Monoclonal antibodies against carcinoembryonic antigens and their uses

Abstract

The present invention provides anti-carcinoembryonic antigen (CEA) antibodies for use in detecting CEA, treating disorders associated with CEA expression, diagnosing cancers characterized by aberrant CEA expression, and predicting the efficacy of cancer chemotherapy. Anti-CEA antibodies, antibody fragments, monoclonal antibodies, antibody conjugates, compositions comprising the described antibodies, and methods of their use are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 63 / 336,676, filed April 29, 2022, and U.S. Application No. 63 / 275,998, filed November 5, 2021, the entire contents of which are incorporated by reference herein for all purposes.

[0002] The present invention relates to monoclonal antibodies (mAbs) and antigen-binding fragments thereof that bind carcinoembryonic antigen (also referred to herein as "CEA"). The present invention thus encompasses these mAbs, and in particular their use for detecting CEA and for diagnosing and treating diseases and conditions associated with, or known to be associated with, aberrant CEA expression, such as cancer. [Background technology]

[0003] Carcinoembryonic antigen (CEA) belongs to the family of immunoglobulins with a molecular weight of about 180,500 daltons (180 kDa). CEA is a protein present in certain tissues of the developing baby (fetus), but by the time the baby is born, its expression drops to very low levels. In adults, CEA is normally present at very low levels in the blood, but can be elevated in certain types of cancer. CEA, also known as CEACAM5 or CD66e, was found in malignant tumors of endoderm-derived epithelium of the gastrointestinal tract and pancreas (Gold et al. The Journal of Experimental Medicine 122:467-481 (1965)). Since its discovery almost 50 years ago, CEA has been found to be overexpressed in the majority of human carcinomas (Gold et al. The Journal of Experimental Medicine 122:467-481 (1965), Blumenthal et al. BMC Cancer 7:1-15 (2007)). The function of CEA is to transport iron from the intestine, reticuloendothelial system, and hepatocytes to all proliferating cells in the body. CEA may also have a physiological role as a granulocyte / pollen binding protein (GPBP) involved in the removal of certain organics and allergens from serum, and may have an additional role in stimulating cell proliferation. Human CEA is described in the database UniProtKB / Swiss-Prot:P06731. CEA has immunoglobulin-like structural characteristics and has many glycosylation modification sites (Beauchemin et al. Cancer and Metastasis Reviews 32:643-671(2013)). CEA can also be overexpressed in some non-cancer-related conditions, such as inflammation, liver cirrhosis, peptic ulcer, ulcerative colitis, rectal polyps, emphysema, and benign breast disease, as well as in smokers. For this reason, detection of CEA is generally considered to be useful not as a general cancer screening tool, but rather to evaluate response to cancer treatment. See The Dynamic Monitoring of CEA in Response to Chemotherapy and Prognosis of MCRC Patients. Yu, et al. BMC Cancer. (2018) 18: 1076, and ASCO 2006 Update of Recommendations for the Use of Tumor Markers in Gastrointestinal Cancer. Gershon et al. Journal of Clinical Oncology (2006) 24: 5313. For example, when an individual is diagnosed with cancer, an initial baseline test for CEA can be performed. Subsequent serial testing of CEA can be performed to monitor cancer progression as the individual undergoes treatment and / or when the individual goes into remission. See Roberto et al. British Journal of Cancer (2021) 124:839. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Gold et al.The Journal of Experimental Medicine 122:467-481(1965) [Non-Patent Document 2] Blumenthal et al.BMC Cancer 7:1-15(2007) [Non-Patent Document 3] Beauchemin et al.Cancer and Metastasis Reviews 32:643-671(2013) [Non-Patent Document 4] The Dynamic Monitoring of CEA in Response to Chemotherapy and Prognosis of MCRC Patients.Yu,et al.BMC Cancer.(2018)18:1076 [Non-Patent Document 5] ASCO 2006 Update of Recommendations for the Use of Tumor Markers in Gastrointestinal Cancer.Gershon et al.Journal of Clinical Oncology(2006)24:5313 [Non-Patent Document 6] Roberto et al.British Journal of Cancer(2021)124:839 [Brief description of the drawings]

[0005] [Figure 1] The heavy chain amplicon of ADx-CEA is shown. [Diagram 2] Showing the NCBI_Ig Blast Tool_IMGT view of the heavy chain of ADx-CEA. Single letter coded amino acid positions, CDR positions, and inclusion of gaps (*) to maximize homology within human IgHVDJ are from the IMGT database. The nucleotide and predicted protein sequence are shown below for the heavy chain of the VDJ region. [Diagram 3] NCBI_Ig Tool_IMGT blast sample of the light chain of ADx-CEA is displayed. Single letter coded amino acid positions, CDR positions, and inclusion of gaps (*) to maximize homology within human IgKVJ are from the IMGT database. The nucleotide and predicted protein sequence are shown below for the K chain of the VJ region. [Figure 4] 1 shows SDS-PAGE analysis of isolated ADx-CEA heavy and light chains. [Diagram 5]1 shows a Western blot analysis of CEA detected by isolated ADx-CEA. [Figure 6A] Representative images of indirect immunofluorescence staining of CEA detected by isolated ADx-CEA localized in vesicles in a punctate manner are shown in Figure 6A. Figure 6B shows a phase contrast image. [Figure 6B] Representative images of indirect immunofluorescence staining of CEA detected by isolated ADx-CEA localized in vesicles in a punctate manner are shown in Figure 6B. [Figure 7] 1 shows a Western blot analysis of isolated ADx-CEA in patient samples, where the patient has pancreatic cancer compared to a control. [Figure 8] 1 shows IHC staining analysis of ADx-CEA in pancreatic cancer tissues at various stages. [Figure 9A] Figure 9 shows the overall sensitivity and specificity of ADx-CEA. Figure 9A shows the sensitivity compared to Dako Omnis (Agilent) anti-CEA mAb (clone II-7). [Figure 9B] Figure 9B shows the overall sensitivity and specificity of ADx-CEA, and Figure 9B shows the specificity compared to the Dako antibody. [Figure 10A] Figure 10 shows the sensitivity and specificity of ADx-CEA at early stages (I, II). Figure 10A shows the sensitivity compared to Dako Omnis (Agilent) anti-CEA mAb (clone II-7). [Figure 10B] Figure 10 shows the sensitivity and specificity of ADx-CEA at early stages (I, II). Figure 10B shows the specificity compared to Dako Omnis (Agilent) anti-CEA mAb (clone II-7). [Figure 11A] Figure 11 shows the sensitivity and specificity of ADx-CEA at late stages (III, IV). Figure 11A shows the sensitivity compared to Dako Omnis (Agilent) anti-CEA mAb (clone II-7). [Figure 11B]Figure 11B shows the sensitivity and specificity of ADx-CEA at late stages (III, IV).Figure 11B shows the specificity compared to Dako Omnis (Agilent) anti-CEA mAb (clone II-7). [Figure 12A] Soft agar coronary artery formation and related data: Figure 12A shows a representative low magnification image of colony formation in untreated BxPC-3 cells. [Figure 12B] Soft agar coronary artery formation and related data are shown. Figure 12B shows representative high magnification images of colony formation in ADx-CEA treated and untreated BxPC-3 cells. Images of wells are representative of three independent experiments. [Figure 12C] Soft agar coronary artery formation and related data are shown. Figure 12C shows quantification of BxPC-3 colony numbers after ADx-CEA treatment. [Figure 13A] Figure 13 shows CEA expression in various formalin-fixed paraffin-embedded (FFPE) human tissues via IHC staining. CEA protein expression was verified in healthy and cancerous tissues from healthy individuals and cancer patients using ADx-CEA. Pancreatic, colonic and liver cancer tissues showed positive membrane and cytoplasmic staining for CEA expression. Figure 13A shows the results for pancreatic tissue. [Figure 13B] Figure 13 shows CEA expression in various formalin-fixed paraffin-embedded (FFPE) human tissues via IHC staining. CEA protein expression was verified in healthy and cancerous tissues from healthy individuals and cancer patients using ADx-CEA. Pancreatic, colon and liver cancer tissues showed positive membrane and cytoplasmic staining for CEA expression. Figure 13B shows the results for colon tissue. [Figure 13C] Figure 13 shows CEA expression in various formalin-fixed paraffin-embedded (FFPE) human tissues via IHC staining. CEA protein expression was verified in healthy and cancerous tissues from healthy individuals and cancer patients using ADx-CEA. Pancreatic, colonic and liver cancer tissues showed positive membrane and cytoplasmic staining for CEA expression. Figure 13C shows the results for liver tissue. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] I. Definition In this application, the use of "or" means "and / or" unless otherwise specified. In the context of multiple dependent claims, the use of "or" refers back to two or more preceding independent or dependent claims, in the alternative only. The terms "comprising", "including", and "having" may be used interchangeably herein. In accordance with the present invention, an "isolated" molecule is one that has been removed from its natural environment. Thus, the term "isolated" does not necessarily reflect the extent to which a molecule has been purified.

[0007] As used herein, CEA is also known as carcinoembryonic antigen-related cell adhesion molecule 5, CECAM5, cell adhesion molecule 5, and CD66e. See, e.g., UniProtKB / Swiss-Prot:P06731.

[0008] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Recombinant antibodies, chimeric antibodies, and humanized antibodies are also included.

[0009] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0010] The term "antigen" refers to one or more molecules or one or more portions of a molecule that can be bound by an antibody and can induce an animal to produce an antibody that can bind to the epitope of the antigen. An antigen may have one or more epitopes. The specific reaction referred to above means that the antigen reacts very preferentially with its corresponding antibody and not with the multitude of other antibodies that may be elicited by other antigens. The binding of the antigen to the antibody must be above background levels.

[0011] The term "cancer" is used herein to refer to a group of cells that exhibits abnormally high levels of proliferation and growth. Cancers can be benign (also called benign tumors), pre-malignant, or malignant. Cancer cells can be solid cancer cells or leukemic cancer cells.

[0012] The term "complementarity determining region, or CDR" refers to an amino acid sequence which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. Each of the light and heavy chains of an immunoglobulin has three CDRs.

[0013] The "class" of an antibody refers to the type of constant domain or region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these are further subdivided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0014] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain having a structure substantially similar to a native antibody structure or containing an Fc region as defined herein.

[0015] A "human antibody" is one that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0016] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of a natural antibody generally have a similar structure, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6 th ed., W. H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind a specific antigen may be isolated using the VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0017] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In some embodiments, for VL, the subgroup is subgroup kappa I as in Kabat et al. (see above). In some embodiments, for VH, the subgroup is subgroup III as in Kabat et al. (see above).

[0018] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts"). Generally, antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).

[0019] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0020] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are identical and / or bind the same epitope, except for variant antibodies that may, for example, contain naturally occurring mutations or arise during the production of a monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0021] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or the heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or the light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0022] "Stage" of pancreatic cancer refers to the American Joint Committee on Cancer (AJCC) clinical stages: 0, IA, IB, IIA, IIB, III, and IV, as defined by the American Joint Committee on Cancer. Exocrine Pancreas.In: AJCC Cancer Staging Manual.8 th ed. New York, NY: Springer; 2017:337. As used herein, "early stage" pancreatic cancer refers to stages I and II, and "late stage" pancreatic cancer refers to stages III and IV.

[0023] As used herein, the term "negative control" in relation to assays and detections referred to herein refers to a control commonly used in the art by a professional for the type of assay and type of cancer being detected or assayed. In one example of detecting CEA levels in human blood, normal blood (plasma or serum) CEA levels may be about 0-2.5 ng / mL. If the detected CEA level is higher than this level, the level may be said to be higher than the negative control.

[0024] II. Compositions and Methods Anti-CEA antibodies, antibody fragments, monoclonal antibodies, antibody conjugates, compositions comprising the described antibodies, and methods of their use are provided.

[0025] A. Exemplary Anti-CEA Antibodies The following sequence listing provides the sequences of the antibodies (ADx-CEA) disclosed and claimed herein, see SEQ ID NOs: 1-12.

[0026] Provided herein are antibodies and antibody fragments that specifically bind to human CEA, including ADx-CEA, and fragments and variants thereof.

[0027] In certain embodiments, anti-CEA antibodies and antibody fragments are provided that comprise a heavy chain variable region ("VH") comprising a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 1, 2, and 3, respectively, and a light chain variable region ("VL") comprising a VL CDR1, CDR2, and / or CDR3 of any one of SEQ ID NOs: 4, 5, and 6, respectively.

[0028] In some embodiments, an antibody is provided that comprises: (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO:1; (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:2; (c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:3; (d) an LCDR1 (with or without) comprising the amino acid sequence of SEQ ID NO:4; (e) an LCDR2 comprising the amino acid sequence of SEQ ID NO:5; and (f) an LCDR3 comprising the amino acid sequence of SEQ ID NO:6; or (b) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7 and the VL is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8; or (c) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL comprises the amino acid sequence of SEQ ID NO: 8; or (d) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, (c) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, (d) an LCDR1 (with or without) comprising the amino acid sequence of SEQ ID NO: 4, (e) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (f) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, and a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7, and the VL is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0029] In some embodiments, the isolated antibody or antibody fragment described herein is a monoclonal antibody. In further embodiments, the isolated antibody or antibody fragment is a fully human antibody. In some embodiments, the isolated antibody or antibody fragment is an antibody fragment. In further embodiments, the antibody fragment is a Fab, Fab', Fv, scFv, or (Fab') 2 In some embodiments, the isolated antibody or antibody fragment is a full length antibody. In some embodiments, the isolated antibody or antibody fragment comprises an Fc region, and the Fc region of the antibody comprises IgG1, IgG2, IgG3, IgG4, IgG4.1, or IgG4.2.

[0030] In some embodiments, a composition is provided that includes an antibody or antibody fragment and a pharma- ceutically acceptable carrier.

[0031] In some embodiments, the isolated antibody or antibody fragment is immobilized on a solid phase. In some embodiments, the isolated antibody or antibody fragment is detectably labeled. In some embodiments, the isolated antibody or antibody fragment is conjugated to a cytotoxic radionuclide. In some embodiments, the isolated antibody or antibody fragment is conjugated to a cytotoxic drug. In some embodiments, the isolated antibody or antibody fragment is conjugated to a cytotoxic protein.

[0032] In some embodiments, an isolated DNA sequence is provided that encodes an antibody or antibody fragment. In some embodiments, the DNA comprises SEQ ID NO:11 or a fragment thereof. In some embodiments, the DNA comprises SEQ ID NO:12 or a fragment thereof. In some embodiments, the DNA comprises SEQ ID NO:11 or a fragment thereof and SEQ ID NO:12 or a fragment thereof. In some embodiments, the DNA comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:11, or a fragment thereof. In some embodiments, the DNA comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:12, or a fragment thereof. In some embodiments, the DNA comprises SEQ ID NO:11 or a fragment thereof and SEQ ID NO:12 or a fragment thereof. In some embodiments, the DNA comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:11 or a fragment thereof, and a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:12 or a fragment thereof.

[0033] In some embodiments, a vector is provided that comprises a DNA sequence encoding an antibody or antibody fragment.

[0034] In some embodiments, a host cell is provided that is transformed with a vector containing a DNA sequence encoding an antibody or antibody fragment.

[0035] In some embodiments, a process for the production of an antibody is provided, comprising culturing a host cell transformed with a vector containing a DNA sequence encoding an antibody or antibody fragment, and isolating the antibody molecules.

[0036] In certain embodiments, the anti-CEA antibody or antibody fragment comprises an isolated antibody comprising a VH at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and a VL at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, (d) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, (e) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (f) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 (with or without).

[0037] In some embodiments, the present invention includes a method of treating cancer, comprising administering a therapeutically effective amount of an anti-CEA antibody or antibody fragment, or a composition thereof, or a DNA or vector thereof, described herein, to a subject in need thereof. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer.

[0038] In certain embodiments, an immunoassay for detecting CEA antigen is provided, comprising: (a) contacting a sample with an effective binding amount of an antibody or antibody fragment described herein, such as ADx-CEA; and (b) detecting the antigen by detecting binding of the antibody to the CEA antigen. In some embodiments, the assay is used to detect cancer cells expressing the CEA antigen. In some embodiments, the assay is used to detect solid tumors. In further embodiments, the cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer.

[0039] In some embodiments, the invention includes a method of detecting cancer in a subject, comprising contacting a sample isolated from a subject having or suspected of having cancer with an antibody or antibody fragment described herein. In some embodiments, the cancer is a solid tumor. In further embodiments, the solid tumor is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer.

[0040] In certain embodiments, the present invention provides a kit for immunohistochemical detection of solid tumor cancers containing cells expressing the CEA antigen, comprising: (a) an antibody or antibody fragment as described herein, such as ADx-CEA; and (b) a secondary antibody conjugated to a detectable label, which binds to and can detect the antibody of (a) when bound to the CEA antigen. Alternatively, the secondary antibody is not conjugated to a detectable label, and instead a label that binds to or interacts with the secondary antibody is used for detection. Methods of detecting CEA using the kit are included.

[0041] In some embodiments, the invention is a method for determining the status of a solid tumor cancer in a subject, comprising: (a) removing a sample from a subject having a solid tumor cancer; (b) contacting the sample with an antibody or antibody fragment described herein, such as ADx-CEA, thereby forming a complex between the CEA antigen and the antibody or antibody fragment; (c) labeling the specimen with a label specific for the antigen-antibody complex; and (d) detecting the presence of the antigen-antibody complex by detecting the label. In some embodiments, the sample is blood, including whole blood, serum, or plasma, or tissue or cells.

[0042] In some embodiments, steps (a)-(d) described herein are repeated at a later time point. In some embodiments, this time point is after the subject has started a therapy. In some embodiments, this time point is after the subject has started a new or second (co)therapy. In some embodiments, the solid tumor cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer. In some embodiments, the solid tumor cancer is pancreatic cancer. In some embodiments, the subject has been diagnosed with cancer and is undergoing or about to undergo a therapy. In some embodiments, the sample is isolated from the subject before the initiation of the therapy. In some embodiments, the sample is isolated from the subject after the initiation of the therapy. In some embodiments, the therapy is chemotherapy or radiation. In some embodiments, the chemotherapy is erlotinib, fluorouracil, or oxaliplatin. In some embodiments, the therapy is surgery, including Whipple procedure, distal pancreatectomy, and total pancreatectomy, radiation therapy, proton beam therapy, stereotactic radiation therapy (SBRT) or Cyberknife, immunotherapy, targeted therapy, or chemotherapy.

[0043] In some embodiments, the invention includes a method for determining the status of a solid tumor cancer in a subject as described herein, in which CEA levels are determined, and a higher CEA level at a later time point indicates that the therapy is not completely effective, and the same or lower CEA antigen level at a later time point indicates that the therapy is at least partially effective. In some embodiments, the subject is in remission. In some embodiments, the method described herein is repeated at a time point after the subject is in remission, and CEA levels are determined, and a higher CEA level at a later time point indicates that the cancer is no longer in remission, and the same or lower CEA level at a later time point indicates that the cancer continues to be in remission. In some embodiments, the method is an alternative to computed tomography.

[0044] In certain embodiments, the present invention includes a method for detecting cancer characterized by expression of the gene product of CEA and its homologs, comprising: (a) identifying gene products and homologs expressed by CEA in a human patient with cancer by isolating a biological sample from the subject, the sample containing the CEA gene product if the CEA gene is expressed; (b) utilizing the gene product as a biomarker by contacting the biological sample with an anti-CEA antibody or antibody fragment described herein, such as ADx-CEA, or a composition thereof, to generate a sample-antibody complex; (c) removing any unbound antibody and then contacting the sample-antibody complex with an antibody or a label specific for the sample-antibody complex; and (d) detecting the presence of an antigen-antibody complex by detecting the label, the detection of the label being indicative of detection of cancer.

[0045] In some embodiments, the invention provides methods for determining the state of cells in a sample, comprising: (a) obtaining the sample from a subject; contacting the sample with an antibody or antibody fragment described herein, such as ADx-CEA, or a composition thereof; and (c) determining the amount of CEA detected by the antibody or antibody fragment.

[0046] In some embodiments, the present invention provides a method for detecting pancreatic cancer in a patient, the method comprising: (a) removing a pancreas or blood specimen from a patient suspected of or having pancreatic cancer; (b) contacting the specimen with an antibody or antibody fragment, or composition thereof, described herein, thereby forming an antigen-antibody complex in the specimen; (c) labeling the specimen with a label specific for the antibody or antigen-antibody complex; (d) detecting the presence of the antigen-antibody complex by detecting the label; and (e) determining a CEA antigen level compared to a negative control, where a higher CEA antigen level than the negative control is indicative of pancreatic cancer. In some embodiments, the method is performed in vitro. In further embodiments, the pancreatic cancer is at an early stage, and the method is capable of detecting CEA antigen at this early stage. In further embodiments, the pancreatic cancer is at stage I, II, IIA, or IIB, and the method is capable of detecting CEA antigen at this early stage. In some embodiments, the pancreatic cancer is at stage II, IIA, or IIB.

[0047] In some embodiments, the present invention provides a kit for the detection of cancer, comprising: (a) an antibody or antibody fragment having heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 4, 5 and 6, respectively, and (b) a secondary antibody conjugated to a detectable label or a separate unconjugated detectable label, wherein the detectable label or the separate unconjugated detectable label binds to the secondary antibody, antibody-antigen complex, or monoclonal antibody of (a). In some embodiments, the kit is for immunohistochemical detection of pancreatic cancer.

[0048] In some embodiments, the invention includes an immunohistochemical method for detecting cancer in a tissue specimen taken from a patient, comprising the steps of: (a) obtaining a tissue specimen; (b) contacting the tissue specimen with an antibody of an antibody fragment described herein, or a composition thereof; (c) after step (b), contacting the tissue specimen with a secondary antibody, antibody-antigen complex, or a detectable label that binds to the antibody; and (d) staining the tissue specimen with an immunohistochemical stain, wherein the staining indicates antibody binding and the presence of cancer in the tissue specimen. In some embodiments, the cancer is pancreatic cancer.

[0049] In some embodiments, compositions are provided comprising a tissue, cell, or blood specimen, an antibody or antibody fragment, or composition thereof, as described herein, and an antibody-antigen complex with a CEA antigen in the tissue, cell, or blood sample, wherein the sample is from a patient suffering from cancer. In certain embodiments, the cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer.

[0050] In some embodiments, there is provided a use of an antibody or antibody fragment, or composition thereof, described herein, for detecting cancer, hi further embodiments, the cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer.

[0051] In some embodiments, a method for monitoring the progression of cancer and / or therapeutic effectiveness of a cancer therapeutic comprises: (a) obtaining an initial sample from a human patient having cancer at a first time point; (b) contacting the sample with an antibody or antibody fragment, or composition thereof, described herein, to form an antigen-antibody complex; (c) labeling the specimen with a label specific for the antigen-antibody complex; (d) detecting the presence of the antigen-antibody complex by detecting the label; and (e) determining the level of CEA antigen detected by the antibody or antibody fragment to determine the level of CEA antigen associated with the patient's cancer. (f) obtaining a second sample at a second (later) time point, optionally after the subject has been undergoing therapy; (g) repeating steps (b)-(e) to determine a second CEA antigen level associated with the patient's cancer; and (h) determining that the patient's cancer has progressed if the CEA antigen level at the second time point is higher than the baseline level, and determining that the patient's cancer has regressed if the CEA antigen level at the second time point is lower than the baseline level. In some embodiments, the cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer. In some embodiments, the solid tumor cancer is pancreatic cancer. In some embodiments, an increase in CEA at the second time point indicates cancer progression. In some embodiments, a decrease in CEA at the second time point indicates cancer regression. In some embodiments, the first time point is pre-therapy or at initial diagnosis. In some embodiments, the first time point is after therapy or at initial diagnosis. In some embodiments, the second time point is after receiving therapy. In some embodiments, the method is performed in vitro. In some embodiments, the therapy is surgery, including Whipple procedure, distal pancreatectomy, and total pancreatectomy, radiation therapy, proton beam therapy, stereotactic radiation therapy (SBRT) or Cyberknife, immunotherapy, targeted therapy, and chemotherapy.

[0052] B. Glycosylation variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0053] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides that are generally attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may comprise a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the present invention may be performed to generate antibody variants with certain improved properties.

[0054] In some embodiments, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 compared to the sum of all glycan structures (e.g., complex, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2008 / 077546. "Asn297" refers to an asparagine residue located at about position 297 (Eu numbering of Fc region residues) in the Fc region. However, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Publication No. 2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 01321 40, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986), U.S. Patent Application No. 2003 / 0157108 A1 (Presta, L), and WO2004 / 056312 A1 (Adams et al., especially in Example 11), as well as knockout cell lines such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004), Kanda, Y. et al. al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO2003 / 085107).

[0055] Antibody variants are further provided with bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants include, for example, WO2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US2005 / 0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. For such antibody variants, see, e.g., WO1997 / 30087 (Patel et al.), WO1998 / 58964 (Raju, S.), and WO1999 / 22764 (Raju, S.).

[0056] C. Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0057] In certain embodiments, the present invention contemplates antibody variants that have some, but not all, effector functions, making them desirable candidates for applications where the half-life of the antibody in vivo is important, but where certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcgR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcgRIII, whereas monocytes express FcgRI, FcgRII, and FcgRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., for flow cytometry, see ACTI™ Non-Radioactive Cytotoxicity Assay (CellTechnology, Inc. Mountain View, CA, and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al. Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0058] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions of two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0059] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0060] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0061] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0062] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), have been described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions which improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., a substitution at Fc region residue 434 (e.g., U.S. Patent No. 7,371,826).

[0063] See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0064] In some embodiments, the antibody is provided according to the sequence listing and the isotype is human IgG1. In some embodiments, the antibody is provided according to the sequence listing and the isotype is human IgG2. In some embodiments, the antibody is provided according to the sequence listing and the isotype is human IgG3. In some embodiments, the antibody is provided according to the sequence listing and the isotype is human IgG4. [Table 1-1] [Table 1-2]

[0065] D. Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioM antibodies," in which one or more residues of an antibody are substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0066] E. Antibody derivatives In certain embodiments, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties that are known and readily available in the art. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0067] In other embodiments, conjugates of antibodies and nonproteinaceous moieties are provided that can be selectively heated by exposure to radiation. In some embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that are not harmful to normal cells but heat the nonproteinaceous moiety to a temperature that kills cells in close proximity to the antibody-nonproteinaceous moiety.

[0068] F. Recombinant Methods The antibodies may be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In some embodiments, an isolated nucleic acid is provided that encodes an anti-CEA antibody described herein. Such a nucleic acid may encode an amino acid sequence comprising the VL of the antibody, and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In further embodiments, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In some embodiments, the host cell is eukaryotic, for example, a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In some embodiments, a method of making an anti-CEA antibody is provided, comprising culturing a host cell comprising nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0069] For recombinant production of an anti-CEA antibody, for example, nucleic acid encoding the antibody as described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0070] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0071] In addition to prokaryotes, eukaryotes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0072] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0073] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0074] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed by SV40, human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK, buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals of Other useful mammalian host cell lines include TRI cells, MRC 5 cells, and FS4 cells, as described in NYAcad.Sci.383:44-68 (1982). - These include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0075] G. Immunoconjugates The present invention also provides immunoconjugates comprising an anti-CEA antibody herein conjugated to one or more other therapeutic agents or radioisotopes.

[0076] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 When used for detection, the radioconjugate may comprise a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0077] Conjugates of antibodies may be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6 diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). For example, carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug in cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (see Chari et al., Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0078] The immunoconjugates or ADCs herein expressly contemplate such conjugates prepared with cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0079] H. Pharmaceutical Formulations and Compositions Pharmaceutical formulations or compositions of anti-CEA antibodies described herein are prepared by mixing the antibody having the desired purity, in the form of a lyophilized formulation or aqueous solution, with one or more optional pharma- ceutically acceptable carriers, diluents, and / or excipients (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers, diluents, and excipients are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, sterile water, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of suitable pharmacokinetic and / or therapeutic agents include, but are not limited to, peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmacokinetic and therapeutic agents herein include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0080] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including histidine-acetate buffers.

[0081] The formulations or compositions herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose.

[0082] The active ingredient may be encapsulated in microcapsules prepared, for example, by droplet formation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0083] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include translucent matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0084] Formulations or compositions to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0085] I. Embodiment The following embodiments are provided: Embodiment 1. An isolated antibody or antibody fragment comprising: (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO:1; (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:2; (c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:3; (d) an LCDR1 comprising the amino acid sequence of SEQ ID NO:4; (e) an LCDR2 comprising the amino acid sequence of SEQ ID NO:5; and (f) an LCDR3 comprising the amino acid sequence of SEQ ID NO:6. Embodiment 2. The antibody comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and V L is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8. Embodiment 3. The antibody comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H comprises the amino acid sequence of SEQ ID NO:7, and V L 3. The isolated antibody of embodiment 1 or 2, wherein said antibody comprises the amino acid sequence of SEQ ID NO:8. Embodiment 4. An isolated antibody or antibody fragment, comprising: (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO:1; (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:2; (c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:3; (d) an LCDR1 comprising the amino acid sequence of SEQ ID NO:4; (e) an LCDR2 comprising the amino acid sequence of SEQ ID NO:5; and (f) an LCDR3 comprising the amino acid sequence of SEQ ID NO:6; and a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and V Lan isolated antibody or antibody fragment which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8. Embodiment 5. The isolated antibody or antibody fragment of any one of the preceding embodiments, wherein the antibody is a monoclonal antibody. Embodiment 6 The isolated antibody or antibody fragment of any one of the preceding embodiments, wherein the antibody is a fully human antibody. Embodiment 7. The isolated antibody or antibody fragment of any one of the preceding embodiments, wherein the antibody is an antibody fragment. Embodiment 8. The fragment is a Fab, Fab', Fv, scFv, or (Fab') 2 8. The isolated antibody or antibody fragment of embodiment 7, wherein Embodiment 9. The isolated antibody or antibody fragment of any one of embodiments 1-5, wherein the antibody is a full-length antibody. Embodiment 10. The isolated antibody or antibody fragment of any one of embodiments 1-9, wherein the Fc region of the antibody, if present, comprises IgG1, IgG2, IgG3, IgG4, IgG4.1, or IgG4.2. Embodiment 11. A composition comprising the antibody or antibody fragment of any one of embodiments 1 to 10 and a pharma- ceutically acceptable carrier. Embodiment 12. An isolated antibody or antibody fragment of any one of embodiments 1 to 10, immobilized on a solid phase. Embodiment 13. An isolated antibody or antibody fragment of any one of embodiments 1 to 10, which is detectably labeled. Embodiment 14. An isolated antibody or antibody fragment of any one of embodiments 1 to 10, conjugated to a cytotoxic radionuclide. Embodiment 15. An isolated antibody or antibody fragment of any one of embodiments 1 to 10 conjugated to a cytotoxic drug. Embodiment 16. An isolated antibody or antibody fragment of any one of embodiments 1 to 10 conjugated to a cytotoxic protein. Embodiment 17. An isolated DNA sequence encoding the antibody or antibody fragment of any one of embodiments 1 to 10. Embodiment 18. The DNA of embodiment 17, comprising SEQ ID NO:11 or a fragment thereof, or a sequence that is at least 80, 90, or 95% identical to SEQ ID NO:11. Embodiment 19. The DNA of embodiment 17, comprising SEQ ID NO:12 or a fragment thereof, or a sequence that is at least 80, 90, or 95% identical to SEQ ID NO:12. Embodiment 20. The DNA of embodiment 17, comprising SEQ ID NO:11 or a fragment thereof and SEQ ID NO:12 or a fragment thereof, or comprising a sequence that is at least 80, 90, or 95% identical to SEQ ID NO:11 and a sequence that is at least 80, 90, or 95% identical to SEQ ID NO:12. Embodiment 21. A vector comprising any one of the DNA sequences of embodiments 17 to 20. Embodiment 22. A host cell transformed with the vector of embodiment 21. Embodiment 23. A process for the production of an antibody, comprising culturing the host cell of embodiment 22 and isolating the antibody molecules. Embodiment 24. A V that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7. H and V that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8. L and an isolated antibody comprising: Embodiment 25. A method for treating cancer, comprising administering a therapeutically effective amount of an anti-CEA antibody described in any one of claims 1 to 10 or 14 to 16, a composition described in claim 11, or a DNA or vector described in any one of claims 17 to 21 to a subject in need thereof. Embodiment 26. An immunoassay for detecting a CEA antigen, comprising: (a) contacting the sample with an effective binding amount of the antibody or antibody fragment of any one of embodiments 1 to 10; and (b) detecting the CEA antigen by detecting binding of the antibody to the antigen. Embodiment 27 The immunoassay of embodiment 26, wherein the assay is used to detect cancer cells expressing the CEA antigen. Embodiment 28 The immunoassay of embodiment 26, wherein the assay is used to detect solid tumors. Embodiment 29. The immunoassay of embodiment 27 or 28, wherein the cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer. Embodiment 30. A method for detecting cancer in a subject, comprising contacting a sample isolated from a subject having or suspected of having cancer with an antibody of any one of embodiments 1 to 10. Embodiment 31 The method of embodiment 30, wherein the cancer is a solid tumor. Embodiment 32 The method of embodiment 30, wherein the solid tumor is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer. Embodiment 33. A kit for immunohistochemical detection of solid tumor cancers containing cells expressing the CEA antigen, comprising: (a) an antibody according to any one of embodiments 1 to 10; and (b) a secondary antibody conjugated to a detectable label, the secondary antibody binding to and capable of detecting the antibody of (a) when bound to the CEA antigen. Embodiment 34. A method for determining the status of a solid tumor cancer in a subject, comprising: (a) removing a sample from a subject having a solid tumor cancer; (b) contacting the sample with an antibody or antibody fragment of any one of embodiments 1 to 10, thereby forming a complex between the CEA antigen and the antibody or antibody fragment; (c) labeling the specimen with a label specific for the antigen-antibody complex; and (d) detecting the presence of the antigen-antibody complex by detecting the label. Embodiment 35. The method of embodiment 34, wherein the solid tumor cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer. Embodiment 36 The method of embodiment 34, wherein the solid tumor cancer is pancreatic cancer. Embodiment 37. The method of any one of embodiments 34-36, wherein the subject has been diagnosed with cancer and is undergoing or about to undergo therapy. Embodiment 38 The method of embodiment 37, wherein the sample is isolated from the subject prior to initiation of therapy. Embodiment 39 The method of embodiment 37, wherein the sample is isolated from the subject after initiation of therapy. Embodiment 40. The method of any one of embodiments 34-39, further comprising repeating steps (a)-(d) at a later time. Embodiment 41 The method of embodiment 40, wherein the later time point is after the subject has begun therapy. Embodiment 42. The method of embodiment 40, wherein the later time point is after the subject has started a new or second (co-)therapy. Embodiment 43. The method of embodiment 41 or 42, wherein the therapy is chemotherapy or radiation. Embodiment 44. The method of claim 43, wherein the chemotherapy is erlotinib, fluorouracil, or oxaliplatin. Embodiment 45. The method of claim 41 or 42, wherein the therapy is surgery, including Whipple procedure, distal pancreatectomy, and total pancreatectomy, radiation therapy, proton beam therapy, stereotactic radiation therapy (SBRT) or Cyberknife, immunotherapy, targeted therapy, or chemotherapy. Embodiment 46. The method of any one of embodiments 34 to 45, wherein the detected CEA antigen level is determined, and a higher CEA level at a later time point indicates that the therapy is completely effective, and the same or a lower CEA antigen level at a later time point indicates that the therapy is at least partially effective. Embodiment 47. The method of any one of embodiments 34-46, wherein the subject is in remission. Embodiment 48. The method of embodiment 47, further comprising repeating steps (a)-(d) at a time point after the subject has gone into remission, wherein the CEA level is determined, and a higher CEA level at the later time point indicates that the cancer is no longer in remission, and the same or a lower CEA level at the later time point indicates that the therapy is continuing in remission. Embodiment 49. The method of any one of embodiments 34 to 48, wherein the sample is blood, including whole blood, serum, or plasma, tissue, or cells. Embodiment 50. The method of any one of embodiments 34 to 49, wherein the method is an alternative to computed tomography. Embodiment 51. A method for detecting cancer characterized by expression of the gene product of CEA and its homologues, comprising: (a) identifying the gene product and its homologues expressed by CEA in a human patient having cancer by isolating a biological sample from the subject, the sample containing the CEA gene product if the CEA gene is expressed; (b) contacting the biological sample with an anti-CEA antibody according to any one of claims 1 to 10 or 14 to 16, or the composition according to claim 11, to generate a sample-antibody complex, thereby utilizing the gene product as a biomarker; (c) removing any unbound antibody, and then contacting the sample-antibody complex with an antibody or a label specific for the sample-antibody complex; and (d) detecting the presence of an antigen-antibody complex by detecting the label, the detection of the label indicating detection of cancer. Embodiment 52. A method for determining the state of cells in a sample, comprising: a. obtaining said sample from a subject; b. contacting the sample with an antibody or antibody fragment of any one of embodiments 1-10 or 14-16, or a composition of embodiment 11; c. determining the amount of CEA detected by the antibody or antibody fragment. Embodiment 53. A method for detecting pancreatic cancer in a patient, comprising: (a) removing a pancreas or blood specimen from a patient suspected of or having pancreatic cancer; (b) contacting the specimen with an antibody or antibody fragment of any one of embodiments 1-10 or 14-16, or a composition of claim 11, thereby forming an antigen-antibody complex in the specimen; (c) labeling the specimen with a label specific for the antibody or antigen-antibody complex; (d) detecting the presence of the antigen-antibody complex by detecting the label; and (e) determining the CEA antigen level compared to a negative control, wherein a higher CEA antigen level than the negative control is indicative of pancreatic cancer. Embodiment 54 The method of embodiment 53, wherein the method is carried out in vitro. Embodiment 55 The method of embodiment 53 or 54, wherein the pancreatic cancer is at an early stage and the method is capable of detecting CEA antigen at this early stage. Embodiment 56. The method of any of embodiments 53 to 55, wherein the pancreatic cancer is at stage I, II, III, or IV, and the method is capable of detecting CEA antigen at this early stage. Embodiment 57. A kit for immunohistochemical detection of pancreatic cancer, comprising: (a) a monoclonal antibody having heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 4, 5, and 6, respectively; and (b) a secondary antibody conjugated to a detectable label or a separate unconjugated detectable label, wherein the detectable label or the separate unconjugated detectable label binds to the secondary antibody, the antibody-antigen complex, or the monoclonal antibody of (a). Embodiment 58. An immunohistochemical method for detecting pancreatic cancer in a tissue specimen taken from a patient, comprising the steps of: (a) obtaining a tissue specimen; (b) contacting the tissue specimen with an antibody of any one of embodiments 10 or 14 to 16, or the composition of claim 11; (c) after step b), contacting the tissue specimen with a secondary antibody, an antibody-antigen complex, or a detectable label that binds to the antibody; and (d) staining the tissue specimen with an immunohistochemical stain, wherein the staining indicates antibody binding and the presence of pancreatic cancer in the tissue specimen. Embodiment 59. A composition comprising a tissue specimen and an antibody-antigen complex with the antibody or antibody fragment of any one of embodiments 10 or 14 to 16, or the composition of claim 11, wherein the tissue specimen is from a patient suffering from cancer. Embodiment 60. The composition of embodiment 59, wherein the cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer. Embodiment 61. Use of the composition of embodiment 11 or the antibody or antibody fragment of any one of embodiments 1 to 10 or 11 to 14 for detecting cancer. Embodiment 62. The composition of embodiment 61, wherein the cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer. Embodiment 63. A method for monitoring the progression of cancer and / or the therapeutic efficacy of a cancer therapeutic comprising obtaining an initial sample from a human patient having cancer at a first time point, contacting the sample with an antibody or antibody fragment of any one of embodiments 10 or 14 to 16, or a composition according to claim 11 to form an antigen-antibody complex, labeling the specimen with a label specific for the antigen-antibody complex, detecting the presence of the antigen-antibody complex by detecting the label, and determining the level of CEA antigen detected by the antibody or antibody fragment to identify the patient's cancer. A method comprising determining a baseline level of the relevant CEA antigen, obtaining a second sample at a second (later) time point, optionally after a period during which the subject has been receiving therapy, and repeating steps (b)-(e) to determine a second CEA antigen level associated with the patient's cancer, determining that the patient's cancer has progressed if the CEA antigen level at the second time point is higher than the baseline level, and determining that the patient's cancer has regressed if the CEA antigen level at the second time point is lower than the baseline level. Embodiment 64. The method of embodiment 63, wherein the cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer. Embodiment 65 The method of embodiment 63, wherein the solid tumor cancer is pancreatic cancer. Embodiment 66 The method of embodiment 63, wherein an increase in CEA at the second time point indicates progression of the cancer. Embodiment 67 The method of embodiment 63, wherein a decrease in CEA at the second time point indicates regression of the cancer. Embodiment 68 The method of embodiment 63, wherein the first time point is pre-therapy or at the time of initial diagnosis. Embodiment 69. The method of embodiment 63, wherein the first time point is post-therapy or at initial diagnosis. Embodiment 70 The method of embodiment 63, wherein the second time point is after receiving therapy. Embodiment 71 The method of embodiment 63, wherein the method is carried out in vitro. Embodiment 72. The method of embodiment 69, wherein the therapy is surgery, including Whipple procedure, distal pancreatectomy, and total pancreatectomy, radiation therapy, proton therapy, stereotactic radiation therapy (SBRT) or Cyberknife, immunotherapy, targeted therapy, and chemotherapy. EXAMPLES

[0086] Example 1. Antibodies were prepared using native (wt) CEA antigen as an immunogen, and approximately 19,200 hybridomas were produced and screened. Approximately 20 promising candidates were further evaluated, and finally five clones were selected for follow-up as follows: After determining the IgG isotypes (IgG1, G2, G2a, G2b) of the hybridoma clones, the hybridoma cell line cultures (2 × 10 6Total RNA was extracted from the 100-kDa guinea pigs. RNA was reverse transcribed into complementary DNA (cDNA) using the SuperScript® III First Strand Synthesis System (ThermoFisher Scientific, Carlsbad, CA, USA). One microgram of cDNA from each sample was subjected to polymerase chain reaction (PCR). Mouse universal Ig heavy or light chain primers were used to amplify the V-regions of the cDNA samples of the hybridoma clone (ADx-CEA). Universal primers were designed from highly conserved regions described in Dattamajumdar, Anupam K., et al. "Rapid cloning of any rearranged mouse immunoglobulin variable genes." Immunogenetics 43.3 (1996): 141-151. Primer pair HF and HR were used to amplify gene H, and pair KF and KR were used to amplify gene K. See Table 2. The heavy chain primer sequences were as follows: VH 5'-tgaggtgcagctggaggagtc-3' (SEQ ID NO: 13) and JH 5'-gtgaccgtggtcccttggccccag-3' (SEQ ID NO: 14). The light chain primer sequences were as follows: VK 5'-gacattctgatgacccagtct-3' (SEQ ID NO: 15) and JK 5'-ttttatttccagcttggtccc-3' (SEQ ID NO: 16). The pairs are listed in Table 2 below. PCR cycling was performed with an initial 6 cycles of 94°C for 15 s, 62°C (-1.2°C / cycle) for 30 s, and 72°C for 30 s, followed by 30 cycles of 94°C for 15 s, 56°C for 30 s, and 72°C for 30 s. One hybridoma was selected as the lead for further testing and produced the antibody ADx-CEA having CDRs of SEQ ID NOs: 1-6, VH of SEQ ID NO: 7, VL of SEQ ID NO: 8, full heavy of SEQ ID NO: 9, and full light of SEQ ID NO: 10, as described herein. Throughout, this lead antibody is referred to as ADx-CEA. The amplicon for ADx-CEA is shown in Figure 1, with the heavy and light chains running at approximately 380-500 bp on 1% agarose. [Table 2-1] [Table 2-2]

[0087] Example 2. PCR products were purified and cloned into pCR4-TOPO vector using a TA cloning strategy (ThermoFisher Scientific, Carlsbad, CA, USA) according to the manufacturer's instructions. Three to five single colonies were selected and plasmid DNA was amplified using primers specific to the vector DNA sequence. Subcloning was performed as necessary. Plasmids from each colony were isolated using a Miniprep Kit (Qiagen Inc). PCR inserts were verified by restriction analysis using EcoR1 digestion and sequenced on both strands using M13 universal primers using cycle sequencing reactions with fluorescent dye terminators and capillary-based electrophoresis. DNA sequence data from all constructs were analyzed to determine consensus sequences for the heavy and light chains. The consensus sequences were compared to all known variable region sequences to rule out artifacts and / or process contamination. The consensus sequences were then analyzed using an online tool to verify that the sequences were capable of encoding productive immunoglobulins. Sequence comparisons with mouse and human databases (GenBank) were performed using BLAST (Basic Local Alignment Search Tool) from NCBI (NIH, Bethesda, MD) against the Kabat database (to identify CDR1 and CDR2) and IMGT / V Quest (to detect CDR3) programs, where the query was the DNA sequence of the ADx-CEA clone. Representative BLAST results for the heavy and light chains of ADx-CEA are shown in Figures 2 and 3. For validation purposes and as shown in Figures 2 and 3, the reference genome sequences of IGHV4-4*02; IGHV3-33*08 and IGHV4-30-4*01 proved to be the most reliable sequences (66-67%) for detecting homology in the V region when blasted against Homo Sapiens Ig germline sequences. The most reliable variable (V) gene match was found to be IGHV4-4*02, and the most reliable diversity (D) gene match was found to be IGHD2-8*01.The most reliable joining (J) gene segment match was found to be IGHJ6*02.

[0088] Example 3. A lead candidate monoclonal antibody, referred to herein as "ADx-CEA", was selected. Approximately 1 μg and 5 μg of purified ADx-CEA were suspended in PBS and applied to an SDS-PAGE gel under reducing conditions (boiled for 3 minutes in sample buffer containing beta-mercaptoethanol and 10% SDS) and to a 10% Bis-Tris gel under non-reducing conditions (lanes 3, 5 and 7, 9, respectively). The gels were run at 129 volts and then stained with Coomassie Blue R350 (0.1 w / v), 20% v / v methanol, and 10% v / v acetic acid, and destained in 50% v / v methanol in water with 10% v / v acetic acid.

[0089] As shown in Figure 4, lane 3 contained 1 μg of ADx-CEA mAb (reduced). Lane 5 contained 5 μg of ADx-CEA mAb (reduced). Lane 7 contained 1 μg of ADx-CEA mAb (non-reduced). Lane 9 contained 5 μg of ADx-CEA mAb (non-reduced). Under denaturing conditions, the heavy chain of ADx-CEA (IgG1 kappa) was detected at approximately 50 kDa. The light chain of ADx-CEA was detected at approximately 25 kDa (Figure 4).

[0090] Example 4. 25 μl of sample buffer containing 50 μg of recombinant CEA (Mybiosource Carcinoembryonic Recombinant Protein, Cat. No. MBS142843) was boiled for 3 min and loaded onto a 4-12% Tris-Glycine gel along with 5 μl of molecular marker. The gel was run at 125 V for 1.5 h. The gel was then transferred to a PVDF membrane. The membrane was incubated with ADx-CEA mAb overnight at 4°C. After rinsing the membrane with PBST, it was incubated with secondary antibody (sheep anti-mouse IgG-HRP, 1:1000 dilution in PBST) for 1 h and rinsed with PBST. Bands visualized with 1-Step NBT / BCIP solution.

[0091] Under reducing conditions, based on the manufacturer's instructions, ADx-CEA detected two bands at the molecular weights of human CEA protein of 16 kDa and 25 kDa, as indicated by the arrows in Figure 5. Figure 5 represents a repeat experiment.

[0092] Example 5. PANC-1 cells (human pancreatic cancer cell line isolated from ductal cell-derived pancreatic cancer; purchased from ATCC) were cultured on glass-bottom wells for 18 hours. Cells were fixed with 10% paraformaldehyde and permeabilized with Triton® X-100. Cells were washed with PBS and incubated with ADx-CEA (5 μg / ml) for 15 minutes. Cells were washed with PBS and subsequently incubated with FITC-conjugated mouse IgG (4 μg / ml) for 15 minutes. Cells were washed and staining was visualized under an immunofluorescence microscope.

[0093] Figures 6A-B show representative images of indirect immunofluorescence staining of CEA detected by ADx-CEA localized in vesicles in a punctate manner. Figure 6A shows a phase contrast image. Figure 6B shows an immunofluorescence staining image.

[0094] Example 6. Plasma samples from healthy donors (n=9) and patients (n=17) who were untreated and diagnosed with pancreatic cancer at various stages (IA, IB, IIA, IIB, III, IIIA, IV) were diluted in PBS. Sample buffer was added in a final volume of 25 μl and boiled for 3 min. Samples were loaded onto a 4-12% Tris-Glycine gel along with 5 μl of molecular markers. The gel was run at 125 V for 1.5 h. The gel was then transferred to a PVDF membrane. The membrane was incubated with ADx-CEA overnight at 4°C. After rinsing the membrane with PBST, it was incubated with secondary antibody (sheep anti-mouse IgG-HRP, diluted 1:1000 in PBST) for 1 h and rinsed with PBST. Bands visualized with 1-Step NBT / BCIP solution.

[0095] ADx-CEA detected CEA protein in plasma blood samples from healthy donors and patients diagnosed with pancreatic cancer. Figure 7. CEA protein expression showed various levels, with low levels of CEA detected in healthy blood samples (n=9). Blood analysis of pancreatic cancer patients diagnosed at various stages showed that the levels of CEA protein increased with stage (n=17).

[0096] Example 7. Tissue array slides with formalin-fixed paraffin-embedded (FFPE) tissue sections (Biocore USA, Cat. No. BC001157) were used. Slides were deparaffinized with xylene and hydrated through a series of alcohols. Antigen retrieval was performed in a steamer in citrate-EDTA buffer (pH 6.0) for 20 min. Slides were then washed with H 2 0 2 The slides were treated with 10 min with PBS and then with Background Sniper (Biocare Medical, Cat. No. BS966) for 10 min. Primary antibodies were diluted 1:50 in NDB's Antibody Diluent Buffer for 60 min. Detection of antibody staining was achieved using Biocare Mach 3 reagent with 10 min incubation for each component. Counterstaining was performed using Gill's II Hematoxylin. Slides were then dehydrated in alcohol, cleared in xylene, and mounted with Permount. The presence of staining was determined using a Lab Vision IHC 360 Autostainer and a Dako Link plus IHC System.

[0097] ADx-CEA detected early stage pancreatic cancer. Figure 8. As shown in Figure 8, DAKO anti-human commercial CEA mAb showed false negative staining in various stages of pancreatic cancer tissues that showed partial positivity, staining only stage II tissues, but not stages I, III, and IV. On the other hand, ADx-CEA showed positive staining in the same tissues, including stage I-IV tissues. The staining showed membranous, cytoplasmic, and pattern, indicating increasing expression of CEA detected as the stage increased. A pathologist confirmed that the stained tissues were diagnosed as pancreatic cancer.

[0098] Example 8. Immunohistochemistry (IHC) staining in a total of 215 healthy and pancreatic cancer tissues in the pathology laboratory (John's Hopkins and CLIA Pathology) was performed using ADx-CEA on a total of 215 tissue samples, including healthy normal human tissues (n=19), and patient tissues diagnosed with various stages of pancreatic cancer (n=196), and compared in parallel using a commercially available FDA-approved mouse anti-human CEA antibody from DAKO.

[0099] The calculated sensitivity of ADx-CEA was 99.5%, while the commercial mAb was 34%, as shown in Tables 3-5 and Figures 9A-B. The sensitivity and specificity of early stages (I, II) are shown in Figures 10A-B. The sensitivity and specificity of late stages (III, IV) are shown in Figures 11A-B. [Table 3]

[0100] Tables 4-5 display the frequency distributions and the corresponding sensitivity and specificity. [Table 4]

[0101] Sensitivity of ADx-CEA (estimated using data from Table 4): 195 / 196 = 0.9948 x 100 = 99.5%

[0102] Specificity of ADx-CEA (estimated using data from Table 4): 19 / 19 = 1.00 x 100 = 100% [Table 5]

[0103] DAKO-CEA sensitivity (estimated using data from Table 5): 66 / 196 = 0.3367 x 100 = 34%

[0104] Specificity of DAKO-CEA (estimated using data from Table 5): 19 / 19 = 1.00 x 100 = 100%

[0105] Example 9. The human BxPC-3 pancreatic adenocarcinoma cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 (American Type Culture Collection, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Thermostat Scientific, Inc.) and maintained at 37°C in a 5% CO2 atmosphere.

[0106] Soft agar colony formation assay: BxPC-3 cells (2,000 cells) were suspended with ADx-CEA in 0.3% Noble agar (Difco; BD Biosciences, Franklin Lakes, NJ, USA) in RPMI-1640 supplemented with 10% FBS or in PBS alone and overlaid on 0.8% Noble agar in 12-well tissue culture plates. Colonies were grown in growth medium for 2.5 weeks and observed for colony formation and counted for data analysis.

[0107] BxPC-3 cells were cultured in soft agar in the absence of ADx-CEA (PBS only) and in the presence of different concentrations of ADx-CEA. After 2.5 weeks, colonies were imaged using an inverted microscope (Figure 12A-B). Images of wells are representative of three independent experiments.

[0108] BxPC-3 cells were grown in soft agar in PBS or 10, 25, and 50 μg / mL ADx-CEA. After 2.5 weeks, individual colonies larger than 70 μm were counted. Experiments were repeated three times. Statistical significance of the difference in total colony counts between control and treated samples was determined by Student's t-test (*p<0.005) (FIG. 12C). BxPC-3 cells showed a significant dose-dependent decrease in colony formation in the presence of 10 μg / mL, 25 μg / mL, and 50 μg / mL ADx-CEA.

[0109] Example 10. CEA protein expression was verified in healthy and cancerous tissues from healthy individuals and cancer patients using ADx-CEA described herein using IHC staining. The antibody was tested on tissues from breast, lymph, cervix, prostate, pancreas, ovary, bladder, kidney, uterus, colon, liver, brain, rectum, esophagus, stomach, lung, and uterus. Cancer tissues from pancreas (Figure 13A), colon (Figure 13B), and liver (Figure 13C) showed positive membranous cytoplasmic staining for CEA expression. Breast and kidney cancers showed weak staining (data not shown). Tissues from lymph, cervix, prostate, ovary, esophagus, bladder, uterus, brain, stomach, uterus, lung, and rectum showed negative staining for CEA expression (data not shown).

Claims

1. An isolated antibody or antibody fragment comprising: (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; (c) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; (d) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; (e) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) an LCDR3 comprising the amino acid sequence of SEQ ID NO:

6.

2. The antibody comprises a heavy chain variable region (V Ｈ ) and the light chain variable region (V Ｌ ) and V Ｈ comprises the amino acid sequence of SEQ ID NO: 7, Ｌ The isolated antibody of claim 1, wherein the antibody comprises the amino acid sequence of SEQ ID NO:

8.

3. 2. The isolated antibody or antibody fragment of claim 1, wherein the antibody is a monoclonal antibody, a fully human antibody, Fab, Fab', Fv, scFv, or (Fab')2, a full-length antibody, or wherein the Fc region of the antibody comprises IgG1, IgG2, IgG3, IgG4, IgG4.1, or IgG4.2, and / or the isolated antibody or antibody fragment is immobilized on a solid phase, detectably labeled, conjugated to a cytotoxic radionuclide, conjugated to a cytotoxic drug, or conjugated to a cytotoxic protein.

4. A composition comprising the antibody or antibody fragment of any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. 4. An isolated DNA sequence encoding the antibody or antibody fragment of any one of claims 1 to 3, optionally comprising SEQ ID NO:11 or a fragment thereof, SEQ ID NO:12 or a fragment thereof, or SEQ ID NO:11 or a fragment thereof and SEQ ID NO:12 or a fragment thereof.

6. A vector comprising the isolated DNA sequence of claim 5.

7. A host cell transformed with the vector of claim 6.

8. 10. A process for the production of antibodies, comprising culturing the host cell of claim 7 and isolating the antibody molecules.

9. A composition for treating cancer in a subject in need thereof, comprising an anti-CEA antibody according to any one of claims 1 to 3, or an antibody or antibody fragment according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier, or an isolated DNA sequence encoding the antibody or antibody fragment according to any one of claims 1 to 3, optionally comprising SEQ ID NO: 11 or a fragment thereof, SEQ ID NO: 12 or a fragment thereof, or SEQ ID NO: 11 or a fragment thereof and SEQ ID NO: 12 or a fragment thereof, or a vector comprising said isolated DNA.

10. 1. A composition for use in an immunoassay for detecting a CEA antigen, comprising the antibody or antibody fragment of any one of claims 1 to 3, the immunoassay comprising: (a) contacting a sample with an effective binding amount of the antibody or antibody fragment of any one of claims 1 to 3; and (b) detecting the CEA antigen by detecting binding of the antibody to the antigen, optionally wherein the assay is used to detect cancer cells expressing a CEA antigen or to detect solid tumors, preferably wherein the cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer.

11. 10. A composition for use in a method for detecting cancer in a subject, the composition comprising the antibody of any one of claims 1 to 3, the method comprising contacting the antibody with a sample isolated from a subject having or suspected of having cancer, preferably wherein the cancer is a solid tumor, such as colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer.

12. A kit for the immunohistochemical detection of solid tumor cancers comprising cells expressing the CEA antigen, comprising: (a) an antibody according to any one of claims 1 to 3; and (b) a secondary antibody conjugated to a detectable label, the secondary antibody binding to and capable of detecting the antibody of (a) when bound to the CEA antigen; or 1. A kit for immunohistochemical detection of pancreatic cancer, comprising: (a) a monoclonal antibody having heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 4, 5, and 6, respectively; and (b) a secondary antibody conjugated to a detectable label or a separate, unconjugated detectable label, wherein the detectable label or the separate, unconjugated detectable label binds to the secondary antibody, the antibody-antigen complex, or the monoclonal antibody of (a).

13. 10. A composition for use in a method for determining the status of solid tumor cancer in a subject, comprising the antibody or antibody fragment of any one of claims 1 to 3, the method comprising: (a) removing a sample from a subject having solid tumor cancer; (b) contacting the sample with the antibody or antibody fragment, thereby forming a complex between a CEA antigen and the antibody or antibody fragment; (c) labeling the specimen with a label specific for antigen-antibody complexes; and (d) detecting the presence of the antigen-antibody complex by detecting the label, optionally wherein the solid tumor cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer, and / or the subject has colorectal cancer, liver cancer, stomach cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer. , has been diagnosed with cancer and is undergoing or about to undergo therapy, and / or optionally, the method further comprises repeating steps (a) to (d) at a later time, for example after the subject has started therapy or after the subject has started a new or second (co-)therapy, and / or optionally, the therapy is chemotherapy or radiation, and / or optionally, the chemotherapy is erlotinib, fluorouracil, or oxaliplatin, and / or optionally, the therapy is surgery, including Whipple procedure, distal pancreatectomy, and total pancreatectomy, radiation therapy, proton beam therapy, stereotactic radiation therapy (SBRT) or Cyberknife, immunotherapy, targeted therapy, or chemotherapy.

14. 14. The composition of claim 13, wherein the subject is in remission, and preferably the method further comprises repeating steps (a)-(d) at a time point after the subject is in remission, wherein the CEA level is determined, and wherein a higher CEA level at the later time point indicates that the cancer is no longer in remission, and wherein the same or lower CEA level at the later time point indicates that the therapy continues to result in at least partial remission.

15. 10. A composition for use in a method for detecting cancer characterized by expression of a CEA gene product and its homologs, comprising the anti-CEA antibody of any one of claims 1 to 3, or the antibody or antibody fragment of any one of claims 1 to 3, and a pharmaceutically acceptable carrier, the method comprising: (a) identifying a gene product and its homologs expressed by CEA in a human patient with cancer by isolating a biological sample from the subject, wherein the sample contains a CEA gene product if the CEA gene is expressed; (b) utilizing the gene product as a biomarker by contacting the biological sample with the anti-CEA antibody or the composition to create a sample-antibody complex; (c) removing any unbound antibody and then contacting the sample-antibody complex with a label specific for the antibody or sample-antibody complex; and (d) detecting the presence of the antigen-antibody complex by detecting the label, wherein detection of the label indicates detection of cancer.

16. 1. A composition for use in a method for determining the status of a subject having cancer, the composition comprising the antibody or antibody fragment of any one of claims 1 to 3, or the antibody or antibody fragment of any one of claims 1 to 3, and a pharmaceutically acceptable carrier, the method comprising: (a) obtaining a sample from a subject; (b) contacting the sample with the antibody or antibody fragment, or the composition; (c) determining the amount of CEA detected by the antibody or antibody fragment, wherein positive staining indicates malignant or benign cancer and negative staining indicates the cells are not cancerous.

17. 1. A composition for use in a method for detecting pancreatic cancer in a patient, the composition comprising the antibody or antibody fragment of any one of claims 1 to 3, or the antibody or antibody fragment of any one of claims 1 to 3, and a pharmaceutically acceptable carrier, the method comprising: (a) removing a pancreas or blood specimen from a patient suspected of having or having pancreatic cancer; (b) contacting the specimen with the antibody or antibody fragment, or the composition, thereby forming an antigen-antibody complex in the specimen; (c) if the antibody of step (b) is unlabeled, labeling the specimen with a label specific for the antibody or antigen-antibody complex; and (d) detecting the presence of the antigen-antibody complex by detecting the label. and (e) determining the CEA antigen level in comparison to a negative control, wherein a CEA antigen level higher than the negative control is indicative of pancreatic cancer, preferably wherein the sample is pancreatic and the negative control is a similar pancreatic sample from a subject without cancer, or wherein the sample is blood and the negative control is a blood sample having 0-2.5 ng / mL of CEA when analyzed, or wherein the method is performed in vitro, or wherein the pancreatic cancer is in an early stage and the method is capable of detecting CEA antigen at this early stage, or wherein the pancreatic cancer is in stage I, II, III, or IV and the method is capable of detecting CEA antigen at this early stage.

18. 1. A composition for use in an immunohistochemical method for detecting pancreatic cancer in a tissue specimen taken from a patient, the composition comprising the antibody or antibody fragment of any one of claims 1 to 3, or the antibody or antibody fragment of any one of claims 1 to 3, and a pharmaceutically acceptable carrier, the method comprising: a) obtaining a tissue specimen; b) contacting the tissue specimen with the antibody of the antibody fragment or the composition; c) after step b), contacting the tissue specimen with the second antibody, the antibody-antigen complex, or a detectable label that binds to the antibody; d) staining the tissue specimen with an immunohistochemical stain; The composition, wherein the staining indicates antibody binding and the presence of pancreatic cancer in the tissue specimen.

19. 1. A composition for use in a method for monitoring the progression of cancer and / or the therapeutic efficacy of a cancer therapeutic, comprising the antibody or antibody fragment of any one of claims 1 to 3, or the antibody or antibody fragment of any one of claims 1 to 3, and a pharmaceutically acceptable carrier, said method comprising: (a) obtaining an initial sample from a human patient with cancer at a first time point; (b) contacting the sample with the antibody or antibody fragment, or the composition, to form an antigen-antibody complex; (c) labeling the specimen with a label specific for the antigen-antibody complex; (d) detecting the presence of the antigen-antibody complex by detecting the label; (e) determining the level of CEA antigen detected by the antibody or antibody fragment to determine a baseline level of CEA antigen associated with cancer in the patient; (f) obtaining a second sample at a second (later) time point, optionally after the subject has been receiving therapy; (g) repeating steps (b) through (e) to determine a second CEA antigen level associated with cancer in said patient; (h) determining that the patient's cancer has progressed if the CEA antigen level at the second time point is higher than the baseline level, and determining that the patient's cancer has regressed if the CEA antigen level at the second time point is lower than the baseline level, optionally wherein the cancer is colorectal cancer, liver cancer, gastric cancer, ovarian cancer, thyroid cancer, lung cancer, breast cancer, or pancreatic cancer, or wherein an increase in the CEA at the second time point indicates progression of the cancer or a decrease in the CEA at the second time point indicates regression of the cancer.

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