RU2828374C2 - Use of ceacam5 immunoconjugates for treating lung cancer - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, in particular to the use of an antibody or an immunoconjugate containing the antibody for treating non-squamous non-small cell lung cancer. Also disclosed is a method of treating non-squamous non-small cell lung cancer.

EFFECT: invention provides effective treatment of non-squamous non-small cell lung cancer.

30 cl, 4 dwg, 9 tbl, 4 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to the field of therapeutic intervention in cancer types, such as non-squamous non-small cell lung cancer (NSQ NSCLC), in which CEACAM5 is expressed. Certain aspects of the present invention relate to the use of CEACAM5 antagonists, such as antibodies and immunoconjugates to CEACAM5, for the treatment of lung cancer.

BACKGROUND OF THE INVENTION

The mechanism of action of antibody drug conjugates (ADCs) begins with its binding to a specific antigen expressed in sufficient quantities on tumor cells to achieve selective and efficient internalization of the drug. Effective tumor-cell-selective cytotoxic agents using ADCs have recently been shown to be part of an effective cancer treatment strategy, as demonstrated by the recent approvals of brentuximab vedotin for the treatment of Hodgkin lymphoma and trastuzumab emtansine (T-DM1) for the treatment of recurrent metastatic HER2+ breast cancer. Many other malignancies with unmet medical need, such as solid tumor cancers, could benefit from such potential treatments.

Lung cancer, for example, is an aggressive form of cancer that causes hundreds of thousands of deaths in the United States. Unfortunately, it tends to recur after initial treatment and become more resistant to subsequent treatment. Although complex treatments are used to treat individuals with lung cancer, more effective treatments are needed.

BRIEF DESCRIPTION

The present invention provides , inter alia, methods for treating lung cancer (e.g., NSQ NSCLC) in a subject in need thereof, comprising administering an effective amount of an antibody or immunoconjugate (comprising an antibody) that specifically binds CEACAM5.

The present invention provides , inter alia, antibodies or immunoconjugates comprising antibodies (also referred to as ADCs or antibody drug conjugates) and methods of treating a cancer that expresses CEACAM5 in a subject in need thereof, comprising administering an effective amount of an antibody or immunoconjugate that specifically binds CEACAM5. For example, the cancer expresses human carcinoembryonic antigen-related cell adhesion molecule 5 (hCEACAM5). In various embodiments, the cancer expresses hCEACAM5 at a high level. For example, the cancer expresses the A3-B3 domain of hCEACAM5 comprising SEQ ID NOs: 10 and 11, such that the antibody or immunoconjugate binds the domain.

The present invention provides an antibody or an immunoconjugate comprising an antibody for use in the treatment of a cancer characterized by high levels of carcinoembryonic antigen-related cell adhesion molecule 5 in a subject in need thereof. In various embodiments, the antibody specifically binds human carcinoembryonic antigen-related cell adhesion molecule 5 (hCEACAM5) and the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 and wherein the VL comprises three CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 3 (GFVFSSYD); HCDR2 comprises the amino acid sequence of SEQ ID NO: 4 (ISSGGGIT); HCDR3 comprises the amino acid sequence of SEQ ID NO: 5 (AAHYFGSSGPFAY); LCDR1 comprises the amino acid sequence of SEQ ID NO: 6 (ENIFSY); LCDR2 comprises the amino acid sequence of NTR; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 7 (QHHYGTPFT).

The present invention provides an antibody or an immunoconjugate comprising an antibody for use in the treatment of non-squamous non-small cell lung cancer (NSQNSCLC) in a subject in need thereof, wherein the antibody specifically binds hCEACAM5 and wherein the antibody comprises a VH and a VL, wherein the VH comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 and wherein the VL comprises three CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 4; HCDR3 comprises the amino acid sequence of SEQ ID NO: 5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 6; LCDR2 comprises the amino acid sequence of NTR; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 7.

The present invention provides an antibody or an immunoconjugate comprising an antibody for use in a subject who has been previously treated with an anticancer therapeutic agent, wherein the antibody specifically binds hCEACAM5, and wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 and wherein the VL comprises three CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 4; HCDR3 comprises the amino acid sequence of SEQ ID NO: 5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 6; LCDR2 comprises the amino acid sequence of NTR; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the subject is a subject that expresses carcinoembryonic antigen-related cell adhesion molecule at high levels. In other embodiments, the subject has been previously treated with an agent or medicament for treating non-small cell lung cancer. In other embodiments, the agent or medicament is selected from the group consisting of: a chemotherapeutic agent, an angiogenesis inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an anaplastic lymphoma kinase (ALK) inhibitor, a receptor tyrosine kinase (ROS1) inhibitor, and an immune checkpoint inhibitor. In certain aspects of these embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor and/or a PD-L1 inhibitor.

In various embodiments, the cancer is NSQ NSCLC.

In various embodiments, the VH comprises SEQ ID NO: 1

In various embodiments, the heavy chain comprises SEQ ID NO: 8

In various embodiments, the VL comprises SEQ ID NO: 2

In various embodiments, the light chain comprises SEQ ID NO: 9

In various embodiments, the antibody is conjugated or linked to at least one growth inhibitory agent. In an embodiment, the growth inhibitory agent is a cytotoxic agent. In various embodiments of the antibody, the growth inhibitory agent is selected from the group consisting of chemotherapeutic agents, enzymes, antibiotics and toxins such as small molecule toxins or enzymatically active toxins, taxoids, vinca alkaloids, taxanes, maytansinoid or maytansinoid analogs, tomaimycin or pyrrole benzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatin and dolastatin analogs, prodrugs, topoisomerase II inhibitors, DNA alkylating agents, antitubulin agents, and CC-1065 or CC-1065 analogs. In various embodiments of the antibody, the growth inhibitory agent is N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) or N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4). For example, the growth inhibitory agent is DM4.

In various embodiments, the antibody is covalently attached via a cleavable or non-cleavable linker to at least one growth inhibitory agent. In various embodiments of the antibody, the linker is selected from the group consisting of N-succinimidyl pyridyl dithiobutyrate (SPDB), 4-(pyridin-2-yl disulfanyl)-2-sulfobutyric acid (sulfo-SPDB), and succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). For example, the linker is SPDB.

In various embodiments, the antibody is huMAb2-3.

In various embodiments of the present invention, the subject has a percentage hCEACAM5 expression score that is greater than or equal to 50 (consisting of 2+ or 3+ intensities) in tumor cell populations, such as the percentage hCEACAM5 expression is greater than at least about 50, at least about 50 to about 80, at least about 80, or about 100 (consisting of 2+ or 3+ intensities). In various embodiments, the percentage hCEACAM5 expression is at least about 50% to about 80% in a tumor cell population, at least about 80%, at least about 90%, at least about 95%, or about 100% in a tumor cell population. In various embodiments, the cancer is a non-squamous non-small cell lung carcinoma in which hCEACAM5 is highly expressed in at least about 50%, at least about 50% to about 80%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the tumor cell population.

In various embodiments, the antibody or immunoconjugate comprising the antibody is administered intravenously, such as by intravenous infusion.

In various embodiments, the antibody or immunoconjugate comprising the antibody is administered at a rate of 2.5 mg/min for the first 30 minutes. In various embodiments, after about 30 minutes, the rate of administration of the antibody is increased to 5 mg/min.

In various embodiments, the antibody or immunoconjugate comprising the antibody is administered at a dose level of 5, 10, 20, 30, 40, 60, 80, 100, 120, 150, 180 or 210 mg/ ^m2 based on the subject's body surface area. According to an embodiment, the immunoconjugate comprising the antibody is administered at a dose level of 100 mg/ ^m2 based on the subject's body surface area, which corresponds to the maximum tolerated dose (MTD) determined during the dose escalation phase.

In various embodiments, the antibody or immunoconjugate comprising the antibody is administered at a dose of about 2.5 mg/m ² to about 5 mg/m ² . For example, the antibody or immunoconjugate comprising the antibody is administered over one hour at a dose of about 2.5 mg/m ² to about 5 mg/m ² . The dose includes 2.5 mg/m ² of the antibody, 5 mg/m ² of the antibody, and all doses from 2.5 mg/m ² to 5 mg/m ² , such as 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, and 4.75 mg/m ² . In various embodiments, the body surface area is calculated using the height and actual body weight of the subject.

In various embodiments, the antibody or immunoconjugate comprising the antibody is administered every 14 days. In various embodiments, the antibody or immunoconjugate comprising the antibody is administered every three weeks.

In an embodiment, the present invention provides a huMAb2-3-SPDB-DM4 immunoconjugate for use in the treatment of non-squamous non-small cell lung cancer (NSQNSCLC) in a subject in need thereof, wherein the huMAb2-3-SPDB-DM4 immunoconjugate is administered every two weeks at a dosage level of 100 mg/ ^m2 .

In an embodiment, the present invention provides a huMAb2-3-SPDB-DM4 immunoconjugate for use in the treatment of non-squamous non-small cell lung cancer (NSQNSCLC) in a subject in need thereof, wherein the huMAb2-3-SPDB-DM4 immunoconjugate is administered every three weeks at a dosage level of 100 mg/ ^m2 .

In another embodiment, the present invention provides a huMAb2-3-SPDB-DM4 immunoconjugate for use in the treatment of non-squamous non-small cell lung cancer (NSQNSCLC) in a subject in need thereof, wherein the huMAb2-3-SPDB-DM4 immunoconjugate is administered at a first dose level of 150 mg/ ^m2 or 170 mg/ ^m2 and then every two weeks at a dose level of 100 mg/ ^m2 .

In various embodiments, prior to administration of the antibody or immunoconjugate comprising the antibody, a premedication agent is administered to the subject. For example, the premedication agent is an H1-histamine receptor antagonist.

In various embodiments, the H1-histamine receptor antagonist is diphenhydramine or dexchlorpheniramine.

In various embodiments, the subject has previously been treated with an agent or a drug for the treatment of non-small cell lung cancer. For example, the subject has previously been heavily and/or ineffectively treated with the agent or drug. In various embodiments of the present invention, the agent or drug is selected from the group consisting of: a chemotherapeutic agent, an angiogenesis inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an anaplastic lymphoma kinase (ALK) inhibitor, a receptor tyrosine kinase (ROS1) inhibitor, and an immune checkpoint inhibitor. In various embodiments of the present invention, the immune checkpoint inhibitor is a PD-1 inhibitor and/or a PD-L1 inhibitor.

In some embodiments of the antibody or immunoconjugate comprising the antibody for use described above, the progression of at least one symptom of cancer is reduced, slowed, stopped or otherwise reduced. In certain aspects of these embodiments, the rate of tumor growth or the size of the tumor is reduced after treatment with the antibody. In other aspects of these embodiments, the expression profile, intensity and proportion of expressing cells indicate a reduction, slowing or stopping of the manifestation of symptoms of cancer.

The present invention provides a pharmaceutical composition comprising an antibody as described herein, or an immunoconjugate comprising the antibody, and a pharmaceutically acceptable carrier.

The present invention provides an immunoconjugate for use in the treatment of a cancer characterized by high levels of carcinoembryonic antigen-related cell adhesion molecule in a subject in need thereof, wherein the immunoconjugate comprises an antibody drug conjugate (ADC) that specifically binds hCEACAM5 and comprises an antibody as described above, wherein the progression of at least one symptom of the cancer is reduced, slowed, or otherwise reduced.

The present invention provides an immunoconjugate for use in the treatment of NSQ NSCLC in a subject in need thereof, wherein the immunoconjugate comprises an ADC that specifically binds hCEACAM5 and comprises an antibody as described above, wherein the progression of at least one symptom of cancer is reduced, slowed, or otherwise reduced.

The present invention provides an immunoconjugate for use in treating a subject, which is a heavily pretreated subject, in whose body a carcinoembryonic antigen-related cell adhesion molecule is expressed at high levels, wherein the immunoconjugate comprises an ADC that specifically binds hCEACAM5 and comprises an antibody as described above, wherein the progression of at least one symptom of cancer is reduced, slowed, or otherwise reduced.

In various embodiments, the immunoconjugate comprises a huMAb2-3 antibody,

wherein the growth inhibitory agent comprises DM4; and wherein the linker comprises SPDB. In various embodiments, the ADC comprises huMAb2-3-SPDB-DM4.

In various embodiments, the rate of tumor growth or tumor size is reduced following treatment with the immunoconjugate.

In various embodiments, the expression profile, intensity, and proportion of expressing cells indicate a reduction, slowing, or cessation of the manifestation of cancer symptoms.

The present invention provides a pharmaceutical composition comprising an antibody as described herein or an immunoconjugate as described and a pharmaceutically acceptable carrier.

The present invention provides a method for treating a cancer characterized by high levels of carcinoembryonic antigen-related cell adhesion molecule 5 in a subject in need thereof, the method comprising administering an antibody that specifically binds hCEACAM5, wherein the antibody comprises a VH and a VL, wherein the VH comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 and wherein the VL comprises three CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 4; HCDR3 comprises the amino acid sequence of SEQ ID NO: 5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 6; LCDR2 comprises the amino acid sequence of NTR; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 7.

The present invention provides a method of treating NSQ NSCLC in a subject in need thereof, the method comprising administering an antibody that specifically binds hCEACAM5, wherein the antibody comprises a VH and a VL, wherein the VH comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 and wherein the VL comprises three CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 4; HCDR3 comprises the amino acid sequence of SEQ ID NO: 5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 6; LCDR2 comprises the amino acid sequence of NTR; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 7.

The present invention provides a method of treating a subject that has been previously treated with an anti-cancer therapeutic agent, wherein the antibody specifically binds hCEACAM5, wherein the antibody comprises a VH and a VL, wherein the VH comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 and wherein the VL comprises three CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 4; HCDR3 comprises the amino acid sequence of SEQ ID NO: 5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 6; LCDR2 comprises the amino acid sequence of NTR; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the subject is a subject that expresses carcinoembryonic antigen-related cell adhesion molecule at high levels. In other embodiments, the subject has previously been treated with an agent or drug for treating non-small cell lung cancer. In other embodiments, the agent or drug is selected from the group consisting of: a chemotherapeutic agent, an angiogenesis inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an anaplastic lymphoma kinase (ALK) inhibitor, a receptor tyrosine kinase (ROS1) inhibitor, and an immune checkpoint inhibitor. In certain aspects of these embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor and/or a PD-L1 inhibitor.

In various embodiments of the methods described above, the cancer is NSQ NSCLC.

In various embodiments of the method, the VH comprises SEQ ID NO: 1. In various embodiments of the method, the heavy chain comprises SEQ ID NO: 8.

In various embodiments of the method, the VL comprises SEQ ID NO: 2. In various embodiments of the method, the light chain comprises SEQ ID NO: 9.

In various embodiments of the method, the antibody is conjugated or linked to at least one growth suppressor. For example, the growth suppressor is a cytotoxic agent.

In various embodiments of the method, the growth suppressor is selected from the group consisting of chemotherapeutic agents, enzymes, antibiotics and toxins such as small molecule toxins or enzymatically active toxins, taxoids, vinca alkaloids, taxanes, maytansinoid or maytansinoid analogs, tomaimycin or pyrrole benzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatin and dolastatin analogs, prodrugs, topoisomerase II inhibitors, DNA alkylating agents, antitubulin agents and CC-1065 or CC-1065 analogs. In various embodiments of the method, the growth suppressor is DM1 or DM4. For example, the growth suppressor is DM4.

In various embodiments of the method, the antibody is covalently attached via a cleavable or non-cleavable linker to at least one growth inhibitory agent. In various embodiments of the method, the linker is selected from the group consisting of SPDB, sulfo-SPDB, and SMCC. In one embodiment, the linker is SPDB.

In various embodiments of the method, the antibody is huMAb2-3. In various embodiments of the method, the subject has a percentage expression of hCEACAM5 that is greater than or equal to 50 (comprising 2+ and 3+ intensities) in tumor cell populations. For example, the percentage expression of hCEACAM5 is greater than or equal to at least about 50, at least about 50 to about 80, at least about 80, or about 100 (comprising 2+ and 3+ intensities). In various embodiments, the percentage expression of hCEACAM5 is at least about 50% to about 80% of the tumor cell population, at least about 80%, or about 100% of the tumor cell population. In various embodiments, the cancer is a non-squamous non-small cell lung carcinoma in which hCEACAM5 is highly expressed in at least about 50%, at least about 50% to about 80%, at least about 80%, or about 100% of the tumor cell population.

In various embodiments, the antibody is administered intravenously, such as by intravenous infusion.

In various embodiments of the method, the antibody or immunoconjugate containing the antibody is administered at a rate of 2.5 mg/min. during the first 30 minutes. In various embodiments of the method, after 30 minutes, the rate of administration of the antibody is increased to 5 mg/min.

In various embodiments of the method, the antibody or immunoconjugate comprising the antibody is administered at a dosage level of 5, 10, 20, 30, 40, 60, 80, 100, 120, 150, 180, or 210 mg/ ^m2 based on the subject's body surface area. In various embodiments of the method, the antibody or immunoconjugate comprising the antibody is administered at a dosage of about 2.5 mg/ ^m2 to about 5 mg/ ^m2 . For example, the antibody or immunoconjugate comprising the antibody is administered over one hour at a dosage of about 2.5 mg/ ^m2 to about 5 mg/ ^m2 . The dose includes 2.5 mg/ ^m2 of the antibody or immunoconjugate comprising the antibody, 5 mg/ ^m2 of the antibody or immunoconjugate comprising the antibody, and all doses from 2.5 mg/ ^m2 to 5 mg/ ^m2 , such as 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5 and 4.75 mg/ ^m2 . In various embodiments, the body surface area is calculated using the height and actual body weight of the subject.

In various embodiments of the method, the antibody or immunoconjugate comprising the antibody is administered every 14 days. In various embodiments, the antibody or immunoconjugate comprising the antibody is administered every three weeks.

In various embodiments of the method, before administering the antibody or immunoconjugate comprising the antibody, the subject is administered a premedication agent, for example, in the case of premedication of the subject with an H1-histamine receptor antagonist. In various embodiments of the method, the H1-histamine receptor antagonist is diphenhydramine or dexchlorpheniramine.

In various embodiments of the method, the subject has previously been treated with an agent or drug for the treatment of non-small cell lung cancer. For example, the agent or drug is selected from the group consisting of: a chemotherapeutic agent, an angiogenesis inhibitor, an EGFR inhibitor, an anaplastic lymphoma kinase (ALK) inhibitor, a tyrosine kinase (ROS1) receptor inhibitor, and an immune checkpoint inhibitor. For example, the immune checkpoint inhibitor is a PD-1 inhibitor and/or a PD-L1 inhibitor.

In some embodiments of the method described above, the progression of at least one symptom of cancer is reduced, slowed, stopped, or otherwise reduced. In certain aspects of these embodiments, the rate of tumor growth or the size of the tumor is reduced after treatment with the antibody. In other aspects of these embodiments, the expression profile, intensity, and proportion of expressing cells indicate a reduction, slowing, or stopping of the manifestation of symptoms of cancer.

The present invention provides a pharmaceutical composition comprising an antibody as described herein, or an immunoconjugate comprising the antibody, and a pharmaceutically acceptable carrier.

In various embodiments of the method, the antibody is administered as an ADC that specifically binds hCEACAM5 and includes any antibody described herein, wherein the progression of at least one symptom of cancer is reduced, slowed, stopped, or otherwise reduced following administration/treatment with the ADC.

In various embodiments of the method, the ADC comprises a huMAb2-3 antibody;

wherein the growth inhibitory agent comprises DM4; and wherein the linker comprises SPDB. In various embodiments, the ADC comprises huMAb2-3-SPDB-DM4.

In various embodiments, the tumor growth rate and/or tumor size are reduced following treatment with the antibody and/or immunoconjugate.

In various embodiments of the method, the expression profile, intensity and proportion of expressing cells indicate a reduction, slowing down or stopping of the manifestation of cancer symptoms after treatment with the antibody and/or immunoconjugate.

In various embodiments of the method, the antibody and/or immunoconjugate is administered as a pharmaceutical composition containing a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1A, 1B and 1C show examples of staining intensities 1+, 2+, 3+, respectively.

Figure 2 shows a histogram of the greatest relative tumor size reduction in patients treated with huMAb2-3-SPDB-DM4 according to the category of central CEACAM5 expression in the archived sample. CEACAM5 expression (2+/3+) of patients is <50%, 50% to 80%, or ≥ or. PR means partial response. SD means stable disease. PD means progressive disease.

Figure 3 shows the greatest relative tumor size reduction observed in 32 patients treated in the cohort with high CEACAM5 expression levels (mild) and in patients in the cohort with moderate levels of expression. CEACAM5 expression (2+/3+) of patients is <50% or ≥50%. PR means partial response. SD means stable disease. PD means progressive disease.

Figure 4 shows the time to disease progression (TTP) in 32 patients treated in the cohort expressing high levels of CEACAM5 (lung cohort).

DETAILED DESCRIPTION

The present invention provides pharmaceutical compositions and methods of using these compositions for the treatment of NSQ NSCLC and amelioration of at least one symptom of the disease. These compositions comprise at least one antibody that specifically binds (CEACAM5), for example, the antibody is the huMAb2-3 antibody. The huMAb2-3-SPDB-DM4 ADC is an immunoconjugate combining the huMAb2-3 antibody (an anti-CEACAM5 antibody) and maytansinoid derivative 4 (DM4), a potent antifungal agent that inhibits microtubule assembly. DM4 is covalently linked to huMAb2-3 via an optimized SPDB [N-succinimidyl-4-(2-pyridyldithio)-butyrate] linker, which is stable in plasma and cleavable within cells. Following binding and internalization into target cancer cells, huMAb2-3-SPDB-DM4 is degraded, releasing cytotoxic DM4 metabolites.

As used herein, the phrase "a cancer that produces high levels of CEACAM5" refers to several types of cancer, including lung cancer. In some embodiments, the lung cancer is non-squamous non-small cell lung cancer. In certain embodiments, subjects that express high levels of CEACAM5 have an intensity of greater than 2+ in at least 50% of the expressing tumor cell population. Subjects that express high levels of CEACAM5 represent ~20% of lung cancer cases. An ADC described herein comprising a DM4 cytotoxic agent, an SPDB linker, and a humanized huMAb2-3 antibody was administered in a proof-of-concept study. The data demonstrate that the ADC achieved proof-of-concept in a subset of lung cancers.

The ADC was evaluated in a phase 1/2 study in heavily pretreated subjects expressing high levels of CEACAM5. The ADC demonstrated competitive overall response rates (ORR) and duration of response (DoR) for the 3L setting. The most common adverse drug reactions (ADRs) were ophthalmologic toxicity (reversible without treatment discontinuation) and minimal hematologic/nervous system toxicity.

As used herein, the term "heavily pretreated" refers to pretreating a subject for more than 1 month. In other embodiments, a subject that has been heavily pretreated has been treated for more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In certain embodiments, the pretreatment is administration of one or more anticancer therapeutic agents.

Non-small cell lung cancer (NSCLC)

Non-small cell lung cancer is a disease in which malignant (cancer) cells form in the tissues of the lung. Smoking is the main cause of the disease. It is a type of epithelial lung cancer, different from small cell lung carcinoma. There are several types of non-small cell lung cancer. Each type of non-small cell lung cancer has different types of cancer cells. Each type of cancer cell grows and spreads differently. The types of non-small cell lung cancer are named based on the types of cells found in the cancer and how the cells look under a microscope. (1) Squamous cell carcinoma. Cancer that starts from squamous cells, which are thin, flat cells that look like fish scales. It is also called epidermoid carcinoma. (2) Large cell carcinoma. Cancer that can start from several types of large cells. And (3) adenocarcinoma. A cancer that begins in the cells that line the air sacs and produce substances such as mucus.

Effective tumor-selective cytotoxic agents using ADCs have recently been shown to be part of an effective cancer treatment strategy, as demonstrated by the recent approvals of brentuximab vedotin for the treatment of Hodgkin lymphoma and trastuzumab emtansine (T-DM1) for the treatment of recurrent metastatic HER2+ breast cancer. Many other malignancies with unmet medical need could benefit from such potential treatments. The mechanism of action of an ADC begins with its binding to a specific antigen expressed in sufficient quantities on tumor cells to achieve selective and efficient internalization of the drug.

Radical surgery is the standard of care for patients who meet stage I NSCLC criteria (eg, pneumonectomy, lobectomy, segmentectomy or wedge resection, circumferential resection). Treatment with adjuvant drugs should be offered only as part of an investigational trial. Cisplatin-based adjuvant chemotherapy for stages II and IIIA remains the accepted standard for completely resected NSCLC. Other chemotherapeutic agents used in combination with cisplatin or with each other may include carboplatin, paclitaxel (Taxol), albumin-stabilized paclitaxel (nab-paclitaxel, Abraxane), docetaxel (Taxotere), gemcitabine (Gemzar), vinorelbine (Navelbine), irinotecan (Camptosar), etoposide (VP-16), vinblastine, and pemetrexed (Alimta). Radiation therapy may also be considered in patients with N2 lymph nodes. In patients with advanced stage IIIB/IV or unresectable NSCLC, treatment may include multiple cycles of cisplatin-based chemotherapy with a third-generation cytotoxic agent or cytostatic drug (anti-EGFR, anti-VEGFR).

Treatments for cancers, including lung cancers, may include angiogenesis inhibitors, epidermal growth factor receptor (EGFR) inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, ROS1 receptor tyrosine kinase inhibitors, and immune checkpoint inhibitors.

Angiogenesis inhibitors may include, but are not limited to, axitinib (Inlyta), bevacizumab (Avastin), cabozantinib (Cometriq), everolimus (Afinitor, Zortress), lenalidomide (Revlimid), pazopanib (Votrient), ramucirumab (Cyramza), regorafenib (Stivarga), sorafenib (Nexavar), sunitinib (Sutent), thalidomide (Sinovir, Thalomid), vandetanib (Caprelsa), aflibercept, and ziv-aflibercept (Zaltrap).

Epidermal growth factor receptor inhibitors may include, but are not limited to, gefitinib (Iressa), erlotinib (Tarceva), lapatinib (Tykerb), cetuximab (Erbitux), neratinib (Nerlinx), osimertinib (Tagrisso), panitumumab (Vectibix), vandetanib (Caprelsa), necitumumab (Protrazza), and dacomitinib (Vizimpro).

Immune checkpoint inhibitors may include, but are not limited to, programmed death receptor 1 (PD-1) binding agents (e.g., pembrolizumab, nivolumab, cemiplimab), programmed death ligand-1 (PD-L1) binding agents (eg, atezolizumab, durvalumab), CTLA-4 binders (eg, ipilimumab), OX40 or OX40L binders, adenosine A2A receptor binders, B7-H3 binders, B7-H4 binders, BTLA binders, indoleamine 2,3-dioxygenase binders, killer cell immunoglobulin-like receptor (KIR) binders, lymphocyte activation gene-3 (LAG-3) binders, nicotinamide adenine dinucleotide phosphate oxidase isoform (NOX2) binders, T-cell immunoglobulin domain and mucin domain 3 (TIM-3) binders, V-domain Ig suppressor of T-cell activation (VISTA) binders, glucocorticoid-induced TNFR-related gene (GITR) binders, and immunoglobulin-type lectin 7 binders, sialic acid binding peptide (SIGLEC7).

CEA and CEACAM

Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion. CEA was first identified in 1965 (Gold and Freedman, J Exp Med, 121, 439, 1965) as a protein normally expressed in the fetal intestine during the first six months of pregnancy and has been found in pancreatic, liver, and colon cancers. The CEA family belongs to the immunoglobulin superfamily. The CEA family, which consists of 18 genes, is divided into two subgroups of proteins: the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) subgroup and the pregnancy-specific glycoprotein subgroup.

In humans, the CEACAM subgroup consists of seven members: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, and CEACAM8. Numerous studies have demonstrated that CEACAM5, identical to the originally defined CEA, is expressed at high levels on the surface of tumor cells in the colon, stomach, lung, breast, prostate, ovary, cervix, and bladder and is expressed at low levels in several normal epithelial tissues, such as columnar epithelial cells and goblet cells in the colon, mucosal cervical cells in the stomach, and squamous epithelial cells in the esophagus and cervix. Thus, CEA-CAM5 may represent a therapeutic target suitable for tumor-specific targeting approaches, such as immunoconjugate approaches. The present invention provides antibodies directed against CEACAM5 and demonstrates that they can be conjugated to cytotoxic agents using a linker in vivo and safely administered to subjects with NSQ NSCLC. The data demonstrate that the percentage expression of hCEACAM5 in various cancer cells in lung cancer was greater than about 50, from about 50 to about 80, or about 100 (comprising 2+ and 3+ intensities) or equal to the indicated values. The extracellular domains of CEACAM family members consist of repeating immunoglobulin-like (Ig-like) domains that have been classified into 3 types, A, B, and N, according to sequence homology. CEACAM5 contains seven such domains, namely N, A1, B1, A2, B2, A3, and B3.

The A1, A2 and A3 domains of CEACAM5 on the one hand, and the B1, B2 and B3 domains of CEACAM5 on the other hand, display high levels of sequence homology, the A domains of human CEACAM5 representing 84% to 87% pairwise sequence similarities and the B domains representing 69% to 80%. Furthermore, other members of human CEACAM representing the A and/or B domains in their structure, namely CEACAM1, CEACAM6, CEACAM7 and CEACAM8, display homology to human CEACAM5. In particular, the A and B domains of human CEACAM6 display levels of sequence homology with the A1 and A3 domains and either of the B1 and B3 domains of human CEACAM5, respectively, that are even higher than those observed among the A domains and B domains of human CEACAM5.

Numerous anti-CEA antibodies have been produced for diagnostic and therapeutic purposes targeting CEA. Specificity to cognate antigens has always been mentioned as a concern in the art, such as Sharkey et al. (1990, Cancer Research 50, 2823). Due to the above-mentioned levels of homology, some of the previously described antibodies may exhibit binding to repeated epitopes of CEACAM5 present in different domains of the immunoglobulin, exhibit cross-reactivity with other CEACAM members such as CEACAM1, CEACAM6, CEACAM7 or CEACAM8, without having specificity for CEACAM5. Specificity of the antibody to CEACAM5 is necessary for therapies targeting CEA, such that it binds to human tumor cells expressing CEACAM5, but does not bind to some normal tissues expressing other CEACAM members. Notably, CEACAM1, CEACAM6, and CEACAM8 have been described as expressed by neutrophils from human primates and all non-human primates, where they have been shown to regulate granulopoiesis and play a role in the immune response.

An antibody-drug conjugate to CEACAM6 was described as a maytansinoid-antibody conjugate developed by Genentech (Strickland et al, 2009 J Pathol, 218, 380), which has been shown to induce CEACAM6-dependent hematopoietic toxicity in all non-human primates. This toxicity, associated with accumulation of the antibody-drug conjugate in the bone marrow and depletion of granulocytes and their progenitor cells, was considered a major safety concern by the authors. More specifically, for therapeutic purposes, cross-reactivity of the antibody to CEACAM5 with CEACAM1, CEACAM6, CEACAM7, or CEACAM8 may reduce the therapeutic index of the compound by increasing toxicity in normal tissues. Thus, the production of antibodies specifically targeting CEACAM5 that will not cross-react with other molecules of the CEACAM family provides a strong advantage, especially for use as an antibody-drug conjugate (ADC) or with any other mechanism of action leading to the killing of target cells.

Furthermore, since CEACAM5 has been described to be expressed, albeit at low levels, in some normal cellular tissues, the development of anti-CEACAM5 antibodies capable of binding to human CEACAM5 as well as cynomolgus monkey ( Macaca fascicularis ) CEACAM5 is critically important, as such antibodies can be readily tested in preclinical toxicity studies in cynomolgus monkeys to assess their safety profile. Since it has been shown that the efficacy of therapeutic antibodies can depend on the localization of the epitope within the target, both in the case of functional antibodies (Doern et al. 2009, J. Biol. Chem 284 10254) and in the case where the effector or function is involved (Beers et al. Semin Hematol 47:107-114), the human/monkey cross-reactive antibody must be shown to bind epitopes within the same repeat Ig-like homology domain of the human and cynomolgus monkey proteins.

The combination of the need for interspecies cross-reactivity of such antibodies with the specificity for human CEACAM5 andMacaca fascicularis, i.e. without cross-reactivity with other members of Macaca fascicularis and human CEACAM, adds an additional degree of complexity expressed in the overall levels of sequence homology between human andMacaca fascicularis.

Indeed, a global pairwise alignment of the Macaca fascicularis CEACAM5 sequence with the human CEACAM5 sequence (AAA51967.1/GI:180223, 702 amino acids) indicated only 78.5% identity. The Macaca fascicularis CEACAM1, CEACAM5, and CEACAM6 genes were cloned and a global alignment of the human and Macaca fascicularis A, B, and N domains was performed. This alignment predicted that there were only a few regions, if any, to localize the ideal epitope that would be common to human and macaque CEACAM5 and not shared with any other members of the family. For these reasons, developing antibodies cross-reactive between human and Macaca fascicularis CEACAM5 without cross-reactivity with other human or Macaca fascicularis CEACAM members were expected to have a low probability of success. Notably, previously described antibodies to CEACAM5 have almost never been documented to cross-react with Macaca fascicularis with very few exceptions (MT111).

Antibodies to human CEACAM5 have already been used in clinical trials, such as labetuzumab from Immunomedics (also known as hMN14, Sharkey et al, 1995, Cancer Research 55, 5935). This antibody was shown to be non-binding to cognate antigens but does not cross-react with CEACAM5 from Macaca fascicularis . Notably, the MT111 antibody from Micromet (also known as the MEDI-565 antibody from MedImmune) is a bispecific antibody that binds to human CEA-CAM5 and human CD3 (Peng et al. , PLoS ONE 7(5):e3641; WO 2007/071426). MT111 is thought to be generated by fusing a single-chain variable fragment (scFv) of an antibody that recognizes human and cynomolgus macaque CEACAM5 with an scFv of an antibody that recognizes human CD3. MT111 has also been reported not to bind other members of the CEACAM family (Peng et al. , PLoS ONE 7(5): e3641). MT111 binds to a conformational epitope in the A2 domain of human CEACAM5. This conformational epitope is missing in a splice variant of human CEACAM5 that is coexpressed alongside full-length CEACAM5 on tumors (Peng et al. , PLoS ONE 7(5): e3641). In addition, there is no evidence that MT111 binds to the same epitope in Macaca fascicularis CEACAM5.

In an attempt to obtain new antibodies to the CEACAM5 surface protein with optimal characteristics for therapeutic purposes, mice were immunized with recombinant proteins and tumor cells. Hundreds of hybridomas were screened using ELISA on several recombinant proteins of the CEACAM family and flow cytometry with appropriate cell lines to select only immunoglobulins with a preferential profile. Surprisingly, it was possible to select hybridoma clones and obtain the corresponding mature IgGs that have all the desired features. They specifically bind to the A3-B3 domain of human CEACAM5 with high affinity and do not recognize the human proteins CEACAM1, CEACAM6, CEACAM7 and CEACAM8. In a cellular environment, these antibodies show high affinity for tumor cells (in the nanomolar range). In addition, these antibodies also bind to the Macaca fascicularis CEACAM5 protein with a monkey/human affinity ratio of less than or equal to 10.

By targeting the A3-B3 domain of CEACAM5, these antibodies enhance tumor targeting potential as they have the ability to bind to both full-length human CEACAM5 and its splice variant identified by Peng et al. see

Finally, CEACAM5 is described in the literature as a poorly absorbed surface protein (reviewed in Schmidt et al, 2008, Cancer Immunol. Immunother. 57, 1879) and therefore may not be a favorable target for antibody-drug conjugates. Despite what has been reported in the prior art, the present inventors have demonstrated that antibodies prepared by them are able to absorb the CEACAM5-antibody complex after binding and induce cytotoxic activity on tumor cells in vitro in combination with a cytotoxic agent. The same antibodies in combination with a cytotoxic agent are also able to markedly suppress tumor growth in mice bearing primary human colon and gastric tumors. See WO2014079886, which is incorporated herein in its entirety.

Definitions

As used herein, the term "about" in quantitative terms refers to a value that is plus or minus 10% of the value it modifies (rounded to the nearest whole number if the value is not divisible, such as a number of molecules or nucleotides). For example, the phrase "about 100 mg" would cover 90 mg to 110 mg, inclusive, and the phrase "about 2500 mg" would cover 2250 mg to 2750 mg. When using a percentage, the term "about" refers to a value that is plus or minus 10% of that percentage. For example, the phrase "about 20%" would cover 18-22%, and the phrase "about 80%" would cover 72-88%, inclusive. In addition, where the term "approximately" is used herein in conjunction with a quantitative term, it is to be understood that in addition to the meaning of plus or minus 10%, the exact meaning of the quantitative term is also intended and described. For example, the term "approximately 23%" clearly intends, describes, and includes exactly 23%.

It should be noted that the singular form refers to one or more of such entities; for example, the term "symptom" refers to one or more symptoms. In this regard, the singular forms, the terms "one or more" and "at least one" may be used interchangeably herein.

Furthermore, the expression "and/or" as used herein is intended to specifically disclose each of the two stated features or components, with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, the term "and/or" as used in a phrase such as "A, B and/or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone) and C (alone).

It should be understood that in all cases where aspects are described in this document using the words "comprising", other similar aspects are also provided for by using the terms "consisting of" and/or "consisting essentially of".

As used herein, the term "CEACAM5" refers to "carcinoembryonic antigen-related cell adhesion molecule 5", also known as "CD66e" (cluster of differentiation 66e) or CEA. CEACAM5 is a glycoprotein involved in cell adhesion. CEACAM5 is highly expressed, particularly on the surface of colon, gastric, lung and uterine tumor cells.

The reference sequence of full-length human CEACAM5, including the signal peptide (positions 1-34) and propeptide (positions 686-702) is available from the GenBank database under the accession number AAA51967.1 (SEQ ID NO:52). Five non-synonymous SNPs were detected with a frequency higher than 2% in the Caucasian population, four of them are localized in the N domain (at positions 80, 83, 112, 113), the last one - in the A2 domain (at position 398) of human CEACAM5 (SEQ ID NO:58). GenBank accession number AAA51967.1 contains the major haplotype (I80, V83, I112, I113 and E398).

The sequence of the extracellular domain of Macaca fascicularis CEACAM5 cloned by the present inventors is disclosed under SEQ ID NO: 12. See also WO2014079886, which is incorporated herein by reference in its entirety.

SEQ ID NO:12

QLTIESRPFNVAEGKEVLLLAHNVSQNLFGYIWYKGERVDASRRIGSCVIRTQQITPGPAHSGRETIDFNASLLIQNVTQSDTGSYTIQVIKEDLVNEEATGQFRVYPELPKPYITSNNSNPIEDKDAVALTCEPETQDTTYLWWVNNQSLPVSPRLELSSDNRTLTVFNIPRNDTTSYKCETQNPVSVRRSDPVTLNVLY GPDAPTISPLNTPYRAGEYLNLTCHAASNPTAQYFWFVNGTFQQSTQELFIPNITVNNSGSYMCQAHNSATGLNRTTVTAITVYAELPKPYITSNNSNPIEDKDAVTLTCEPETQDTTYLWWVNNQ RLSVSSRLELSNDNRTLTVFNIPRNDTTFYECETQNPVSVRRSDPVTLNVLYGPDAPTISPLNTPYRAGENLNLSCHAASNPAAQYFWFVNGTFQQSTQELFIPNITVNNSGSYMCQAHNSATGLNRTTVTAITVYVELPKPYISSNNSNPIEDKDAVTLTCEPVAENTTYLWWVNNQSLSVSPRLQLSNGNRILTLLSVTRNDTGPYECGIQ NSESAKRSDPVTLNVTYGPDTPIISPPDLSYRSGANLNLSCHSDSNPSPQYSWLINGTLRQHTQVLFISKITSNNNGAYACFVSNLATGRNNSIVKNISVSSGDSAPGSSGLSA

A "domain" may be any region of a protein, typically defined on the basis of sequence homology and often associated with some structural or functional unity. Members of the CEACAM family are known to be composed of Ig-like domains. The term "domain" is used in this document to refer to individual Ig-like domains, such as the "N domain", or to a group of consecutive domains, such as the "A3-B3 domain".

The domain organization of human CEACAM5 is as follows (based on data from GenBank AAA51967.1; SEQ ID NO: 13):

SEQ ID NO: 13 MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWW VNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQN TTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLF NVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI

Table 1A. Human CEACAM5 domains

Human CEACAM5 domains Positions in SEQ ID NO: 13 Domain N 35-142 Domain A1 143-237 Domain B1 238-320 Domain A2 321-415 Domain B2 416-498 Domain A3 499-593 Domain B3 594-685

Accordingly, the A3-B3 domain of human CEACAM5 consists of amino acids 499-685 of SEQ ID NO:13.

The domain organization of Macaca fascicularis CEACAM5 is as follows (based on the cloned extracellular domain sequence; SEQ ID NO:12).

Table 1B. Macaca fascicularis CEACAM5 domains

Macaca fascicularis CEACAM5 domains Positions in SEQ ID NO: 12 Domain N-A1-B1 -1-286 Domain A2-B2 -287-464 Domain A3-B3 465-654

Accordingly, the A3-B3 domain of Macaca fascicularis CEACAM5 consists of amino acids 465-654 of SEQ ID NO:53.

A "coding sequence" or a sequence "encoding" an expression product such as an RNA, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, results in the production of an RNA, polypeptide, protein, or enzyme, i.e. The nucleotide sequence encodes the amino acid sequence for that polypeptide, protein, or enzyme. The coding sequence for a protein may contain an initiation codon (usually ATG) and a stop codon.

References to specific proteins (e.g., antibodies) may include a polypeptide comprising a native amino acid sequence, as well as variants and modified forms, regardless of their origin or method of production. A protein comprising a native amino acid sequence is a protein comprising the same amino acid sequence as that obtained in nature. Such native sequence proteins may be isolated from a natural source or may be produced by standard recombinant or synthetic methods. Native sequence proteins specifically encompass truncated and soluble forms of natural origin, variant forms of natural origin (e.g., alternatively spliced forms), allelic variants, and naturally occurring forms, including post-translational modifications. Native sequence proteins include proteins that carry post-translational modifications, such as those resulting from glycosylation, or phosphorylation, or other modifications of certain amino acid residues.

The term "gene" means a DNA sequence that codes for or corresponds to a specific sequence of amino acids that represents all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, that determine, for example, the conditions under which the gene is expressed. Some genes that are not structural genes may be transcribed from DNA into RNA but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term "gene" may be reserved for a genomic sequence that codes for a protein, i.e., sequences containing the sequences of the regulator, promoter, introns and exons.

The percentage of "sequence identity" can be determined by comparing two sequences optimally aligned within a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e. gaps) compared to a reference sequence (which does not contain any additions or deletions) to optimally align the two sequences. The percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. Optimal alignment of sequences for comparison is performed by global pairwise alignment, e.g., using the algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970). The percentage of sequence identity can be easily determined, for example, using the Needle program with the BLOSUM62 matrix and the following parameters: gap opening penalty=10, gap extension penalty=0.5.

A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue containing a side chain R group with similar chemical properties (e.g., charge, size, or hydrophobicity). In general, a conservative amino acid substitution will have little or no effect on the functional properties of the protein. Examples of amino acid groups that contain side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic side chains with hydroxyl groups: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acid substitution groups can also be defined based on amino acid size.

The present invention provides methods that comprise administering to a subject an antibody or antigen-binding fragment thereof that specifically binds to CEACAM5. As used herein, the term "hCEACAM5" means a human cytokine receptor that specifically binds human CEACAM5. In certain embodiments, the antibody that is administered to the patient specifically binds to at least one domain of hCEACAM5.

Studies were conducted to generate, screen, and select specific antibodies to mouse CEACAM5 that exhibit high affinity for both human and Macaca fascicularis CEACAM5 protein and that are not significantly cross-reactive with human CEACAM1, CEACAM6, CEACAM7, and CEACAM8 proteins and Macaca fascicularis CEACAM1, CEACAM6, and CEACAM8 proteins.

The so-called "MAb1 antibody" contains:

- a heavy chain variable domain consisting of the sequence EVMLVESGGGLVKPGGSLKLSCAAS GFTFSSYA MSWVRQTPEKRLEWVAT ISSGGSYI YYLDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYC ARPAYYGNPAMDY WGQGTSVTVSS (SEQ ID NO:14, with the CDRs shown in boldface characters), wherein FR1-H spans amino acid positions 1 to 25, CDR1-H spans amino acid positions 26 to 33, FR2-H spans amino acid positions 34 to 50, CDR2-H spans amino acid positions 51 to 58, FR3-H spans amino acid positions 59 to 96, CDR3-H spans amino acid positions 97 to 109 and FR4-H spans amino acid positions 110 to 120, and

- a light chain variable domain consisting of the sequence DILMTQSQKFMSTSVGDRVSVTCKAS QNVGTN VAWYQQKPGQSPKPLIY SAS YRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC QQYNsYPLYT FGGGTKLEIK (SEQ ID NO:15, with CDRs shown in boldface characters), wherein FR1-L spans amino acid positions 1 to 26, CDR1-L spans amino acid positions 27 to 32, FR2-L spans amino acid positions 33 to 49, CDR2-L spans amino acid positions 50 to 52, FR3-L spans amino acid positions 53 to 88, CDR3-L spans amino acid positions 89 to 98, and FR4-L spans amino acid positions 99 to 108.

The so-called "MAb2 antibody" contains:

- a heavy chain variable domain consisting of the sequence EVQLQESGGVLVKPGGSLKLSCAAS GFvFSSYD MSWVRQTPEKRLEWVAY IsSgGGiT YFPDTVQGRFTVSRDNAKNTLYLQMNSLKSEDTAIYYC aAHYFGsSGPFAY WGQGTLVTVSA (SEQ ID NO:16, with CDRs shown in boldface characters), wherein FR1-H spans amino acid positions 1 to 25, CDR1-H spans amino acid positions 26 to 33, FR2-H spans amino acid positions 34 to 50, CDR2-H spans amino acid positions 51 to 58, FR3-H spans amino acid positions 59 to 96, CDR3-H spans amino acid positions 97 to 109, and FR4-H spans amino acid positions 110 to 120, and

- a light chain variable domain consisting of the sequence DIQMTQSPASLSASVGETVTITCRAS ENIFSY LAWYQQKQGKSPQLLVY NTK TLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC QHHYGTPFT FGSGTKLEIK (SEQ ID NO:17, with CDRs shown in boldface characters), wherein FR1-L spans amino acid positions 1 to 26, CDR1-L spans amino acid positions 27 to 32, FR2-L spans amino acid positions 33 to 49, CDR2-L spans amino acid positions 50 to 52, FR3-L spans amino acid positions 53 to 88, CDR3-L spans amino acid positions 89 to 97, and FR4-L spans amino acid positions 98 to 107.

[001] A variant of the MAb2 antibody was also produced by introducing a K52R substitution into CDR2-L. This variant, referred to herein as "Mab2 _K52R ", has substantially the same affinity for human and Macaca fascicularis CEACAM5 as MAb2.

[002] The so-called "MAb3 antibody" contains:

- a heavy chain variable domain consisting of the sequence EVKLVESGGGLVKPGGSLTLPCAAS GFTFSrYA MSWVRQTPEKRLEWVAS ISSGGdt YYPDSVKGRFTVSRDNARNILFLQMSSLRSEDTGMYYC ARvnYYdssflDw WGQGTTLTVSS (SEQ ID NO:18, with CDRs shown in boldface characters), wherein FR1-H spans amino acid positions 1 to 25, CDR1-H spans amino acid positions 26 to 33, FR2-H spans amino acid positions 34 to 50, CDR2-H spans amino acid positions 51 to 57, FR3-H spans amino acid positions 58 to 95, CDR3-H spans amino acid positions 96 to 108, and FR4-H spans amino acid positions 109 to 119, And

- a light chain variable domain consisting of the sequence DIVMTQSQRFMSTLEGDRVSVTCKAS QNVGTN VAWYQQKPGQSPKALIY SAS YRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC QQYNnYPLYT FGGGTKLEIK (SEQ ID NO:19, with CDRs shown in boldface characters), wherein FR1-L spans amino acid positions 1 to 26, CDR1-L spans amino acid positions 27 to 32, FR2-L spans amino acid positions 33 to 49, CDR2-L spans amino acid positions 50 to 52, FR3-L spans amino acid positions 53 to 88, CDR3-L spans amino acid positions 89 to 98, and FR4-L spans amino acid positions 99 to 108.

[003] The so-called "MAb4 antibody" contains:

- a heavy chain variable domain consisting of the sequence EVQLVESGGGLVKPGGSLKLSCAAS GFtFSSYD MSWVRQTPEKRLEWVAF IsSyGGrT YYADTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMFYC aAHYFGtSGPFAY WGQGTLVTVSA (SEQ ID NO:20, with CDRs shown in boldface characters), wherein FR1-H spans amino acid positions 1 to 25, CDR1-H spans amino acid positions 26 to 33, FR2-H spans amino acid positions 34 to 50, CDR2-H spans amino acid positions 51 to 58, FR3-H spans amino acid positions 59 to 96, CDR3-H spans amino acid positions 97 to 109, and FR4-H spans amino acid positions 110 to 120, and

- a light chain variable domain consisting of the sequence DIQMTQSPASLSASVGETVTITCRAS ENIYSY FAWYQQKQGKSPQLLVY NaK ILAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYC QHHYGiPFT FGSGTKLELK (SEQ ID NO:21, with CDRs shown in boldface characters), wherein FR1-L spans amino acid positions 1 to 26, CDR1-L spans amino acid positions 27 to 32, FR2-L spans amino acid positions 33 to 49, CDR2-L spans amino acid positions 50 to 52, FR3-L spans amino acid positions 53 to 88, CDR3-L spans amino acid positions 89 to 97, and FR4-L spans amino acid positions 98 to 107.

[004] The so-called "MAb5 antibody" contains:

- a heavy chain variable domain consisting of the sequence ELQLVESGGVLVKPGGSLKLSCAAS GFaFSSYD MSWVRQTPEKRLEWVTY InSgGGiT YYPDTVKGRFTISRDNARNTLYLQMSSLKSEDTAIYYC tAHYFGsSGPFAY WGQGTLVTVSA (SEQ ID NO:22, with CDRs shown in boldface characters), wherein FR1-H spans amino acid positions 1 to 25, CDR1-H spans amino acid positions 26 to 33, FR2-H spans amino acid positions 34 to 50, CDR2-H spans amino acid positions 51 to 58, FR3-H spans amino acid positions 59 to 96, CDR3-H spans amino acid positions 97 to 109, and FR4-H spans amino acid positions 110 to 120, and

- a light chain variable domain consisting of the sequence DIQMTQSPASLSASVGETVTITCRAS ENIYSY LAWYQQKQGKSPQLLVY NaK TLTEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC QHHYGtPFT FGSGTKLEIK (SEQ ID NO:23, with CDRs shown in boldface characters), wherein FR1-L spans amino acid positions 1 to 26, CDR1-L spans amino acid positions 27 to 32, FR2-L spans amino acid positions 33 to 49, CDR2-L spans amino acid positions 50 to 52, FR3-L spans amino acid positions 53 to 88, CDR3-L spans amino acid positions 89 to 97, and FR4-L spans amino acid positions 98 to 107.

Numerous variations and variants of these antibodies are described in WO2014079886, which is incorporated by reference in its entirety. In an embodiment, the antibody of the present invention is the huMAb2-3 antibody or a variant thereof, i.e. an isolated antibody that binds to the A3-B3 domain of human and Macaca fascicularis CEACAM5 proteins and that contains:

a) a heavy chain consisting of the sequence of SEQ ID NO:8 or a sequence at least 85% identical thereto; or

b) a light chain consisting of the sequence of SEQ ID NO:9 or a sequence at least 85% identical thereto, or a heavy chain and a light chain.

In various embodiments, the present invention relates to an antibody that binds to human and Macaca fascicularis CEACAM5. In an embodiment, an antibody of the present invention binds to the A3-B3 domains of human and Macaca fascicularis CEACAM5. More specifically, the antibody can bind to the A3-B3 domains of human and Macaca fascicularis regardless of whether it is expressed as an isolated form, or is present as a soluble extracellular domain or membrane-anchored, full-length CEACAM5 protein.

The specificity of antibodies for the human CEACAM5 domain A3-B3 is advantageous because no SNPs have been reported with a frequency greater than 2% in the Caucasian population in this domain, minimizing the risk that the antibody epitope(s) on CEACAM5 may be altered in a subset of the population.

According to an embodiment, the antibody of the present invention is specific for the surface proteins CEACAM5 of human and Macaca fascicularis . In an embodiment, the antibody of the present invention does not bind to or is not significantly cross-reactive with the proteins CEACAM1 of human, CEACAM6 of human, CEACAM7 of human, CEACAM8 of human, CEACAM1 of Macaca fascicularis , CEACAM6 of Macaca fascicularis and CEACAM8 of Macaca fascicularis .

In various embodiments, the antibody does not bind to or is not significantly cross-reactive with the extracellular domain of the aforementioned human and Macaca fascicularis CEACAM proteins.

The full-length human CEACAM1 protein is available in GenBank under accession number NP_001703.2 The extracellular domain of human CEACAM1 consists of amino acids at positions 35-428 of this protein. The full-length human CEACAM6 protein is available in GenBank under accession number NP_002474.3 The extracellular domain of human CEACAM6 consists of amino acids at positions 35-327 of this protein.

The full-length human CEACAM7 protein is available in the GenBank database under accession number NP_008821.1 The extracellular domain of human CEACAM7 consists of amino acids at positions 36–248 of the protein.

The full-length human CEACAM8 protein is available in the GenBank database under accession number NP_001807.2 The extracellular domain of human CEACAM8 consists of amino acids at positions 35–332 of the protein.

Extracellular domain of CEACAM1M. fascicularisconsists of amino acids in positions 35-428 of the full-length protein, i.e. amino acids 1-394 of protein.

Extracellular domain of CEACAM6M. fascicularisconsists of amino acids in positions 35-327 of the full-length protein, i.e. amino acids 1-293 of protein.

Extracellular domain of CEACAM8M. fascicularisconsists of amino acids in positions 35-332 of the full-length protein, i.e. amino acids 1-298 of protein.

The term "antibody" as used herein refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, linked together by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FR of the antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be altered naturally or artificially. The amino acid consensus sequence may be determined based on a side-by-side analysis of two or more CDRs.

The term "antibody" as used herein also includes antigen-binding fragments of entire antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. as used herein include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody can be produced, for example, from whole antibody molecules by any suitable standard techniques such as proteolytic cleavage or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the variable and optionally constant domains of antibodies. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or it can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains in a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv molecules (scFv); (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementarity determining region (CDR) such as a CDR3 peptide or a peptide with limited conformational freedom FR3-CDR3-FR4. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies), small sized modular protein (SMIP) based immunopharmaceuticals and shark IgNAR variable domains are also included in the expression "antigen binding moiety" as used herein.

An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will typically comprise at least one CDR that is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments comprising a VH domain linked to a VL domain, the VH and VL domains may be arranged relative to one another in any suitable order. For example, the variable region may be dimeric and comprise VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may comprise a monomeric VH or VL domain.

In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3 and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked via an entire or partial hinge or linker region. The hinge region in various embodiments may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in the formation of a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of an antibody in various embodiments may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric VH or VL domains (e.g., using disulfide bond(s).

In particular embodiments, an antibody or antibody fragment for use in the method of the present invention may be a multispecific antibody, which may be specific for different epitopes of a single target polypeptide or may comprise antigen-binding domains specific for epitopes of more than one target polypeptide. An exemplary antibody in a bispecific format that may be used in the context of the present invention includes the use of a C _H3 domain of a first immunoglobulin (Ig) and a C _H3 domain of a second immunoglobulin Ig, wherein the C _H3 domains of the first and second Ig differ from each other by at least one amino acid, and wherein the difference of at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody in which there is no amino acid difference. In one embodiment, the first Ig _CH3 domain binds protein A and the second Ig _CH3 domain comprises a mutation that reduces or eliminates binding to protein A, such as the H95R modification (by IMGT exon numbering; H435R by EU numbering). The second _CH3 may further comprise the Y96F modification (by IMGT; Y436F by EU). Additional modifications that may be identified in the second _CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I by EU) for IgG2 antibodies and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) for IgG4 antibodies. Variations of the bispecific antibody format described above are contemplated within the scope of the present invention. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, in various embodiments can be adapted for use in the context of an antigen-binding fragment of an anti-CEACAM5 antibody using standard techniques available in the art.

The CEACAM5 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the variable heavy and light chain domains compared to the corresponding germline sequences. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and/or CDR regions are backmutated to the corresponding germline residue(s) or to a conservative amino acid substitution (naturally occurring or non-naturally occurring) of the corresponding germline residue(s) (such sequence changes are referred to herein as "germline backmutations"). One skilled in the art, starting from the heavy and light chain variable region sequences disclosed herein, can readily produce a variety of antibodies and antigen-binding fragments that comprise one or more individual germline backmutations or combinations thereof. In certain embodiments, all framework residues and/or CDR residues in the VH and/or VL domains are backmutated to the germline sequence. In other embodiments, only certain residues are backmutated to the germline sequence, such as only mutated residues found in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2 or CDR3. Additionally, the antibodies of the present invention may comprise any combination of two or more germline backmutations within the framework and/or CDR regions, i.e. wherein certain individual residues are backmutated to the germline sequence, while certain other residues that differ from the germline sequence are maintained. Once produced, antibodies and antigen-binding fragments that contain one or more germline backmutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced biological antagonist or agonist properties (if desired), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general method are encompassed by the present invention.

The constant region of an antibody is important in terms of the ability of the antibody to bind complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody can be selected based on whether the antibody is required to mediate cytotoxicity.

The term "human antibody" as used herein is intended to include antibodies comprising variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention, in various embodiments, may further comprise amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesisin vitroor somatic mutationin vivo), such as in the CDRs and, in some embodiments, in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

The term "recombinant human antibody" as used herein is intended to include all human antibodies that are produced, expressed, created, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a combinatorial library of recombinant human antibodies (described further below), antibodies isolated from an animal (e.g., mice), which is transgenic for human immunoglobulin genes (see, for example, Tayloret al.(1992) Nucl. Acids Res. 20:6287-6295, incorporated herein by reference in its entirety), or antibodies produced, expressed, generated, or isolated by any other means that involves splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are mutagenizedin vitro(or, if an animal transgenic for human Ig sequences is used, then somatic mutagenesisin vivo) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies represent sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist within the human germline antibody repertoire.in vivo.

Human antibodies may occur in two forms, due to the heterogeneity of the hinge regions. In one embodiment, the immunoglobulin molecule comprises a stable four-chain structure of approximately 150-160 kDa, in which the dimers are held together by an interchain disulfide bond that links the heavy chains. In another embodiment, the dimers are not linked by interchain disulfide bonds, and a molecule of approximately 75-80 kDa is formed, consisting of covalently linked light and heavy chains (a half-antibody). These embodiments/forms are extremely difficult to separate, even after affinity purification.

The term "humanized antibody" refers to an antibody that is not of human origin, in whole or in part, and that has been modified to replace certain amino acids, such as in the framework regions of the VH and VL domains, to avoid or minimize the immune response in humans. The constant domains of a humanized antibody are, in most cases, the human CH and CL domains.

Numerous methods for humanizing an antibody sequence are known in the art; see, for example, reviewed in Almagro & Fransson (2008) Front Biosci. 13: 1619–1633. One widely used technique is CDR grafting, or antibody reengineering, which involves grafting CDR sequences from a donor-type antibody, typically a murine antibody, into the framework region backbone of a human antibody with differing specificity. Since CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of the CDR-grafted non-human antibody, backmutations can be introduced at selected positions of the CDR-grafted antibody to maintain the specificity and binding affinity of the original antibody. Identification of positions for possible backmutations can be accomplished using information available in the literature and in antibody databases. The amino acid residues that are candidates for backmutation are typically those that are exposed to the surface of the antibody molecule, while residues that are buried or have low surface exposure will typically not be altered. An alternative humanization technique to CDR grafting and backmutation is surface alteration, in which non-surface residues of non-human origin are retained while surface residues are altered to human residues. Another alternative technique is known as "directed selection" (Jesperset al.(1994) Biotechnology 12, 899), and can be used to produce a fully human antibody from a mouse antibody while maintaining the epitope and binding characteristics of the original antibody.

The frequency of occurrence of the second form in different intact IgG isotypes is due, without limitation, to structural differences associated with the isotype of the antibody hinge region. A single amino acid substitution in the hinge region of human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. , (1993) Molecular Immunology 30:105, incorporated by reference in its entirety) to levels typically observed with human IgG1 hinge. The present invention encompasses, in various embodiments, antibodies with one or more mutations in the hinge, CH2, or CH3 region that may be necessary, for example, in production, to increase the yield of the desired antibody form.

The term "isolated antibody" as used herein means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody is originally present or naturally produced, is an "isolated antibody." In various embodiments, an isolated antibody also includes an antibody in situ in a recombinant cell. In other embodiments, isolated antibodies are antibodies that have undergone at least one purification or isolation step. In various embodiments, an isolated antibody may be substantially free of other cellular material and/or other chemical compounds.

The term "specifically binds" etc. means that the antibody or its antigen-binding fragment forms a complex with the antigen that is relatively stable under physiological conditions. Methods for determining the presence of specific binding of an antibody to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds" CEACAM5 as used herein includes antibodies that bind CEACAM5 or a portion thereof with a KD of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured in a surface plasmon resonance assay. Specific binding can also be characterized by a dissociation constant of at least about 1 x 10 ^-6 M or less. In other embodiments, the dissociation constant is at least about 1 x 10 ^-7 M, 1 x 10 ^-8 M, or 1 x 10 ^-9 M. An isolated antibody that specifically binds human CEACAM5 may, however, be cross-reactive to other antigens, such as CEACAM5 molecules from other (non-human) species.

The term "surface plasmon resonance" as used in this document refers to an optical phenomenon that enables real-time analysis of interactions by detecting changes in protein concentrations in a biosensor array, such as those provided by the BIACORE system (Biacore Life Sciences Division of GE Healthcare, Piscataway, NJ).

The term "KD" as used in this document is intended to refer to the equilibrium dissociation constant of the interaction between antibody and antigen.

Theoretically, "affinity" is defined by the equilibrium binding between the whole antibody and the antigen. It can be estimated experimentally by a variety of known methods, such as measuring the rate of association and dissociation using surface plasmon resonance or measuring the EC50 (or apparent KD) in an immunoassay (ELISA, FACS). In these assays, the EC50 is the concentration of antibody that induces a response that is midway between the response at baseline and the maximal response after a specified time of exposure to a specified concentration of antigen by ELISA or cell expression of antigen by FACS.

Binding of a monoclonal antibody to antigen 1 (Ag1) is "cross-reactive" with antigen 2 (Ag2) when the EC50s are in a similar range for both antigens. In this application, binding of a monoclonal antibody to Ag1 is cross-reactive with Ag2 when the ratio of affinity for Ag2 to affinity for Ag1 is 10 or less (e.g., 5, 2, 1, or 0.5), wherein the affinity values are measured by the same method for both antigens.

Affinity for human CEACAM5 or Macaca fascicularis CEACAM5 can be determined as the EC50 value in an ELISA using a soluble recombinant form of CEACAM5 as the capture antigen.

The antibody of the present invention may also have an apparent dissociation constant (apparent KD), which can be determined by FACS analysis on the MKN45 tumor cell line (DSMZ, ACC 409) or patient-derived tumor cell xenografts (CR-IGR-034P available from Oncodesign Biotechnology, CReMEC Tumor Collection), which is ≤25 nM, such as ≤20 nM, ≤10 nM, ≤5 nM, ≤3 nM, or ≤1 nM. The apparent KD may be in the range of 0.01-20 nM, or may be in the range of 0.1-20 nM, 0.1-10 nM, or 0.1-5 nM.

Additionally, the antibodies of the present invention have been shown to be able to detect CEACAM5 expression in frozen, formalin-fixed, paraffin-embedded (FFPE) tissue sections using immunohistochemistry.

The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and may exert different biological effects. Epitopes may be conformational or linear. A conformational epitope is formed by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. Under certain conditions, an epitope may include fragments of saccharides, phosphoryl groups, or sulfonyl groups of the antigen.

Anti-CEACAM5 antibodies useful for the methods described herein may, in various embodiments, include one or more amino acid substitutions, insertions, and/or deletions in the framework regions and/or CDR regions of the variable domains of the heavy and light chains, compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. In various embodiments, the present invention includes methods comprising the use of antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated relative to the corresponding residue(s) of the germline sequence from which the antibody is derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). Numerous antibodies and antigen-binding fragments can be engineered that contain one or more individual germline mutations, or combinations thereof. In certain embodiments, all framework and/or CDR residues in the VH and/or VL domains are backmutated to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, such as only residues located in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4 are mutated, or only residues located in CDR1, CDR2, or CDR3 are mutated. In other embodiments, one or more framework region and/or CDR residues are mutated to the corresponding residue(s) of a different germline sequence (i.e., germline sequence that is different from the germline sequence from which the antibody was originally derived). Additionally, antibodies may contain any combination of two or more germline mutations in the framework regions and/or CDR regions, such as wherein certain individual residues are mutated to the corresponding residue of a certain germline sequence, while certain other residues that differ from the original germline sequence are retained or mutated to the corresponding residue of a different germline sequence. Once produced, antibodies and antigen-binding fragments that contain one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced biological antagonist or agonist properties (if desired), reduced immunogenicity, etc. The present invention encompasses the use of antibodies and antigen-binding fragments produced by this general method.

The present invention also includes methods comprising using antibodies to CEACAM5 comprising variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention includes using antibodies to CEACAM5 with HCVR, LCVR and/or CDR amino acid sequences such as, with 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

According to the present invention, in various embodiments, an anti-CEACAM5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and/or complementarity determining regions (CDRs) comprising any of the amino acid sequences of anti-CEACAM5 antibodies described in Intl. Patent Pub. No. WO 2014/079886 A1, incorporated herein by reference in its entirety.

Modification(s) of the amino acid sequence of the antibodies described herein are contemplated. For example, it may be necessary to enhance the binding affinity and/or other biological properties of the antibody. It is known that when a humanized antibody is produced by simply grafting only the CDRs of the VH and VL of an antibody derived from a non-human animal into the FRs of the VH and VL of a human antibody, the antigen-binding activity may be reduced compared to that of the original antibody derived from a non-human animal. It is believed that several amino acid residues of the VH and VL of a non-human antibody, not only in the CDRs but also in the FRs, may be directly or indirectly associated with the antigen-binding activity. Therefore, replacing these amino acid residues with other amino acid residues derived from the FRs of the VH and VL of a human antibody will reduce the binding activity. To address the problem of human antibodies grafted with non-human CDRs, attempts have been made to identify, among the amino acid sequences of the FRs of VH and VL human antibodies, an amino acid residue that is directly associated with antibody binding, or that interacts with a CDR amino acid residue, or that maintains the three-dimensional structure of the antibody and that is directly associated with antigen binding. Reduced antigen-binding activity can be improved by replacing the identified amino acids with amino acid residues of the parent antibody obtained from a non-human animal.

It is possible to make modifications and changes in the structure of antibodies in this way

the invention and the DNA sequences encoding them, and still obtain functional antibodies or polypeptides with the desired characteristics.

A further object of the present invention also encompasses variants with conserved function of the polypeptides of the present invention. For example, certain amino acids can be replaced by other amino acids in the protein structure without significant loss of activity. Since the interaction ability and nature of a protein determine its biological functional activity, it is possible to make certain amino acid substitutions in the protein sequence and, of course, in its coding DNA sequence, while still obtaining a protein with similar properties. Thus, it is envisaged that various changes can be made in the sequences of the antibodies of the present invention or the corresponding DNA sequences that code for said polypeptides, without significant loss of their biological activity. It is known from the prior art that certain amino acids can be replaced by other amino acids characterized by a similar hydropathicity index or score, and still obtain a protein with similar biological activity, i.e. still obtain a protein with equivalent biological function. It is also possible to use well-known technologies, such as alanine scanning approaches, to identify in an antibody or polypeptide of the present invention all amino acids that can be replaced without significant loss of antigen binding. Such residues can be characterized as neutral, since they are not involved in antigen binding or in maintaining the structure of the antibody. One or more of these neutral positions can be substituted with alanine or another amino acid without changing the essential characteristics of the antibody or polypeptide of the present invention.

Neutral positions can be observed as positions where any amino acid substitution can be included in the antibodies. Indeed, in the alanine scanning principle, alanine is selected because its residue has no specific structural or chemical features. It is generally accepted that if alanine can be replaced by a specific amino acid without changing the properties of the protein, many other, if not all, amino acid substitutions are likely to be neutral as well. In the opposite case, where alanine is a wild-type amino acid, if a specific substitution can be shown to be neutral, it is likely that other substitutions will also be neutral. Therefore, as indicated above, amino acid substitutions are typically based on the relative similarity of the substituents in the amino acid side chain, such as their hydrophobicity, hydrophilicity, charge, size, etc. Illustrative substitutions that take into account any of the above features are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

It may also be necessary to modify the antibody of the present invention taking into account the effector function, for example, to enhance the antigen-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively or additionally, cysteine residue(s) can be introduced into the Fc region, thereby allowing for the formation of an interchain disulfide bond in this region. The homodimeric antibody thus obtained can be characterized by improved internalization capacity and/or increased complement-mediated cell killing and/or increased antibody-dependent cellular cytotoxicity (ADCC) (Caron, P.C.et al.1992; and Shopes B. 1992).

Another type of amino acid modification of the antibody of the present invention may be useful for altering the original glycosylation profile of the antibody, i.e. by removing one or more carbohydrate moieties present in the antibody and/or by adding one or more glycosylation sites that are not present in the antibody. The presence of one of the tripeptide sequences: asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, creates a potential glycosylation site. The addition or deletion of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).

Another type of modification involves the removal of sequences identified either in silico or experimentally as potentially leading to the formation of degradation products or heterogeneity in antibody-based products. As examples, deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface accessibility. Asparagine residues are particularly susceptible to deamidation, primarily when found in the Asn-Gly sequence, and to a lesser extent in other dipeptide sequences such as Asn-Ala. If such a deamidation site, particularly Asn-Gly, is present in an antibody or polypeptide of the present invention, it may therefore be necessary to remove the site, typically by a conservative substitution to remove one of the residues involved. Such substitutions in the sequence to remove one or more of the residues involved are also intended to be encompassed by the present invention.

Another type of covalent modification involves the coupling of glycosides to the antibody by chemical or enzymatic means. These procedures have the advantage that they do not require the production of antibodies in a host cell that has the ability to glycosylate for the purpose of N- or O-linked glycosylation. Depending on the coupling mode employed, the sugar(s) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in WO87/05330.

Removal of any carbohydrate moieties present in the antibody can be achieved chemically or enzymatically. In chemical deglycosylation, the antibody is treated with trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all of the sugars except the cross-linking sugars (N-acetyl glucosamine or N-acetyl galactosamine), while leaving the antibody intact. Chemical deglycosylation is described in Sojahr H. et al. (1987) and in Edge, AS. et al. (1981). Enzymatic cleavage of carbohydrate moieties of antibodies can be achieved using various endo- and exoglycosidases described in Thotakura, NR. et al. (1987).

Another type of covalent modification of an antibody involves linking the antibody to one of a variety of non-protein polymers, such as with polyethylene glycol, polypropylene glycol or polyoxyalkylenes, using the methods set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

In an embodiment, the antibody of the present invention is the huMAb2-3 antibody or a variant thereof. The various amino acid sequences for the huMAb2-3 antibody are shown below under SEQ ID NOs: 1-9.

The amino acid sequence of the heavy chain variable domain under SEQ ID NO: 1 is

EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSS.

The amino acid sequence of the light chain variable domain under SEQ ID NO: 2 is

DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIK.

The CDR sequences SEQ ID NO: 1 and 2 are listed below and cover SEQ ID NO: 3-7.

The amino acid sequence of SEQ ID NO: 3 is GFVFSSYD.

The amino acid sequence of SEQ ID NO: 4 is ISSGGGIT.

The amino acid sequence of SEQ ID NO: 5 is AAHYFGSSGPFAY.

The amino acid sequence under SEQ ID NO: 6 is ENIFSY.

The amino acid sequence of light chain CDR2 is the NTR.

The amino acid sequence of SEQ ID NO: 7 is QHHYGTPFT.

The amino acid sequence of the heavy chain under SEQ ID NO: 8 is

EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSS GPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

The amino acid sequence of the light chain under SEQ ID NO: 9 is

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQQANSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC.

According to the present invention, the antibodies disclosed herein bind to an epitope of the A3-B3 domain of the human CEACAM5 protein. The amino acid sequences of the epitopes of the A3-B3 domain bound by the antibodies of the present invention include SGANLNL (SEQ ID NO: 10) and INGIPQQHTQVLF (SEQ ID NO: 11).

The term "biological equivalent" as used herein refers to a molecule that exhibits similar bioavailability (rate and extent of availability) after administration at the same molar dose and under similar conditions (e.g. the same route of administration), so that, taking into account both efficacy and safety, the expected effect is essentially the same as that of the compared molecule. Two pharmaceutical compositions containing an antibody to CEACAM5 or an immunoconjugate containing the antibody are biologically equivalent if they are pharmaceutically equivalent, i.e. this means that they contain the same amount of active ingredient in the same dosage form, by the same route of administration, and meet the same or comparable standards.

The present invention, in certain embodiments, relates to methods comprising administering to a subject an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 1 and a light chain variable region comprising the sequence of SEQ ID NO: 2.

The present invention provides pharmaceutical compositions containing such an antibody and methods of using these compositions.

In various embodiments, the antibody comprises a CDR in the heavy chain variable region/domain of SEQ ID NO: 1 and a CDR in the light chain variable region/domain of SEQ ID NO: 2. In various embodiments, an antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region comprising the sequence of SEQ ID NO: 2 is an antibody that specifically binds CEACAM5. See International Publication Number WO 2014/079886 A1, incorporated herein by reference in its entirety. In an embodiment, the antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 8 and a light chain variable region comprising the sequence of SEQ ID NO: 9.

Immunoconjugates

The present invention also includes cytotoxic conjugates, or immunoconjugates, or antibody-drug conjugates, or conjugates. As used herein, all of these terms have the same meaning and are used interchangeably.

Accordingly, the present invention provides "immunoconjugates" comprising an antibody of the present invention linked or conjugated to at least one growth inhibitory agent, such as a cytotoxic agent or a radioactive isotope.

The expressions "growth inhibitory agent" or "antiproliferative agent", which may be used interchangeably, refer to a compound or composition that inhibits the growth of a cell, especially a tumor cell, in vitro or in vivo .

The term "cytotoxic agent" as used herein refers to a substance that inhibits or blocks cell function and/or causes cell destruction. The term "cytotoxic agent" is intended to include chemotherapeutic agents, enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and/or variants thereof, and various antineoplastic or anticancer agents disclosed below. In some embodiments, the cytotoxic agent is a taxoid, a vinca alkaloid, a maytansinoid or a maytansinoid analog such as DM1 or DM4, a small molecule drug, a tomaimycin or pyrrole benzodiazepine derivative, an auristatin or dolastatin analog, a prodrug, a topoisomerase II inhibitor, a DNA alkylating agent, an antitubulin agent, a CC-1065 analog, or CC-1065.

As used in this document, the term "maytansinoids" refers to maytansinoids and maytansinoid analogues. Maytansinoids are drugs that inhibit microtubule formation and are highly toxic to mammalian cells.

Examples of suitable maytansinoids include maytansinol and maytansinol analogues.

Examples of suitable maytansinol analogs include those having a modified aromatic ring and those having modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; and 5,846,545.

Specific examples of suitable maytansinol analogues having a modified aromatic ring include:

(1) C-19-dechloro derivative (US Patent No. 4,256,746) (prepared by reduction of ansamitocin P2 with LAH);

(2) C-20-hydroxy derivative (or C-20-demethyl derivative) +/-C-19-dechloro derivative (U.S. Patent Nos. 4,361,650 and 4,307,016) (obtained by demethylation with Streptomyces or Actinomyces or dechlorination with LAH); and

(3) C-20-demethoxy derivative, C-20-acyloxy derivative (-OCOR), +/-dechloro derivative (US Patent No. 4,294,757) (obtained by acylation with acid chlorides).

Specific examples of suitable maytansinol analogues having modifications at other positions include:

(1) C-9-SH derivative (US Patent No. 4,424,219) (obtained by reaction of maytansinol with H ₂ S or P ₂ S ₅ );

(2) C-14-alkoxymethyl (demethoxy/CH ₂ OR) derivative (US Patent No. 4,331,598);

(3) C-14-hydroxymethyl or acyloxymethyl ( _CH2OH or _CH2OAc ) derivative (US Patent No. 4,450,254) (obtained from Nocardia );

(4) C-15-hydroxy/acyloxy derivative (US Patent No. 4,364,866) (obtained by conversion of maytansinol by Streptomyces );

(5) C-15-methoxy derivative (US Patent Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudiflora );

(6) C-18- N -demethyl derivative (U.S. Patent Nos. 4,362,663 and 4,322,348) (obtained by demethylation of maytansinol using Streptomyces ); and

(7) 4,5-deoxy derivative (US Patent No. 4,371,533) (prepared by reduction of maytansinol with titanium trichloride/LAH).

In an embodiment of the present invention, the cytotoxic conjugates of the present invention employ a thiol-containing maytansinoid (DM1), formally named N2' ^- deacetyl- N2 ^' -(3-mercapto-1-oxopropyl)-maytansine, as a cytotoxic agent. DM1 is represented by the following structural formula (I):

In another embodiment, the cytotoxic conjugates of the present invention employ the thiol-containing maytansinoid DM4, formally named N2' ^- deacetyl- N2 ^' -(4-methyl-4-mercapto-1-oxopentyl)-maytansine, as the cytotoxic agent. DM4 is represented by the following structural formula (II):

In additional embodiments of the present invention, other maytansines can be used, including thiol- and disulfide-containing maytansinoids that have a mono- or dialkyl substitution of the carbon atom bearing the sulfur atom. These include a maytansinoid having at C-3 C-14-hydroxymethyl, C-15-hydroxy or C-20-desmethyl, an acylated amino acid side chain with an acyl group bearing a sterically hindered sulfhydryl group, wherein the carbon atom of the acyl group bearing the thiol functionality has two or three substituents, wherein said _substituents are _CH3 , _C2H5 , linear or branched alkyl or alkenyl having from 1 to 10 reactants, and any aggregate that may be present in solution.

Examples of these cytotoxic agents and conjugation methods are further provided in WO2008/010101, which is incorporated by reference.

The term "radioactive isotope" is intended to include radioactive isotopes suitable for treating cancer, such as At ²¹¹ , Bi ²¹² , Er ¹⁶⁹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , In ¹¹¹ , P ³² , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Sr ⁸⁹ and radioactive isotopes of Lu. Such radioactive isotopes typically emit predominantly beta radiation. In an embodiment, the radioactive isotope is an alpha-emitting isotope, more particularly thorium 227, which emits alpha radiation. Immunoconjugates according to the present invention can be prepared as described in WO2004/091668.

In various embodiments, the antibodies of the present invention are covalently attached, directly or via a cleavable or non-cleavable linker, to at least one growth inhibitory agent.

"Linker" as used herein means a chemical moiety that contains a covalent bond or chain of atoms that covalently attaches a polypeptide to a drug moiety.

Conjugates can be prepared using in vitro methods. A linking group is used to link the drug or prodrug to the antibody. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. Conjugation of the antibody of the present invention to cytotoxic or growth inhibitory agents can be accomplished using a variety of bifunctional protein coupling agents including, but not limited to, N-succinimidyl pyridyl dithiobutyrate (SPDB), 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl butanoate ester (nitro-SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfobutyric acid (sulfo-SPDB), N-succinimidyl (2-pyridyl dithio) propionate (SPDP), succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate-HCL), active esters esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate) and bisfluoro compounds of active fluorine (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al. (1987). Carbon-labeled 1-isothiocyanatobenzyl-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating a radionucleotide to an antibody (WO 94/11026).

The linker may be a "cleavable linker" that facilitates the release of the cytotoxic or growth inhibitory agent into the cell. For example, an acid-labile linker, a peptidase-labile linker, an esterase-labile linker, a photolabile linker, or a disulfide-containing linker may be used (see, e.g., (US Patent No. 5,208,020). The linker may also be a "non-cleavable linker" (e.g., an SMCC linker), which may in some cases lead to better tolerability.

Alternatively, a fusion protein comprising an antibody of the present invention and a cytotoxic or growth-suppressing polypeptide may be produced by recombinant techniques or peptide synthesis. The DNA may contain in its length the corresponding regions encoding the two parts of the conjugate adjacent to each other or separated by a region encoding a linker peptide that does not deprive the conjugate of the desired properties.

The antibodies of the present invention can be used in enzyme-mediated prodrug dependent therapy by conjugating the polypeptide to a prodrug activating enzyme that converts the prodrug (e.g., peptidyl chemotherapeutic agent, see WO81/01145) into an active anticancer drug (see, for example, WO88/07378 and U.S. Patent No. 4,975,278). The enzyme component of an immunoconjugate useful in ADEPT includes any enzyme capable of acting on a prodrug so as to convert it into its more active cytotoxic form. Enzymes useful in the method of the present invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic fluorocytosine into the anticancer drug 5-fluorouracil; proteases such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L) useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases useful for converting prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes such as O-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; P-lactamase useful for converting drugs derivatized with P-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogen atoms with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. The enzymes can be covalently linked to the polypeptides of the present invention using techniques well known in the art, such as the use of heterobifunctional crosslinking reagents discussed above.

According to an embodiment, in the conjugate of the present invention, the growth inhibitory agent may be a maytansinoid, in an embodiment DM1 or DM4.

In said conjugate, the antibody is conjugated to said at least one growth inhibitory agent using linking groups. In an embodiment, said linking group is a cleavable or non-cleavable linker, such as SPDB, sulfo-SPDB or SMCC.

The conjugate can be selected from the group consisting of:

antibody-SPDB-DM4 conjugate of formula (III):

(III);

antibody-sulfo-SPDB-DM4 conjugate of formula (IV)

(IV);

And

antibody-SMCC-DM1 conjugate of formula (V)

(V).

In an embodiment, the conjugate is a conjugate of formula (III), (IV) or (V) as defined above, wherein the antibody is an antibody as described herein.

Typically, the conjugate can be prepared by a method comprising the steps of:

(i) providing an optionally buffered aqueous solution of a cell binding agent (e.g. antibodies according to the present invention), in contact with solutions of the linker and the cytotoxic compound;

(ii) subsequently optionally separating the conjugate formed in (i) from the unreacted cell binding agent.

The aqueous solution of the cell binding agent can be buffered with buffers such as, for example, potassium phosphate, acetate, citrate, or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer depends on the nature of the cell-binding agent. The cytotoxic compound is in solution in an organic polar solvent, such as dimethyl sulfoxide (DMSO) or dimethyl acetamide (DMA).

The reaction temperature is usually between 20 and 40°C. The reaction time can vary from 1 to 24 hours. The reaction between the cell-binding agent and the cytotoxic agent can be monitored by size-exclusion chromatography (SEC) with refractometric and/or UV detection. If the conjugate yield is too low, the reaction time can be increased.

A person skilled in the art may use a number of different chromatographic techniques to carry out the separation in step (ii): the conjugate may be purified, for example, by SEC, adsorption chromatography (such as ion exchange chromatography, IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed media chromatography such as hydroxyapatite chromatography, or high performance liquid chromatography (HPLC). Purification by dialysis or diafiltration may also be used.

As used herein, the term "aggregates" refers to the aggregations that two or more cell-binding agents may form, whether or not the agents have been modified by conjugation. Aggregates can be formed under the influence of a wide variety of parameters, such as high concentration of cell-binding agent in solution, pH of the solution, high shear, the number of associated dimers and their hydrophobic nature, and temperature (see Wang and Gosh (2008) J. Membrane Sci. 318: 311-316 and references cited therein); it should be noted that the relative influence of some of these parameters is not well established. In the case of proteins and antibodies, one skilled in the art is referred to Cromwell et al. (2006) AAPS Journal , 8(3): E572-E579). The content of aggregates can be determined using techniques well known to those skilled in the art, such as SEC (see Walter et al ., 1993, Anal. Biochem ., 212(2): 469-480).

After step (i) or (ii), the solution containing the conjugate may be subjected to an additional step (iii) of chromatography, ultrafiltration and/or diafiltration.

The conjugate is recovered at the end of these steps in aqueous solution.

According to an embodiment, the conjugate according to the present invention has a "drug to antibody ratio" (or "DAR") in the range of 1 to 10, such as 2 to 5, in particular 3 to 4. This is typically the case where the conjugates comprise maytansinoid molecules.

This DAR may vary depending on the nature of the antibody and drug (i.e. growth inhibitory agent) applied together with the experimental conditions applied for conjugation (such as the growth inhibitory agent/antibody ratio, reaction time, nature of the solvent and co-solvent, if any). Thus, contact between the antibody and the growth inhibitory agent results in the formation of a mixture containing several conjugates, differing from each other by different ratios of drug to antibody; not necessarily "naked" antibody, not necessarily aggregates. The DAR determined is thus an average value.

A method that can be used to determine DAR is the spectrophotometric measurement of the absorbance ratio of a solution of substantially purified conjugate at λ_Dand 280 nm. 280 nm is a wavelength commonly used to measure protein concentration, such as antibody concentration. Wavelength λ_Dis chosen in such a way as to ensure that distinctions are made between the drug and the antibody, i.e., as is well known to those skilled in the art, λ_Dis the wavelength at which the drug exhibits high absorption, and the λ value_Dsufficiently distant from 280 nm to avoid significant overlap of the absorption peaks of the drug and antibody. In the case of maytansinoid molecules, λ can be chosen_D, which is 252 nm. The method for calculating DAR can be obtained from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science.

The absorption values for the conjugate at λ _D (A _λD ) and at 280 nm (A ₂₈₀ ) are measured either on the monomer peaks of size exclusion chromatography (SEC) analysis (providing the calculation of the parameter "DAR(SEC)") or using classical spectrophotometric apparatus (providing the calculation of the parameter "DAR(UV)"). The absorption values can be expressed as follows:

A _λD = (c _D x ε _DλD ) + (c _A x ε _AλD )

A ₂₈₀ = (c _D x ε _D280 ) + (c _A x ε _A280 ),

Where:

c _D and c _A represent, respectively, the concentration values of the drug and antibody in the solution,

ε _DλD and ε _D280 are, respectively, the molar extinction coefficients of the drug at λ _D and 280 nm,

ε _AλD and ε _A280 are the molar extinction coefficients of the antibody at λ _D and 280 nm, respectively.

Solving these two equations with two unknowns leads to the following equations:

c _D = [(ε _A280 x A _λD ) - (ε _AλD x A ₂₈₀ )] / [(ε _DλD x ε _A280 ) - (ε _AλD x ε _D280 )]

c _A = [A ₂₈₀ - (c _D x ε _D280 )] / ε _A280 .

The mean DAR is then calculated from the ratio of drug concentration to antibody concentration: DAR=c _D /c _A .

Pharmaceutical compositions

The antibodies and immunoconjugates of the present invention can be combined with pharmaceutically acceptable excipients and optionally sustained release matrices such as biodegradable polymers to form therapeutic compositions.

Thus, another object of the present invention relates to a pharmaceutical composition comprising an antibody or immunoconjugate of the present invention and a pharmaceutically acceptable carrier or excipient.

The present invention also relates to a polypeptide or immunoconjugate according to the present invention for use as a medicinal product.

"Pharmaceutical" or "pharmaceutically acceptable" are molecular entities and compositions that do not cause an adverse, allergic or other undesirable reaction when administered to a mammal, in particular a human, as appropriate. A pharmaceutically acceptable carrier or excipient is a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation excipient of any type.

As used herein, "pharmaceutically acceptable carriers" include all possible solvents, dispersion media, coatings, antibacterial and antifungal agents, etc., which are physiologically compatible. Examples of suitable carriers, diluents, and/or excipients include one or more of water, amino acids, saline, phosphate buffered saline, phosphate buffer, acetate, citrate, succinate; amino acids and derivatives such as histidine, arginine, glycine, proline, glycyl-glycine; inorganic salts of NaCl, calcium chloride; sugars and polyhydric alcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as Polysorbate 80, Polysorbate 20, Poloxamer 188; and the like, as well as combinations thereof. In many cases, it will be preferable to include in the composition isotonic agents such as sugars, polyhydric alcohols or sodium chloride, and the composition may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20.

The form of pharmaceutical compositions, route of administration, dosage and regimen normally depend on the condition to be treated, the severity of the disease, the age, weight and gender of the patient, etc.

The pharmaceutical compositions of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, etc.

In an embodiment, the pharmaceutical compositions contain carrier media that are pharmaceutically acceptable for the composition to be injected. They can be isotonic sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts) or dry, in particular lyophilized compositions that, upon addition, as the case may be, of sterile water or physiological solution, provide injection solutions.

The pharmaceutical composition can be administered using drug combination devices.

The doses used for administration can be adapted depending on various parameters and, for example, depending on the route of administration used, the pathology in question or, alternatively, the required duration of treatment.

To prepare pharmaceutical compositions, an effective amount of an antibody or immunoconjugate of the present invention can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, formulations containing sesame oil, peanut oil or aqueous propylene glycol solution; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and ready for injection by an appropriate delivery device or system without degradation. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water by mixing with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The antibody or immunoconjugate of the present invention can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein), and are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, etc. Salts formed with free carboxyl groups can also be prepared from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, glycine, histidine, procaine, etc.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, a polyhydric alcohol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be ensured by using absorption delaying agents in the compositions, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in an appropriate solvent together with any of the ingredients enumerated above, if required, followed by filter sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients in a sterile carrier medium which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient with any additional required ingredient from a solution thereof previously subjected to filter sterilization.

A preparation with more concentrated or highly concentrated solutions for direct injection is also envisaged, where it is assumed that the use of DMSO as a solvent results in extremely rapid penetration, delivering high concentrations of active agents to a small area of the tumor.

Once formulated, the solutions will be administered in a manner appropriate to the dosage formulation and in an amount that is therapeutically effective. The formulations are readily administered in a variety of dosage forms, such as the type of injection solutions described above, but capsules with drug release, etc., may also be used.

For parenteral administration as an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media which can be used will be known to those skilled in the art in light of the present invention. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected into the intended infusion site (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will inevitably occur depending on the condition of the subject being treated. The person responsible for administration will in any case determine the appropriate dose for the individual subject.

The antibody or immunoconjugate of the present invention can be formulated into a therapeutic mixture to contain about 0.01 to 100 milligrams per dose or so.

In addition to an antibody or immunoconjugate formulated for parenteral administration such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solid substances for oral administration; sustained-release capsules; and any other form currently in use.

In certain embodiments, the use of liposomes and/or nanoparticles is provided for the introduction of polypeptides into host cells. The composition and use of liposomes and/or nanoparticles are known to those skilled in the art.

Nanocapsules can generally retain compounds in a stable and reproducible manner. In order to avoid side effects caused by intracellular polymer overload, such ultrafine particles (about 0.1 μm in size) are generally developed using polymers that can be degraded in vivo . Biodegradable polyalkyl cyanoacrylate nanoparticles or biodegradable polylactide or polylactide-co-glycolide nanoparticles that meet these requirements are provided for use in the present invention, and such particles can be easily prepared.

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilamellar vesicles (also called multilamellar vesicles (MLVs)). MLVs typically have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200-500 Å containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Routes of administration and compositions

The methods described herein comprise administering to a subject a therapeutically effective amount of an antibody to CEACAM5 or an immunoconjugate comprising the antibody. As used herein, the term "effective amount" or "therapeutically effective amount" is a therapeutic dose that results in treatment of lung cancer (e.g., NSQ NSCLC). As used herein, the term "treatment" refers to producing a detectable improvement in one or more symptoms associated with lung cancer or producing a biological effect (e.g., (i.e., a reduction in the level of a specific biomarker) that correlates with the underlying pathological mechanism(s) causing the condition or symptom(s). For example, a dose of an anti-CEACAM5 antibody or an immunoconjugate containing the antibody that produces improvement in any of the following symptoms or conditions associated with lung cancer is considered a "therapeutically effective amount."

In another example, a treatment is not effective when a dose of an anti-CEACAM5 antibody or an immunoconjugate containing the antibody does not result in detectable improvement in one or more parameters or symptoms associated with the cancer (e.g., lung cancer), or which does not produce a biological effect that is correlated with the underlying pathological mechanism(s) causing the cancer condition or symptom(s).

According to some of these embodiments, the anti-CEACAM5 antibody or immunoconjugate comprising the antibody is administered intravenously.

According to the methods of the present invention, a therapeutically effective amount of an antibody to CEACAM5 or an immunoconjugate comprising the antibody that is administered to a subject will vary depending on age and size (e.g., body weight or body surface area) of the subject, as well as the route of administration and other factors well known to those skilled in the art.

In certain embodiments, the dose of the antibody or immunoconjugate comprising the antibody varies depending on the body surface area of the subject. In certain embodiments, the dose of the CEACAM5 antibody or immunoconjugate comprising the antibody administered to the subject is from about 1 mg/m²up to approximately 500 mg/m3². In some embodiments, the dose of the antibody or immunoconjugate comprising the antibody administered to the subject is from about 5 mg to about 300 mg/m². In various embodiments, the dose of the antibody or immunoconjugate comprising the antibody administered to the subject is from about 5 to about 250 mg/m². In various embodiments, the dose is 5, 10, 20, 30, 40, 60, 80, 100, 120, 150, 180, or 210 mg/m²based on the subject's body surface area. In various embodiments, the antibody or immunoconjugate comprising the antibody is administered at a dose of from about 2.5 mg/m²up to approximately 5 mg/m3². For example, an antibody or an immunoconjugate containing an antibody is administered over a period of time (e.g., 30 minutes and one hour) at a dose of approximately 2.5 mg/m²up to approximately 5 mg/m3². The dose includes 2.5 mg/m²antibodies, 5 mg/m²antibody or an immunoconjugate containing the antibody, and all doses from 2.5 mg/m²up to 5 mg/m3², for example, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8 and 4.9 mg/m².

For example, the present invention includes, but is not limited to, methods wherein about 1 mg/ ^m2 , about 5 mg/ ^m2 , about 10 mg/ ^m2 , about 15 mg/ ^m2 , about 20 mg/ ^m2 , about 25 mg/ ^m2 , about 30 mg/ ^m2 , about 35 mg/ ^m2 , about 40 mg/ ^m2 , about 45 mg/ ^m2 , about 50 mg/ ^m2 , about 55 mg/ ^m2 , about 60 mg/ ^m2 , about 65 mg/ ^m2 , about 70 mg/ ^m2 , about 75 mg/ ^m2 , about 80 mg/ ^m2 , about 85 mg/ ^m2 , about 90 mg/ ^m2 , about 95 mg/ ^m2 , about 100 mg/ ^m2 , about 105 mg/ ^m2 , approximately 110 mg/ ^m2 , approximately 115 mg/ ^m2 , approximately 120 mg/ ^m2 , approximately 125 mg/ ^m2 , approximately 130 mg/ ^m2 , approximately 135 mg/ ^m2 , approximately 140 mg/ ^m2 , approximately 145 mg/ ^m2 , approximately 150 mg/ ^m2 , approximately 155 mg/ ^m2 , approximately 160 mg/ ^m2 , approximately 165 mg/ ^m2 , approximately 170 mg/ ^m2 , approximately 175 mg/ ^m2 , approximately 180 mg/ ^m2 , approximately 185 mg/ ^m2 , approximately 190 mg/ ^m2 , approximately 195 mg/ ^m2 , approximately 200 mg/ ^m2 , approximately 205 mg/ ^m2 , approximately 210 mg/ ^m2 , approximately 215 mg/ ^m2 , approximately 220 mg/ ^m2 , approximately 225 mg/ ^m2 , approximately 230 mg/ ^m2 , approximately 235 mg/ ^m2 , approximately 240 mg/ ^m2 , approximately 245 mg/ ^m2 , approximately 250 mg/ ^m2 , approximately 255 mg/ ^m2 , approximately 260 mg/ ^m2 , approximately 265 mg/ ^m2 , approximately 270 mg/ ^m2 , approximately 275 mg/ ^m2 , approximately 280 mg/ ^m2 , approximately 285 mg/ ^m2 , approximately 290 mg/ ^m2 , approximately 295 mg/ ^m2 , approximately 300 mg/ ^m2 , approximately 325 mg/ ^m2 , about 350 mg/ ^m2 , about 375 mg/ ^m2 , about 400 mg/ ^m2 , about 425 mg/ ^m2 , about 450 mg/ ^m2 , about 475 mg/ ^m2 , or about 500 mg/ ^m2 of an anti-CEACAM5 antibody or an immunoconjugate comprising the antibody is administered to the patient weekly or once every two weeks. In various embodiments, the antibody or immunoconjugate comprising the antibody is administered in an amount of about 5, 10, 20, 30, 40, 60, 80, 100, 120, 150, 180, or 210 mg/ ^m2 every two weeks based on the body surface area of the subject.

As used herein, the phrase "administered at from about 1 to about 500 mg/kg" means that the specified substance is administered at any value within the stated range, including the endpoints of the range. For example, the phrase "the dose of the anti-CEACAM5 antibody or immunoconjugate comprising the antibody administered to the patient is from 1 mg/m²up to 500 mg/m3²" includes the introduction of 1 mg/m²antibodies to CEACAM5 or immunoconjugate containing the antibody, 500 mg/m²an anti-CEACAM5 antibody or an immunoconjugate comprising the antibody, and all doses in between. In an embodiment, the anti-CEACAM5 antibody or immunoconjugate comprising the antibody is administered at a dose of about 5, 10, 20, 30, 40, 60, 80, 100, 120, 150, 180, or 210 mg/m²over a period of time, such as once every 14 days (i.e. every two weeks) or 3 weeks. In various embodiments of the method, the antibody or immunoconjugate comprising the antibody is administered at a dose listed in Example 1.

In various embodiments, the dose is administered at a constant rate. Alternatively, the dose is administered at a variable rate. In various embodiments, the dose is administered at a constant rate of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 2.5, or 5 mg/min. In various embodiments, the antibody or immunoconjugate comprising the antibody is administered at a rate for the first 30 minutes or for the first 1 hour. In various embodiments, after about 30 minutes or 1 hour, the rate of administration of the antibody is changed. For example, the rate is decreased. In various embodiments, the rate is increased. In various embodiments, the antibody or immunoconjugate comprising the antibody is administered at a rate of 2.5 mg/min. for the first 30 minutes or 1 hour. In various embodiments, after about 30 minutes or 1 hour, the rate of administration of the antibody or immunoconjugate comprising the antibody is increased to 5 mg/min.

The methods of the present invention comprise administering multiple doses of an anti-CEACAM5 antibody or an immunoconjugate comprising the antibody to a patient over a period of time. For example, the anti-CEACAM5 antibody or immunoconjugate comprising the antibody may be administered about 1-5 times per day, about 1-5 times per week, about 1-5 times every two weeks, about 1-5 times per month, or about 1-5 times per year. In certain embodiments, the methods of the present invention comprise administering a first dose of an anti-CEACAM5 antibody or an immunoconjugate comprising the antibody to the patient at a first time point, followed by administering at least a second dose of the anti-CEACAM5 antibody or an immunoconjugate comprising the antibody to the patient at a second time point. In certain embodiments, the first and second doses may comprise the same amount of an anti-CEACAM5 antibody or an immunoconjugate comprising the antibody. The time period between the first and second doses may be from about several hours to about several weeks. For example, the second time point (i.e. the time when the second dose is administered) may be anywhere from approximately 1 hour to approximately 7 weeks after the first time point (i.e. the time when the first dose is administered). According to certain exemplary embodiments of the present invention, the second time point can be about 1 hour, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 14 weeks or more after the first time point. In certain embodiments, the second time point is about 1 week or about 2 weeks. Third and subsequent doses can be administered in a similar manner over the course of treatment of the patient. The present invention provides methods of using therapeutic compositions comprising anti-CEACAM5 antibodies or antigen-binding fragments thereof, immunoconjugates comprising the antibodies, and optionally one or more additional therapeutic agents. The therapeutic compositions of the present invention will be administered with suitable carriers, excipients and other agents which are included in the formulations to provide improved transport, delivery, tolerability, etc. A large number of suitable formulations can be found in a reference book known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, incorporated herein by reference in its entirety. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as LIPOFECTIN), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. See also Powellet al."Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311, incorporated herein by reference in its entirety.

Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis (see, for example, Wuet al.(1987) J. Biol. Chem. 262:4429-4432, incorporated herein by reference in its entirety). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucosal membranes (e.g., mucous membrane of the oral cavity, mucous membrane of the rectum and intestines, etc.) and can be administered together with other biologically active agents. Administration can be systemic or local. The antibody to CEACAM5 or an immunoconjugate containing the antibody can be administered subcutaneously.

The pharmaceutical composition can also be delivered in a vesicle, such as a liposome (see Langer (1990) Science 249:1527-1533, incorporated herein by reference in its entirety). In certain situations, the pharmaceutical composition can be delivered in a controlled release system, such as using a pump or polymeric materials. In another embodiment, the controlled release system can be placed near the target of the composition, requiring only a fraction of the systemic dose.

Injectable drugs may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, for local injection, drip infusions, etc. These injectables can be obtained by known methods. For example, injectables can be obtained, for example, dissolving, suspending or emulsifying the antibody or salt thereof described above in a sterile aqueous medium or oil medium commonly used for injection. As an aqueous medium for injection, there are, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent such as alcohol (e.g. ethanol), polyhydric alcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil), etc.). The following are used as an oil medium, for example: sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection solution obtained in this way can be placed in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in unit dose dosage forms suitable for adjusting the dose of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injection forms (ampoules), suppositories, etc.

According to the methods disclosed herein, the anti-CEACAM5 antibody or immunoconjugate comprising the antibody (or a pharmaceutical formulation comprising the antibody or an immunoconjugate comprising the antibody) can be administered to a patient using any suitable device or mechanism. For example, administration can be performed using a syringe and needle, or using a reusable syringe pen and/or an autoinjector delivery device. The methods of the present invention include the use of multiple reusable syringe pens and/or autoinjector delivery devices to administer the anti-CEACAM5 antibody or immunoconjugate comprising the antibody (or a pharmaceutical formulation comprising the antibody or an immunoconjugate comprising the antibody). Examples of such devices include, but are not limited to, AUTOPEN (Owen Mumford, Inc., Woodstock, UK), DISETRONIC pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75/25 pen, HUMALOG pen, HUMALIN 70/30 pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR (Novo Nordisk, Copenhagen, Denmark), BD pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN, OPTIPEN PRO, OPTIPEN STARLET, and OPTICLIK (Sanofi-Aventis, Frankfurt, Germany). Examples of single-use injection pens and/or autoinjector delivery devices useful in the subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly) injection pen, the SURECLICK autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and the HUMIRA (Abbott Labs, North Chicago, Ill.) injection pen, to name but a few.

In one embodiment, the antibody or immunoconjugate comprising the antibody is administered using a pre-filled syringe. In another embodiment, the antibody or immunoconjugate comprising the antibody is administered using a pre-filled syringe with a safety device. For example, the safety device prevents accidental injury to a health care worker. In various embodiments, the antibody is administered using a pre-filled syringe with a safety device ÈRIS (West Pharmaceutical Services Inc.). See also U.S. Patent Nos. 5,215,534 and 9,248,242, incorporated herein by reference in their entireties.

In another embodiment, the antibody or immunoconjugate comprising the antibody is administered using an autoinjector. In various embodiments, the antibody or immunoconjugate comprising the antibody is administered using an autoinjector with PUSHCLICK technology (SHL Group). In various embodiments, the autoinjector is a device comprising a syringe that delivers a dose of the composition and/or antibody to a subject. See also U.S. Patent Nos. 9,427,531 and 9,566,395, incorporated herein by reference in their entireties.

The use of a microinfusor for delivering an anti-CEACAM5 antibody or an immunoconjugate comprising the antibody (or a pharmaceutical formulation comprising the antibody or an immunoconjugate comprising the antibody) to a patient is also contemplated herein. As used herein, the term "microinfusor" means a subcutaneous delivery device designed to slowly administer large volumes (e.g., up to approximately 2.5 ml or more) of a therapeutic formulation over an extended period of time (e.g. approximately 10, 15, 20, 25, 30 or more minutes). See, for example, U.S. 6629949; US 6659982; and Meehan et al., J. Controlled Release 46:107-116 (1996), incorporated herein by reference in their entireties. Microinfusors are particularly useful for delivering large doses of therapeutic proteins contained in highly concentrated and/or viscous solutions.

All publications referenced in this document are hereby incorporated by reference in their entirety for all purposes.

EXAMPLES

Example 1. TED13751 - a first-in-human study to evaluate the safety, pharmacokinetics, and antitumor activity of huMAb2-3-SPDB-DM4 in patients with advanced solid tumors - study design

A first in human (FIH) study to evaluate the safety and pharmacokinetics of an ADC identified as huMAb2-3-SPDB-DM4 administered as a single agent every 2 weeks (q2w; 14 days) via the intravenous route was conducted in adults with advanced unresectable or metastatic solid tumors.

The study was divided into two parts: a dose escalation phase and a stable dose phase.

During the dose escalation phase, the treatment population was enriched without restriction to patients with tumor types known to express CEACAM5; confirmation of CEACAM5 expression was performed retrospectively using IHC on the most recent archival tissue samples and in a central laboratory. Circulating CEACAM5 expression was also used for enrichment. A maximum tolerated dose (MTD) of 100 mg/ ^m2 was determined based on subject body surface area during the dose escalation phase.

During the stable dose phase, the treatment population was limited to patients with NSQ NSCLC, with CEACAM5 expression score ≥2+ in intensity, with documented involvement of 50% of the tumor cell population during pre-screening on the most recent archival tissue sample, and using local IHC assessment for patients potentially eligible for treatment in the gastric adenocarcinoma cohort.

There were 2 independent cohorts of NSQ NSCLC in the stable dose phase. The first (lung cohort) included patients with CEACAM5 expression score ≥2+ in intensity involving at least 50% of the tumor population. The second cohort (lung cohort 2) included patients who had a positive prescreening result with intensity ≥2+ in 21% to <50% of the tumor population. For sites that were negative for CEACAM5 on the huMAb2-3-SPDB-DM4 monoclonal antibody based on their local IHC platform, prescreening for CEACAM5 tumor expression was performed centrally. Full screening was performed for archival cases meeting the above definition.

The expression level of CEACAM5 was documented essentially retrospectively and centrally in both archival and fresh (original sample) tumor tissues.

Confirmation of CEACAM5 expression in tumors was performed retrospectively in a central laboratory on fresh tumor tissues collected at baseline (mandatory biopsy only in the stable-dose phase of NSQ NSCLC). If sufficient archival tumor material was available, central assessment was also performed retrospectively to gain knowledge of variability in expression assessment. Results of retrospective analyses did not influence patient treatment. This was performed to better interpret overall response and as a baseline for comparison with CEACAM5 expression at progression (investigations of CEACAM5 loss as a mechanism of acquired resistance).

A maximum of 60 patients were included in the NSQ NSCLC (lung) cohort and a maximum of 28 patients were included in the NSQ NSCLC (secondary lung cohort). Only patients with measurable parameters in malignancy were eligible. NSQ NSCLC patients with malignancy that met stringent CEACAM5 expression criteria were additionally screened for his or her eligibility for treatment with the maximum tolerated dose (MTD) without a loading dose during the pre-screening procedure.

The safety of the first 6 patients enrolled in the stable dose phase was reviewed by the study committee when the 6th patient had been treated for 2 cycles before enrolling subsequent patients. The MTD (no loading dose) was confirmed if one third or less of the treated patients (included in 3 cohorts) experienced dose-limiting toxicity (DLT) at the end of cycle 2 at the planned dose of huMAb2-3-SPDB-DM4. At the same time, the pre-treatment assessment was the presence or absence of benefit from primary prevention of corneal toxicity in preventing corneal toxicity. In addition, the occurrence of cumulative toxicity, if any, was assessed. Antitumor activity of the drug was assessed according to RECIST 1.1.

The study duration for an individual patient included an inclusion period of no more than 4 weeks (baseline period), a treatment period of at least 1 cycle (2 weeks), an end-of-treatment (EOT) visit approximately 30 days after the last administration of the investigational medical device (lMP), and at least one follow-up visit (approximately 30 days after the EOT visit) to assess immunogenicity.

INCLUSION CRITERIA

I 1. Locally advanced or metastatic solid malignancy for which, in the investigator's judgment, no standard alternative therapy is available and which meets the subsequent inclusion criteria.

I 2. At least 6 x 5 μm slides and additional slides that can be either 3 x 10 μm (best) or 6 x 5 μm or equivalent to preserve the same total amount of required archival FFPE tissue material should be available for local on-site testing and/or transport to the clinical trial sponsor or to a laboratory designated by the clinical trial sponsor, assessment of CEACAM5 expression in tumor (retrospectively in the dose escalation phase and prospectively in the stable dose phase), and testing for other predictive biomarkers of response. If less material is available, the patient may still be eligible after discussion with the clinical trial sponsor who has assessed and confirmed that there is sufficient appropriate material for the primary assessments.

I 3. For participants in the dose-escalation phase cohorts (primary and secondary). Inclusion was expanded (although not limited) to tumors expressing or capable of expressing CEACAM5, which included malignancies with a high prevalence of CEACAM5 expression (i.e. (NSQ NSCLC). For stable-dose phase cohort participants, enrollment was limited to patients with either NSQ NSCLC or other lung cancer subtypes. There were two independent stable-dose phase NSQ NSCLC cohorts based on local or central assessment of CEACAM5 expression on archived tumor tissue. The first (lung cohort), which began enrollment before Amendment #4, included patients with CEACAM5 expression score ≥2+ in intensity involving at least 50% of the tumor cell population. The second independent cohort (lung cohort 2) included patients who had a positive prescreening result with intensity ≥2+ in 21% to <50% of the tumor cell population.

I 4. At least one measurable lesion according to RECIST v1.1 only during the stable dose phase.

I 5. At least one biopsyable lesion (stable-dose phase cohort only). Patient must consent to a baseline biopsy to retrospectively confirm CEACAM5 expression in the tumor prior to treatment unless NSCLC or SCLC is lesion-free).

STUDY DRUG

Dosage form

huMAb2-3-SPDB-DM4 ADC was supplied as a 25 mL extractable volume concentrate for infusion of 125 mg (5 mg/mL) contained in a 30 mL Type I glass vial.

Dose of drug per administration

The drug is administered every 2 weeks at the MTD (100 mg/m²) as determined during the dose escalation phase.

The patients' body surface area (BSA) was calculated using their height and actual body weight. For patients with BSA >2.2 ^m2, the dose will be calculated based on 2.2 ^m2 BSA.

Premedication with an H1-histamine receptor antagonist (diphenylhydramine 50 mg PO or equivalent [eg, dexchlorpheniramine], administered approximately 1 hour prior to huMAb2-3-SPDB-DM4 administration), is required for all patients.

Using a controlled infusion pump, huMAb2-3-SPDB-DM4 will be administered by intravenous infusion at a rate of 2.5 mg/min for the first 30 minutes and then titrated up to 5 mg/min in the absence of hypersensitivity reactions.

The exact dose and time of IMP administration (day/month/year h:min) will be documented in the eCRF.

Duration of treatment

huMAb2-3-SPDB-DM4 was administered on day 1 and repeated every 14 days; this 14-day period constituted one treatment cycle (1 cycle). Patients may continue treatment until disease progression, unacceptable toxicity, or readiness to discontinue treatment.

Method of dilution and infusion

There was no bacteriostatic agent present in the product; therefore, aseptic technique was required. Before dosing, the investigational pharmacist should prepare a separate dose for each patient, starting with a pre-filled packet of diluent (0.9% sodium chloride).

Once the solution was prepared, the dose was administered to the patient within 7.5 hours from the time the bag was prepared until the end of the dose infusion.

Two types of administration were used.

· Infusion via syringe pump for low doses (up to 30 mg/ ^m2 ).

· Infusion via pump for other doses.

An intravenous administration line with a 0.2-micron filter device attached to it was used for infusions. Study drug was not administered with any other intravenous fluids. However, the infusion tubing was optionally primed with isotonic saline or huMAb2-3-SPDB-DM4. For infusion volumes ≤25 mL, flushing and destruction of 25 mL of huMAb2-3-SPDB-DM4 was required prior to infusion of the dose. At the end of the pump infusion, the drip line was flushed with isotonic saline as needed to ensure delivery of the full dose. At the end of the syringe pump infusion, the remaining huMAb2-3-SPDB-DM4 in the syringe was destroyed.

TUMOR RESPONSE ASSESSMENT

To assess objective response or future progression, it was necessary to estimate the total tumour burden at baseline and use this as a comparator for subsequent measurements. Only patients with measurable disease at baseline were included in protocols where objective tumour response was the primary endpoint. Measurable disease was defined by the presence of at least one measurable lesion. In studies where the primary endpoint was tumour progression (either time to progression or proportion progressing at a specified time), the protocol specified if inclusion was limited to patients with measurable disease or if patients with unmeasurable disease were also eligible. See Table 2 and Table 3.

Response criteria

Table 2

Assessment of target lesions

Complete response (CR) Disappearance of all target lesions. Any abnormal lymph nodes (both target and non-target) must demonstrate a decrease in short axis to <10 mm.

Partial response (PR): at least 30% reduction in the sum of the diameters of target lesions, using the sum of the diameters at baseline as the reference.

Disease progression (PD): at least a 20% increase in the sum of target lesion diameters, using the lowest sum in the study as the reference (this includes the sum at baseline if that is the lowest sum in the study). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progression).

Stable disease (SD): there is neither sufficient reduction in size to meet PR criteria nor sufficient increase in size to meet PD criteria, using the smallest sum of diameters during the study as the reference.

Table 3

Evaluation of non-target lesions

Disappearance of all non-target lesions and normalization of tumor marker levels. All lymph nodes should be of non-pathological size (short axis < 10 mm).

Insufficient Preservation of one or more non-target lesions and/or maintenance of tumor marker levels.

Disease response/stabilization above normal range.

(SD):

Disease progression (PD): Apparent progression (see comments below) of existing non-target lesions. (Note: the appearance of one or more new lesions is also considered progression.)

Although pure progression of only "non-target" lesions is an exception, in such circumstances the judgment of the treating physician should prevail and the progression status should be confirmed later by the expert panel (or study director).

ASSESSING THE BEST OVERALL ANSWER

The following table provides a summary of the calculation of overall response status at each time point for patients with measurable disease manifestations at baseline.

Table 4

Target lesions Non-target lesions New foci of defeat General answer CR CR No CR CR No CR/No PD No PR CR Not rated No PR PR No PD or not all assessed No PR SD No PD or not all assessed No SD Not all are appreciated No PD No NE P.D. Any Yes or no P.D. Any P.D. Yes or no P.D. Any Any Yes P.D.

Table 5

General Answer General Answer BEST General Answer

First moment in time Subsequent moment in time

CR CR CR

CR PR SD, PD or PR a

CR SD SD provides minimum criteria for the duration of SD, otherwise consistent with PD

CR PD SD provides minimum criteria for the duration of SD, otherwise consistent with PD

CR NE SD provides minimum criteria for the duration of SD, otherwise consistent with PD

PRCRPR

PR PR PR

PR SD SD

PR PD SD provides minimum criteria for the duration of SD, otherwise consistent with PD

PR NE SD provides minimum criteria for SD duration otherwise consistent with PD

NE NE NE

a If the CR is exactly right at time 1, then any disease observed at a subsequent time, even disease that meets the criteria for PR relative to baseline, creates a PD of the disease at that time (since the disease would have reappeared after the CR). The best response will depend on whether the minimum duration for SD was met. However, sometimes a 'CR' may be required when subsequent imaging suggests that small lesions are likely still present and, more accurately, the patient had a PR rather than a CR at time 1. In these circumstances, the initial CR should be changed to a PR and the best response is a PR.

The best overall response was determined once all patient data were known.

Determination of best response in trials where confirmation of complete or partial response was not required. The best response in these trials is defined as the best response across all time points (e.g., a patient with SD at the first assessment, PR at the second assessment, and PD at the last assessment has the best overall response PR). When SD is considered to represent the best response, it also meets the protocol-specified minimum time from baseline. If the minimum time is not met, when SD otherwise represents the best response at a time point, the patient's best response depends on subsequent assessments. For example, a patient with SD at the first assessment, PD at the second, and who did not meet the minimum duration for SD would have the best response PD. The same patient lost to the study after the first SD assessment would be considered not evaluable.

Determining the best response in trials where confirmation of a complete or partial response

required. Complete or partial responses may only be required if the criteria for each are met at a subsequent time point as specified in the protocol (usually 4 weeks). In these circumstances, the best overall response has been interpreted in Table 5.

CEACAM5 - (IHC) IMMUNOHISTOCHEMICAL COUNTING METHOD

The stained immunohistochemical preparations with CEACAM5 are evaluated by a pathologist using light microscopy.

A positive CEACAM5 assay is defined as the percentage of viable tumor cells expressing CEACAM5 and being positive for membrane staining.

Tumor cells are CEACAM5 positive if they show partial or complete circumferential staining of the cytoplasmic membrane with intensities of 2+ and 3+. They are considered negative if they show staining with an intensity of 1+ (weak staining) or do not show staining (intensity 0).

All tumor cells observed in the section were assessed for CEACAM5.

A minimum of 100 viable tumor cells must be present per section to determine the percentage of CEACAM5-positive cells.

The count includes the percentage of tumor cells stained with each intensity measured as follows:

% CEACAM5-positive cells = 100 × # of membrane-stained tumor cells expressing CEACAM5 with an intensity ≥ 2+/total # of viable tumor cells present in the section.

Fig. 1A, 1B and 1C show examples of staining intensities 1+, 2+, 3+, respectively. Membrane staining intensities of 2+ and 3+ are considered CEACAM5 positive. A general description of the IHC method including counting is provided by So-Woon Kim et al (Journal of Pathology and Translational Medicine 2016; 50: 411-418).

Example 2. TED13751 - stable dose phase - NSQ NSCLC cohort - initial interim analysis phase

As discussed in Example 1, based on local or central assessment of CEACAM5 expression in archival tumor tissue, there were two independent cohorts in the stable-dose phase of non-NSQ NSCLC: the first cohort included patients with CEACAM5 expression ≥2+ in intensity involving at least 50% of the tumor cell population. The second independent cohort (lung cohort 2) included patients who had a positive pre-screen result with an intensity of ≥2+ in 21% to <50% of the tumor cell population. Both cohorts were selected to receive 100 mg/ ^m2 huMAb2-3-SPDB-DM4.

For the NSQ NSCLC (lung) cohort with CEACAM5 expression ≥50% in the tumor cell population with an intensity ≥2+, a minimum of 30 patients pretreated with anti-PDL1 antibodies were included to evaluate the antitumor activity of huMAb2-3-SPDB-DM4 in patients pretreated with anti-PDL1 antibodies and to provide minimal power for subgroup analyses in this subset. For the NSQ NSCLC cohort (second lung cohort) with CEACAM5 expression ≥1% to ≥50% in the tumor cell population with intensity ≥2+, the relationship between CEACAM5 expression level and efficacy outcome can be assessed by adding a cohort of 28 treated patients in whom CEACAM5 is expressed in tumor cells with mild membrane staining (at least 1% of positive tumor cells and <50% of tumor cells with intensity >2+).

As shown in Tables 1 and 2 , an objective response of 25.9% was observed in the cohort with high CEACAM5 expression levels (lung cohort), while no objective response was observed in the cohort with low expression levels (second lung cohort). Tables 1 and 2 summarize the objective response results of NSCLC patients treated with huMAb2-3-SPDB-DM4. The tables compare the objective response between NSCLC patients with hCEACAM5 expression percentage greater than or equal to 50 (consisting of intensities 2+ and 3+) and patients with hCEACAM5 expression percentages between 1 and 49.

Table 1. Objective response of patients with hCEACAM5 expression percentage greater than or equal to 50 (composed of intensities 2+ and 3+)

(CEACAM5 > 50%) (N=27) Objective response (CR+PR) 7 (25.9%) Full answer 0 Partial answer 7 (*25.9%) Disease stabilization 12 (44.4%) Disease progression 8 (29.6%) Disease control rate (PR+SD) 19 (70.3%)

* 90% confidence interval: 14.7% - 41.52%.

Table 2. Objective response of patients with hCEACAM5 expression score in percentage equal to 1-49 (consisting of intensities 2+ and 3+)

(CEACAM5 1- 49%) (N=11) Objective response (CR+PR) 0 Disease stabilization 5 (45.5%) Disease progression 6 (54.6%)

Additionally, as shown in Fig. 2 , the best relative tumor mass reduction was observed in patients with CEACAM5 expression scores of 50-80%, or greater than 80%.

As shown in Tables 3 and 4 , duration of response (DoR) and time to progression (TTP) also improved in the high expression cohort (lung cohort).

Table 3. Duration of response for NSCLC patients treated with huMAb2-3 - SPDB-DM4 with a hCEACAM5 expression percentage greater than or equal to 50 (composed of 2+ and 3+ intensities)

Response time (months) (N=27) Number of responses 7 Median value 4.5 90% CI 3.68 to 8.51

Table 4. Time to progression for huMAb2-3-SPDB-DM4-treated NSCLC patients with hCEACAM5 expression percentage greater than or equal to 50 (composed of 2+ and 3+ intensities)

Time to progression (months) (N=27) Median value 3.7 90% CI 2.60 to 5.39

Furthermore, of those patients with high expression levels (hCEACAM5 expression percentage greater than or equal to 50), similar objective responses were obtained in those patients who were pretreated with anti-PD1/PDL1 antibodies and those who were not pretreated with anti-PD1/PDL1 antibodies ( Tables 5 and 6 ).

Table 5. Best objective response to ICI (anti-PD1/PDL1 antibody) in pretreated NSCLC patients (CEACAM5)

Previously treated with anti-PD1/PDL1 antibodies (N=17) Objective response (CR+PR) 4 (23.5%) Full answer 0 Partial answer 4 (*23.5%) Disease stabilization 9(52.9%) Disease progression 4 (23.6%) Disease control rate (PR+SD) 13 (76.4%)

* 90% confidence interval: 11.3% - 43.30%

Table 6. Best objective response to ICI (anti-PD1/PDL1 antibodies) in pre-treated NSCLC patients (CEACAM5 ≥ 50%)

Previously untreated with anti-PD1/PDL1 antibodies (N=10) Objective response (CR+PR) 3 (30.0%) Full answer 0 Partial answer 3 (**30.0%) Disease stabilization 3 (30.0%) Disease progression 4 (40.0%) Disease control rate (PR+SD) 6 (60.0%)

* 90% confidence interval: 12.69% - 55.83%.

Example 3. TED13751 - stable dose phase - high expression cohort with NSQ NSCLC - initial phase full cohort analysis in 32 treated patients

The full analysis of the 32-patient cohort confirms the interim analysis of Example 2.

It demonstrates encouraging antitumor activity in heavily pretreated patients with NSQ NSCLC CEACAM5 ≥ 50%. This antitumor activity was associated with a response rate of 25% by RECIST1.1 (90% CI 14.70-39.20%).

As shown in Table 7 , an objective response of 25% was observed in 32 patients treated in the cohort with high CEACAM5 expression levels (lung cohort).

Table 7. Best objective response in 32 patients treated in the cohort with high CEACAM5 expression levels (lung cohort).

(CEACAM5 > 50%)
"Subjects in whose organisms expression occurs at high levels" (N=32) Objective response (CR+PR)
Full answer
Partial answer 8 (*25.0%)
0
8 (*25.0%) Disease stabilization
Disease progression
Disease control rate (PR+SD) 12 (37.5%)
12** (37.5%)
20 (62.5%)

Additionally, as shown in Fig. 3 , the best relative tumor mass reduction was generally observed in patients with a CEACAM5 expression score of 50-80%, or greater than 80%. As shown in Fig. 4 , time to progression (TTP) was also improved in the cohort with high expression levels (lung cohort). Furthermore, among these patients with high expression levels, similar objective responses were obtained in those patients who were pretreated with anti-PD1/PDL1 antibodies and those who were not pretreated with anti-PD1/PDL1 antibodies, as shown in Table 8 below.

Table 8. Best objective response in patients pretreated or untreated with PD1/PDL1 antibodies

Prognostic factor Quantity Answers (n[%]) 90%CIa Any prior anti-PD1/PDL1 antibody therapy Quantity 32 Pre-existing PD1/PDL1 antibodies 20 5 (25.0%) (from 12.74% to 43.22%) Without pre-existing antibodies to PD1/PDL1 12 3 (25.0%) (from 10.47% to 48.73%) Estimated by Wilson's interval counting
*Excluding primary tumor focus
Subgroup analyses will only be performed if there are sufficient patients in each subcategory.

Example 4. TED13751 - stable dose phase - moderately expressing cohort with NSQ NSCLC

As shown in Table 9 , only one objective response (in 20 patients) was observed in the cohort with low expression levels (second lung cohort)

Table 9. Objective response in 20 patients observed in the cohort with moderate expression levels (second lung cohort)

(CEACAM5 1- 49%)
"Subjects in whose organisms expression occurs at moderate levels" (N=20) Objective response (CR+PR) 1 (**5.0%) Full answer 0 Partial answer 1 (**5.0%) Disease stabilization 11 (55.0%) Disease progression 7 (35.0%) Not subject to assessment 1 (5.0%) Disease control rate (PR+SD) 12 (60.0%)

In conclusion, these data demonstrate that proof of concept has been achieved in a subset of NSCLC lung cancer types treated with huMAb2-3-SPDB-DM4. Specifically, these data support the conclusion that huMAb2-3-SPDB-DM4 is effective in the treatment of NSQ NSCLC, a subtype that accounts for approximately 60% of lung cancer types. Additionally, these data support the conclusion that huMAb2-3-SPDB-DM4 is particularly effective in the treatment of NSQ NSCLC with high levels of CEACAM5 expression, tumor types that account for approximately 20% of NSQ NSCLC cancer types.

--->

SEQUENCE LIST

<110> SANOFI

<120> APPLICATION OF IMMUNOCONJUGATES TO CEACAM5 FOR THE TREATMENT OF LUNG CANCER

<130> FR2019/003 EP

<160> 23

<170> PatentIn version 3.5

<210> 1

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH1a from humanized MAb2 antibody

<400> 1

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Arg Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Gly Gly Ile Thr Tyr Ala Pro Ser Thr Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala His Tyr Phe Gly Ser Ser Gly Pro Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 2

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL1c from humanized MAb2 antibody

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Phe Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val

35 40 45

Tyr Asn Thr Arg Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 3

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223>HCDR1

<400> 3

Gly Phe Val Phe Ser Ser Tyr Asp

1 5

<210> 4

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223>HCDR2

<400> 4

Ile Ser Ser Gly Gly Gly Ile Thr

1 5

<210> 5

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223>HCDR3

<400> 5

Ala Ala His Tyr Phe Gly Ser Ser Gly Pro Phe Ala Tyr

1 5 10

<210> 6

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR1

<400> 6

Glu Asn Ile Phe Ser Tyr

1 5

<210> 7

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR3

<400> 7

Gln His His Tyr Gly Thr Pro Phe Thr

1 5

<210> 8

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy Chain

<400> 8

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Arg Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Gly Gly Ile Thr Tyr Ala Pro Ser Thr Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala His Tyr Phe Gly Ser Ser Gly Pro Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 9

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain

<400> 9

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 10

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> A3-B3 domain of human CEACAM5 protein

<400> 10

Ser Gly Ala Asn Leu Asn Leu

1 5

<210> 11

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> epitopes of domain A3-B3

<400> 11

Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu Phe

1 5 10

<210> 12

<211> 654

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Macaca fascicularis CEACAM5 extracellular domain

<400> 12

Gln Leu Thr Ile Glu Ser Arg Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Ala His Asn Val Ser Gln Asn Leu Phe Gly Tyr Ile

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Ala Ser Arg Arg Ile Gly Ser Cys

35 40 45

Val Ile Arg Thr Gln Gln Ile Thr Pro Gly Pro Ala His Ser Gly Arg

50 55 60

Glu Thr Ile Asp Phe Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Ser Asp Thr Gly Ser Tyr Thr Ile Gln Val Ile Lys Glu Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Tyr Ile Thr Ser Asn Asn Ser Asn Pro Ile Glu Asp Lys Asp Ala

115 120 125

Val Ala Leu Thr Cys Glu Pro Glu Thr Gln Asp Thr Thr Tyr Leu Trp

130 135 140

Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Glu Leu Ser

145 150 155 160

Ser Asp Asn Arg Thr Leu Thr Val Phe Asn Ile Pro Arg Asn Asp Thr

165 170 175

Thr Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Val Arg Arg Ser

180 185 190

Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro Thr Ile

195 200 205

Ser Pro Leu Asn Thr Pro Tyr Arg Ala Gly Glu Tyr Leu Asn Leu Thr

210 215 220

Cys His Ala Ala Ser Asn Pro Thr Ala Gln Tyr Phe Trp Phe Val Asn

225 230 235 240

Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr

245 250 255

Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala His Asn Ser Ala Thr

260 265 270

Gly Leu Asn Arg Thr Thr Val Thr Ala Ile Thr Val Tyr Ala Glu Leu

275 280 285

Pro Lys Pro Tyr Ile Thr Ser Asn Asn Ser Asn Pro Ile Glu Asp Lys

290 295 300

Asp Ala Val Thr Leu Thr Cys Glu Pro Glu Thr Gln Asp Thr Thr Tyr

305 310 315 320

Leu Trp Trp Val Asn Asn Gln Arg Leu Ser Val Ser Ser Arg Leu Glu

325 330 335

Leu Ser Asn Asp Asn Arg Thr Leu Thr Val Phe Asn Ile Pro Arg Asn

340 345 350

Asp Thr Thr Phe Tyr Glu Cys Glu Thr Gln Asn Pro Val Ser Val Arg

355 360 365

Arg Ser Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro

370 375 380

Thr Ile Ser Pro Leu Asn Thr Pro Tyr Arg Ala Gly Glu Asn Leu Asn

385 390 395 400

Leu Ser Cys His Ala Ala Ser Asn Pro Ala Ala Gln Tyr Phe Trp Phe

405 410 415

Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn

420 425 430

Ile Thr Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala His Asn Ser

435 440 445

Ala Thr Gly Leu Asn Arg Thr Thr Val Thr Ala Ile Thr Val Tyr Val

450 455 460

Glu Leu Pro Lys Pro Tyr Ile Ser Ser Asn Asn Ser Asn Pro Ile Glu

465 470 475 480

Asp Lys Asp Ala Val Thr Leu Thr Cys Glu Pro Val Ala Glu Asn Thr

485 490 495

Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Ser Val Ser Pro Arg

500 505 510

Leu Gln Leu Ser Asn Gly Asn Arg Ile Leu Thr Leu Leu Ser Val Thr

515 520 525

Arg Asn Asp Thr Gly Pro Tyr Glu Cys Gly Ile Gln Asn Ser Glu Ser

530 535 540

Ala Lys Arg Ser Asp Pro Val Thr Leu Asn Val Thr Tyr Gly Pro Asp

545 550 555 560

Thr Pro Ile Ile Ser Pro Pro Asp Leu Ser Tyr Arg Ser Gly Ala Asn

565 570 575

Leu Asn Leu Ser Cys His Ser Asp Ser Asn Pro Ser Pro Gln Tyr Ser

580 585 590

Trp Leu Ile Asn Gly Thr Leu Arg Gln His Thr Gln Val Leu Phe Ile

595 600 605

Ser Lys Ile Thr Ser Asn Asn Asn Gly Ala Tyr Ala Cys Phe Val Ser

610 615 620

Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Asn Ile Ser Val

625 630 635 640

Ser Ser Gly Asp Ser Ala Pro Gly Ser Ser Gly Leu Ser Ala

645 650

<210> 13

<211> 702

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Human CEACAM5

<400> 13

Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln

1 5 10 15

Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr

20 25 30

Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly

50 55 60

Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile

65 70 75 80

Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser

85 90 95

Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile

100 105 110

Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp

115 120 125

Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu

130 135 140

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys

145 150 155 160

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr

165 170 175

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

180 185 190

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn

195 200 205

Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg

210 215 220

Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro

225 230 235 240

Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn

245 250 255

Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe

260 265 270

Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn

275 280 285

Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser

290 295 300

Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala

305 310 315 320

Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu

325 330 335

Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr

340 345 350

Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg

355 360 365

Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr

370 375 380

Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Glu Leu Ser

385 390 395 400

Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp

405 410 415

Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn

420 425 430

Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser

435 440 445

Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile

450 455 460

Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn

465 470 475 480

Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val

485 490 495

Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro

500 505 510

Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln

515 520 525

Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser

530 535 540

Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn

545 550 555 560

Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser

565 570 575

Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly

580 585 590

Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly

595 600 605

Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln

610 615 620

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

625 630 635 640

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

645 650 655

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

660 665 670

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr

675 680 685

Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu Ile

690 695 700

<210> 14

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MAb1 heavy chain variable domain

<400> 14

Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Leu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Pro Ala Tyr Tyr Gly Asn Pro Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 15

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MAb1 light chain variable domain

<400> 15

Asp Ile Leu Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 16

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MAb2 heavy chain variable domain

<400> 16

Glu Val Gln Leu Gln Glu Ser Gly Gly Val Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Gly Gly Ile Thr Tyr Phe Pro Asp Thr Val

50 55 60

Gln Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ala His Tyr Phe Gly Ser Ser Gly Pro Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 17

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MAb2 light chain variable domain

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Phe Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Thr Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 18

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MAb3 heavy chain variable domain

<400> 18

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Pro Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Asp Thr Tyr Tyr Pro Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Arg Asn Ile Leu Phe Leu

65 70 75 80

Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Arg Val Asn Tyr Tyr Asp Ser Ser Phe Leu Asp Trp Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 19

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MAb3 light chain variable domain

<400> 19

Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Ser Thr Leu Glu Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Leu

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 20

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MAb4 heavy chain variable domain

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Phe Ile Ser Ser Tyr Gly Gly Arg Thr Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys

85 90 95

Ala Ala His Tyr Phe Gly Thr Ser Gly Pro Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 21

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MAb4 light chain variable domain

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Phe Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Ala Lys Ile Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His His Tyr Gly Ile Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 22

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MAb5 heavy chain variable domain

<400> 22

Glu Leu Gln Leu Val Glu Ser Gly Gly Val Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Thr Tyr Ile Asn Ser Gly Gly Gly Ile Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Thr Ala His Tyr Phe Gly Ser Ser Gly Pro Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 23

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MAb5 light chain variable domain

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Ala Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<---

Claims (112)

1. Use of an antibody or an immunoconjugate comprising the antibody for the treatment of non-squamous non-small cell lung cancer (NSQ NSCLC) in a subject in need thereof, wherein the antibody specifically binds hCEACAM5 and wherein the antibody comprises a VH and a VL, wherein the VH comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3, and wherein the VL comprises three CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 3 ( GFVFSSYD) ; HCDR2 comprises the amino acid sequence of SEQ ID NO: 4 ( ISSGGGIT) ; HCDR3 comprises the amino acid sequence of SEQ ID NO: 5 ( AAHYFGSSGPFAY) ; LCDR1 comprises the amino acid sequence of SEQ ID NO: 6 ( ENIFSY) ; LCDR2 comprises the amino acid sequence of NTR ; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 7 ( QHHYGTPFT) , wherein the subject is characterized by hCEACAM5 expression, consisting of 2+ and 3+ intensities, in greater than or equal to 50% of tumor cell populations, wherein the immunoconjugate is formulated for administration at a dose level of 80, 100, or 120 mg/ ^m2 based on the subject's body surface area. 2. The use according to claim 1, wherein the subject is a subject in whose body carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is expressed at high levels. 3. The use according to any one of claims 1 or 2, wherein the subject has previously been treated with an agent or drug for the treatment of non-small cell lung cancer. 4. The use according to claim 3, wherein the agent or medicinal agent is selected from the group consisting of: a chemotherapeutic agent, an angiogenesis inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an anaplastic lymphoma kinase (ALK) inhibitor, a tyrosine kinase receptor (ROS1) inhibitor, and an immune checkpoint inhibitor. 5. The use according to claim 4, wherein the immune checkpoint inhibitor is a PD-1 inhibitor and/or a PD-L1 inhibitor. 6. The use according to any one of claims 1 to 5, wherein VH comprises SEQ ID NO: 1 (EVQLQESGPGLVKPGGSLSLSCAAS GFVFSSYD MSWVRQTPERGLEWVAY ISSGGGIT YAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYC AAHYFGSSGPFAY WGQGTLVTVSS). 7. Use according to claim 6, wherein the heavy chain comprises SEQ ID NO: 8 (EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWV AYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFG SSGPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG). 8. The use according to any one of claims 1 to 7, wherein VL comprises SEQ ID NO: 2 (DIQMTQSPASLSASVGDRVTITCRAS ENIFSY LAWYQQKPGKSPKLLVY NTR TLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYC QHHYGTPFT FGSGTKLEIK). 9. Use according to claim 8, wherein the light chain comprises SEQ ID NO: 9 (DIQMTQSPASLSSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVY NTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C). 10. The use according to any one of claims 1 to 9, wherein the antibody in the immunoconjugate is conjugated or linked to at least one growth suppressive agent. 11. The use according to claim 10, wherein said growth suppressant is a cytotoxic agent. 12. The use according to claim 10 or 11, wherein said growth suppressant is selected from the group consisting of chemotherapeutic agents, enzymes, antibiotics and toxins such as low molecular weight toxins or enzymatically active toxins, taxoids, vinca alkaloids, taxanes, maytansinoid or maytansinoid analogues, tomaimycin or pyrrole benzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatin and dolastatin analogues, prodrugs, topoisomerase II inhibitors, DNA alkylating agents, antitubulin agents and CC-1065 or CC-1065 analogues. 13. The use according to claim 10 or 11, wherein said growth suppressant is N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) or N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4). 14. The use according to any one of claims 10-13, wherein the antibody is covalently attached via a cleavable or non-cleavable linker to at least one growth suppressive agent. 15. The use according to claim 14, wherein said linker is selected from the group consisting of N-succinimidyl pyridyl dithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfobutyric acid (sulfo-SPDB) and succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). 16. The use according to any one of claims 1-15, wherein the immunoconjugate is formulated for administration every 14 days or every 3 weeks. 17. A method of treating non-squamous non-small cell lung cancer (NSQ NSCLC) in a subject in need thereof, the method comprising administering an antibody or an immunoconjugate comprising an antibody that specifically binds hCEACAM5, wherein the antibody comprises a VH and a VL, wherein the VH comprises the three complementarity determining regions HCDR1, HCDR2 and HCDR3, and wherein the VL comprises the three CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 3 (GFVFSSYD); HCDR2 comprises the amino acid sequence of SEQ ID NO: 4 (ISSSGGGIT); HCDR3 comprises the amino acid sequence of SEQ ID NO: 5 (AAHYFGSSGPFAY); LCDR1 contains the amino acid sequence of SEQ ID NO: 6 (ENIFSY); LCDR2 contains the amino acid sequenceNTR; and LCDR3 comprises the amino acid sequence under SEQ ID NO: 7 (QHHYGTPFT), wherein the subject is characterized by hCEACAM5 expression, consisting of 2+ and 3+ intensities, in greater than or equal to 50% of tumor cell populations, wherein the antibody is administered at a dose level of 80, 100, or 120 mg/ ^m2 based on the subject's body surface area. 18. The method of claim 17, wherein the subject is a subject in whose body carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is expressed at high levels. 19. The method of claim 17, wherein the subject has previously been treated with an agent or drug for the treatment of non-small cell lung cancer. 20. The method according to claim 17, wherein the agent or medicinal agent is selected from the group consisting of: a chemotherapeutic agent, an angiogenesis inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an anaplastic lymphoma kinase (ALK) inhibitor, a receptor tyrosine kinase (ROS1) inhibitor, and an immune checkpoint inhibitor. 21. The method according to claim 20, wherein the immune checkpoint inhibitor is a PD-1 inhibitor and/or a PD-L1 inhibitor. 22. The method according to any one of claims 17-21, wherein the antibody is conjugated or linked to at least one growth suppressive agent. 23. The method of claim 22, wherein said growth suppressant is N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) or N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4). 24. The method according to any one of claims 17-23, wherein the antibody is covalently attached via a cleavable or non-cleavable linker to at least one growth suppressive agent. 25. The method of claim 24, wherein said linker is selected from the group consisting of N-succinimidyl pyridyl dithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfobutyric acid (sulfo-SPDB), and succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). 26. The method according to any one of claims 17-25, wherein the antibody is administered every 14 days or every 3 weeks. 27. Use of an immunoconjugate comprising an antibody for the treatment of non-squamous non-small cell lung cancer (NSQ NSCLC) in a subject in need thereof, wherein the antibody specifically binds to hCEACAM5, and wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 8 (EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISS GGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGP FAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG), and wherein the light chain comprises SEQ ID NO: 9 (DIQMTQSPASLSSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTL AEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC), wherein the immunoconjugate is administered at a dose level of 80, 100, or 120 mg/ ^m2 _, based on the subject's body surface area, every 14 days or every 3 weeks. 28. A method for treating non-squamous non-small cell lung cancer (NSQ NSCLC), wherein the method comprises administering an immunoconjugate comprising an antibody, wherein the antibody specifically binds to hCEACAM5, and wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 8 (EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISS GGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGP FAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG), and wherein the light chain comprises SEQ ID NO: 9 (DIQMTQSPASLSSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTL AEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC), wherein the immunoconjugate is administered at a dose level of 80, 100, or 120 mg/ ^m2 , based on the subject's body surface area, every 14 days or every 3 weeks. 29. Use of an antibody or an immunoconjugate comprising the antibody for the treatment of non-squamous non-small cell lung cancer (NSQ NSCLC) in a subject in need thereof, wherein the antibody specifically binds hCEACAM5 and wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 8 (EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWV AYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCA AHYFGSSGPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG), the light chain comprises SEQ ID NO: 9 (DIQMTQSPASLSSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVY NTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGS GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC), wherein the antibody is covalently attached via a cleavable or non-cleavable linker to at least one growth inhibitory agent, and said linker is selected from the group consisting of N-succinimidyl pyridyl dithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfobutyric acid (sulfo-SPDB) and succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), wherein the growth inhibitory agent is (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) DM1 or N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4), where the immunoconjugate is administered as a separate agent, wherein the subject is characterized by hCEACAM5 expression, consisting of 2+ and 3+ intensities, in greater than or equal to 50% of tumor cell populations, wherein the subject has previously been treated with an agent or drug for the treatment of non-small cell lung cancer, wherein the agent or drug is selected from a chemotherapeutic agent and an immune checkpoint inhibitor, wherein the immunoconjugate is administered at a dose level of 80, 100, or 120 mg/ ^m2 based on the subject's body surface area, every 14 days or every 3 weeks. 30. A method for treating non-squamous non-small cell lung cancer (NSQ NSCLC) in a subject in need thereof, the method comprising administering an immunoconjugate comprising an antibody, wherein the antibody specifically binds hCEACAM5 and wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 8 (EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWV AYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCA AHYFGSSGPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG), the light chain comprises SEQ ID NO: 9 (DIQMTQSPASLSSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVY NTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGS GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC), wherein the antibody is covalently attached via a cleavable or non-cleavable linker to at least one growth inhibitory agent, and said linker is selected from the group consisting of N-succinimidyl pyridyl dithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfobutyric acid (sulfo-SPDB) and succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), wherein the growth inhibitory agent is (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) DM1 or N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4), where the immunoconjugate is administered as a separate agent, wherein the subject is characterized by hCEACAM5 expression, consisting of 2+ and 3+ intensities, in greater than or equal to 50% of tumor cell populations, wherein the subject has previously been treated with an agent or drug for the treatment of non-small cell lung cancer, wherein the agent or drug is selected from a chemotherapeutic agent and an immune checkpoint inhibitor, wherein the immunoconjugate is administered at a dose level of 80, 100, or 120 mg/ ^m2 based on the subject's body surface area, every 14 days or every 3 weeks.