Anti-CECAM6 antibody with reduced side effects

Abstract

The present invention provides an antibody that can bind to human CEACAM6 and alleviate CEACAM6-mediated immunosuppression, and has reduced side effects during treatment.The present invention further provides an isolated nucleic acid encoding the antibody and a vector containing the same, an isolated cell expressing the antibody, a method for producing the antibody, and a pharmaceutical composition and a kit containing the antibody.The antibody of the present invention can be used to treat cancer, and can be used to treat other disorders and conditions associated with the expression of CEACAM6.

Description

[Technical field]

[0001] The present invention provides an antibody that can bind to human CEACAM6 and alleviate CEACAM6-mediated immunosuppression, and has reduced side effects during treatment.The present invention further provides an isolated nucleic acid encoding the antibody and a vector containing the same, an isolated cell expressing the antibody, a method for producing the antibody, and a pharmaceutical composition and a kit containing the antibody.The antibody of the present invention can be used to treat cancer, and can be used to treat other disorders and conditions associated with the expression of CEACAM6. [Background technology]

[0002] Prior Art Several cancer types have the ability to block effector functions of T cells, limiting the effectiveness of cancer immunotherapy. However, antibody blockade of immune checkpoint molecules is a clinically validated approach to reactivate immune cells. The most prominent example is the blockade of the programmed cell death protein 1 / programmed death ligand 1 (PD-1 / PD-L1) axis. Several agents targeting this axis are approved or currently in clinical development, and impressive clinical responses have been reported in diseases such as melanoma, renal cell carcinoma, and lung cancer. Despite the success of these approaches, several groups of patients do not respond to or develop resistance to PD-1 / PD-L1 inhibitors, necessitating the need for novel immunotherapeutic solutions.

[0003] CEACAM6 (also known as carcinoembryonic antigen-related cell adhesion molecule 6, CD66c, nonspecific cross-reacting antigen, NCA, or NCA 50 / 90) is an attractive target for therapeutic intervention in cancer immunotherapy. In humans, CEACAM6 is expressed in cells of several cancer types. The highest prevalence of membrane-localized CEACAM6 expression has been identified in lung, colon, pancreatic, and gastric adenocarcinomas and has been found to correlate with tumor progression and adverse clinical outcomes. Furthermore, tumor-infiltrating myeloid cells, particularly granulocytes and, to a lesser extent, macrophages, express high levels of CEACAM6. Under normal conditions, CEACAM6 is expressed in blood myeloid cells, with the highest levels identified in granulocytes, resident myeloid cells, and epithelial cells of the lung and intestine. Orthologs of CEACAM6 exist in humans and nonhuman primates, but no orthologs are known in rodents.

[0004] It has been shown that blocking CEACAM6 with monoclonal antibodies (mAbs) or silencing with small interfering ribonucleic acid (siRNA) restores T cell activity against malignant plasma cells from multiple myeloma as well as other solid tumors (Witzens-Harig et al., Blood 2013 May 30;121(22):4493-503; WO 2016 / 150899 A2). This suggests that CEACAM6 expressed on the surface of malignant cells is involved in the regulation of antitumor responses mediated by CD8+ T cells, which is consistent with the fact that CEACAM6 acts as an immunosuppressant in solid tumors.

[0005] There are several anti-CEACAM6 antibodies. Most of them are non-human reagent antibodies, many of them polyclonal. Their specificity and selectivity for human CEACAM6 and cross-reactivity for monkey CEACAM6 are mostly undisclosed or unknown. Therapeutic antibodies against CEACAM6 are also known in the art. Some are not selective for human CEACAM6 (e.g., MN-3 from Immunomedics, Neo201 / h16C3 from Neogenix; both bind in addition to human CEACAM5). The single domain antibody 2A3 and its fusion variants (WO 2012 / 040824 A1 and Niu et al., J Control Release. 2012 Jul. 10;161(1):18-24) have not been characterized with respect to selectivity and cross-reactivity for monkey CEACAM6.

[0006] The murine antibody 9A6 (Genovac / Aldevron) was the first antibody described that can modulate the immunosuppressive activity of CEACAM6 (Witzens-Harig et al., Blood 2013 May 30; 121(22):4493-503). 9A6 inhibits the immunosuppressive activity of CEACAM6, leading to enhanced cytokine secretion by T cells in vitro and antitumor effects in vivo (Khandelwal et al., Poster Abstract 61, 22nd Annual International Cancer Immunotherapy Symposium 2014 October 6-8, New York City, USA). The murine antibody 9A6 shows no cross-reactivity with monkey CEACAM6 (WO 2016 / 150899 A2). Furthermore, its murine nature makes direct therapeutic use in humans difficult.

[0007] WO 2016 / 150899 A2 discloses a series of human anti-CEACAM6 antibodies useful in therapeutic applications that alleviate the immunosuppressive activity of CEACAM6, which can be therapeutically applied in human cancer patients. These antibodies are specific for human and cynomolgus monkey (Macaca fascicularis) CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6, CD66c, nonspecific cross-reacting antigen, NCA, NCA-50 / 90) and do not significantly cross-react with the closely related human CEACAM1, human CEACAM3, and human CEACAM5. The anti-CECAM6 antibody TPP-3310 disclosed in WO 2016 / 150899 A2 is a preferred embodiment of these antibodies.

[0008] Combination therapy of anti-CEACAM6 antibodies with other immunotherapeutic approaches has been disclosed in WO 2020 / 099230 A1 (in combination with anti-PD1 and anti-PD-L1 antibodies) and WO 2020 / 126808 A1 (in combination with anti-TIM3 antibodies).

[0009] It is currently known that the clinical efficacy of many therapeutic antibodies is achieved only in a fraction of patients. Therefore, the selection of the antibody isotype format is an important step toward improving patient outcomes (Vukovic et al., Clin Exp Immunol. March 2021; 203(3): 351-365). Many Fc engineering options exist to modulate the effector function or half-life of natural antibody isotypes (Wang et al., Protein Cell. January 2018; 9(1): 63-73).

[0010] For example, several mutation variants that enhance CDC effector function have been described. Similarly, several mutations that enhance FcγR-dependent effector functions such as ADCC and ADCP are known. These enhancements can be brought about not only by amino acid mutations but also by glycoengineering. A prominent example is the non-fucosylation of antibodies, which correlates with strong binding to FcγRIIIa, thus enhancing ADCC by NK cells.

[0011] If the mAb is intended to bind to a cell surface receptor and block the receptor-ligand interaction (i.e., an antagonist), it may be desirable to reduce or eliminate effector functions, for example to prevent cell death of normal cells expressing the target or to prevent undesired cytokine secretion. It is recognized that the four human IgG subclasses each have different abilities to induce immune effector functions. For example, IgG1 and IgG3 can recruit complement much more effectively than IgG2 and IgG4, whereas IgG2 and IgG4 have very limited ability to induce ADCC. Examples of Fc engineering include the human IgG4 variants L235E or F234A / L235A, and the human IgG1 variant L234A / L235A ("LALA"; Xu et al., Cell Immunol 2000 Feb. 25; 200(1):16-26). Another early approach intended to reduce effector function was to mutate the glycosylation site at N297 with mutations such as N297A, N297Q, and N297G ("aglycosylation"; Bolt et al., Eur J Immunol. 1993 February; 23(2):403-11; Tao and Morrison, J Immunol. 1989 October 15; 143(8):2595-601; Walker et al., Biochem J. 1989 April 15; 259(2):347-53; Leabman et al., MAbs 2013 November-December; 5(6):896-903). Another variation is a cross-subclass approach to reduce effector function, as exemplified by the approved anti-C5 therapeutic eculizumab, which has a CH1 and hinge region from IgG2 but a CH2 and CH3 from IgG4.Other examples include L234F / L235E / P331S of human IgG1 ("FES"; Oganesyan et al., Acta Crystallogr D Biol Crystallogr. 2008 June; 64(Pt 6):700-4), P329G / L234A / L235A of human IgG1 ("PG-LALA"; Schlothauer et al., Protein Eng Des Sel 2016 October; 29(10):457-466), "IgG1sigma" (L234A / L235A / G237A / P238S / H268A / A330S / P331S, Tam et al., Antibodies (Basel) 2017 September 1; 6(3):12), and "IgG1-NNAS" (S298N / T299A / Y300S, Zhou et al., MAbs 2020 Jan-Dec;12(1):1814-583).

[0012] Furthermore, mutations have been reported that increase co-engagement of antigens with FcγR, for example by enhancing binding to FcγRIIb or FcγRIIa on antigen-positive cells bearing FcγR.

[0013] Finally, addressing the Fc-FcRn interaction can modulate the half-life of antibodies in vivo. When the interaction is abrogated, for example by H435A, the half-life is dramatically shortened as the antibody is no longer protected from lysosomal degradation by FcRn recycling. In contrast, "YTE" (M252Y / S254T / T256E) and equivalent mutations have been shown to significantly extend half-life in both preclinical species and humans due to more efficient recycling from endosomes. Summary of the Invention [Problem to be solved by the invention]

[0014] technical issues To study the therapeutic potential of anti-CEACAM6 antibody TPP-3310 (disclosed in WO 2016 / 150899 A2) in cancer patients in clinical trials, a human IgG2 format with reduced effector function was selected based on its favorable preclinical safety profile. Quite unexpectedly, cancer patients treated with low doses of TPP-3310 developed neutropenia as a side effect (see Example 2).

[0015] Therefore, there is a strong demand for antibodies suitable for therapeutic use that can bind to human CEACAM6 and relieve CEACAM6-mediated immunosuppression, and that have reduced side effects during treatment. [Means for solving the problem]

[0016] Resolving the problem As shown in this application, it is quite surprising that neutrophils can be activated by TPP-3310 in a whole blood assay, at least partially reproducing the clinical findings (see Example 3). However, this activation requires a very finely interwoven dependency on pre-stimulation, epitope and antibody isotype. Moreover, the effect is Fc-dependent and involves FcγR. This is completely unpredictable, since the strict dependency on the Fc part, as well as even the involvement of FcγRII, rather suggests that human IgG1 is a very potent molecule.

[0017] Contrary to previous teachings, the present inventors found that for TPP-3310 (human IgG2), changing the isotype to human IgG1 actually completely blocked neutrophil activation in a whole blood assay. The human IgG2 isotype, which is thought to be more silent, is in fact a molecule capable of exerting neutrophil activating effects (see Example 3).

[0018] On the other hand, the human IgG1 format has strong interactions with FcγR, which precludes its use in therapeutic antibody formats simply because of its strong and undesirable effector potential, such as ADCC, ADCP, and CDC activity.

[0019] The antibodies of the invention comprise an IgG1-based engineered format (L234A L235A, preferably N297A in combination with aglycosylation) that meets the requirements of being devoid of FcγR interactions and therefore devoid of effector function while at the same time being unable to activate neutrophils in the blood under prestimulation conditions.

[0020] Thus, therapeutic intervention in cancer patients with an anti-CEACAM6 IgG1-based engineered antibody (TPP-21518) may avoid neutropenia as an adverse event.

[0021] Summary of the Invention These and other objects are accomplished by the teachings of the present invention.

[0022] First aspect of the invention: anti-CECAM6 antibodies In a first aspect, the present invention relates to an anti-CECAM6 antibody comprising an IgG1 Fc region lacking a glycan attached to a conserved N-linked site in the CH2 domain of the Fc region, said IgG1 Fc region comprising at least the amino acid substitutions L234A and L235A, numbered according to the EU index of Kabat.

[0023] In certain embodiments of the first aspect, the invention provides an anti-CECAM6 antibody comprising an IgG1 Fc region, wherein the IgG1 Fc region comprises the amino acid substitutions N297A, N297G, or N297Q, numbered according to the EU index of Kabat.

[0024] In certain embodiments of the first aspect, the invention provides an anti-CECAM6 antibody comprising an IgG1 Fc region, wherein the IgG1 Fc region comprises amino acid substitutions N297A, N297G, or N297Q, and at least amino acid substitutions L234A and L235A, numbered according to the EU index of Kabat.

[0025] In certain embodiments of the first aspect, the invention provides an anti-CECAM6 antibody comprising an IgG1 Fc region, wherein the IgG1 Fc region comprises at least the amino acid substitutions N297A, L234A, and L235A, numbered according to the EU index of Kabat.

[0026] In certain embodiments of the first aspect, the invention provides an anti-CECAM6 antibody comprising an IgG1 Fc region, wherein the IgG1 Fc region comprises the following amino acid substitutions: N297A, L234A, and L235A, numbered according to the EU index of Kabat.

[0027] In certain embodiments of the first aspect, the above-mentioned anti-CECAM6 antibody competes for CEACAM6 binding with an antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 67.

[0028] In a certain embodiment of the first aspect, the above-mentioned anti-CECAM6 antibody comprises a heavy chain variable region H-CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a heavy chain variable region H-CDR2 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain variable region H-CDR3 comprising the amino acid sequence of SEQ ID NO: 66, a light chain variable region L-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, a light chain variable region L-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a light chain variable region L-CDR3 comprising the amino acid sequence of SEQ ID NO: 70.

[0029] In certain embodiments of the first aspect, the anti-CECAM6 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 67.

[0030] In certain embodiments of the first aspect, the anti-CECAM6 antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:71, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:72.

[0031] In certain embodiments, the invention provides an anti-CECAM6 antibody comprising a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:71 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:72.

[0032] Further aspects of the present invention: In a further aspect, the present invention provides a nucleic acid encoding an anti-CECAM6 antibody of the first aspect, and a vector comprising said nucleic acid.

[0033] In a further aspect, the present invention provides an isolated cell expressing an anti-CECAM6 antibody of the first aspect. In a preferred embodiment, the cell is a prokaryotic or eukaryotic cell.

[0034] In a further aspect, the present invention provides a method of producing an anti-CECAM6 antibody of the first aspect.

[0035] In a further aspect, the present invention provides an anti-CECAM6 antibody of the first aspect for use as a medicament, in particular for use as a medicament for the treatment of cancer. In certain embodiments of this aspect, a method for treating a cancer associated with the undesired presence of CEACAM6 is provided, comprising administering to a subject in need thereof an effective amount of an anti-CECAM6 antibody of the first aspect.

[0036] In a further aspect, the invention provides an anti-CEACAM6 antibody of the first aspect for use in combination, either simultaneously, separately or sequentially, with an anti-PD-1 antibody or an anti-PD-L1 antibody in the treatment of cancer. In certain embodiments, the anti-PD-1 antibody is nivolumab or pembrolizumab, and the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab. In certain embodiments of this aspect, there is provided a method of treating cancer comprising administering to a patient in need thereof an effective amount of an anti-CEACAM6 antibody of the first aspect, either simultaneously, separately or sequentially in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody, preferably wherein the anti-PD-1 antibody is nivolumab or pembrolizumab, and the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

[0037] In a further aspect, the invention provides an anti-CEACAM6 antibody of the first aspect for use in simultaneous, separate or sequential combination with an anti-TIM-3 antibody in the treatment of cancer. In certain embodiments, the anti-TIM-3 antibody is covolimab, MBG-453, BMS-986258, Sym-023, LY-3321367 or INCAGN-2390. In certain embodiments of this aspect, a method of treating cancer is provided, comprising administering to a patient in need thereof an effective amount of an anti-CEACAM6 antibody of the first aspect in simultaneous, separate or sequential combination with an anti-TIM-3 antibody, preferably wherein the anti-TIM-3 antibody is covolimab, MBG-453, BMS-986258, Sym-023, LY-3321367 or INCAGN-2390.

[0038] In a further aspect, the present invention provides a pharmaceutical composition comprising an anti-CECAM6 antibody of the first aspect. [Brief description of the drawings]

[0039] [Figure 1]Figure 1 shows TNF-alpha plasma levels at various time points after the start of intravenous infusion of anti-CEACAM6 antibody TPP-3310 in cancer patients. Three patients per dose cohort were treated with either 2.5 mg, 5 mg, 10 mg or 30 mg of the clinical formulation of TPP-3310 over 1 hour. Mean values ​​and standard deviations are shown. X-axis: time after start of infusion (in hours), Y-axis: TNF-alpha concentration in plasma [pg / mL]. [Diagram 2] Figure 1 shows IL-6 plasma levels at various time points after the start of intravenous infusion of anti-CEACAM6 antibody TPP-3310 in cancer patients. Three patients per dose cohort were infused over 1 hour with either 2.5 mg, 5 mg, 10 mg or 30 mg of the clinical formulation of TPP-3310. Mean values ​​and standard deviations are shown. X-axis: time after initiation of infusion (in hours), Y-axis: plasma IL-6 concentration [pg / mL]. [Diagram 3] Figure 1 shows IL-10 plasma levels at various time points after the start of intravenous infusion of anti-CEACAM6 antibody TPP-3310 in cancer patients. Three patients per dose cohort were infused over 1 hour with either 2.5 mg, 5 mg, 10 mg or 30 mg of the clinical formulation of TPP-3310. Mean values ​​and standard deviations are shown. X-axis: time after initiation of infusion (in hours), Y-axis: plasma IL-10 concentration [pg / mL]. [Figure 4] Figure 1 shows plasma levels of myeloperoxidase (MPO) at various time points following intravenous infusion of anti-CEACAM6 antibody TPP-3310 in cancer patients. Three patients per dose cohort were infused over 1 hour with either 2.5 mg, 5 mg, 10 mg or 30 mg of the clinical formulation of TPP-3310. Relative values ​​compared to pretreatment are shown as mean percentages and standard deviations. X-axis: time after start of infusion (in hours), Y-axis: plasma MPO concentration [% of 0 hour pretreatment levels]. [Diagram 5]FIG. 1 shows neutrophil counts at selected time points in patients treated with 30 mg TPP-3310. Values ​​below 0.5 / nL are considered severe neutropenia according to CTCAE criteria. N / A: no sample taken. X-axis: time after infusion. 1: pre-dose; 2: 24 hours; 3: 48 hours; 4: 7 days; 5: 14 days; 6: 21 days. Y-axis: neutrophils per nL. [Figure 6] Myeloperoxidase (MPO) release by anti-CEACAM6 antibody TPP-3310 (human IgG2 format) (black bars) and corresponding isotype control antibody (white bars) with (+fMLP) and without (w / o fMLP) suboptimal fMLP stimulation. X-axis: antibody concentration [μM]; Y-axis: MPO pg / ml. [Figure 7] Myeloperoxidase (MPO) release by anti-CEACAM6 antibody TPP-5468 (human IgG1 format) (black bars) and corresponding isotype control antibody (white bars) with (+fMLP) and without (w / o fMLP) suboptimal fMLP stimulation. X-axis: antibody concentration [μM]; Y-axis: MPO pg / ml. [Figure 8] Myeloperoxidase (MPO) release by anti-CEACAM6 antibody 9A6 TPP-3470 (human IgG2 format) (black bars) and corresponding isotype control antibody (white bars) with suboptimal fMLP stimulation (+fMLP) and without fMLP stimulation (w / o fMLP). X-axis: antibody concentration [μM]; Y-axis: MPO pg / ml. [Figure 9] Myeloperoxidase (MPO) release by anti-CEACAM6 antibody Neo201 TPP-1173 (human IgG1 format) (black bars) and corresponding isotype control antibody (white bars) with (+fMLP) and without (w / o fMLP) suboptimal fMLP stimulation. X-axis: antibody concentration [μM]; Y-axis: MPO pg / ml. [Figure 10]Myeloperoxidase (MPO) release by anti-CEACAM6 antibody Neo201 TPP-3688 (human IgG2 format) (black bars) and corresponding isotype control antibody (white bars) with (+fMLP) and without (w / o fMLP) suboptimal fMLP stimulation. X-axis: antibody concentration [μM]; Y-axis: MPO pg / ml. [Figure 11] Myeloperoxidase (MPO) release by anti-CEACAM6 antibody Fab fragment APP-1574 (human IgG1 derived) (black vertical bars) and the corresponding isotype control antibody fragment (white vertical bars) with (+fMLP) and without (w / o fMLP) suboptimal fMLP stimulation. X-axis: antibody concentration [μM]; Y-axis: MPO pg / ml. [Figure 12] Myeloperoxidase (MPO) release by anti-CEACAM6 antibody F(ab)2 fragment APP-6036 (human IgG1 derived) (black vertical bars) and the corresponding isotype control antibody fragment (white vertical bars) with suboptimal fMLP stimulation (+fMLP) and without fMLP stimulation (w / o fMLP). X-axis: antibody concentration [μM]; Y-axis: MPO pg / ml. [Figure 13] Myeloperoxidase (MPO) release by anti-CEACAM6 antibody F(ab)2 fragment APP-60849 (human IgG2 derived) (black vertical bars) and the corresponding isotype control antibody fragment (white vertical bars) with suboptimal fMLP stimulation (+fMLP) and without fMLP stimulation (w / o fMLP). X-axis: antibody concentration [μM]; Y-axis: MPO pg / ml. [Figure 14] Myeloperoxidase (MPO) release by anti-CEACAM6 antibody TPP-3310 (black bars) and the corresponding isotype control antibody TPP-1238 (white bars) with (+fMLP) and without (w / o fMLP) suboptimal fMLP stimulation. A non-binding F(ab)2 fragment matched to AT10 was added at a concentration of 1.4 μM prior to the addition of anti-CEACAM6 antibody TPP-3310 or its isotype control antibody TPP-1238. [Figure 15] Myeloperoxidase (MPO) release by anti-CEACAM6 antibody TPP-3310 (black bars) and the corresponding isotype control antibody TPP-1238 (white bars) with (+fMLP) and without (w / o fMLP) suboptimal fMLP stimulation. Blocking anti-CD32 antibody F(ab)2 fragment AT10 was added at a concentration of 1.4 μM prior to the addition of anti-CEACAM6 antibody TPP-3310 or its isotype control antibody TPP-1238. X-axis: antibody concentration [μM]; Y-axis: MPO pg / ml. [Figure 16] Myeloperoxidase (MPO) release by the indicated anti-CEACAM6 antibody TPP-21518 (human IgG1-LALAaglyco) (black bars) and the corresponding isotype control antibody (white bars) with (+fMLP) and without (w / o fMLP) suboptimal fMLP stimulation. X-axis: antibody concentration [μM]; Y-axis: MPO pg / ml. [Figure 17] Figure 1 shows the percentage phagocytosis of labeled neutrophils by M2c macrophages measured by flow cytometry after 2 hours of co-culture in the presence of anti-CEACAM6 antibodies [TPP-3310 (human IgG2); TPP-21518 (human IgG1-LALAaglyco); TPP-5468 (human IgG1); TPP-1745 (9A6 human IgG1); TPP-1173 (Neo201 human IgG1)] and corresponding isotype controls [TPP-1238 (human IgG2); TPP-21501 (human IgG1-LALAaglyco); TPP-754 (human IgG1)]. Mouse anti-huCD47 is included as a positive control for phagocytosis. X-axis: test articles with the respective protein identifiers at 1 μM, 100 nM, 10 nM and 1 nM concentrations. "-" = no antibody added. CD47 = mouse anti-huCD47; mIgG1 = non-binding isotype control for mouse anti-huCD47. Y-axis: Percentage of viable CD206-APC-positive M2c macrophages that became CFSE positive from phagocytosis of CFSE-labeled neutrophils. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Detailed Description of the Invention definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs. However, the following references can provide those skilled in the art to which the present invention belongs with general definitions of many of the terms used in the present invention, and can be referenced and used to the extent that such definitions are consistent with the meanings commonly understood in the art. Such references include, but are not limited to, Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); Hale & Marham, The Harper Collins Dictionary of Biology (1991); and Lackie et al., The Dictionary of Cell & Molecular Biology (3rd ed. 1999); and Cellular and Molecular Immunology, Abbas, ed., Lichtman and Pober, 2nd ed., WB Saunders Company. Reference can be made to any additional technical sources available to those skilled in the art that provide definitions of terms used herein that have the meanings commonly understood in the art. For purposes of the present invention, the following terms are further defined. Additional terms are defined elsewhere in the specification. As used in this specification and the appended claims, the singular forms "a" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art, and so forth.

[0041] In the context of the present invention, the term "comprises" or "comprising" means "including, but not limited to." The term is intended to be open-ended and specifies the presence of any described features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more restrictive terms "consisting of" and "essentially consisting of." In one embodiment, the term "comprising" as used throughout this application, and particularly in the claims, can be replaced with the term "consisting of."

[0042] In this context, the term "about" or "approximately" means within 80%-120%, or within 90%-110%, such as within 95%-105%, of a given value or range.

[0043] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0044] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

[0045] "ADCP" or antibody-dependent cell-mediated phagocytosis, as used herein, refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.

[0046] The term "antibody" as used herein is intended to mean an immunoglobulin molecule. An antibody may comprise four polypeptide chains, two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa), typically interconnected by disulfide bonds. In certain embodiments, an antibody is composed of two identical pairs of polypeptide chains. The amino-terminal portion of each chain comprises a "variable" region of about 100-110 or more amino acids primarily responsible for antigen recognition. The heavy chain variable region is abbreviated herein as VH, and the light chain variable region is abbreviated herein as VL. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector functions. The heavy chain constant region may comprise, for example, three domains CH1, CH2, and CH3. The light chain constant region is composed of one domain (CL). The VH and VL regions are further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and up to four FRs, arranged, for example, from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0047] In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By "immunoglobulin (Ig) domain" herein is meant a region of an immunoglobulin that has a characteristic tertiary structure. Of interest in the present invention are the heavy chain domains that contain the constant heavy (CH) domain and the hinge domain. For IgG antibodies, each IgG isotype has three CH regions. Thus, the "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-220 according to the EU index in Kabat; "CH2" refers to positions 237-340 according to the EU index in Kabat; and "CH3" refers to positions 341-447 according to the EU index in Kabat.

[0048] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The IgG1 Fc region includes the CH2 and CH3 domains of the IgG1 heavy chain. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG1 heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0049] As used herein, the term "complementarity determining region" (CDR; e.g., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain whose presence is necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3. Each complementarity determining region may be selected from amino acid residues from a "complementarity determining region" as defined by Kabat (e.g., for residues 23-36 (L1), 52-58 (L2), and 91-101 (L3) of the light chain variable domain, and residues 31-35 (H1), 50-65 (H2), and 98-110 (H3) of the heavy chain variable domain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from a "hypervariable loop" (e.g., for residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain (Chothia and Lesk; J Mol Biol 196:901-917 (1987). In some cases, the complementarity determining regions may comprise amino acids from both the CDR regions and the hypervariable loops as defined according to Kabat. "Framework" or FR residues are those variable domain residues other than the hypervariable region residues.

[0050] Immunoglobulins can be divided into different classes depending on the amino acid sequence of the constant domain of their heavy chains. Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α) and epsilon (ε), which define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. In a particular embodiment, the antibody of the invention is an IgG antibody. Some of these can be further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. In a particular embodiment, the antibody of the invention is an IgG1. Different isotypes may have different effector functions. Human light chains are classified as kappa (κ) light chains and lambda (λ) light chains. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

[0051] As used herein, a "functional fragment" or "antigen-binding antibody fragment" of an antibody / immunoglobulin is defined as a fragment of an antibody / immunoglobulin (e.g., the variable region of an IgG) that retains the antigen-binding region. The "antigen-binding region" of an antibody is typically identified in one or more hypervariable regions of the antibody, such as the CDR1, CDR2, and / or CDR3 regions. However, variable "framework" regions may also play an important role in antigen binding, such as providing a scaffold for the CDRs. Preferably, the "antigen-binding region" comprises at least amino acid residues 4-103 of the variable light chain (VL) and amino acid residues 5-109 of the variable heavy chain (VH), more preferably amino acid residues 3-107 of the VL and amino acid residues 4-111 of the VH, and particularly preferably the complete VL and VH chains (amino acids 1-109 of VL and amino acids 1-113 of VH; numbering according to WO 97 / 08320).

[0052] Non-limiting examples of "functional fragments" or "antigen-binding antibody fragments" include Fab, Fab', F(ab'), Fv fragments, domain antibodies (dAbs), complementarity determining region (CDR) fragments, single chain antibodies (scFv), single chain antibody fragments, diabodies, triabodies, tetrabodies, minibodies, linear antibodies (Zapata et al., Protein Eng: pp. 1057-1062 (1995)); chelating recombinant antibodies, tribodies or bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies, or muteins or derivatives thereof, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide, such as CDR sequences, so long as the antibody retains the desired biological activity; and multispecific antibodies formed from antibody fragments, such as bispecifics and trispecifics (CA K Borrebaeck, ed. (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel (eds.) (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag). An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. F(ab') 2 or Fab, C H1 Domain and C LThey can be engineered to minimize or completely eliminate intermolecular disulfide interactions that occur between domains. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, whose name reflects its ability to crystallize easily. Pepsin treatment produces an F(ab')2 fragment with two "Fv" fragments. The "Fv" fragment is the smallest antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen.

[0053] "Single-chain Fv" or "sFv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.

[0054] Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the Fv to form the desired structure for antigen binding. For a general review of Fv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0055] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as a pair of Fab' fragments which had hinge cysteine ​​residues between them.

[0056] The terms "mutein" or "variant" can be used interchangeably and refer to an antibody or antigen-binding fragment that contains at least one amino acid substitution, deletion, or insertion in the variable region or a portion equivalent to the variable region, provided that the mutein or variant retains the desired binding affinity or biological activity. A variant of an antibody or antigen-binding antibody fragment contemplated in the present invention is a molecule in which the binding activity of the antibody or antigen-binding antibody fragment is maintained.

[0057] A "chimeric antibody" or antigen-binding fragment thereof is defined herein as one in which the variable domains are of non-human origin and some or all of the constant domains are of human origin.

[0058] A "humanized antibody" contains CDR regions from a non-human species, such as mouse, grafted onto V regions from human sequences with the necessary framework backmutations. Thus, in most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. See, e.g., U.S. Pat. Nos. 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, each of which is incorporated herein by reference. In some cases, framework residues of human immunoglobulins are replaced by corresponding non-human residues (see, e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762, each of which is incorporated herein by reference). Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain a desired affinity). In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable regions corresponding to those of a non-human immunoglobulin and all or substantially all of the framework regions being those of a human immunoglobulin sequence. A humanized antibody optionally will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 331:522-25 (1986); Riechmann et al., Nature 332:323-27 (1988); and Presta, Curr. Opin. Struct. Biol. 2:593-96 (1992), each of which is incorporated herein by reference.

[0059] A "human antibody" or "fully human antibody" comprises CDRs of human origin, i.e., CDRs of human origin. A fully human antibody may contain a small number of germline deviations compared to the closest human germline reference as determined based on the IMGT database (www.imgt.org). For example, a fully human antibody according to the invention may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 germline deviations in the CDRs compared to the closest human germline reference. Fully human antibodies can be developed from B cells of human origin by cloning techniques combined with cell enrichment or immortalization steps. However, the majority of fully human antibodies are isolated from immunized mice transgenic for the human IgG locus or from high-performance combinatorial libraries by phage display (Bruggemann M., Osborn MJ, Ma B., Hayre J., Avis S., Lundstrom B. and Buelow R., Human Antibody Production in Transgenic Animals, Arch Immunol Ther Exp (Warsz.) 63 (2015), pp. 101-108; Carter PJ, Potent antibody therapeutics by design, Nat Rev Immunol 6 (2006), pp. 343-357; Frenzel A., Schirrmann T. and Hust M., Phage display-derived human antibodies in clinical development and therapy, MAbs 8 (2016), pp. 1177-1194; Nelson AL, Dhimolea E. and Reichert JM, Development trends for human monoclonal antibody therapeutics, Nat Rev Drug Discov 9 (2010), pp. 767-774).

[0060] Several techniques are available for making fully human antibodies (see WO2008 / 112640 A3). Cambridge Antibody Technologies (CAT) and Dyax have obtained antibody cDNA sequences from peripheral B cells isolated from immunized humans and devised a phage display library to identify human variable region sequences of particular specificity. Briefly, antibody variable region sequences are fused to either gene III or gene VIII structures of M13 bacteriophage. These antibody variable region sequences are expressed as either Fab or single chain Fv (scFv) structures at the tip of phages carrying the respective sequences. By repeating the panning method using different levels of antigen binding conditions (stringency), phages expressing Fab or scFv structures specific for the antigen of interest can be selected and isolated. The antibody variable region cDNA sequences of the selected phages can then be elucidated using standard sequencing procedures. These sequences can then be used to reconstruct complete antibodies with the desired isotype using established antibody engineering techniques. The antibodies constructed according to this method are considered to be fully human antibodies (including CDRs). To improve the immunoreactivity (antigen binding affinity and specificity) of the selected antibody, in vitro maturation methods can be introduced, including combinations of different heavy and light chains, deletions / additions / mutations in CDR3 of heavy and light chains (to mimic VJ and VDJ recombination), and random mutations (to mimic somatic hypermutation). An example of a "fully human" antibody produced by this method is the anti-tumor necrosis factor alpha antibody Humira (adalimumab).

[0061] "Human Engineered™" antibodies were produced by modifying the parent sequences according to the methods described in Studnicka et al., US Pat. No. 5,766,886.

[0062] The antibodies of the invention may be derived from a recombinant antibody gene library. The development of techniques for generating repertoires of recombinant human antibody genes and displaying the encoded antibody fragments on the surface of filamentous bacteriophage provides a recombinant means for directly generating and selecting human antibodies, which can also be applied to humanized, chimeric, murine or mutein antibodies. Antibodies produced by phage technology lack effector functions because they are produced in bacteria as antigen-binding fragments, usually Fv or Fab fragments. Effector functions can be introduced by one of two strategies: Fragments can be engineered to be either complete antibodies for expression in mammalian cells or bispecific antibody fragments with a second binding site capable of eliciting effector functions. Typically, antibody heavy (e.g., VH-CH1) and light (e.g., VL-CL) chain fragments can be cloned separately by PCR, randomly recombined in a combinatorial phage display library, and then selected for binding to a specific antigen. Fab fragments are expressed on the phage surface, i.e., physically associated with the genes that encode them. Thus, selection of Fabs by antigen binding co-selects the sequences encoding the Fabs, which can then be amplified. Through several rounds of antigen binding and reamplification, a procedure called panning, Fabs specific for the antigen are enriched and finally isolated.

[0063] Various procedures have been described for human antibodies derived from phage display libraries. Such libraries can be constructed on a single master framework into which the diverse CDRs formed in vivo (i.e. of human origin) can be recombined as described by Carlsson and Soderlind Exp. Rev. Mol. Diagn. 1 (1), pp. 102-108 (2001), Soderlin et al., Nat. Biotech. 18, pp. 852-856 (2000) and U.S. Pat. No. 6,989,250. Alternatively, such antibody libraries can be designed in silico and based on amino acid sequences encoded by synthetically produced nucleic acids. In silico design of antibody sequences can be achieved, for example, by analyzing a database of human sequences and using the data obtained therefrom to devise polypeptide sequences. Methods for designing and obtaining in silico generated sequences are described, for example, in Knappik et al., J. Mol. Biol. (2000) 296:57; Krebs et al., J. Immunol. Methods. (2001) 254:67; and U.S. Patent No. 6,300,064. For a review of phage display screening (see, for example, Hoet RM et al., Nat Biotechnol 2005; 23(3):344-8), for well-established hybridoma technology (see, for example, Kohler and Milstein Nature. 1975 Aug. 7; 256(5517):495-7), or for immunization of mice, particularly hMAb mice (e.g., VelocImmune mouse®).

[0064] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible minor mutations, e.g., naturally occurring mutations. Thus, the term "monoclonal" indicates the character of the antibody as not being a mixture of separate antibodies. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The term "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used may be made by the hybridoma method first described by Kohler et al., Nature, 256:495

[1975] , or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). A "monoclonal antibody" may also be, for example, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, a Human Engineered™ antibody, or an antibody fragment.

[0065] An "isolated" antibody is one which has been identified and separated from components of the cells in which it is expressed. Contaminating components of the cells are substances which would interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones and other proteinaceous or nonproteinaceous solutes.

[0066] An "isolated" nucleic acid is one that is identified and separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0067] An "anti-antigen" antibody refers to an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, and an anti-CECAM6 antibody specifically binds to CECAM6.

[0068] As used herein, an antibody "specifically binds to," "is specific for," or "specifically recognizes" an antigen of interest, e.g., CEACAM6, and is useful as a therapeutic agent to target cells or tissues expressing the antigen, and binds the antigen with sufficient affinity such that it does not exhibit significant cross-reactivity with proteins other than orthologs and variants (e.g., mutants, splice variants, or proteolytically truncated forms) of the aforementioned antigen target. As used herein, the terms "specifically recognize" or "specifically bind to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target refer to, for example, an antibody that is specific for, e.g., an antibody that is specific for, e.g., CEACAM6, and is useful as a therapeutic agent to target cells or tissues expressing the antigen, and that binds the antigen with sufficient affinity such that it does not exhibit significant cross-reactivity with proteins other than orthologs and variants (e.g., mutants, splice variants, or proteolytically truncated forms) of the aforementioned antigen target. -4 Less than M or about 10 -5 Less than M or about 10 -6 Less than M or about 10 -7 Less than M or about 10 -8 Less than M or about 10 -9 Less than M or about 10 -10 Less than M or about 10 -11 Less than M or about 10 -12An antibody or antigen-binding fragment thereof with a monovalent KD for an antigen that is less than or equal to M can be exhibited. An antibody "specifically binds to", "is specific for" or "specifically recognizes" an antigen if such an antibody can distinguish such an antigen from one or more reference antigens. In its most common form, "specific binding", "specifically binds to", "is specific for" or "specifically recognizes" refers to the ability of an antibody to distinguish between an antigen of interest and unrelated antigens, for example as determined according to any of the following methods: Such methods include, but are not limited to, surface plasmon resonance (SPR), Western blot, ELISA test, RIA test, ECL test, IRMA test and peptide scan. For example, a standard ELISA assay can be performed. Scoring can be performed by standard color development (e.g., secondary antibody with horseradish peroxidase and tetramethylbenzidine with hydrogen peroxide). The reaction in a particular well is scored by optical density at, for example, 450 nm. A typical background (=negative reaction) can be 0.1 OD. A typical positive reaction can be 1 OD. This means that the positive / negative difference is greater than 5-fold, 10-fold, 50-fold, and preferably greater than 100-fold. Typically, the determination of binding specificity is not performed using a single reference antigen, but rather using a combination of about 3-5 unrelated antigens, such as milk powder, BSA, transferrin, etc.

[0069] "Binding affinity" or "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The dissociation constant "K D" is commonly used to indicate the affinity between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen), i.e., how tightly the ligand binds to a particular protein. Ligand-protein affinity is influenced by non-covalent intermolecular interactions between the two molecules. Affinity can be measured by common methods known in the art, including those described herein. In one embodiment, the term "K" is used in the present invention to indicate the affinity between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Ligand-protein affinity is influenced by non-covalent intermolecular interactions between the two molecules. Affinity can be measured by common methods known in the art, including those described herein. D " or "K D The "value" is measured by using a surface plasmon resonance assay using a Biacore T200 instrument (GE Healthcare Biacore, Inc.). Other suitable instruments are the BIACORE T100, BIACORE®-2000, BIACORe 4000, BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ), or the ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.).

[0070] As used herein, the term "epitope" includes a protein determinant capable of specific binding to an antibody, an immunoglobulin, or a T-cell receptor. Epitopic determinants usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, or combinations thereof, and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0071] The terms "antibody that binds to the same epitope" or "antibody that competes for binding" as a reference antibody, or the term "competes" when used in the context of antigen binding proteins (e.g., antibodies) that compete for the same epitope, refer to competition between the antigen binding proteins as determined by an assay in which the antigen binding protein being tested (e.g., an antibody or an immunologically functional fragment thereof) inhibits or suppresses (e.g., reduces) the specific binding of the reference antigen binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., CEACAM6 or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, including, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct label assays, solid-phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using an I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176:546-552); and direct label RIA (see, e.g., Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually, the test antigen binding protein is present in excess.Antigen-binding proteins identified by competitive assays (competitor antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein, and antigen-binding proteins that bind to adjacent epitopes close enough to sterically hinder the epitope bound by the reference antigen-binding protein. Typically, when a competing antigen-binding protein is present in excess, it inhibits (e.g., reduces) specific binding of the reference antigen-binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0072] The term "mature antibody" or "mature antigen-binding fragment", e.g., mature Fab variant or "optimized" variant, includes derivatives of antibodies or antibody fragments that exhibit stronger binding, i.e., higher affinity binding, to a given antigen, e.g., the extracellular domain of a target protein. Maturation is the process of identifying a small number of mutations within the six CDRs of an antibody or antibody fragment that result in this increased affinity. The maturation process is a combination of molecular biology techniques of introducing mutations into an antibody and screening to identify improved binders.

[0073] "Percent sequence identity" with respect to a reference polynucleotide or polypeptide sequence, respectively, is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical to the nucleic acid or amino acid residues in the reference polynucleotide or polypeptide sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Conservative substitutions are not considered part of the sequence identity. Preference is given to alignment without gaps. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0074] "Sequence homology" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions.

[0075] An "antagonist" or "blocking" antibody is one that significantly inhibits (partially or completely) the biological activity of the antigen to which it binds. In certain embodiments, the antibody or antigen-binding fragment of the invention is a CEACAM6 blocking antibody or antigen-binding fragment.

[0076] The term "antibody conjugate" refers to one or more molecules, including drugs (in which case the antibody conjugate is referred to as an "antibody-drug conjugate" ("ADC")), and large molecular weight molecules, such as peptides or proteins, conjugated to the antibody.

[0077] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted one-letter codes.

[0078] The term "vector" as used herein means a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0079] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which at least one exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants," "transformed cells," "transfectants," "transfected cells," and "transduced cells," including the primary transformed / transfected / transduced cell and its progeny, regardless of the number of passagings. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0080] As used herein, the phrase "therapeutically effective amount" is meant to refer to an amount of therapeutic or prophylactic antibody adequate to elicit a desired therapeutic or prophylactic effect or response, including alleviating some or all of such symptoms of a disease or reducing predisposition to a disease, when administered in accordance with a desired treatment regimen.

[0081] The term "pharmaceutical formulation" / "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0082] As used herein, "CEACAM6" refers to "carcinoembryonic antigen-related cell adhesion molecule 6," also known as "CD66c" (cluster of differentiation 66c) or nonspecific cross-reacting antigen or NCA or NCA-50 / 90. CEACAM6 is a glycosylphosphatidylinositol (GPI)-linked cell surface protein involved in cell-cell adhesion. The term "CEACAM6" as used herein includes human CEACAM6 (hCEACAM6), variants, isoforms, and species homologs (orthologs) of hCEACAM6. The reference sequence of human CEACAM6 (hCEACAM6) is available from the UniProtKB / Swiss-Prot database under accession number P40199.3 and from NCBI under reference sequence: NP_002474.4. The mature extracellular region of human CEACAM6 consists of amino acids 35-320 of SEQ ID NO:75. Domain 1 (also known as the N domain, also known as N-terminal domain 1) of human CEACAM6 consists of amino acids 35 to 142 of SEQ ID NO:75.

[0083] The terms "anti-CEACAM6 antibody" and "antibody that binds to CEACAM6" refer to an antibody capable of binding to human CEACAM6 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting CEACAM6. In one embodiment, the extent of binding of the anti-CEACAM6 antibody to an unrelated non-CEACAM6 protein is less than about 10%, less than about 5%, or less than about 2% of the binding of the antibody to CEACAM6 as measured, for example, by standard ELISA procedures. In certain embodiments, an antibody that binds to CEACAM6 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10-8 M~10 -13 M, for example 10 -9 M~10 -13 M) of binding activity (EC50). In certain embodiments, the anti-CEACAM6 antibody binds to an epitope of CEACAM6 that is conserved among CEACAM6 of different species.

[0084] "Programmed Death-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed primarily on activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs that share at least one common epitope with hPD-1. The complete hPD-1 sequence can be found at GenBank Accession No. U64863.

[0085] "Programmed Death Ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPDL1), variants, isoforms, and species homologs of hPD-L1, and analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found at GenBank Accession No. Q9NZQ7.

[0086] As used herein, "TIM-3" refers to the TIM family member "T cell immunoglobulin domain and mucin domain 3" (also known as HAVCAR2). TIM-3 is a cell surface transmembrane protein. It has been described as an activation-induced inhibitory molecule involved in tolerance and has been shown to induce T cell exhaustion. As used herein, the term "TIM-3" includes human TIM-3 (hTIM-3), variants, isoforms, and species homologs of hTIM-3, and analogs that share at least one common epitope with hTIM-3. The reference sequence of human TIM-3 can be obtained from the UniProtKB / Swiss-Prot database under accession number UniProtKB Q8TDQ0 (HAVR2_HUMAN) and from the NCBI database under NCBI reference sequence: NP_116171.3.

[0087] TIFF2024534186000001.tif252168TIFF2024534186000002.tif202169TIFF2024534186000003.tif254164 TIFF2024534186000004.tif202169TIFF2024534186000005.tif255164TIFF2024534186000006.tif253161 TIFF2024534186000007.tif253161TIFF2024534186000008.tif186169TIFF2024534186000009.tif249169 TIFF2024534186000010.tif249169TIFF2024534186000011.tif198169TIFF2024534186000012.tif159168

[0088] First aspect of the invention: anti-CECAM6 antibodies The present invention relates to an antibody (anti-CECAM6 antibody) that can bind to human CEACAM6 and alleviate CEACAM6-mediated immunosuppression, and which has reduced side effects during treatment.

[0089] Of particular interest in the present invention is the Fc region of said anti-CEACAM6 antibody. "Fc" or "Fc region" as used herein means a polypeptide comprising the constant region of an antibody heavy chain excluding the first constant region immunoglobulin domain CH1 and optionally a portion of the hinge. Fc thus refers to the last two constant region immunoglobulin domains CH2 and CH3. Although the boundaries of the Fc region vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 at its carboxyl terminus, where numbering is according to the EU index as in Kabat et al. An IgG1 Fc region is the Fc region of an antibody of the IgG1 isotype.

[0090] In a first aspect, the present invention relates to an anti-CECAM6 antibody comprising an IgG1 Fc region lacking a glycan attached to the conserved N-linked site in the CH2 domain of the Fc region, said IgG1 Fc region comprising at least the amino acid substitutions L234A and L235A numbered according to the EU index of Kabat. Antibodies lacking a glycan attached to the conserved N-linked site of the CH2 domain are also called aglycosylated or aglyco antibodies. The conserved N-linked glycosylation occurs at N297 numbered according to the EU index of Kabat.

[0091] In one embodiment of the invention, the modification comprises a mutation at the heavy chain glycosylation site to prevent glycosylation at that site. Thus, in a preferred embodiment of the invention, an aglycosylated antibody or antibody derivative is prepared by mutation of the heavy chain glycosylation site, i.e., N297 using Kabat EU numbering, and expressed in a suitable host cell.

[0092] In certain embodiments of the first aspect, the invention provides an anti-CECAM6 antibody comprising an IgG1 Fc region, the IgG1 Fc region comprising the amino acid substitutions N297A, N297G, or N297Q, numbered according to the EU index of Kabat. The anti-CECAM6 antibody comprising an IgG1 Fc region, the IgG1 Fc region comprising the amino acid substitutions N297A, N297G, or N297Q, numbered according to the EU index of Kabat, is an antibody that lacks a glycan attached to the conserved N-linked site of the CH2 domain, not further mentioned as being glycan-deleted.

[0093] In another embodiment of the invention, the aglycosylated antibody is produced by a method comprising expressing the antibody in a host cell that is unable to attach glycans to Asn residues, for example by using a prokaryotic host cell, or by using a eukaryotic host cell modified to lack the necessary enzymes.

[0094] In another embodiment of the invention, the aglycosylated antibody is produced by an in vitro method comprising expressing an antibody that does not have N-glycosylation capacity.

[0095] In another embodiment of the present invention, aglycosylated antibodies are produced by a method that includes removing CH2 domain-linked glycans, i.e., deglycosylation. These aglycosylated antibodies can be produced by conventional methods and then enzymatically deglycosylated. Methods for enzymatic deglycosylation of antibodies are well known in the art (e.g., Winkelhake & Nicolson (1976), J Biol Chem. 251 (4): 1074-80).

[0096] In another embodiment of the invention, deglycosylation can be achieved using the glycosylation inhibitor tunicamycin (Nose & Wigzell (1983), Proc Natl Acad Sci USA, 80(21):6632-6). Briefly, this modification prevents glycosylation at the conserved N-linked site of the CH2 domain of the Fc portion of the antibody.

[0097] In certain embodiments of the first aspect, the invention provides an anti-CECAM6 antibody comprising an IgG1 Fc region, wherein the IgG1 Fc region comprises amino acid substitutions N297A, N297G, or N297Q, and at least amino acid substitutions L234A and L235A, numbered according to the EU index of Kabat.

[0098] In certain embodiments of the first aspect, the invention provides an anti-CECAM6 antibody comprising an IgG1 Fc region, wherein the IgG1 Fc region comprises at least the amino acid substitutions N297A, L234A, and L235A, numbered according to the EU index of Kabat.

[0099] In certain embodiments of the first aspect, the invention provides an anti-CECAM6 antibody comprising an IgG1 Fc region, wherein the IgG1 Fc region comprises the following amino acid substitutions: N297A, L234A, and L235A, numbered according to the EU index of Kabat.

[0100] In certain embodiments of the first aspect, the above-mentioned anti-CECAM6 antibody competes for CEACAM6 binding with an antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 67.

[0101] In a certain embodiment of the first aspect, the above-mentioned anti-CECAM6 antibody comprises a heavy chain variable region H-CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a heavy chain variable region H-CDR2 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain variable region H-CDR3 comprising the amino acid sequence of SEQ ID NO: 66, a light chain variable region L-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, a light chain variable region L-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a light chain variable region L-CDR3 comprising the amino acid sequence of SEQ ID NO: 70.

[0102] In certain embodiments of the first aspect, the anti-CECAM6 antibody comprises a heavy chain variable region H-CDR1 amino acid sequence of SEQ ID NO: 64, a heavy chain variable region H-CDR2 amino acid sequence of SEQ ID NO: 65, a heavy chain variable region H-CDR3 amino acid sequence of SEQ ID NO: 66, a light chain variable region L-CDR1 amino acid sequence of SEQ ID NO: 68, a light chain variable region L-CDR2 amino acid sequence of SEQ ID NO: 69, and a light chain variable region L-CDR3 amino acid sequence of SEQ ID NO: 70.

[0103] In certain embodiments of the first aspect, the anti-CECAM6 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 67.

[0104] In certain embodiments of the first aspect, the anti-CECAM6 antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:71, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:72.

[0105] In certain embodiments, the anti-CECAM6 antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:71 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:72.

[0106] In certain preferred embodiments of the first aspect, the anti-CECAM6 antibody is an isolated antibody.

[0107] In certain preferred embodiments of the first aspect, said anti-CECAM6 antibody is a monoclonal antibody.

[0108] In certain preferred embodiments of the first aspect, said anti-CECAM6 antibodies are human or humanized antibodies.

[0109] In certain preferred embodiments of the first aspect, the anti-CECAM6 antibodies bind to a CEACAM6 comprising the amino acid sequence of SEQ ID NO:75.

[0110] In certain preferred embodiments of the first aspect, the anti-CECAM6 antibodies bind to CEACAM6 domain 1 comprising amino acids 35-142 of SEQ ID NO:75.

[0111] Antibody production A further aspect of the present invention provides a method for producing an antibody of the first aspect. A detailed description of the method for providing an antibody with certain binding properties is disclosed in WO 2016 / 150899 A2.

[0112] The antibodies of the invention can be obtained from recombinant antibody libraries based on amino acid sequences isolated from the antibodies of a large number of healthy volunteers, for example using n-CoDeR® technology (Carlson & Soderlind, Expert Rev Mol Diagn. 2001 May; 1(1):102-8) which recombines fully human CDRs into new antibody molecules. Alternatively, CEACAM6-specific antibodies can be isolated using antibody libraries, such as the fully human antibody phage display library described in Hoet RM et al., Nat Biotechnol 2005; 23(3):344-8. Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0113] Human antibodies can also be prepared by administering immunogens to transgenic animals that have been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, which either replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. For example, immunization of genetically engineered mice can be performed, particularly immunization of hMAb mice (e.g., VelocImmune mouse® or XENOMOUSE®).

[0114] Further antibodies can be produced using hybridoma technology (see, e.g., Kohler and Milstein Nature. 1975 Aug. 7; 256(5517):495-7), to obtain, for example, mouse, rat or rabbit antibodies which can be converted into chimeric or humanized antibodies. Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall' Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osboum et al., Methods 36:61-68 (2005), and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0115] Peptide variants The antibody of the present invention is not limited to the specific peptide sequence provided herein.Rather, the present invention also encompasses variants of these polypeptides.By referring to this disclosure and the techniques and references available in the past, the skilled artisan can prepare, test and utilize functional variants of the antibody disclosed herein, and it will be understood by the skilled artisan that these variants that have the ability to bind to CEACAM6 are within the scope of the present invention.

[0116] Variants may include, for example, antibodies with at least one altered complementarity determining region (CDR) (hypervariable) and / or framework (FR) (variable) domain / position relative to the peptide sequences disclosed herein.

[0117] By altering one or more amino acid residues in the CDR or FR regions, one skilled in the art can routinely generate mutated or diversified antibody sequences, which can be screened, for example, against an antigen for new or improved properties.

[0118] A further preferred embodiment of the present invention is an antibody or antigen-binding fragment in which the VH and VL sequences are selected from the sequences provided. This can be used by those skilled in the art to design peptide variants that are within the scope of the present invention. The variants are preferably constructed by changing amino acids in one or more CDR regions. The variants can also have one or more modified framework regions. Changes can also be made in the framework regions. For example, the peptide FR domain can be changed if there is a deviation in the residues compared to the germline sequence.

[0119] Alternatively, one skilled in the art can perform a similar analysis by comparing the amino acid sequences disclosed herein with known sequences of the same class of such antibodies, for example using the procedures described in Knappik A. et al., JMB 2000, 296:57-86.

[0120] Furthermore, variants can be obtained by using an antibody as a starting point for further optimization by diversifying one or more amino acid residues of the antibody, preferably one or more amino acid residues of the CDRs, and screening the resulting collection of antibody variants for variants with improved properties. Particularly preferred is diversification of one or more amino acid residues in the CDR3 of VL and / or VH. Diversification can be performed, for example, by synthesizing a collection of DNA molecules using trinucleotide mutagenesis (TRIM) technology (Virnekas B. et al., Nucl. Acids Res. 1994, 22: 5600). Antibodies or antigen-binding fragments thereof include molecules with modifications / variations, including, but not limited to, modifications that result in altered half-life (e.g., modifications of the Fc portion or attachment of additional molecules, such as PEG), altered binding affinity, or altered ADCC or CDC activity.

[0121] Conservative amino acid variants Polypeptide variants can be made that preserve the overall molecular structure of the antibody peptide sequences described herein. Some rational substitutions will be understood by those skilled in the art, given the properties of individual amino acids. Amino acid substitutions, i.e. "conservative substitutions", can be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved.

[0122] For example, (a) non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine, (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, (c) positively charged (basic) amino acids include arginine, lysine, and histidine, and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions can typically be made within groups (a)-(d). Additionally, glycine and proline can be substituted for one another based on their ability to disrupt α-helices. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine, and lysine, are more commonly found in α-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophan, and threonine are more commonly found in β-pleated sheets. Glycine, serine, aspartic acid, asparagine, and proline are generally found in order. Some preferred substitutions can be made between the following groups: (i) S and T, (ii) P and G, and (iii) A, V, L, and I. With the known genetic code and recombinant and synthetic DNA techniques, one of skill in the art can readily construct DNAs that encode conservative amino acid variants.

[0123] Antibody-drug conjugates (ADCs) The present invention also provides antibody-drug conjugates (ADCs, immunoconjugates) comprising an anti-CEACAM6 antibody of the first aspect conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g. protein toxins, enzymatically active toxins of bacterial, fungal, plant, human or animal origin, or fragments thereof), or radioactive isotopes.

[0124] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including, but not limited to, maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent EP 0425235); auristatins, such as monomethyluristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof; anthracyclines, such as daunomycin or doxorubicin; methotrexate; vindesine taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0125] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alphasarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (P API, P APII, and PAP-S), momordica charantia inhibitor, curtin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0126] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the generation of radioconjugates. Examples include: 227 Th, 225Ac, 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Radioactive isotopes include Pb and Lu. When radioactive conjugates are used for detection, they may contain radioactive atoms for scintigraphy studies, such as Tc99m, or spin labels for nuclear magnetic resonance (NMR) imaging, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0127] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and diactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0128] The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug inside the cell, such as an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52: 127-131 (1992)).

[0129] The immunoconjugates or ADCs herein expressly contemplate such conjugates prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), available commercially (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0130] In a further aspect, the invention provides an anti-CEACAM6 antibody of the first aspect conjugated to one or more cytotoxic agents as described above to form an ADC.

[0131] The DNA molecule of the present invention The present invention also relates to DNA molecules encoding the antibodies of the present invention. The DNA sequences used for the expressed antibodies are shown in Table 0 and in the sequence listing, for example for TPP-21518. These sequences are optimized for mammalian expression in certain cases. The DNA molecules of the present invention are not limited to the sequences disclosed herein, but also include variants thereof. DNA variants within the present invention can be described by reference to physical properties in hybridization. Those skilled in the art will understand that nucleic acid hybridization techniques can be used to identify DNA to identify its complement and, since DNA is double-stranded, its equivalent or homologue. It will also be understood that hybridization can occur with less than 100% complementarity. However, hybridization techniques can be used to distinguish DNA sequences based on their structural relatedness to a particular probe, provided that the conditions are appropriately selected. For guidance regarding such conditions, see Sambrook et al., 1989, supra, and Ausubel et al., 1995 (Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Sedman, JG, Smith, JA, & Struhl, K., eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons).

[0132] The structural similarity between two polynucleotide sequences can be expressed as a function of the "stringency" of the conditions under which the two sequences hybridize to each other.As used herein, the term "stringency" refers to the degree to which conditions prevent hybridization.Stringent conditions highly prevent hybridization, and only the most structurally related molecules hybridize to each other under such conditions.Conversely, non-stringent conditions favor the hybridization of molecules with a lower degree of structural relatedness.Thus, the stringency of hybridization is directly related to the structural relationship of two nucleic acid sequences.

[0133] The stringency of hybridization is a function of many factors, including total DNA concentration, ionic strength, temperature, probe size, and the presence of agents that disrupt hydrogen bonds. Factors that promote hybridization include high DNA concentration, high ionic strength, low temperature, long probe size, and the absence of agents that disrupt hydrogen bonds. Hybridization is typically carried out in two steps: a "binding" step and a "washing" step.

[0134] Functionally equivalent DNA variants Yet another class of DNA variants within the scope of the present invention can be described with reference to the products they encode: These functionally equivalent polynucleotides are characterized by the fact that, due to the degeneracy of the genetic code, they code for the same peptide sequence.

[0135] It is recognized that variants of the DNA molecules provided herein can be constructed in several different ways. For example, they can be constructed as fully synthetic DNA. Methods for efficient synthesis of oligonucleotides are widely available. See Ausubel et al., section 2.11, supplement 21 (1993). Overlapping oligonucleotides can be synthesized and constructed as originally reported by Khorana et al., J. Mol. Biol. 72:209 217 (1971). See also Ausubel et al., supra, Section 8.2. Synthetic DNA is preferably designed with engineered restriction sites at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector.

[0136] As indicated, a method for producing variants is to start with one of the DNAs disclosed herein and then perform site-directed mutagenesis. See Ausubel et al., supra, chapter 8, supplement 37 (1997). In a typical method, the target DNA is cloned into a single-stranded DNA bacteriophage vehicle. The single-stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide change(s). The complementary strand is synthesized, and the double-stranded phage is introduced into a host. Some of the resulting progeny contain the desired mutation, which can be confirmed using DNA sequencing. In addition, various methods are available to increase the probability that the progeny phage are the desired mutation. These methods are well known to those skilled in the art, and kits for generating such mutations are commercially available.

[0137] Recombinant DNA constructs and expression of anti-CEACAM6 antibodies The present invention further provides recombinant DNA constructs encoding the antibodies of the present invention. These recombinant constructs of the present invention can be used in combination with vectors, such as plasmids, phagemids, phages or viral vectors, into which a DNA molecule encoding an antibody or antigen-binding fragment thereof or variant thereof of the present invention is inserted.

[0138] The antibodies, antigen-binding portions or variants thereof provided herein can be prepared by recombinantly expressing nucleic acid sequences encoding the light and heavy chains or portions thereof in a host cell. To recombinantly express an antibody, antigen-binding portion or variant thereof, a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the light and heavy chains or portions thereof, such that the light and heavy chains are expressed in the host cell. Standard recombinant DNA techniques are used to prepare and / or obtain nucleic acids encoding the heavy and light chains, incorporate these nucleic acids into recombinant expression vectors, and introduce the vectors into host cells, as described in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY, (1989), Ausubel, FM et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), and U.S. Patent No. 4,816,397 to Boss et al.

[0139] Furthermore, the nucleic acid sequences encoding the variable regions of the heavy and / or light chains can be converted into nucleic acid sequences encoding, for example, full-length antibody chains, Fab fragments, or scFvs. The DNA fragments encoding the VL or VH can be operably linked to another DNA fragment encoding, for example, an antibody constant region or a flexible linker (such that the amino acid sequences encoded by the two DNA fragments are in frame). The sequences of human heavy and light chain constant regions are known in the art (see, for example, Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification.

[0140] To generate a polynucleotide sequence encoding an scFv, the nucleic acids encoding the VH and VL can be operably linked to another fragment encoding a flexible linker, and the VH and VL sequences expressed as a contiguous single-chain protein in which the VL and VH regions are joined by the flexible linker (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

[0141] Standard recombinant DNA expression methods can be used to express antibodies, antigen-binding fragments thereof, or variants thereof (see, e.g., Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, DNA encoding the desired polypeptide can be inserted into an expression vector, which is then transfected into a suitable host cell. Suitable host cells are prokaryotic and eukaryotic cells. Examples of prokaryotic host cells are, for example, bacteria, and examples of eukaryotic host cells are yeast, insects and insect cells, plants and plant cells, transgenic animals, or mammalian cells. In some embodiments, DNA encoding the heavy and light chains is inserted into separate vectors. In other embodiments, DNA encoding the heavy and light chains is inserted into the same vector. It will be appreciated that the design of the expression vector, including the selection of regulatory sequences, will be influenced by factors such as the choice of the host cell, the level of expression of the desired protein, and whether expression is constitutive or inducible.

[0142] Thus, one embodiment of the present invention is also a host cell comprising the vector or nucleic acid molecule, whereby the host cell may be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or a prokaryotic cell, such as a bacterial cell.

[0143] Another embodiment of the invention is a method of using host cells to produce antibodies and antigen-binding fragments, comprising culturing the host cells under suitable conditions and recovering the antibodies.

[0144] Accordingly, another embodiment of the invention is the production of antibodies according to the invention using the host cells of the invention and purification of these antibodies to at least 95% homogeneity by weight.

[0145] Bacterial expression Useful expression vectors for bacteria are constructed by inserting a DNA sequence encoding a desired protein in operable reading phase with a functional promoter, along with appropriate translation initiation and termination signals. The vector contains one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, provide amplification within the host. Suitable prokaryotic hosts for transformation include, but are not limited to, E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0146] Bacterial vectors can be, for example, bacteriophage-, plasmid- or phagemid-based. These vectors can contain a selectable marker and a bacterial origin of replication from a commercially available plasmid, which typically contains elements of the well-known cloning vector pBR322 (ATCC 37017). After transforming a suitable host strain and growing the host strain to an appropriate cell density, the selected promoter is derepressed / induced by appropriate means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.

[0147] In bacterial systems, a number of expression vectors may be advantageously selected depending on the intended use of the expressed protein, for example, if large quantities of such proteins are to be produced, for example, for the production of antibodies or screening of peptide libraries, vectors which direct high level expression of fusion proteins that are easily purified may be desired.

[0148] Thus, one embodiment of the present invention is an expression vector comprising a nucleic acid sequence encoding the novel antibody of the present invention.

[0149] Antibodies or antigen-binding fragments or variants thereof of the present invention include naturally occurring purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from prokaryotic hosts including, for example, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species belonging to the genera Pseudomonas, Streptomyces, and Staphylococcus, preferably from E. coli cells.

[0150] Mammalian Expression Preferred control sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), promoters and / or enhancers from Simian Virus 40 (SV40) (e.g., SV40 promoter / enhancer), promoters and / or enhancers from adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma. Antibody expression can be constitutive or regulated (e.g., inducible by addition or removal of small molecule inducers, such as tetracycline, in combination with the Tet system). For further details of viral control elements and sequences thereof, see, for example, U.S. Patent No. 5,168,062 to Stinski, U.S. Patent No. 4,510,245 to Bell et al., and U.S. Patent No. 4,968,615 to Schaffner et al. The recombinant expression vector may also include an origin of replication and a selectable marker (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). Suitable selectable markers include genes that confer resistance to drugs, such as G418, puromycin, hygromycin, blasticidin, zeocin / bleomycin, or methotrexate, on a host cell into which the vector has been introduced, or selectable markers that utilize auxotrophy, such as glutamine synthetase (Bebbington et al., Biotechnology (NY). February 1992; 10(2):169-75). For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, the neo gene confers resistance to G418, the bsd gene from Aspergillus terreus confers resistance to blasticidin, puromycin N-acetyltransferase confers resistance to puromycin, the Sh ble gene product confers resistance to zeocin, and resistance to hygromycin is conferred by the E. coli hygromycin resistance gene (hyg or hph).Selectable markers such as DHFR or glutamine synthetase are also useful in amplification techniques in conjunction with MTX and MSX.

[0151] Transfection of the expression vector into the host cell can be carried out using standard techniques, such as electroporation, nucleofection, calcium phosphate precipitation, lipofection, polycation-based transfection, such as polystyreneimine (PEI)-based transfection, and DEAE-dextran transfection.

[0152] Suitable mammalian host cells for expressing the antibodies, antigen-binding fragments thereof, or variants thereof provided herein include Chinese hamster ovary (CHO cells), such as CHO-K1, CHO-S, CHO-K1SV [e.g., dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. June 1983; 33(2):405-12, used with the DHFR selectable marker, as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621; and Fan et al., Biotechnol Bioeng. April 2012; 109(4):1007-15], NS0 myeloma cells, COS cells, HEK293 cells, HKB11 cells, BHK21 cells, CAP cells, EB66 cells, and SP2 cells.

[0153] Expression can also be transient or semi-stable in expression systems such as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E, HEK293-Freestyle, HKB11, Expi293F, 293EBNALT75, CHO Freestyle, CHO-S, CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7 or CAP-T cells (e.g., Durocher et al., Nucleic Acids Res. 2002 Jan. 15; 30(2): E9).

[0154] In some embodiments, the expression vector is designed so that the expressed protein is secreted into the medium that the host cells are grown in. The antibody, antigen-binding fragment thereof, or variant thereof can be recovered from the culture medium using standard protein purification methods.

[0155] purification The antibodies or antigen-binding fragments or variants thereof of the present invention can be recovered and purified from recombinant cell cultures by well-known methods, including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, protein A chromatography, protein G chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each of which is incorporated herein by reference in its entirety.

[0156] The antibodies or antigen-binding fragments thereof or variants thereof of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from eukaryotic hosts, including, for example, yeast, higher plants, insect and mammalian cells. Depending on the host used in a recombinant production procedure, the antibodies of the present invention can be glycosylated or non-glycosylated. Such methods are described in many standard laboratory manuals, e.g., Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20. In preferred embodiments, the antibody is (1) purified to greater than 95% by weight, and in more preferred embodiments greater than 99% by weight, of the antibody as determined, for example, by the Lowry method, UV-Vis spectroscopy, or by SDS-capillary gel electrophoresis (e.g., on a Caliper LabChip GXII, GX 90, or Biorad Bioanalyzer instrument); (2) purified to a sufficient extent to obtain at least 15 residues of N-terminal or internal amino acid sequence; or (3) purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, or preferably silver staining. An isolated naturally occurring antibody includes an in situ antibody within a recombinant cell, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0157] Treatment method In a further aspect, the invention relates to a method of treatment.

[0158] The therapeutic method includes administering to a subject in need of treatment a therapeutically effective amount of an antibody or its antigen-binding fragment or variant thereof as contemplated by the present invention. A "therapeutically effective" amount according to the present invention is defined as an amount of an antibody or antigen-binding fragment that is sufficient to reduce the proliferation of CEACAM6-positive cells or reduce the size of a CEACAM6-expressing tumor in the treated area of ​​the subject, either as a single dose or according to a multiple dose regimen, alone or in combination with other agents, to alleviate adverse conditions but in a toxicologically acceptable amount. The subject may be a human or a non-human animal (e.g., rabbit, rat, mouse, dog, monkey or other lower primate).

[0159] It is an embodiment of the present invention to provide the present antibody or antigen-binding fragment thereof for use as a medicament for the treatment of cancer. In a preferred embodiment, the cancer is a tumor, and in a highly preferred embodiment, the cancer is a solid tumor.

[0160] The use of the antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of a disease is one embodiment of the present invention.

[0161] Use of the antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of cancer is an embodiment of the invention. In a preferred embodiment, the cancer is a tumor, and in a highly preferred embodiment, the cancer is a solid tumor.

[0162] The antibody of the present invention can be used as a therapeutic or diagnostic tool in various situations involving abnormal CEACAM6 signaling, such as cell proliferation disorders, such as cancer or fibrotic disorders. Particularly suitable disorders and conditions for treatment with the antibody of the present invention are solid tumors, such as breast, respiratory, brain, reproductive, digestive, urinary, eye, liver, skin, head and neck, thyroid, and parathyroid cancers, and their distant metastases. These disorders also include lymphomas, sarcomas, and leukemias.

[0163] Tumors of the digestive tract include, but are not limited to anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer.

[0164] Examples of esophageal cancer include, but are not limited to, esophageal carcinoma and adenocarcinoma, as well as squamous cell carcinoma, leiomyosarcoma, malignant melanoma, rhabdomyosarcoma, and lymphoma.

[0165] Examples of gastric cancer include, but are not limited to, intestinal-type gastric adenocarcinoma and diffuse-type gastric adenocarcinoma.

[0166] Examples of pancreatic cancer include, but are not limited to, pancreatic ductal adenocarcinoma, adenosquamous carcinoma, and pancreatic endocrine tumors.

[0167] Examples of breast cancer include, but are not limited to, triple-negative breast cancer, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.

[0168] Examples of cancers of the respiratory tract include, but are not limited to, small cell and non-small cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma.

[0169] Examples of brain tumors include, but are not limited to, brain stem and pituitary glioma, cerebellar and cerebral astrocytoma, glioblastoma, medulloblastoma, ependymoma, as well as neuroectodermal tumor, and pineal tumor.

[0170] Tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, vulvar cancer, and uterine sarcoma.

[0171] Examples of ovarian cancer include, but are not limited to, serous tumor, endometrioid tumor, mucinous cystadenocarcinoma, granulosa cell tumor, Sertoli-Leydig cell tumor, and Allehenblastoma.

[0172] Examples of cervical cancer include, but are not limited to, squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, small cell carcinoma, neuroendocrine tumors, hyaline cell carcinoma, and choriocarcinoma.

[0173] Tumors of the urinary tract include, but are not limited to bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureter cancer, urethral cancer, and hereditary and sporadic papillary renal cancer.

[0174] Examples of kidney cancer include, but are not limited to, renal cell carcinoma, urothelial cell carcinoma, juxtaglomerular cell tumor (renoma), angiomyolipoma, renal oncocytoma, Bellini duct carcinoma, clear cell sarcoma of the kidney, mesodermal nephroma, and Wilms' tumor.

[0175] Examples of bladder cancer include, but are not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, and small cell carcinoma.

[0176] Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma.

[0177] Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without fibrolamellar mutation), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular-cholangiocarcinoma.

[0178] Skin cancer includes, but is not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.

[0179] Head and neck cancers include, but are not limited to squamous cell carcinoma of the head and neck, laryngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, salivary gland cancer, lip and oral cavity cancer, and squamous cell carcinoma.

[0180] Lymphomas include, but are not limited to AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin's disease, and lymphoma of the central nervous system.

[0181] Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.

[0182] Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

[0183] In a preferred embodiment, the antibodies or antigen-binding fragments thereof of the invention are suitable for therapeutic or diagnostic methods for treating or diagnosing a cancer disease selected from the group consisting of colorectal cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), pancreatic cancer, gastric cancer, breast cancer and multiple myeloma.

[0184] The above disorders are well characterized in humans, but also have similar etiologies in other animals, including mammals, and can be treated by administering the pharmaceutical compositions of the present invention.

[0185] The antibodies of the present invention may be co-administered with known pharmaceutical agents, and in some cases the antibodies themselves may be modified. For example, the antibodies or antigen-binding fragments or variants thereof may be conjugated to cytotoxic drugs or radioisotopes to further enhance their efficacy.

[0186] The antibody or antigen-binding fragment or variant thereof of the present invention can be administered as a pharmaceutical alone or in combination with one or more additional therapeutic agents, provided that the combination does not cause unacceptable side effects. This combination therapy includes administration of a single pharmaceutical formulation containing the antibody or antigen-binding fragment or variant thereof of the present invention and one or more additional therapeutic agents, as well as administration of the antibody of the present invention and each additional therapeutic agent in its own separate pharmaceutical formulation. For example, the antibody or antigen-binding fragment or variant thereof of the present invention and the therapeutic agent may be administered to a patient together in a single liquid composition, or each agent may be administered in a separate dosage form.

[0187] When separate dosage forms are used, the antibody or antigen-binding fragment or variant thereof of the present invention and the one or more additional therapeutic agents may be administered at essentially the same time (e.g., simultaneously) or may be administered separately at a staggered time (e.g., sequentially).

[0188] In particular, the antibodies or antigen-binding fragments or variants thereof of the present invention can be used in combination, either fixed or separate, with other second agent anti-tumor agents, such as alkylating agents, antimetabolites, plant-derived anti-tumor agents, hormonal therapy agents, topoisomerase inhibitors, immunological preparations, antibodies, antibody drugs, biological response modifiers, anti-angiogenic compounds, cellular therapy agents, and other anti-tumor agents, including, but not limited to, camptothecin derivatives, kinase inhibitors, targeted agents.

[0189] In this regard, the following are non-limiting examples of secondary agents that can be used in combination with the antibodies of the invention: 131l-chTNT, abarelix, abemaciclib, abiraterone, acalabrutinib, aclarubicin, adalimumab, adotrastuzumab emtansine, afatinib, aflibercept, aldesleukin, alectinib, alemtuzumab, alendronate, alitretinoin, alpharadin, altretamine, amifostine, aminoglutethimide, hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anetholethiourethion, anetumab ravtansine, angiotensin II, antithrombin III, apalutamide, aprepitant, arcitumomab, aruglavin, arsenic trioxide, asparaginase, Atezolizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, basiliximab, belotecan, bendamustine, besilesomab, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, blinatumomab, bortezomib, bosutinib, buserelin, brentuximab vedotin, brigatinib, busulfan, cabazitaxel, cabozantinib, calcitonin, calcium folinate, calcium levofolinate, capecitabine, capromab, carbapenem Mazepine carboplatin, carboquone, carfilzomib, carmophor, carmustine, catumaxomab, celecoxib, cermoleukin, cemiplimab, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronate, clofarabine, cobimetinib, copanlisib, crisantaspase, crizotinib, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daratumumab, darbe Poetin alfa, dabrafenib, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dianhydrogalactitol, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, dinutuximab, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin + estrone, dronabinol, durvalumab, eculizumab, edrecolomab, elliptinium acetate, elotuzumab,Eltrombopag, enasidenib, endostatin, enocitabine, enzalutamide, epirubicin, epithiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, ethinyl estradiol, etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, Gadobutrol, gadoteridol, gadoterate meglumine, gadoversetamide, gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, glucarpidase, glutoxime, GM-CSF, goserelin, granisetron, granulocyte colony-stimulating factor, histamine dihydrochloride, hitorelin, hydroxycarbamide, I-125 seed, lansoprazole, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, inotuzumab Ozogamicin, interferon alpha, interferon beta, interferon gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, irinotecan, itraconazole, ixabepilone, ixazomib, lanreotide, lansoprazole, lapatinib, iasocholine, lenalidomide, lenvatinib, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine sodium lisulide, lobaplatin, lomustine, lonidamine, lutetium Lu177 dotatate, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate, methoxsalen, methylaminolevulinate, methylprednisolone, methyltestosterone, metyrosine, midostaurin, mifamurtide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol,Mitomycin, Mitotane, Mitoxantrone, Mogamulizumab, Molgramostim, Mopidamol, Morphine hydrochloride, Morphine sulfate, Mubashi, Nabilone, Nabiximol, Nafarelin, Naloxone + Pentazocine, Naltrexone, Nartograstim, Necitumumab, Nedaplatin, Nelarabine, Neratinib, Neridronic acid, Netupitant / Palonosetron, Nivolumab, Pentetreotide, Nilotinib, Nilutamide, Nimorazole, Nimotuzumab, Nimustine, Nintedanib, Niraparib, Nitracrine, Nivolumab, Obinutuzumab , octreotide, ofatumumab, olaparib, olaratumab, omacetaxine mepesuccinate, omeprazole, ondansetron, opelvequin, orgotein, olilotimod, osimertinib, oxaliplatin, oxycodone, oxymethionine, ozogamicin, p53 gene therapy, paclitaxel, palbociclib, palifermin, palladium-103 seed, palonosetron, pamidronate, panitumumab, panobinostat, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxazole), PEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pembrolizumab, pemetrexed, pentazocine, pentostatin, peplomycin, perflubutan, perfosfamide, pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plerixafor, plicamycin, poliglusum, polyestradiol phosphate, polyvinylpyrrolidone + sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium triphosphate , pralatrexate, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, razoxane, refametinib, regorafenib, ribociclib, risedronate, rhenium-186 etidronate, rituximab, rolapitant, romidepsin, romiplostim, romurtide, rucaparib, samarium (153Sm) lexidronam, sargramostim, sarilumab, satumomab, secretin,Siltuximab, Sipuleucel-T, Sizofiran, Sobuzoxane, Glycidazole Sodium, Sonidegib, Sorafenib, Stanozolol, Streptozocin, Sunitinib, Talaporfin, Talimogene Laherparepvec, Tamibarotene, Tamoxifen, Tapentadol, Tasonermin, Teselekin, Technetium (99mTc) Nofetumomab merpentane, 99mTc-HYNIC-[Tyr3]-octreotide, tegafur, tegafur + gimeracil + oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyrotropin alfa, thioguanine, tisagenlecleucel, tislelizumab, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab, trastuzumab Emtansine, treosulfan, tretinoin, trifluridine + tipiracil, trilostane, triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, baratinib, valrubicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin.,

[0190] Additionally, the antibodies of the present invention can be combined with therapeutic modalities that induce immunogenic cell death, including, but not limited to, ultraviolet light, oxidative treatment, heat shock, targeted and non-targeted radiation therapy, shikonin, high hydrostatic pressure, oncolytic viruses, and photodynamic therapy.

[0191] Additionally, the antibodies of the invention may be combined with agents that cause immunogenic cell death, including, but not limited to, sunitinib, JAK2 inhibitors, anthracyclines, doxorubicin, mitoxantrone, oxaliplatin and cyclophosphamide, targeted and non-targeted microtubule destabilizing agents such as auristatins and maytansinoids.

[0192] The compounds of the invention can also be used in the treatment of cancer in combination with radiation therapy and / or surgical intervention.

[0193] Additionally, the antibodies of the present invention may be utilized by themselves or in compositions, in research and diagnostics, or as analytical reference standards, and the like, as are well known in the art.

[0194] In a further aspect, the present invention provides an anti-CECAM6 antibody of the first aspect for use as a medicament, in particular for use as a medicament for the treatment of cancer. In certain embodiments of this aspect, a method for treating a cancer associated with the undesired presence of CEACAM6 is provided, comprising administering to a subject in need thereof an effective amount of an anti-CECAM6 antibody of the first aspect.

[0195] In a further aspect, the invention provides an anti-CEACAM6 antibody of the first aspect for use in simultaneous, separate or sequential combination with an anti-PD-1 antibody or an anti-PD-L1 antibody in the treatment of cancer.

[0196] In certain embodiments, the anti-PD-1 antibody is nivolumab or pembrolizumab, and the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab. In certain embodiments of this aspect, there is provided a method of treating cancer comprising administering to a patient in need thereof an effective amount of an anti-CEACAM6 antibody of the first aspect in simultaneous, separate, or sequential combination with an anti-PD-1 antibody or an anti-PD-L1 antibody, preferably wherein the anti-PD-1 antibody is nivolumab or pembrolizumab, and the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

[0197] In certain embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is nivolumab or has the same CDR regions as nivolumab. Nivolumab (trade name "OPDIVO", formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively inhibits the interaction with PD-1 ligands (PD-L1 and PD-L2), thereby inhibiting downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449). In another embodiment, the anti-PD-1 antibody or fragment thereof cross-competes with nivolumab.

[0198] In other embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is pembrolizumab or has the same CDR regions as pembrolizumab. Pembrolizumab (also known as KEYTRUDA, lambrolizumab, and MK-3475) is a humanized monoclonal IgG4 antibody against the human cell surface receptor PD-1. Pembrolizumab is described, for example, in U.S. Patent No. 8,900,587.

[0199] In other embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is MEDI0608 (formerly known as AMP-514) or has the same CDR regions as MEDI0608. MEDI0608 is a monoclonal antibody against the PD-1 receptor. MEDI0608 is described, for example, in U.S. Patent No. 8,609,089, B2.

[0200] In other embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is BGB-A317, or has the same CDR regions as BGB-A317, a humanized monoclonal antibody described in U.S. Patent Application Publication No. 2015 / 0079109.

[0201] In certain embodiments, the anti-PD-L1 antibody, or antigen-binding portion thereof, is atezolizumab or has the same CDR regions as atezolizumab. Atezolizumab (trade name "TECENTRIQ"), also known as MPDL3280A and RG7446, is described in U.S. Patent No. 8,217,149.

[0202] In another embodiment, the anti-PD-L1 antibody, or antigen-binding portion thereof, is avelumab, or has the same CDR regions as avelumab. Avelumab (trade name "BAVENCIO"), also known as MSB0010718C, is described in U.S. Patent Application Publication No. 2014 / 0341917.

[0203] In other embodiments, the anti-PD-L1 antibody, or antigen-binding portion thereof, is durvalumab or has the same CDR regions as durvalumab. Durvalumab (trade name "IMFINZI", also known as MEDI4736) is described in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2014 / 0356353.

[0204] In another embodiment, the anti-PD-L1 antibody, or antigen-binding portion thereof, is BMS-936559, or has the same CDR regions as BMS-936559. BMS-936559 (formerly known as 12A4 or MDX-1105) is a fully human IgG4 monoclonal antibody that targets the PD-1 ligand PD-L1, and is described in U.S. Patent No. 7,943,743 or WO 2013 / 173223.

[0205] In a further aspect, the invention provides an anti-CEACAM6 antibody of the first aspect for use in combination, simultaneously, separately or sequentially, with an anti-TIM-3 antibody in the treatment of cancer. In certain embodiments, the anti-TIM-3 antibody is covolimab, MBG-453, BMS-986258, Sym-023, LY-3321367 or INCAGN-2390. In certain embodiments of this aspect, a method of treating cancer is provided, comprising administering to a patient in need thereof an effective amount of an anti-CEACAM6 antibody of the first aspect in combination, simultaneously, separately or sequentially, with an anti-TIM-3 antibody, preferably wherein the anti-TIM-3 antibody is covolimab, MBG-453, BMS-986258, Sym-023, LY-3321367 or INCAGN-2390.

[0206] In certain embodiments, the anti-TIM-3 antibody or antigen-binding portion thereof is covolimab (TSR-022, Tesaro) or has the same CDR regions as covolimab. Covolimab is a TIM-3 immune checkpoint inhibitor antibody that selectively inhibits the interaction with some known TIM-3 ligands (HMGB1, galectin-9, phosphatidylserine (PS)), thereby preventing downregulation of anti-tumor T cell function. Covolimab is described, for example, in WO 2016161270 A1 and WO 2018129553 A1. Covolimab is currently in clinical trials; ClinicalTrials.gov identifiers: NCT02817633 and NCT03680508.

[0207] In other embodiments, the anti-TIM-3 antibody or antigen-binding portion thereof is MBG-453 (Novartis) or has the same CDR regions as MBG-453. MBG-453 is a TIM-3 immune checkpoint inhibitor antibody that selectively inhibits the interaction with some known TIM-3 ligands (HMGB1, Galectin-9, Phosphatidylserine (PS)), thereby preventing downregulation of anti-tumor T cell function. MBG-453 is described, for example, in WO 2015117002 A1. MBG-453 is registered under CAS No: 2128742-61-8. MBG-453 is currently in clinical trials; ClinicalTrials.gov Identifiers: NCT02608268 and NCT03066648.

[0208] In other embodiments, the anti-TIM-3 antibody or antigen-binding portion thereof is BMS-986258 (Bristol-Myers Squibb, Five Prime) or has the same CDR regions as BMS-986258. BMS-986258 is a TIM-3 immune checkpoint inhibitor antibody that selectively blocks interaction with some of the known TIM-3 ligands (HMGB1, Galectin-9, Phosphatidylserine (PS)), thereby preventing downregulation of anti-tumor T cell function. BMS-986258 is currently in clinical trials; ClinicalTrials.gov Identifier: NCT03446040. BMS-986258 is described, for example, in WO 2018013818 A2.

[0209] In other embodiments, the anti-TIM-3 antibody or antigen-binding portion thereof is Sym-023 (Symphogen) or has the same CDR regions as Sym-023. Sym-023 is a TIM-3 immune checkpoint inhibitor antibody that selectively blocks interaction with some known TIM-3 ligands (HMGB1, galectin-9, phosphatidylserine (PS) and thereby prevents downregulation of anti-tumor T cell function. Sym-023 is currently in clinical trials; Clinical Trials.gov Identifier: NCT03489343. Sym-023 is described, for example, in WO 2017178493 A1.

[0210] In other embodiments, the anti-TIM-3 antibody or antigen-binding portion thereof is LY-3321367 (Eli Lilly) or has the same CDR regions as LY-3321367. LY-3321367 is a TIM-3 immune checkpoint inhibitor antibody that selectively blocks interaction with some known TIM-3 ligands (HMGB1, galectin-9, phosphatidylserine (PS) and thereby prevents downregulation of anti-tumor T cell function. LY-3321367 is currently in clinical trials; ClinicalTrials.gov Identifier: NCT03099109. LY-3321367 is described, for example, in WO 2018039020 A1.

[0211] In other embodiments, the anti-TIM-3 antibody or antigen-binding portion thereof is INCAGN-2390 (Agenus) or has the same CDR regions as INCAGN-2390. INCAGN-2390 is a TIM-3 immune checkpoint inhibitor antibody that selectively blocks interaction with some known TIM-3 ligands (HMGB1, galectin-9, phosphatidylserine (PS) and thereby prevents downregulation of anti-tumor T cell function. INCAGN-2390 is currently in clinical trials; ClinicalTrials.gov Identifier: NCT03652077. INCAGN-2390 is described, for example, in WO 2017205721 A1.

[0212] In other embodiments, the anti-TIM-3 antibody, or antigen-binding portion thereof, is MAB2365 from R&D Jackson Immunoresearch, or has the same CDR regions as MAB2365. MAB2365 is an rIgG2 antibody.

[0213] Diagnostic methods In a further aspect, the present invention relates to a diagnostic method. Anti-CEACAM6 antibodies or antigen-binding fragments thereof can be used to detect the presence of CEACAM6-expressing tumors. The presence of CEACAM6-containing cells or excreted CEACAM6 in various biological samples, including serum and tissue biopsy specimens, can be detected using anti-CEACAM6 antibodies. Furthermore, anti-CEACAM6 antibodies can be used to detect: 99 It can be used in a variety of imaging procedures using Tc (or other isotope) conjugated antibodies, such as immunoscintigraphy. 111 Imaging protocols similar to those described using In-conjugated anti-PSMA antibodies can be used to detect pancreatic or ovarian cancer (Sodee et al., Clin. Nuc. Med. 21: 759-766, 1997). Another detection method that can be used is positron emission tomography by conjugating the antibody of the present invention with a suitable isotope (see Herzog et al., J. Nucl. Med. 34: 2222-2226, 1993).

[0214] Pharmaceutical Compositions and Administration In a further aspect, the invention relates to pharmaceutical compositions comprising the anti-CEACAM6 antibody of the first aspect, and administration of the anti-CEACAM6 antibody of the first aspect. Pharmaceutical compositions for use according to the invention to treat any of the aforementioned disorders can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. The antibody or antigen-binding fragment thereof of the invention can be administered by any suitable means, which may vary depending on the type of disorder to be treated. Possible routes of administration include parenteral administration (e.g., intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous), intrapulmonary and intranasal administration, including intralesional administration if localized immunosuppressive treatment is desired. Furthermore, the antibody or antigen-binding fragment thereof or variant thereof of the invention can be administered by pulse infusion, e.g., with decreasing doses of the antibody. Preferably, administration is by injection, most preferably by intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Dosage varies depending on various factors, e.g., clinical symptoms, weight of the individual, and the presence or absence of other drugs. One of skill in the art will recognize that the route of administration will vary depending on the disorder or condition being treated.

[0215] One embodiment of the present invention is a pharmaceutical composition comprising the anti-CEACAM6 antibody or its antigen-binding fragment or variant of the first aspect alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, glucose and water. A further embodiment is a pharmaceutical composition comprising a CEACAM6-binding antibody or its antigen-binding fragment and an additional pharma- ceutical active compound suitable for treating a CEACAM6-related disease, such as cancer. Any of these molecules may be administered to a patient alone or in combination with other agents, drugs or hormones in a pharmaceutical composition mixed with excipient(s) or a pharma- ceutical acceptable carrier. In one embodiment of the present invention, the pharma- ceutical acceptable carrier is pharma- ceutical inactive.

[0216] The present invention also relates to the administration of pharmaceutical compositions. Such administration is often performed parenterally. Methods of parenteral delivery include local administration, intra-arterial administration (directly into tumor), intramuscular administration, subcutaneous administration, intramedullary administration, intrathecal administration, intraventricular administration, intravenous administration, intraperitoneal administration, or intranasal administration. In addition to the active ingredient, these pharmaceutical compositions may contain a suitable pharma-ceutical acceptable carrier, including excipients and adjuvants that facilitate the processing of the active compound into a pharmaceutical preparation that can be used. Further details of the techniques for formulation and administration can be found in the latest edition of Remington's Pharmaceutical Sciences (edited by Maack Publishing Co, Easton, Pa.).

[0217] Pharmaceutical preparations for parenteral administration include aqueous solutions of the active compounds. For injection, the pharmaceutical compositions of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspensions can also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0218] Pharmaceutical compositions can be provided as salts, which can be formed with acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or other protic solvents, as is the corresponding free base form. In other cases, a preferred formulation can be a lyophilized powder in 1 mM to 50 mM histidine or phosphate or Tris, 0.1% to 2% sucrose, and / or 2% to 7% mannitol, at a pH range of 4.5 to 7.5, optionally with additional agents such as polysorbates that are combined with a buffer prior to use.

[0219] After pharmaceutical compositions containing a compound of the invention formulated in an acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of an anti-CEACAM6 antibody or antigen-binding fragment thereof, such labeling would include the amount, frequency, and method of administration.

[0220] kit The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Such container(s) may bear a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products reflecting approval by the governmental agency of the manufacture, use, or sale of the product for administration to humans.

[0221] A further preferred embodiment of the present invention is as follows.

[0222] 1. An anti-CECAM6 antibody comprising an IgG1 Fc region lacking a glycan attached to a conserved N-linked site within the CH2 domain of the Fc region, said IgG1 Fc region comprising at least the amino acid substitutions L234A and L235A, numbered according to the EU index of Kabat.

[0223] 2. An anti-CECAM6 antibody described in embodiment 1, wherein the IgG1 Fc region comprises the amino acid substitutions N297A, N297G or N297Q, numbered according to the EU index of Kabat.

[0224] 3. An anti-CECAM6 antibody comprising an IgG1 Fc region, said IgG1 Fc region comprising at least the amino acid substitutions N297A, L234A and L235A numbered according to the EU index of Kabat.

[0225] 4. An anti-CECAM6 antibody described in any one of embodiments 1 to 3, which competes for CEACAM6 binding with an antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 67.

[0226] 5. a. a heavy chain variable region H-CDR1 comprising the amino acid sequence of SEQ ID NO: 64; b. A heavy chain variable region H-CDR2 comprising the amino acid sequence of SEQ ID NO: 65; c. A heavy chain variable region H-CDR3 comprising the amino acid sequence of SEQ ID NO: 66; d. a light chain variable region L-CDR1 comprising the amino acid sequence of SEQ ID NO: 68; e. a light chain variable region L-CDR2 comprising the amino acid sequence of SEQ ID NO:69; and f. Light chain variable region L-CDR3 comprising the amino acid sequence of SEQ ID NO: 70 An anti-CECAM6 antibody according to any one of embodiments 1 to 4, comprising:

[0227] 6. a. the heavy chain variable region H-CDR1 amino acid sequence of SEQ ID NO: 64; b. The heavy chain variable region H-CDR2 amino acid sequence of SEQ ID NO: 65; c. The heavy chain variable region H-CDR3 amino acid sequence of SEQ ID NO: 66; d. The light chain variable region L-CDR1 amino acid sequence of SEQ ID NO:68; e. the light chain variable region L-CDR2 amino acid sequence of SEQ ID NO:69, and f. The light chain variable region L-CDR3 amino acid sequence of SEQ ID NO: 70 An anti-CECAM6 antibody according to any one of embodiments 1 to 4, comprising:

[0228] 7. a. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 63; and b. A light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:67 7. An anti-CECAM6 antibody according to any one of embodiments 1 to 6, comprising:

[0229] 8. a. a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 71; and b. A light chain (LC) comprising the amino acid sequence of SEQ ID NO: 72 An anti-CECAM6 antibody according to any one of embodiments 1 to 7, comprising:

[0230] 9. a. a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 71; and b. A light chain (LC) comprising the amino acid sequence of SEQ ID NO:72 An anti-CECAM6 antibody comprising:

[0231] 10. a. a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 71; and b. A light chain (LC) comprising the amino acid sequence of SEQ ID NO: 72 An anti-CECAM6 antibody consisting of

[0232] 11. An anti-CECAM6 antibody described in any one of embodiments 1 to 10, which is isolated.

[0233] 12. An anti-CECAM6 antibody described in any one of embodiments 1 to 11, which is a monoclonal antibody.

[0234] 13. An anti-CECAM6 antibody described in any one of embodiments 1 to 12, which is a human antibody or a humanized antibody.

[0235] 14. An anti-CECAM6 antibody described in any one of embodiments 1 to 13, which specifically binds to CECAM6 having the amino acid sequence of SEQ ID NO:75.

[0236] 15. An anti-CECAM6 antibody described in any one of embodiments 1 to 14, which specifically binds to CECAM6 domain 1 comprising amino acids 35 to 142 of SEQ ID NO:75.

[0237] 16. A nucleic acid encoding an anti-CECAM6 antibody according to any one of embodiments 1 to 15.

[0238] 17. A vector comprising the nucleic acid described in embodiment 16.

[0239] 18. An isolated cell expressing an anti-CECAM6 antibody described in any one of embodiments 1 to 15 and / or comprising a nucleic acid described in embodiment 16 or a vector described in embodiment 17.

[0240] 19. The isolated cell of embodiment 18, which is a prokaryotic or eukaryotic cell.

[0241] 20. A method for producing an anti-CECAM6 antibody described in any one of embodiments 1 to 15, comprising culturing the cell described in embodiment 18 and purifying the antibody.

[0242] 21. An anti-CECAM6 antibody described in any one of embodiments 1 to 15 for use as a pharmaceutical.

[0243] 22. An anti-CECAM6 antibody described in any one of embodiments 1 to 15, for use as a pharmaceutical for the treatment of cancer.

[0244] 23. Use of an anti-CECAM6 antibody described in any one of embodiments 1 to 15 in the manufacture of a medicament for the treatment of a disease.

[0245] 24. Use of an anti-CECAM6 antibody described in any one of embodiments 1 to 15 in the manufacture of a medicament for the treatment of cancer.

[0246] 25. A method for treating cancer associated with undesirable presence of CECAM6 and / or high presence of membrane-localized CEACAM6, comprising administering to a subject in need thereof an effective amount of an anti-CECAM6 antibody described in any one of embodiments 1 to 15.

[0247] 26. A pharmaceutical composition comprising an anti-CECAM6 antibody described in any one of embodiments 1 to 15.

[0248] 27. An anti-CEACAM6 antibody described in any one of embodiments 1 to 15 for use in simultaneous, separate or sequential combination with an anti-PD-1 antibody or an anti-PD-L1 antibody in the treatment of cancer.

[0249] 28. The anti-CEACAM6 antibody for use according to embodiment 27, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab, and the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

[0250] 29. A method for treating cancer, comprising administering to a patient in need thereof an effective amount of an anti-CEACAM6 antibody described in any one of embodiments 1 to 15 in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody, either simultaneously, separately or sequentially.

[0251] 30. The method of treating cancer of embodiment 29, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab, and the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

[0252] 31. An anti-CEACAM6 antibody described in any one of embodiments 1 to 15 for use in simultaneous, separate or sequential combination with an anti-TIM-3 antibody in the treatment of cancer.

[0253] 32. An anti-CEACAM6 antibody for use according to embodiment 31, wherein the anti-TIM-3 antibody is covolimab, MBG-453, BMS-986258, Sym-023, LY-3321367 or INCAGN-2390.

[0254] 33. A method for treating cancer, comprising administering to a patient in need thereof an effective amount of an anti-CEACAM6 antibody described in any one of embodiments 1 to 15 in combination with an anti-TIM-3 antibody, either simultaneously, individually or sequentially.

[0255] 34. The method of treating cancer of embodiment 32, wherein the anti-TIM-3 antibody is covolimab, MBG-453, BMS-986258, Sym-023, LY-3321367 or INCAGN-2390.

[0256] [Example] The present invention is further illustrated by the following examples, which are provided solely to illustrate the invention by reference to specific embodiments, which illustrate certain aspects of the invention, but do not represent limitations or restrictions on the scope of the disclosed invention.

[0257] All examples were carried out using standard techniques well known and routine to those skilled in the art, unless otherwise specified in detail. Routine molecular biology techniques in the following examples can be carried out as described in standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0258] [Example 1] Generation of antibodies and antibody sequences An overview of the protein sequences of the antibodies and reference compounds used is given in Table 1.

[0259] [Table 1] TIFF2024534186000014.tif239145

[0260] 9A6 mouse IgG1 antibody (GM-0509) was obtained from Genovac and chimerized to human IgG2 or human IgG1. Neo201 protein sequences for either human IgG1 or human IgG2 were based on US Patent Publication No. 2013 / 0189268. TPP-3310 CEACAM6-human IgG2 protein sequence was based on WO 2016 / 150899 A2. All antibodies were expressed in HEK293 cells using standard transient transfection procedures and purified from cell culture supernatants by Protein-A and size exclusion chromatography.

[0261] The non-glycosylated variant (IgG1aglyco) was generated by mutation of asparagine 297 (numbering according to Eu nomenclature; Edelman et al., Proc Natl Acad Sci USA. May 1969; 63(1): 78-85; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed. US Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication 91-3242) to alanine. The LALA mutation refers to the L234A / L235A mutation, and LALAaglyco is a triple mutation of L234A / L235A / N297A.

[0262] Fab and F(ab) 2Proteins were generated by enzymatic cleavage of parent IgG by papain and fabricator cleavage, respectively. Briefly, immobilized papain (Thermo Fisher Scientific No. 20341) was used for Fab generation according to the manufacturer's recommendations. After cleavage, Fabs were purified using size exclusion chromatography with MabSelectSuRe (GE-Healthcare) and Superdex 200 16 / 60. Similarly, FabRICATOR (IdeS) (FragITkit Genovis No. A2-FR2-1000) was used to cleave F(ab) 2 After cleavage, the Fc protein was removed using Capture Select Fc resin (Thermo Scientific), and the F(ab) was purified by size exclusion chromatography using Superdex 200 16 / 60. 2 The protein was further purified.

[0263] [Example 2] Clinical study of TPP-3310 in which neutropenia was observed as a side effect EDTA-anticoagulated peripheral venous blood was collected pretreatment and at various time points after initiation of infusion from patients in four dose cohorts receiving 2.5, 5, 10, or 30 mg of the anti-CEACAM6 antibody TPP-3310. Plasma levels of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-alpha) were determined by Mesoscale ELISA. Myeloperoxidase (MPO) was determined by conventional ELISA.

[0264] As shown in Figures 1, 2, and 3, a transient systemic mixed inflammatory (TNF-alpha, IL-6) / anti-inflammatory (IL-10) response began after 1-2 hours and resolved by 24 hours in all dose cohorts. There was strong interpatient variability in the inflammatory response. No dose-dependence was observed.

[0265] FIG. 4 shows plasma levels of myeloperoxidase at various time points after the start of intravenous infusion of anti-CEACAM6 antibody TPP-3310 in cancer patients.

[0266] The occurrence of neutropenia as a side effect in cancer patients treated with low doses of TPP-3310 (Figure 5) was surprising and unexpected.

[0267] As shown in Figures 1, 2 and 3, an early and transient increase in TNF-alpha, IL-6 and IL-10 was observed, suggesting an inflammatory event. Surprisingly, myeloperoxidase release from neutrophils could be observed with a short delay after the initial inflammatory event (Figure 4). This prompted us to investigate whether pre-stimulation (e.g., by inflammatory cytokines) was necessary to detect a deleterious effect of TPP-3310 on neutrophil activation and eventual depletion that could explain the neutropenic findings in clinical trials (Figure 5).

[0268] [Example 3] Assessment of neutrophil activation by myeloperoxidase release assay in whole blood CEACAM6 is known to be expressed on human neutrophils. Therefore, the effect of TPP-3310 on human peripheral whole blood was also analyzed before conducting clinical trials. In these assays, the amount of myeloperoxidase (MPO) released from neutrophils into the supernatant as an activation marker is determined. The test antibody (TPP-3310) was compared to an isotype-matched non-binding control (TPP-1238). No effects have been observed so far in different donors prior to this clinical trial.

[0269] In order to reproduce the unexpected findings in the clinical trials (especially neutropenia and MPO release), various stimuli in the whole blood assay were tested for their ability to mimic these side effects in vitro. The use of the neutrophil activator fMLP (N-formylmethionine-leucyl-phenylalanine) proved to be particularly useful, as it allowed us to show the deleterious activation effect of the anti-CEACAM6 antibody TPP-3310 in this whole blood assay, an effect that would otherwise go undetected under standard assay conditions. This unusual effect was highly reproducible and consistent across a variety of different blood donors.

[0270] Experimental details Anticoagulated human peripheral whole blood was incubated with titrated concentrations of several different anti-CEACAM6 antibody formats and corresponding isotype control antibodies, with or without prior fMLP (N-formylmethionine-leucyl-phenylalanine) treatment.

[0271] After incubation, the neutrophil activation ability of the antibodies was assessed by determining the amount of myeloperoxidase released in the supernatant.

[0272] Briefly, whole blood was incubated in microtiter plate wells with or without a suboptimal concentration of fMLP (0.01 μM; Sigma Aldrich #F3506) for 15 minutes at room temperature. This suboptimal concentration of fMLP did not yet result in measurable release of MPO by neutrophils. Antibodies were then added at titrated concentrations, followed by incubation at 37° C. for 2 hours. After incubation, cells were pelleted by centrifugation and the supernatant was transferred for a second centrifugation. The supernatant was then stored at −20° C. until analysis. Analysis was performed using a Myeloperoxidase Human Instant ELISA Kit (eBiosciences #BMS2038INST).

[0273] As is evident from Figure 6, the effect of anti-CEACAM6 antibody TPP-3310 (human IgG2 format) on neutrophil activation is not detectable in whole blood under standard assay conditions (without fMLP). However, when pre-stimulation (addition of subactivating fMLP concentrations) is used, a significant and dose-dependent neutrophil activation is observed, which can be reproduced in a variety of human donors. Thus, by using these assay conditions, unexpected findings from clinical studies can be brought to the in vitro assay.

[0274] A control was used in these experiments, an anti-CEACAM6 antibody (TPP-5468) with the same variable sequence but reformatted into a human IgG1 format. Surprisingly, this anti-CEACAM6 human IgG1 format did not result in any neutrophil activation, regardless of the presence or absence of fMLP (Figure 7). This is a major surprise, since even more potent activation was expected, since human IgG1 antibodies are known to mediate effector functions most potently through Fcγ receptor and complement binding.

[0275] To further clarify the influence of isotype and epitope, we tested other unrelated anti-CEACAM6 antibodies with similar and different epitopes. Anti-CEACAM6 antibody 9A6 (TPP-3470 human IgG2) recognizes an epitope that overlaps with TPP-3310 and competes for binding to the membrane-distal N-terminal D1 domain of CEACAM6 (see WO 2016 / 150899 A2). In contrast, Neo201 (TPP-1173 human IgG1 or TPP-3688 human IgG2) recognizes a different membrane-proximal epitope on the D3 domain (also known as the B domain; see WO 2016 / 150899 A2) of CEACAM6.

[0276] As is evident from Figures 8, 9 and 10, the CEACAM6 antibody 9A6, which recognizes a very similar epitope as TPP-3310, was able to exert the same neutrophil activation effect. In contrast, the anti-CEACAM6 antibody Neo201, which recognizes a different epitope, was unable to activate either in human IgG1 or human IgG2 format.

[0277] These results suggested both strong epitope and isotype dependence. As mentioned above, the difference in the effect of anti-CEACAM6 TPP-3310 (human IgG2) and its human IgG1 counterpart (TPP-5468) was unexpected. Therefore, we first sought to analyze whether the Fc portion of the antibody was involved and then whether the Fcγ receptor was also involved. For this purpose, a monomeric Fab fragment (APP-1574) was synthesized by co-transfection with F(ab) fragments prepared from IgG1 (APP-6036) or IgG2 (APP-6849). 2 In another set of experiments, the Fcγ receptor blocking antibody AT10 was used to examine its effect on neutrophil activation by TPP-3310.

[0278] As is apparent from Figures 11, 12 and 13, the anti-CEACAM6 Fab fragment APP-1574 also exhibits F(ab) 2 Fragments APP-6036 or APP-6849 were also unable to mediate MPO release, suggesting the involvement of the Fc portion of the human IgG2 antibody TPP-3310 in the release of MPO and thus in the activation of neutrophils.

[0279] Next, anti-CD32 F(ab') was added prior to the addition of the anti-CEACAM6 antibody TPP-3310. 2 FcγR blocking experiments were performed by introducing antibody AT10 (obtained from Biozol) at a concentration of 1.4 μM. Again, an isotype-matched F(ab) 2 The fragment was used as a control.

[0280] As is clear from Figures 14 and 15, MPO release induced by the anti-CEACAM6 antibody TPP-3310 can be inhibited by a CD32 blocking antibody, suggesting that the MPO release effect is dependent on FcγRII binding.

[0281] In summary, these MPO release experiments demonstrated that anti-CEACAM6 antibodies in human IgG2 isotype format (TPP-3310 and TPP-3470) that recognize a membrane-distal epitope can activate neutrophils, but release MPO only in samples pre-stimulated with fMLP. Samples without fMLP pre-stimulation did not result in MPO release when incubated with TPP-3310 or TPP-3470. Most notably, the human IgG2 format was strictly required, since the same antibody in human IgG1 (TPP-5468) exerted no effect, despite recognizing the same or similar membrane-distal epitope of CEACAM6 as TPP-3310 or TPP-3470.

[0282] This MPO release from the pre-stimulated samples was not obtained with anti-CEACAM6 antibodies (Neo201 TPP-1173 human IgG1, TPP-3688 human IgG2) that recognize more membrane-proximal epitopes of the CEACAM6 molecule regardless of isotype.

[0283] This MPO release from the pre-stimulated sample was determined by Fab or (Fab) 2 This was not achieved with anti-CEACAM6 formats lacking the Fc portion of the antibody, such as APP-1574, APP-6036, and APP-6849. Furthermore, this was not achieved under CD32 blocking conditions (blocked anti-CD32 F(ab') before the addition of the anti-CEACAM6 antibody TPP3310). 2 MPO release experiments performed with IgG10 (by introducing antibody AT10) further demonstrated that the MPO release effect was dependent on FcγRII binding.

[0284] In conclusion, these findings suggest a very finely interwoven dependence of prestimulation, epitope, and isotype requirements for neutrophil activation in whole blood. This is completely unexpected, since the strict dependency on the Fc portion, and even the involvement of FcγRII, would rather suggest that human IgG1 is the more potent molecule, but in fact it is quite inactive in this assay. In contrast, the supposedly more silent human IgG2 isotype is in fact the only molecule capable of exerting a neutrophil activating effect.

[0285] [Example 4] Determination of the affinity of various engineered anti-CEACAM6 antibodies to human Fcγ receptors using surface plasmon resonance To analyze the contribution of Fc-Fcγ receptor interactions, the affinity of the interaction (or its absence) was determined for various antibody formats, each of which has the same variable domains as TPP-3310.

[0286] To evaluate the affinity of the various engineered anti-CEACAM6 antibodies, binding assays to human Fcγ receptors were performed using surface plasmon resonance (SPR).

[0287] Binding assays were performed at 25° C. in a Biacore T200 instrument (Cytiva) using assay buffer HBS EP+ supplemented with 500 mM NaCl. Fcγ receptors were captured via covalently amine-coupled anti-penta-his tag IgG ("His capture kit", order no. 2895056, Cytiva) to an S-series CM5 sensor chip (Cytiva). Amine coupling was performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and ethanolamine HCl, pH 8.5 ("amine coupling kit" BR-1000-50, Cytiva) according to the manufacturer's instructions. Human Fcγ receptor I (R&D Systems, order number 1257-FC), Fcγ receptor IIa (R&D Systems, order number 1330-CD / F), Fcγ receptor IIb / c (R&D Systems, order number 1875-CD), Fcγ receptor IIIa (R&D Systems, order number 4325-FC) and Fcγ receptor IIIb (R&D Systems, order number 1597-FC) were captured up to approximately 30 RU.

[0288] Anti-CEACAM6 engineered antibodies were used as analytes in a concentration series from 0.04 to 25 μM in multi-cycle kinetics mode. The sensor surface was regenerated with glycine pH 1.5 after each analyte injection. The resulting sensorgrams were double-referenced (subtraction of the signal of the reference flow cell and the buffer injection) and fitted to a 1:1 Langmuir binding model to derive steady-state affinity data using Biacore T200 evaluation software.

[0289] A comparison of the anti-CEACAM6 antibody TPP-3310 (human IgG2) with its counterpart as a human IgG1 antibody is shown in Table 2. As expected, the interaction between human IgG1 and various FcγRs is much stronger than that of the supposedly more silent isotype IgG2. Therefore, the results of the whole blood MPO release assay in Example 3 are even more puzzling.

[0290] [Table 2]

[0291] Even if a human IgG1 format, such as TPP-5468, is safe with respect to activating neutrophils in whole blood as exemplified in Example 3, the strong interaction of the IgG1 format with FcγR precludes its use in therapeutic antibodies due to the potential for strong and undesirable effector activity such as ADCC, ADCP, and CDC.

[0292] Therefore, an IgG1-based format lacking FcγR interactions and therefore effector functions was required. To this end, various Fc-engineered variants were tested: an aglycosylated antibody (TPP-10914), an antibody with a "LALA" mutation (TPP-19919), as well as an antibody with a combination of LALA and aglycosylation mutations (TPP-21518). The results are shown in Table 3.

[0293] [Table 3]

[0294] Surprisingly, as can be seen from Table 3, both TPP-10914 ("aglyco") and TPP-19919 ("LALA") still showed binding to Fcγ receptor I, and in the case of TPP-19919, also to Fcγ receptor IIIa. Only the combination of "LALA" and the "aglyco N297A" mutation (TPP-21518) results in a completely silent isotype that does not show any binding to Fcγ receptors in the SPR assay.

[0295] [Example 5] Assessment of neutrophil activation by myeloperoxidase release assay in whole blood Next, Fc-engineered silent isotype anti-CEACAM6 antibody TPP-21518 was tested in the MPO assay to confirm that this modified format also did not show any undesired neutrophil activation. The experimental setup was the same as in Example 3.

[0296] As can be inferred from FIG. 16, TPP-21518 was unable to cause activation in the MPO assay with or without fFMLP prestimulation.

[0297] [Example 6] Assessment of ADCP potency of different antibody variants Next, the Fc-engineered silent isotype anti-CEACAM6 antibody TPP-21518 was tested in the ADCP assay to confirm that this modified antibody also did not exhibit any undesirable ADCP activity and therefore was safe to be used in clinical treatment.

[0298] Flow cytometry-based readout is used to track antibody-dependent cellular phagocytosis (ADCP) of CFSE-labeled neutrophils by primary macrophages. Neutrophils are isolated from freshly drawn whole blood of healthy donors using the StemCell EasySep™ Human Neutrophil Isolation Kit (#17957) and immediately used for ADCP. Primary macrophage effector cells are generated from peripheral blood mononuclear cells (PBMCs) of healthy donors. Briefly, CD14+ monocyte populations are purified from PBMCs using Miltenyi's Pan Monocyte Isolation Kit (#130-096-537) and differentiated in culture for 7-9 days using specific combinations of cytokines and LPS to generate M1, M2a or M2c macrophages.

[0299] Neutrophils are pretreated with 10 nM fMLP for 30 min prior to the experiment. ADCP of approximately 20,000 CFSE-labeled neutrophils is achieved when co-cultured with macrophages (approximately 80,000) in a ratio of approximately 1:4 in the presence of anti-CEACAM6 antibody for 2 h at 37°C. The assay is performed in the presence of 10% normal human serum. The percentage of ADCP is determined from flow cytometry counts of live CFSE-positive macrophages (PI negative, CD206+, CFSE+) relative to total live macrophages (PI negative, CD206+).

[0300] Figure 17 shows representative data (N=4 experiments using unrelated neutrophil and macrophage donors) of the silenced ADCP activity of the human IgG1-LALAglyco version of anti-CEACAM6 (TPP-21518) versus unmodified IgG1 (TPP-5468) and IgG2 anti-CEACAM6 (TPP-3310). The lack of phagocytosis with a non-binding isotype control antibody suggests anti-CEACAM6-dependent phagocytosis. An anti-huCD47 positive control mouse antibody (clone B6H12), which blocks the "do not eat me" signal, confirms the phagocytic activity of the macrophage preps and their susceptibility to neutrophil phagocytosis.

[0301] [Example 7] Evaluation of different antibody variants in T cell potency assays Next, the Fc-engineered silent isotype anti-CEACAM6 antibody TPP-21518 was tested in a T cell potency assay to confirm that this modified format was also capable of mediating the desired pharmacological effects and was therefore suitable for use in clinical therapy.

[0302] In vitro pharmacological efficacy of anti-CEACAM6 antibody on IL-2 secretion by survivin peptide-specific T cells Tumor antigen-specific T cells were generated by the procedure described by Brackertz et al. (Brackertz et al., Blood Cancer J. 2011 March; 1(3):e1). Briefly, survivin-specific CD8+ T cells were isolated from peripheral mononuclear cells by CD8-specific magnetic activated cell sorting. Isolated HLA-A2 CD8+ T cells were stimulated with HLA-A2 dendritic cells loaded with 10g of survivin epitope (ELTLGEFLKL). After stimulation, proliferating T cells were transfected with HLA-A2 / survivin multimers (APC-tagged APC). * 02:0 1 39 1 LMLGEFLKL Survivin 96-1 04, Prolmmune Limited, #F391-4A-E), FACS sorted and cloned by limiting dilution in 96-well plates. Expansion of T cell clones was performed using 2×10 6 5 x 10 T cell clones and 5 x 10 7 irradiated PBMCs (30 Gy) and 1 × 10 7 Feeder cells composed of irradiated (100-150 Gy) LCLs were cultured in 40 ml of RPMI-1 640 medium containing glutamine (Sigma-Aldrich), 10% human serum (human AB serum, Valley Biomedical, Inc., #HP1 022), and 1% penicillin / streptomycin (Life Technologies) at 37°C and 5% CO2. Growth was performed for 14 days in the presence of 50 U / ml IL-2 (Proleukin, Novartis, #1003780), 2.5 ng / ml IL-1 5 (rhlL-1 5-CF R&D #247_IL-025 / CF), and 30 ng / ml anti-human CD3 antibody (OKT3 eBiosciences 16-0037-85). The HCC2935 human lung adenocarcinoma line was cultured in RPMI-1640 (Sigma-Aldrich) containing 10% FCS (FBS Superior, Biochrom) and 1% penicillin / streptomycin at 37°C and 5% CO2.

[0303] To analyze the modulatory activity of anti-CEACAM6 antibodies on the immunosuppressive function of CEACAM6 in vitro, survivin-peptide-specific CD8+ T cell clones were co-cultured with CEACAM6, a lung adenocarcinoma cell line, HCC2935. IFN-gamma secretion was used as a readout for T cell activity. IFN-gamma was measured by supernatant IFN-gamma ELISA. In the co-culture, HCC2936 tumor cells were non-enzymatically detached using PBS-EDTA for 5 min, centrifuged, washed, and counted. 40,000 HCC2936 target cells were directly seeded in triplicate on IFN-gamma U-96-well ELISA plates. Meanwhile, survivin-peptide-specific T cells were collected, washed with X-Vivo-20, and seeded at 80,000 cells per well. IgG1 LALAglycoanti-CEACAM6 antibodies inhibited EC 50 The anti-CEACAM6 antibody was added to the wells at a final concentration of 0.03–7.5 μg / ml to calculate the EC. Co-cultures of tumor cells, anti-CEACAM6 antibody and T cells were incubated at 37°C for 24 h. IFN-gamma-ELISA (BD Human IFN-gamma ELISA Set #5551 42) was developed according to the manufacturer's instructions. The optical density of the ELISA plate was measured using a Tecan Infinite M200 plate reader. Co-culture of HCC2936 tumor cells and survivin-peptide specific CD8+ T cells in the presence of anti-CEACAM6 antibody resulted in a statistically significant increase in IFN-gamma production by T cells compared to samples treated with an isotype-matched control antibody. The EC of the IgG1 LALA aglyco anti-CEACAM6 antibody TPP-21518 in this assay 50 was 0.55 μg / ml.

[0304] In vitro pharmacological effects of anti-CEACAM6 antibody on IFN-gamma secretion by polyclonal tumor-infiltrating T cells Pancreatic cancer tumor-infiltrating lymphocyte cell lines (TILs) were isolated from fresh primary cultures of tumor tissue obtained from surgery. Briefly, fresh primary tissue material was cut into small pieces and cultured with 16000IU / IL-2 in small dishes (Lonza) containing 2% human serum albumin, 2.5μg / ml fungizone, 20μg / ml gentamicin, and 1% penicillin / streptomycin for 0–18 days. Cells were then collected from the supernatant and either cryopreserved or used directly for the "rapid expansion protocol" (REP). To rapidly expand TILs, frozen TILs were gently thawed and cultured at 0.6×10 6 Cells / ml were cultured for 1 day in complete lymphocyte medium CLM RPMI-1640 (Life Technologies #21875034), 10% human AB serum (MILAN Analytica #000083), 1% penicillin / streptomycin (Life Technologies #15140122), 1% ml HEPES (Life Technologies #15630056), 0.01% β-mercaptoethanol [stock 50 mM] (Life Technologies #31350010)) with 6000 IU / ml IL-2. TILs were collected and expanded at a ratio of 1:100 with 60 Gy irradiated feeder PBMCs from three different donors in 400 ml of REP medium (a mix of 50% CLM and 50% AIM-V serum-free medium (Gibco #12055091) containing 3000 IU / ml IL-2 and 30 ng / ml OKT-3 antibody (eBioscience #16-0037-85)) in G-REX-100 flasks (Wilson Wolf #80500S). Cells were cultured and split as described in Jin et al., J Immunother. 2012 Apr; 35(3):283-92. After 14 days, cells were collected and frozen in aliquots. Prior to the co-culture cytotoxicity assay, individual aliquots of TILs were gently thawed and 0.6 × 10 6 Cells / ml were cultured in CLM containing 6000 IU / ml IL-2 for 2 days and in CLM without IL-2 for 1 day.

[0305] For co-culture, HCC2935 tumor cells were non-enzymatically detached using PBS-EDTA for 5 min, centrifuged, washed, and counted. 25,000 HCC2936 target cells were directly seeded in triplicate into U-96 well ELISA plates. Meanwhile, TIL cells were thawed, washed with X-Vivo-20, and seeded at 50,000 cells per well. IgG1 LALA aglyco anti-CEACAM6 antibody was added to the tumor and T cell co-cultures. Bispecific antibody anti-CD3xanti-EPCAM IgG (0.25ng / ml) (Marme et al., Int J Cancer. 2002 Sept. 10;101 (2):183-9; Salnikov et al., J Cell Mol Med. 2009 Sept;13(9B):4023-33) was added to the co-cultures to allow HLA-independent T cell-mediated tumor cell killing and to increase tumor cell recognition by TILs. Co-cultures were incubated at 37°C for 24 hours. IFN-gamma-ELISA (BD human IFN-gamma ELISA set #5551 42) was developed according to the manufacturer's instructions. The optical density of the ELISA plates was measured using a Tecan Infinite M200 plate reader. Co-culture of HCC2936 tumor cells and TIL CD8+ T cells in the presence of anti-CEACAM6 antibody resulted in a statistically significant increase in IFN-gamma production by T cells compared to samples treated with an isotype-matched control antibody. The EC of the IgG1 LALAglyco anti-CEACAM6 antibody TPP-21518 in this assay 50 was 0.5 μg / ml.

[0306] In vitro pharmacological effects of anti-CEACAM6 antibodies on tumor cell killing assays using tumor-infiltrating CD8+ T cells. T cell-mediated cytotoxicity of HCC2935 tumor cells was analyzed with an impedance-based cytotoxicity assay (xCELLigence) system. In this system, cytotoxicity is measured directly and continuously over a long period of time, about 100 hours (real time). Adherent tumor cells attach to microelectrodes at the bottom of a 96-well E-plate (E-Plate VIEW 96 PET; ACEA Biosciences #ID: H000568) and change the electrical impedance of these electrodes, which is monitored as an increase in the dimensionless "cell index." After tumor cell adhesion (24 hours), antibodies and T cells are added to the wells and, as the T cells exert their cytotoxic activity, the tumor cells lyse and detach from the electrodes. This detachment changes the impedance of the wells, measured as a decrease in the "cell index" or "normalized cell index," where the "cell index" is normalized to the time of T cell addition. Only tumor cell cytolysis is measured, since T cells alone do not affect the electrical impedance of the electrodes. (Peper et al. J Immunol Methods. March 2014:405: 192-8).

[0307] The effect of CEACAM6 antibody on the cytolytic activity of TIL cells from patients with pancreatic cancer was tested. Thus, 10,000 cells of CEACAM6-positive lung cancer cell line HCC2935 were added to 96-well plates and cultured for 24 hours. TILs were then added at various ratios in the presence of CEACAM6 antibody (0.03-7.5 μg / ml) and bispecific antibody anti-CD3xanti-EPCAM IgG (0.25 ng / ml) (Marme et al., Int J Cancer. 2002 Sept. 10;101 (2):183-9; Salnikov et al., J Cell Mol Med. 2009 Sept.;13(9B):4023-33) to allow HLA-independent T cell-mediated tumor cell killing. Significant cytolytic killing of the target cell line HCC2935 was observed in the presence of anti-CEACAM6 antibody. In additional experiments, the effect of the CEACAM6 antibody TPP-21518 proved to be dose-dependent, with an EC 50 The value was determined.

[0308] All examples were carried out using standard techniques well known and routine to those skilled in the art, unless otherwise specified in detail.

[0309] The results are shown in Table 4.

[0310] [Table 4]

[0311] In summary, these experiments demonstrate that the CEACAM6 antibody TPP-21518 of the present invention effectively blocks the immunosuppressive receptor CEACAM6 and has the potential to improve the cytotoxicity of not only model T cells but also patient-derived tumor-infiltrating lymphocytes against CEACAM6-positive tumor cells.In this sense, TPP-21518 has similar potency to its human IgG2 counterpart TPP-3310, and is therefore suitable for use in clinical treatment.

Claims

1. an anti-CEACAM6 antibody comprising an IgG1 Fc region lacking a glycan attached to a conserved N-linked site within the CH2 domain of the Fc region, wherein the IgG1 Fc region comprises at least the amino acid substitutions L234A and L235A, numbered according to the EU index of Kabat; the antibody specifically binds to CEACAM6 domain 1 comprising amino acids 35-142 of SEQ ID NO: 75, and / or The antibody a. a heavy chain variable region H-CDR1 comprising the amino acid sequence of SEQ ID NO: 64; b. a heavy chain variable region H-CDR2 comprising the amino acid sequence of SEQ ID NO: 65; c. a heavy chain variable region H-CDR3 comprising the amino acid sequence of SEQ ID NO: 66; d. a light chain variable region L-CDR1 comprising the amino acid sequence of SEQ ID NO: 68; e. a light chain variable region L-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and f. Light chain variable region L-CDR3 comprising the amino acid sequence of SEQ ID NO: 70 an anti-CEACAM6 antibody comprising:

2. The anti-CEACAM6 antibody of claim 1, wherein the IgG1 Fc region comprises the amino acid substitutions N297A, N297G, or N297Q, numbered according to the EU index of Kabat.

3. An anti-CEACAM6 antibody described in claim 1, which competes for CEACAM6 binding with an antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

67.

4. a. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 63, and b. A light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 67 The anti-CEACAM6 antibody of claim 1, comprising:

5. a. a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 71, and b. A light chain (LC) comprising the amino acid sequence of SEQ ID NO: 72 The anti-CEACAM6 antibody of claim 1, comprising:

6. The anti-CEACAM6 antibody of claim 1, which is a monoclonal antibody.

7. The anti-CEACAM6 antibody of claim 1, which is a human antibody or a humanized antibody.

8. A nucleic acid encoding the anti-CEACAM6 antibody of claim 1.

9. An isolated cell expressing the anti-CEACAM6 antibody of claim 1 and / or containing the nucleic acid of claim 8.

10. A method for producing the anti-CEACAM6 antibody of claim 1, comprising culturing the cell of claim 9 and purifying the antibody.

11. A composition comprising an anti-CEACAM6 antibody according to any one of claims 1 to 7 for use as a pharmaceutical.

12. A composition comprising an anti-CEACAM6 antibody according to any one of claims 1 to 7 for use as a medicament for the treatment of cancer.

13. A pharmaceutical composition comprising an anti-CEACAM6 antibody described in any one of claims 1 to 7.

14. A composition comprising the anti-CEACAM6 antibody of any one of claims 1 to 7 for use in simultaneous, separate or sequential combination with an anti-PD-1 antibody or an anti-PD-L1 antibody in the treatment of cancer, wherein preferably the anti-PD-1 antibody is nivolumab or pembrolizumab, and the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

15. A composition comprising an anti-CEACAM6 antibody according to any one of claims 1 to 7 for use in combination with an anti-TIM-3 antibody simultaneously, separately or sequentially in the treatment of cancer, wherein the anti-TIM-3 antibody is preferably covolimab, MBG-453, BMS-986258, Sym-023, LY-3321367 or INCAGN-2390.

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