Gly Ser Asn Arg Ala Thr 1 5 <210> 83 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB13 CDRL3 <400> 83 Gln Gln Phe Asp Tyr Phe Met Asn Lys Asn Thr 1 5 10 <210> 84 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB14 CDRL1 <400> 84 Arg Ala Ser Gln Ser Val Asn Ser Asn Leu Asn 1 5 10 <210> 85 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB14 CDRL2 <400> 85 His Gly Ser Asn Arg Ala Thr 1 5 <210> 86 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB14 CDRL3 <400> 86 Gln Gln Phe Asp Tyr Phe Arg Glu Ser Asn Thr 1 5 10 <210> 87 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB15 CDRL1 <400> 87 Arg Ala Ser Gln Thr Val Asn Asn Asn Leu Asn 1 5 10 <210> 88 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB15 CDRL2 <400> 88 Tyr Ala Ser Asn Arg Ala Thr 1 5 <210> 89 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB15 CDRL3 <400> 89 Gln Gln Phe Asn Tyr His His Glu His Asn Thr 1 5 10 <210> 90 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB17 CDRL1 <400> 90 Arg Ala Ser Gln Ser Val Asn Ser Asn Leu Asn 1 5 10 <210> 91 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB17 CDRL2 <400> 91 His Gly Ser Asn Arg Ala Thr 1 5 <210> 92 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB17 CDRL3 <400> 92 Gln Gln Phe Asp Tyr Phe Lys Glu His Asn Thr 1 5 10 <210> 93 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB20 CDRL1 <400> 93 Arg Ala Ser Gln Ser Val Asn Ser Asn Leu Asn 1 5 10 <210> 94 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB20 CDRL2 <400> 94 His Gly Ser Asn Arg Ala Thr 1 5 <210> 95 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB20 CDRL3 <400> 95 Gln Gln Phe Asp Tyr Phe Arg Glu Leu Asn Thr 1 5 10 <210> 96 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB54 CDRL1 <400> 96 Arg Ala Ser Gln Glu Val His Lys Asn Leu Asn 1 5 10 <210> 97 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB54 CDRL2 <400> 97 His Ser Ser Asn Arg Ala Thr 1 5 <210> 98 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB54 CDRL3 <400> 98 Gln Gln Phe Asn Tyr His His Glu His Asn Thr 1 5 10 <210> 99 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB60 CDRL1 <400> 99 Arg Ala Ser Gln Thr Val Asn Ala Asn Leu Asn 1 5 10 <210> 100 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB60 CDRL2 <400> 100 Tyr Ala Ser Asn Arg Ala Thr 1 5 <210> 101 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB60 CDRL3 <400> 101 Gln Gln Phe Asp Tyr His Asn Glu His Asn Thr 1 5 10 <210> 102 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB66 CDRL1 <400> 102 His Ser Ser Gln Val Val Asn Lys Asn Leu Asn 1 5 10 <210> 103 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB66 CDRL2 <400> 103 His Gly Ser Asn Arg Ala Thr 1 5 <210> 104 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB66 CDRL3 <400> 104 Gln Gln Phe Asp Tyr Phe Arg Glu His Asn Thr 1 5 10 <210> 105 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB71 CDRL1 <400> 105 Arg Ala Ser Gln Ser Val Asn Ser Asn Leu Asn 1 5 10 <210> 106 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB71 CDRL2 <400> 106 His Ser Ser Asn Arg Pro His 1 5 <210> 107 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB71 CDRL3 <400> 107 Gln Gln Phe Asp Tyr Phe Lys Glu His Asn Thr 1 5 10 <210> 108 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB72 CDRL1 <400> 108 Arg Ala Ser Gln Ser Val Asn Ser Asn Leu Asn 1 5 10 <210> 109 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB72 CDRL2 <400> 109 His Ser Thr Asn Arg Pro Arg 1 5 <210> 110 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB72 CDRL3 <400> 110 Gln Gln Phe Asp Tyr Phe Lys Glu Tyr Asn Thr 1 5 10 <210> 111 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB73 CDRL1 <400> 111 Arg Ala Ser Gln Ser Val Asn Ser Asn Leu Asn 1 5 10 <210> 112 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB73 CDRL2 <400> 112 His Ser Asn Asn Arg Pro His 1 5 <210> 113 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB73 CDRL3 <400> 113 Gln Gln Phe Asp Tyr Phe Arg Glu Tyr Asn Thr 1 5 10 <210> 114 <211> 109 <212> PRT <213> Artificial sequence <220> <223> CEA AB13 VL Light Chain Variable Region <400> 114 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Met Asn 85 90 95 Lys Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 115 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> CEA AB14 VL Light Chain Variable Region <400> 115 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Val Asn Asn Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Tyr His His Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 116 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> CEA AB15 VL Light Chain Variable Region <400> 116 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Val Asn Asn Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Tyr His His Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 117 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> CEA AB17 VL Light Chain Variable Region <400> 117 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Lys Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 118 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> CEA AB20 VL Light Chain Variable Region <400> 118 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Arg Glu 85 90 95 Leu Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 119 <211> 109 It should be noted that there may be an error in "ID=48" where it is "0003507" instead of "0003507" in the original text. I have translated it as is while pointing out this potential issue.<212> PRT <213> Artificial sequence <220> <223> CEA AB54 VL light chain variable zone <400> 119 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Glu Val His Lys Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Ser Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Tyr His His Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 120 <211> 109 <212> PRT <213> Artificial sequence <220> <223> CEA AB60 VL light chain variable zone <400> 120 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Val Asn Ala Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr His Asn Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 121 <211> 109 <212> PRT <213> artificial sequence <220> <223> CEA AB66 VL <400> 121 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys His Ser Ser Gln Val Val Asn Lys Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Arg Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 122 <211> 109 <212> PRT <213> Artificial sequence <220> <223> CEA AB71 VL light chain variable region <400> 122 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Ser Ser Asn Arg Pro His Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Lys Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 123 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> CEA AB72 VL light chain variable region <400> 123 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Ser Thr Asn Arg Pro Arg Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Lys Glu 85 90 95 Tyr Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 124 <211> 109 <212> PRT <213> Artificial sequence <220> <223> CEA AB73 VL light chain variable region <400> 124 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Ser Asn Asn Arg Pro His Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Arg Glu 85 90 95 Tyr Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 125 <211> 216 <212> PRT <213> Artificial sequence <220> <223> CEA AB13 LC light chain <400> 125 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Met Asn 85 90 95 Lys Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 126 <211> 216 <212> PRT <213> Artificial sequence <220> <223> CEA AB14 LC light chain <400> 126 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Arg Glu 85 90 95 Ser Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 127 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> CEA AB15 LC Light Chain <400> 127 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Val Asn Asn Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Tyr His His Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 128 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> CEA AB17 LC light chain <400> 128 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Lys Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 129 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> CEA AB20 LC Light Chain <400> 129 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Arg Glu 85 90 95 Leu Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 130 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> CEA AB54 LC light chain <400> 130 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Glu Val His Lys Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Ser Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Tyr His His Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 131 <211> 216 <212> PRT <213> Artificial sequence <220> <223> CEA AB60 LC light chain <400> 131 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Val Asn Ala Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr His Asn Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys<X 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 132 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> CEA AB66 LC Light Chain <400> 132 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys His Ser Ser Gln Val Val Asn Lys Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Gly Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Arg Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 133 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> CEA AB71 LC Light Chain <400> 133 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Ser Ser Asn Arg Pro His Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Lys Glu 85 90 95 His Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 134 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> CEA AB72 LC light chain <400> 134 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Ser Thr Asn Arg Pro Arg Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Lys Glu 85 90 95 Tyr Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 135 <211> 216 <212> PRT <213> Synthetic sequence <220> <223> CEA AB73 LC light chain <400> 135 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45<e> Tyr His Ser Asn Asn Arg Pro His Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Tyr Phe Arg Glu 85 90 95 Tyr Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 136 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB1 CDRL1 mutation <220> <221> misc_feature <222> (1)..(2) <223> Xaa can be any natural amino acid. <220> <221> misc_feature <222> (5)..(5) <223> Xaa can be any natural amino acid. <220> <221> misc_feature <222> (7)..(8) <223> Xaa can be any natural amino acid. <400> 136 Xaa Xaa Ser Gln Xaa Val Xaa Xaa Asn Leu Asn 1 5 10 <210> 137 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CEA AB1 CDRL2 mutation <220> <221> misc_feature <222> (1)..(3) <223> Xaa can be any natural amino acid. <220> <221> misc_feature <222> (6) (7) <223> Xaa can be any natural amino acid. <400> 137 Xaa Xaa Xaa Asn Arg Xaa Xaa 1 5 <210> 138 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CEA AB1 CDRL3 mutation <220> <221> misc_feature <222> (2)..(2) <223> Xaa can be any natural amino acid. <220> <221> misc_feature <222> (4)..(7) <223> Xaa can be any natural amino acid. <220> <221> misc_feature <222> (9)..(9) <223> Xaa can be any natural amino acid. <400> 138 Gln Xaa Phe Xaa Xaa Xaa Xaa Glu Xaa Asn Thr 1 5 10

Claims

1. A bispecific antibody comprising a first binding moiety specifically binding to human CEACAM5 and a second binding moiety specifically binding to human CD3ε, wherein: a) The first binding portion includes a heavy chain variable region (VH), which includes CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO:

4. b) The first binding portion includes a light chain variable region (VL), which contains a selection from the following CDRL group: b1) CDRL1 (SEQ ID NO: 90), CDRL2 (SEQ ID NO: 91), and CDRL3 (SEQ ID NO: 92), b2) CDRL1 (SEQ ID NO: 96), CDRL2 (SEQ ID NO: 97), and CDRL3 (SEQ ID NO: 98) b3) CDRL1 (SEQ ID NO: 99), CDRL2 (SEQ ID NO: 100), and CDRL3 (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, c) The second binding portion includes VH, which includes CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3, and CDRH3 of SEQ ID NO: 4, and d) The second binding portion includes VL, which includes CDRL1 of SEQ ID NO: 18, CDRL2 of SEQ ID NO: 19 and CDRL3 of SEQ ID NO:

20.

2. The bispecific antibody according to claim 1, comprising: a) The heavy chain variable region VH of SEQ ID NO: 1 in the first binding portion, b) The light chain variable region VL in the first binding portion, which is selected from: 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, and c) The heavy chain variable region VH of SEQ ID NO: 1 and the light chain variable region VL of SEQ ID NO: 17 in the second binding portion.

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

4. A bispecific antibody comprising a first binding moiety specifically binding to human CEACAM5 and a second binding moiety specifically binding to human CD3ε, said antibody comprising a) The heavy chain variable region VH of SEQ ID NO: 1 in the first binding portion, b) The light chain in the first bonding portion, selected from: b1) The light chain of SEQ ID NO: 128, b2) The light chain of SEQ ID NO: 130 b3) Light chain of SEQ ID NO: 131 b4) Light chain of SEQ ID NO: 132 b5) Light chain of SEQ ID NO: 133, b6) The light chain of SEQ ID NO: 134, and b7) The light chain of SEQ ID NO: 135, and c) The heavy chain variable region VH of SEQ ID NO: 1 and the light chain of SEQ ID NO: 28 in the second binding portion.

5. The bispecific antibody according to any one of claims 1 to 4, characterized in that... It contains a common heavy chain, which is selected from: a) The heavy chain of SEQ ID NO: 43 b) The heavy chain of SEQ ID NO: 44, and c) Heavy chain of SEQ ID NO:

45.

6. A bispecific antibody comprising a first binding moiety specifically binding to human CEACAM5 and a second binding moiety specifically binding to human CD3ε, said antibody comprising a common heavy chain of SEQ ID NO: 45 and a light chain of SEQ ID NO: 28 in the second binding moiety and a light chain of SEQ ID NO: 128 in the first binding moiety.

7. A bispecific antibody comprising a first binding moiety specifically binding to human CEACAM5 and a second binding moiety specifically binding to human CD3ε, said antibody comprising a common heavy chain of SEQ ID NO: 45 and a light chain of SEQ ID NO: 28 in the second binding moiety and a light chain of SEQ ID NO: 133 in the first binding moiety.

8. A bispecific antibody comprising a first binding moiety specifically binding to human CEACAM5 and a second binding moiety specifically binding to human CD3ε, said antibody comprising a common heavy chain of SEQ ID NO: 45 and a light chain of SEQ ID NO: 28 in the second binding moiety and a light chain of SEQ ID NO: 135 in the first binding moiety.

9. The bispecific antibody according to any one of claims 1 to 4 and 6 to 8, characterized in that... It contains an Fc domain, wherein each subunit of the Fc domain contains amino acid substitutions L234A, L235A, and P329A, which are indexed according to the Kabat EU index.

10. Use of the bispecific antibody according to any one of claims 1 to 4 and 6 to 8 in the preparation of medicaments for treating colorectal cancer, esophageal cancer, pancreatic adenocarcinoma, gastric cancer, non-small cell lung cancer, breast cancer, head and neck cancer, uterine cancer, and bladder cancer.

11. Use of the bispecific antibody according to any one of claims 1 to 4 and 6 to 8 in the preparation of a medicament for use as a monotherapy 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.

12. Use of the bispecific antibody according to any one of claims 1 to 4 and 6 to 8 in the preparation of a medicament for 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 in combination with the bispecific anti-CEAxCD47 antibody simultaneously, alone, or in sequence.

13. Use of the bispecific antibody according to any one of claims 1 to 4 and 6 to 8 in the preparation of a medicament for 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 in combination with a bispecific anti-CEAxCD47 antibody and / or a PD-1 axis antagonist, either simultaneously, alone, or in sequence.

14. Use of the bispecific antibody according to any one of claims 1 to 4 and 6 to 8 in the preparation of a medicament for 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 in combination with PD-1 axis antagonists, either simultaneously, alone, or in sequence.

15. The use according to claim 14, wherein the PD-1 axis antagonist is selected from pembrolizumab, nivolumab, pidilizumab, or lambolizumab.

16. The use according to claim 12, wherein the bispecific antibody and the bispecific anti-CEAxCD47 antibody are used in cancer treatment by alternating administration of the antibody and the bispecific anti-CEAxCD47 antibody at intervals of 6 to 15 days.

17. The use according to claim 10, wherein the antibody is administered to a patient in a single or divided dose or by continuous infusion at a dose of 0.1 to 100 mg / kg body weight per day or week.

18. The use according to claim 11, wherein the antibody is administered to a patient in a single or divided dose or by continuous infusion at a dose of 0.1 to 100 mg / kg body weight per day or per week.

19. The use according to claim 12, wherein the antibody is administered to a patient in a single or divided dose or by continuous infusion at a dose of 0.1 to 100 mg / kg body weight per day or per week.

20. The use according to claim 13, wherein the antibody is administered to a patient in a single or divided dose or by continuous infusion at a dose of 0.1 to 100 mg / kg body weight per day or per week.

21. The use according to claim 14, wherein the antibody is administered to a patient in a single or divided dose or by continuous infusion at a dose of 0.1 to 100 mg / kg body weight per day or per week.

22. The use according to claim 15, wherein the antibody is administered to a patient in a single or divided dose or by continuous infusion at a dose of 0.1 to 100 mg / kg body weight per day or week.

23. The use according to claim 16, wherein the antibody is administered to a patient in a single or divided dose or by continuous infusion at a dose of 0.1 to 100 mg / kg body weight per day or week.

24. The use according to claim 17, wherein the antibody is administered to a patient at a dose of 1 to 20 mg / kg.

25. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 4 and 6 to 8 and a pharmaceutically acceptable carrier.

26. An isolated polynucleotide encoding an antibody or its binding portion as described in any one of claims 1 to 4 and 6 to 8.

27. A vector comprising the isolated polynucleotide of claim 26.

28. A cell comprising the isolated polynucleotide of claim 26 or the vector of claim 27, wherein the cell is not a plant cell.

29. The cell according to claim 28, wherein the cell is selected from Streptomyces, yeast, CHO, YB / 20, NSO, NIH-3T3, BHK, SP2 / 0, R1.1, COS 1, COS 7, BSC1, BSC40, BMT10 cells, insect cells, and human cells in tissue culture.

30. The cell of claim 29, wherein the human cell is PER-C6, HEK-293T, HeLa, or HepG2.

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