# Patent

## Title
Methods and compositions for screening and detecting biomarkers

## Abstract

The present invention is concerned with a novel antigens associated with human tumors, including carcinomas of the colon or lung, as well as novel monoclonal antibodies which specifically bind to said antigen. The antibodies bind to normal human cells to a much lesser degree than to tumor cells. The antigens include 100 kDa glycoprotein that has a colorectal cancer membrane bound and a soluble form, has a UV absorbance peak at about 228 nm, an isoelectric point of about 3.5 to 4, a sialic acid content of about 20%, and does not substantially bind to an antibody specific for ACT. The antibodies find use both in methods such as the detection of malignant cells associated with tumors and in monitoring therapeutic treatment of humans with tumors.

## Background
BACKGROUND
Carcinomas cause millions of deaths annually. Of all cancers, colorectal cancer is the second leading cause of cancer-related deaths in the U.S. Most cases of carcinomas are incurable by chemotherapy and radiation therapy unless detected and treated in the early stages of the disease. The more advanced a cancer is when it is diagnosed; the less likely it is that therapy will be effective. Therefore, despite the advances in cancer research, there remains a need for novel antibodies useful for the early diagnosis and treatment of carcinomas of the colon, and lung.
Generally, antibodies are used as invaluable reagents in diagnostics. In fact, they have played a major role in deciphering the functions of various bio-molecules in biosynthetic pathways. They have also become the reagents of choice for identification and characterization of tumor specific antigens and have become a valuable tool in the classification of cancer. Once tumor-associated antigens have been purified from tissue extracts, such antigens can be used to elicit production of antibodies to the antigen by injection into animals. Monoclonal antibodies can then be produced. Such antibodies are useful both therapeutically and diagnostically.
In vitro diagnostic methods are known in the art and include immunohistiological detection of tumor cells (e.g., on human tissue, cells or excised tumor specimens) or serologic detection of tumor-associated antigens (e.g., in blood samples or other biological fluids). Immunohistiological techniques involve contacting a biological specimen such as a tumor tissue specimen with the antibody of the invention and then detecting the presence on the specimen of the antibody complexed to its antigen. The formation of such antibody-antigen complexes with the specimen indicates the presence of tumor cells in the tissue. Detection of the antibody on the specimen can be accomplished using techniques known in the art, such as the immunoperoxidase staining technique, the avidin-biotin (ABC) technique or immunofluorescence techniques (see Ciocca et al., Meth. Enzymol., 121:562-79 (1986); Kimball (ed.), Introduction To Immunology (2nd Ed.), pp. 113-117, Macmillan Publ. Co. (1986)).
Serologic diagnostic techniques involve the detection and quantitation of tumor-associated antigens that have been secreted or “shed” into the serum or other biological fluids of patients thought to be suffering from carcinoma. Such antigens can be detected in the body fluids using techniques known in the art such as radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA) wherein an antibody reactive with the “shed” antigen is used to detect the presence of the antigen in a fluid sample (see, e.g., Uotila et al., J. Immunol. Methods, 42:11 (1981); Allum et al., “Monoclonal Antibodies in the Diagnosis and Treatment of Malignant Conditions” Surg. Ann., 18:41-64, 48-51 (1986); Sikora et al. (eds.), Monoclonal Antibodies, pp. 32-52, Blackwell Scientific Publications, (1984)).
However, in some cases, serological techniques have not produced results robust enough to be useful for cancer screening. Serum markers such as CEA, CA19-9 and CA242 have all been shown to have poor sensitivity and specificity for colon cancer. CEA was discovered in 1969 and was believed to be a sensitive and specific marker for colon cancer. However, further studies were unable to produce the original results. It was shown that at a cutoff concentration of 2.5 ug/L, CEA screening would yield a sensitivity of 30%-40% and a specificity of 87%. Utilizing these numbers, for every 1 colon cancer patient identified with a CEA-based assay there would be 250 false-positives and 60% of cancers would be missed. Due to these overall poor results these markers are not used for colon cancer screening.
For this reason Fecal Occult Blood (FOB) tests have long been the mainstay of colon cancer screening. Large randomized studies have shown that screening with serial FOB tests reduces mortality from colon cancer. Typical examples of FOB tests come in two types, the guaiac-based tests available under the trade names SKB-Hemoccult II® & Hemocult II SENSA®, and the immune-chemical tests available under the trade names SKB-HemeSelect® and Entrerix-InSure Fit®. Disadvantages of guaiac-based FOB tests include burdensome dietary restrictions, the inconvenient collection process, the limited single application sensitivity, and the costs associated with poor specificity (5-10% rate of false positives). FOB tests have sensitivity of 25%-40% and specificity of 80%-90%. While the immune-chemical based FOB tests have the advantages of improved compliance due to ease of use and no diet restrictions, sensitivity issues remain.
It is thus apparent that antibodies reactive with an antigen expressed at high levels by a variety of tumors may become useful towards an earlier diagnosis of cancers, the immunological monitoring of cancer patients, as well as for development of improved methods for therapy of cancers. It is also apparent that purified antigens associated with carcinomas derived from specific organs and tissues of the body can be of value for creating such monoclonal antibodies, as well as for creating cancer vaccines. It is also apparent that improved materials and methods for serological assays to identify the presence of cancer in humans would be valuable.
SUMMARY OF THE INVENTION
The disclosure provides immunogenic compositions, antibodies, kits, and methods useful in the early detection of cancers or monitoring the treatment of cancer. In embodiments, the cancers are colon-rectal and lung. Immunogenic compositions include an immunogenic composition comprising an isolated fraction from colorectal and/or liver cancer cell samples having a component of about a 100 kDa glycoprotein that has a membrane bound and a soluble form (e.g. cancer cell membrane bound form), has a UV absorbance peak at about 228 nm, an isoelectric point of about 3.5 to 4, a sialic acid content of about 20%, and does not substantially bind to antibodies specific for anti-chymotrypsin (ACT). In embodiments, the 100 kDa protein has an amino acid sequence of CEACAM 5 isoform 2 (SEQ ID NO:1; FIG. 14). In embodiments, the 100 kDa glycoprotein has a deletion of an alanine at position 320 and an altered glycosylation pattern as compared to 180 kDa CEA.
In some cases, the immunogenic composition is obtained by a method comprising contacting colorectal and/or liver tumor cells with an acid to form an extract; separating components of the extract by molecular weight to form a first fraction comprising components with a molecular weight of about 60 kDa or greater; isolating components of the first fraction that have a molecular weight of about 75 kDa or greater and less than about 200 kDa, and further isolating a fraction that has a molecular weight of about 100 kDa.
In embodiments, the composition further comprises an adjuvant. In specific embodiments, the adjuvant is selected from the group consisting of alum, muramyl dipeptide, and Freund's complete adjuvant. Immunogenic compositions are useful for obtaining antibodies. Antigens described herein may also be detectably labeled or attached to a solid substrate for use in a screening assay.
The disclosure includes antibodies that specifically bind a 100 kDa glycoprotein that has a membrane bound and a soluble form, has a UV absorbance peak at about 228 nm, an isoelectric point of about 3.5 to 4, a sialic acid content of about 20%, and do not substantially bind to ACT are described herein.
In embodiments, an antibody or antigen binding fragment specifically binds to 100 kDa glycoprotein comprising an amino acid sequence of SEQ ID NO:1 and does not bind to one or more linear peptides consisting of a linear peptide of 15 amino acids from amino acids 1-60 of SEQ ID NO:1 as shown in SEQ ID NO:2, a linear peptide of amino acids 111 to 125 of SEQ ID NO:1 as shown in SEQ ID NO:3 and/or a linear peptide of 15 amino acids from amino acids 150-701 of SEQ ID NO:1 as shown in SEQ ID NO:4. In embodiments, the antibodies do not substantially bind to epitopes in domains of CEACAM5 selected from the group consisting of amino acids 146-237 (A1 Ig-2), amino acids 238-323 (B1 Ig-3), amino acids 324-415 (A2 Ig-4), amino acids 416-498 (B2 Ig-5), amino acids 502-593 (A3 Ig-6) and amino acids 594-677 (B3 Ig-7) and combinations thereof. Amino acid numbering of CEACAM5 corresponds to that of in NP_004354; gI 98986445; Uniprot P06731-1.
In embodiments, an antibody or antigen binding fragment does not substantially cross react with CEA of a molecular weight of 180 kDa, CEACAM 6(NCA), CEACAM5 lacking all glycosylation, and/or 100 kDa glycoprotein having a sequence of SEQ ID NO:1 lacking all glycosylation.
In embodiments, an antibody or antigen binding fragment specifically binds to an epitope on a 100 kDa glycoprotein comprising an amino acid sequence of SEQ ID NO:1, the epitope comprising, consisting essentially of, or consisting of amino acids 61-69 HLFGYSWYK (SEQ ID NO:6), 73-77 VDGNR (SEQ ID NO:7) or 69-82 KGERVDGNRQIIGY (SEQ ID NO:8), and 96-107 SGREIIYPNASL (SEQ ID NO:9) of SEQ ID NO:1. In embodiments, an antibody specifically binds to an epitope on a 100 kDa glycoprotein comprising an amino acid sequence of SEQ ID NO:1, the epitope comprising, consisting essentially of, or consisting of amino acids 61-69(SEQ ID NO:6), 78-98 QIIGYVIGTQQAT PGPAYSGR (SEQ ID NO:10), 96-107(SEQ ID NO:9), and 127-139 SDLVNEEATGQFR (SEQ ID NO:11). Amino acid numbering corresponding to that of SEQ ID NO:1. In embodiments, the epitope is a conformational epitope in the N terminal amino acids (amino acids 1-150; SEQ ID NO:5) of the 100 kDa glycoprotein. In embodiments, the antibodies do not substantially bind to epitopes in other domains of CEACAM5.
In embodiments, an antibody or antigen binding fragment thereof comprises heavy chain CDRs comprising: CDRH1: (TDSYIN SEQ ID NO:14) or GNTFTDSYIN(SEQ ID NO:15); CDRH2: EIYPGNGDVYYNENFK (SEQ ID NO:16); CDRH3: TTVFAY (SEQ ID NO:17) or LCAGSNMITTVFAY(SEQ ID NO:18). In embodiments, an antibody or antigen binding fragment thereof comprises heavy chain CDRs comprising: CDRH1: GYTFTNYWIN(SEQ ID NO:19) or NYWIN(SEQ ID NO:20); CDRH2: NIYPGSTRANYNEKFK(SEQ ID NO:21); CDRH3: YCTRTHSI(SEQ ID NO:22).
In embodiments, an antibody or antigen binding fragment thereof comprises light chain CDRs comprising: CDRL1: RASQDIRNYLN(SEQ ID NO:26); CDRL2: YTSRLHS (SEQ ID NO:27); CDRL3: QQGNTLPW (SEQ ID NO:28). In embodiments, an antibody or antigen binding fragment thereof comprises light chain CDRs comprising: CDRL1: RASQSISSYLN(SEQ ID NO:31); CDRL2: AASSLQS (SEQ ID NO:32); CDRL3: QQTVVAPP (SEQ ID NO:33).
In embodiments, an antibody or antigen binding fragment thereof comprises HCDR1/HCDR2/HCDR3 of SEQ ID NOs:14/16/17, SEQ ID NOs:15/16/17, SEQ ID NOs:14/16/18, or SEQ ID NOs:15/16/18; and LCDR1/LCDR2/LCDR3 of SEQ ID NOs:26/27/28. In embodiments, an antibody or antigen binding fragment thereof comprises HCDR1/HCDR2/HCDR3 of SEQ ID NOs:20/21/22 or SEQ ID NOs:19/21/22, and LCDR1/LCDR2/LCDR3 of SEQ ID NOs:31/32/33.
In embodiments, an antibody or antigen binding fragment thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO:119 and light chain variable region having a sequence of SEQ ID NO:120. In other embodiments, an antibody or antigen binding fragment comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO:121 and light chain variable region having an amino acid sequence of SEQ ID NO:122.
Antibodies can be polyclonal, monoclonal, or recombinant. Antibodies can be an antigen binding fragment such as of Fab, single chain antibodies, scFv, F(ab)2 and Fv fragments. Antibodies can be human, synthetic, (eg. phage display), chimeric, or humanized antibodies. In embodiments, antibodies are labeled with a signal generating element. In other embodiments, the label is selected from the group consisting of biotin, fluorescent dyes, chemiluminescent tags, radioactive tags, enzymes, and combinations thereof. In yet other embodiments, the antibody is immobilized on a solid surface.
The disclosure provides immunoassays. In embodiments, the method further comprises testing the subject for a cancer selected from the group consisting of colon-rectal and lung. In embodiments, the testing involves detecting a biomarker of the cancer, imaging of the body of the subject such as a CAT, Pet or MRI scan, and/or conducting a colonoscopy.
In embodiments, the immunoassay is a sandwich assay. An immunoassay comprises a first antibody that specifically binds that specifically binds to a 100 kDa glycoprotein having an amino acid sequence of SEQ ID NO:1, and does not substantially bind to ACT attached to a solid substrate (e.g. a capture antibody), and a second antibody that specifically binds to a 100 kDa glycoprotein that has an amino acid sequence of SEQ ID NO:1, that is labeled with a signal generating element. In embodiments, the first antibody is a monoclonal antibody or a polyclonal antibody. In embodiments, the second antibody is a monoclonal antibody or polyclonal antibody.
The disclosure also provides kits. In embodiments, a kit comprises: a) a first antibody that specifically binds to a 100 kDa glycoprotein that has an amino acid sequence of SEQ ID NO:1 and that does not substantially bind ACT; b) a second antibody that specifically binds to a 100 kDa glycoprotein that has an amino acid sequence of SEQ ID NO:1 and is labeled with a signal generating element; and c) a calibrator. In embodiments, the first antibody is a monoclonal antibody or a polyclonal antibody. In embodiments, the second antibody is a monoclonal antibody or polyclonal antibody. In embodiments, the first antibody has different epitope specificity than the second antibody. In embodiments, the kit also provides instructions for identifying a subject as “at risk” for colorectal cancer if the amount of antigen detected is about 6.5 units/ml or greater.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a summary of all data from the 422 patient clinical study. Panel A shows the combined data for all non-cancerous and cancerous patients. The box covers the middle 50% of the data while the line is the median. The whiskers cover the middle 90% of the data (from 5% to 95%). Panel B is a receiver operator characteristic (ROC) curve comparing the cancerous vs. non-cancerous samples.
FIG. 2 shows SDS-PAGE and goat anti-CA11-19 polyclonal antibodies Conjugate Western of tumor and normal tissue perchloric acid extracts. CODES 1, 2, 7, 8, 15, 23, 25, 27, 32, 38, 39, 41, 42, 45, 50 are Normal Tissue Perchloric acid extracts. Panels A & B show the results of  ̃50 tumor extracts.
FIG. 3 shows fractionation of a perchloric acid extract of two separate colorectal tumor cell samples on Sepharose B. Absorbance at 280 for each sample is shown by (▴; ▪) The fraction of one of the samples were also evaluated in the ELISA assay (♦).
FIG. 4 shows methods for N-glycan profiling by MALDI-MS of glycoprotein 100 kDa obtained from an acid extract of a colon cancer cell.
FIG. 5A shows localization of target glycoprotein 100 kDa band and an additional band at 45 kDa observed in the CA11-19 168B. FIG. 5B shows pictures of actual gels used for sample analysis.
FIG. 6A shows the MALDI-full mass spectra of the 100 kDa band. FIG. 6B shows the MALDI-full mass spectra of the 45 kDa band.
FIG. 7 shows the sample preparation methods for O-glycan profiling by MALDI-MS.
FIG. 8 shows CA11-19 168BNC and CA11-19 168BSTK separated by SDS-PAGE for in-gel digestion.
FIG. 9 shows NSI-FTMS spectra of O-glycan from the 100 kDa band from the both samples. FIG. 9A) is the profile for antigen sample NC. FIG. 9B) is the profile for antigen sample STK.
FIG. 10 A) The MSMS spectra of the permethylated O-glycan at m/z 936, m/z 953, and m/z 1157 derived from the O-glycan spectrum of CA 11-19 168BNC (FIG. 10 upper spectrum). The MSMS profile of the molecular ion at m/z 936 (NeuAc-HexNAc-HexNAc), the diagnostic b ion is shown at m/z 398, together with the complementary y ions at m/z 561, m/z 659, and m/z 677. FIG. 10B shows The MSMS profile of the molecular ion at m/z 953 (Hex-DHex-HexNAc2) (10B) and FIG. 10C shows the MSMS profile of the molecular ion at m/z 1157 (Hex2-DHex-HexNAc2) (10C).
FIG. 11 Analysis of binding of monoclonal antibodies specific for the 100 kDa antigen isolated from colon or liver cancer cells designated antibody 5E5-1 and Antibody 5A1-1 to the 100 kDa antigen designated as 168B. FIG. 11A) Analysis of the mix A: 5E5-1/168B, showed that the antigen and the antibody were detected with observed MH+=92.879 kDa (168B-antigen) and MH+=158.498 kDa. (5E5-1 antibody); FIG. 11B) The cross-linking experiment was completed after 180 minutes incubation time with the crosslinking reagent K200. After cross-linking, we detected two additional peaks with MH+=258.945 kDa [5E5-1•B168] and MH+=354.164 kDa [5E5-1•2B168]. FIG. 11C) Using Complex Tracker software, we overlaid the control and cross-link spectra. The overlay confirmed the detection of two non covalent complexes [5E5-1•B168] and [5E5-1•2B168].
FIG. 12 A) Analysis of the Mix C (both monoclonal antibodies plus antigen): 5E5-1/5A1-1/168B showed that the antigen and the antibody were detected with MH+=92.910 kDa (168B) and MH+=158.988 kDa (5E5, 5A1-1). FIG. 12B) The cross-linking experiment was completed after 180 minutes incubation time with the crosslinking reagent K200. After cross-linking, we detected two additional peaks with MH+=260.155 kDa and MH+=356.012 kDa. FIG. 12C) Using Complex Tracker software, we overlaid the control and cross-link spectra. The overlay confirmed the detection of two non covalent complexes [5E5-1•B168] and [5E5-1•2B168].
FIG. 13 A) After competition with tryptic peptides, we did not detect inhibition of the binding of the antibody 5E5-1 on the antigen 168B. FIG. 13B) After competition, we observed two non covalent complexes: [5E5-1•B168] and [5E5-1•2B168] FIG. 13C) Using Complex Tracker software, we overlaid the control and cross-link spectra. The overlay confirmed the detection of two non covalent complexes [5E5-1•B168] and [5E5-1•2B168].
FIG. 14 shows the sequence of the 168B antigen. Overlap mapping of the trypsin, chymotrypsin and ASP-N peptides covered, 85.6% of the sequence.
FIG. 15 shows the map of the epitope bound by antibody 5E5-1. Amino acids in bold indicate potential points of contact between antibody 5E5-1 and antigen 168B.
FIG. 16 shows the map of the epitope bound by antibody 5A1-1. Amino acids in bold indicate potential points of contact between antibody 5A1-1 and antigen 168B.
FIG. 17 shows the amino acid sequence of the heavy and light chain of antibody 5E5-1. CDR regions are highlighted in bold. N terminal sequence of the heavy and light variable domains are identified by italics.
FIG. 18 shows the nucleic acid sequence of the heavy and light chain of antibody 5E5-1.
FIG. 19 shows the amino acid sequence of the heavy and light chain of antibody 5A1-1. CDR regions are highlighted in bold. N terminal sequence of the heavy and light variable domains are identified by italics.
FIG. 20 shows the nucleic acid sequence of the heavy and light chain of antibody 5A1-1.



DETAILED DESCRIPTION OF INVENTION
It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
As used herein and in the claims, the singular forms “a,” “an”, and “the” include the plural reference unless the context clearly indicates otherwise. Thus, for example, the reference to an antibody is a reference to one or more such antibodies, including equivalents thereof known to those skilled in the art. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with numerical values means±20% and with percentages means±1%.
All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains.
For the purposes of this application the following terms shall have the following meanings:
An “epitope” refers to that portion of any molecule capable of being recognized by, and bound by, an antibody (the corresponding antibody binding region may be referred to as a paratope). In general, epitopes comprise chemically active surface groupings of molecules, for example, amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics.
An “antigen” is a molecule or a portion of a molecule capable of being bound by an antibody. An antigen may have one or more than one epitope. An antigen will bind in a highly selective manner with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
An “antibody” includes both intact immunoglobulin molecules as well as portions, fragments, peptides and derivatives thereof, such as, for example, Fab, Fab′, F(ab′)2, Fv, scFv, CDR regions, or any portion or peptide sequence of the antibody that is capable of binding antigen or epitope. An antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
Antibody also includes chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments, portions, regions, peptides or derivatives thereof, provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis, phage display, or recombinant techniques. Antibody fragments or portions may lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. Examples of antibody may be produced from intact antibodies using methods well known in the art, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). See e.g., Wahl et al., 24 J. Nucl. Med. 316-25 (1983). Portions of antibodies may be made by any of the above methods, or may be made by expressing a portion of the recombinant molecule. For example, the CDR region(s) of a recombinant antibody may be isolated and subcloned into the appropriate expression vector. See, e.g., U.S. Pat. No. 6,680,053.
A “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.
The term “human antibody” means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.
The term “humanized antibody” refers to forms of non-human (e.g. murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. No. 5,225,539.
The term “chimeric antibodies” refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g. mouse, rat, rabbit, etc) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.
As used herein, “alpha-1-antichymotrypsin”, or “ACT” refers to a polypeptide having an amino acid sequence the same as that derived from a naturally occurring noncomplexed polypeptide and that has serine protease inhibitory activity. ACT is also known as SERPINA3, AACT, growth inhibiting protein 24 (GIG24), growth inhibiting protein 25 (GIG25), cell growth inhibiting gene 24/25 protein, and serine proteinase inhibitor clade A, member 3. A representative sequence of ACT is NP_001076/gI 50659080.
As used herein, “carcinoembryonic antigen” or “CEA” refers to a family of polypeptides having an amino acid sequence the same as a naturally occurring polypeptide, and is glycosyl phosphotidyl inositol cell surface anchored glycoprotein. CEA is generally understood to refer to a glycoprotein of a molecular weight of 180 kDa.
As used herein “CEACAM5” refers to a carcinoembryonic antigen that is a member of a family of carcinoembryonic antigens. CEACAM5, without any glycosylation, has an exemplary amino acid sequence found in NP_004354; gI 98986445; Uniprot P06731-1. An isoform of CEACAM5 has an amino acid deletion at amino acid position 320 and has an amino acid sequence of SEQ ID NO:1. (Exemplary sequence found in Uniprot P06731-2). Unglycosylated CEACAM5 contains a signal peptide of 1-34, a protein of 35-685, and amino acids 686-702 that are removed in the mature form. Within the protein several domains are found at amino acids 35-145 (N terminal Ig-1), amino acids 146-237 (A1 Ig-2), amino acids 238-323 (B1 Ig-3), amino acids 324-415 (A2 Ig-4), amino acids 416-498 (B2 Ig-5), amino acids 502-593 (A3 Ig-6) and amino acids 594-677 (B3 Ig-7). Amino acid numbers refer to those of found in NP_004354; gI 98986445; Uniprot P06731-1. The amino acid numbers will be shifted one number from that of amino acid reference sequence of SEQ ID NO:1 because of the deletion of amino acid 320. Antibodies to the domains of CEACAM5 have been characterized as Gold epitope designations: Gold 1: A3B3; Gold 2-A2B2; Gold 3-A3B3; Gold 4-A1B1; and Gold 5-N terminal. CEACAM5 can be produced as a heavily glycosylated form having a molecular weight of 180 kDa and has 28 potential N glycosylation sites.
As used herein, the term “not substantially bind” means that the detectable signal from the binding of the antibody to a component in a sample is within one or two standard deviations of the signal generated due to the presence of an unrelated polypeptide control such as bovine serum albumin.
As used herein “specific binding” refers to an antibody that reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to an epitope or protein than with alternative substances, including unrelated proteins. In certain embodiments, “specifically binds” means, for instance, that an antibody binds to a protein with a KD of about 0.1 mM or less, but more usually less than about 1 μM. In certain embodiments, “specifically binds” means that an antibody binds to a protein at times with a KD of at least about 0.1 μM. or less, and at other times at least about 0.01 μM. or less. It is understood that an antibody or binding moiety that specifically binds to a first target may or may not specifically bind to a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, i.e. binding to a single target. Thus, an antibody may, in certain embodiments, specifically binds to more than one target. In certain alternative embodiments, an antibody may be bispecific and comprise at least two antigen-binding sites with differing specificities.
The term “comprising” refers to a composition, compound, formulation, or method that is inclusive and does not exclude additional elements or method steps.
The term “consisting of” refers to a compound, composition, formulation, or method that excludes the presence of any additional component or method steps.
The term “consisting essentially of” refers to a composition, compound, formulation or method that is inclusive of additional elements or method steps that do not materially affect the characteristic(s) of the composition, compound, formulation or method.
The term “isolated” refers to the separation of a material from at least one other material in a mixture or from materials that are naturally associated with the material.
The terms “patient” or “subject” are used interchangeably and refer to any member of Kingdom Animalia. Preferably a subject is a mammal, such as a human, domesticated mammal or a livestock mammal.
The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ration.
The phrase “pharmaceutically-acceptable carrier” refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the compound or analogue or derivative from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which may serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
The term “purified” or “to purify” or “substantially purified” refers to the removal of inactive or inhibitory components (e.g., contaminants) from a composition to the extent that 10% or less (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less) of the composition is not active compounds or pharmaceutically acceptable carrier.
As used herein, the term “at risk for disease” (e.g., at risk for, cancer, etc.) refers to a subject (e.g., a human) that is predisposed to experiencing a particular disease (e.g., cancer, etc.). This predisposition may be genetic (e.g., a particular genetic tendency to experience the disease, such as heritable disorders), or due to other factors (e.g., hypertension, age, weight, environmental conditions, exposures to detrimental compounds present in the environment, etc.).
Immunogenic Compositions
The disclosure provides immunogenic compositions comprising an isolated fraction from colorectal and/or liver cancer cell samples having components with a molecular weight that comprises a 100 kDa glycoprotein that has a membrane bound and a soluble thin′ (e.g. cancer cell membrane bound form), has a UV absorbance peak at about 228 nm, an isoelectric point of about 3.5 to 4, a sialic acid content of about 20%, and does not substantially bind to antibodies specific for ACT.
In embodiments, the immunogenic compositions and/or the isolated fraction are obtained by a method comprising contacting colorectal and/or liver cancer cells with an acid to form an extract; separating components of the extract by molecular weight to form a first fraction having components with a molecular weight of about 60 kDa or greater; isolating components of the first fraction that have a molecular weight of about 75 kDa or greater and less than about 200 kDa, and further isolating a 100 kDa protein. In embodiments, the acid is selected from the group consisting of trichloroacetic acid, dichloroacetic acid, tribromic acid, trifluoric acid, sodium trichloroacetate, hydrochloric acid, and perchloric acid. In embodiments, the acid concentration ranges from about 1 to about 50%. In embodiments, the components are separated by gel filtration. In embodiments, the gel filtration medium is selected from the group consisting of Sepharose, and Superdex gel filtration medium.
In embodiments, the immunogenic composition comprises a 100 kDa glycoprotein isolated from a colorectal or liver cancer cell sample having an amino acid sequence of SEQ ID NO:1. In embodiments, the 100 kDa has a sequence that does not include the leader sequence of amino acids 1-34 of SEQ ID NO:1. While not meant to limit the scope of the disclosure it is thought that the 100 kDa is a form of CEACAM5 with a deletion of an amino acid at position 320 and altered glycosylation as compared to the 180 kDa CEA.
Immunogenic compositions are optionally combined with an adjuvant. An immunogenic effective amount of the antigen is that amount that stimulates an immune response. Immunogenic effective amounts can be determined based on the animal used for immunization. Adjuvants include alum, muramyl peptides, monophosphoryl lipid a, liposomes, incomplete Freunds' adjuvant, and complete Freund's adjuvant.
A typical immunization protocol involves injecting an animal such as mice, goats, sheep, or primates with the immunogenic composition. At least one booster is administered about two weeks after the initial immunization.
Alternatively, the immunogenic composition can be attached to a solid substrate and used to bind antibodies produced in animal models or by phage display. Solid surfaces include membranes, multiwell plates, chromatography media, glass slides, latex beads, magnetic beads, microarray chips, capillary tubes and chips. Similarly, the immunogenic composition may be detectably labeled with a signal generating element. Such labels include fluorescent markers, biotin, enzymes, radioactive labels and the like.
Antibodies
The present disclosure includes novel antibodies, methods for producing the antibodies, and diagnostic and therapeutic methods employing the antibodies.
In embodiments, an antibody specifically binds to a 100 kDa glycoprotein that has a colorectal cancer membrane bound and a soluble form, has a UV absorbance peak at about 228 nm, an isoelectric point of about 3.5 to 4, a sialic acid content of about 20%, and does not substantially bind to 180 kDa glycosylated CEA and/or ACT. In embodiments, antibodies are further screened for binding to human cancer cells, particularly from the lung and/or colon. The antibodies are selected to react with a range of cancers while showing essentially no reactivity with normal human tissues or other types of tumors such as lymphomas.
In embodiments, an isolated antibody or antigen-binding fragment thereof specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, but does not bind to glycosylated 180 kDa CEA and one or more linear peptides consisting of a linear peptide of 15 amino acids from amino acids 1-60 of SEQ ID NO:2, a linear peptide of amino acids 110 to 125 of SEQ ID NO:3, and/or a linear peptide of 15 amino acids from amino acids 150-701 of SEQ ID NO:4. In embodiments, the antibodies are further selected to react with colorectal cancer cells.
In embodiments, the antibody or antigen binding fragment binds to a conformational epitope. In some embodiments, the antibody or antigen binding fragment specifically binds to and/or competes for binding to an epitope comprising, consisting essentially of, consisting of amino acids 61-69 (HLFGYSWYK; SEQ ID NO:6), 73-77 (VDGNR; SEQ ID NO:7) or 69-82 KGERVDGNRQIIGY (SEQ ID NO:8), and 96-107(SGREIIYPNASL; SEQ ID NO:9) of SEQ ID NO:1. In other embodiments, the antibody or antigen binding fragment binds to an epitope comprising, consisting essentially of, consisting of amino acids 61-69(SEQ ID NO:6), 78-98(QII GYVIGTQQAT PGPAYSGR; SEQ ID NO:10), 96-107(SEQ ID NO:9), and 127-139(SDLVNEEATGQFR; SEQ ID NO:11) of SEQ ID NO:1. In embodiments, the conformational epitope retains glycosylation.
The antibody or antigen binding fragments thereof does not substantially bind to one or more of the linear peptides of an amino acid sequence selected from the group consisting of SEQ ID NOs:40-101.
In embodiments, the antibody or antigen binding fragment does not substantially bind to epitopes in one or all other domains of CEACAM5 selected from the group consisting of amino acids 146-237 (A1 Ig-2), amino acids 238-322 (B1 Ig-3), amino acids 324-415 (A2 Ig-4), amino acids 416-498 (B2 Ig-5), amino acids 502-593 (A3 Ig-6) and amino acids 594-677 (B3 Ig-7).
In embodiments, an isolated antibody or antigen-binding fragment thereof specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, but does not bind to glycosylated 180 kDa CEA, CEACAM6 (NCA) or unglycosylated CEACAM5 and/or one or more linear peptides consisting of a linear peptide of 15 amino acids from amino acids 1-60 of SEQ ID NO:2, a linear peptide of amino acids 111 to 125 of SEQ ID NO:3, a linear peptide of 15 amino acids from amino acids 150-701 of SEQ ID NO:4, a peptide of amino acids 1-32 including the leader sequence (SEQ ID NO:35), a peptide of amino acids 1-32 without the leader sequence (SEQ ID NO:36), a peptide of amino acids 42-60 (SEQ ID NO:37), a peptide of amino acids 117-127 with the leader (SEQ ID NO:38), a peptide of amino acids 117-127 with the leader (SEQ ID NO:39), and combinations thereof.
In embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, but does not bind to glycosylated 180 kDa CEA and comprises: (a) heavy chain CDRs (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO:119 and SEQ ID NO:121, and (b) light chain CDRs (LCDR1, LCDR2 and LCDR3) from a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO:120 and SEQ ID NO:122.
In other embodiments, an isolated antibody or antigen-binding fragment thereof comprises: a) HCDR1, HCDR2, and HCDR3 selected from the group consisting of SEQ ID NOs:15/16/17, 14/16/17, 18/20/21 and SEQ ID NO:19/20/21; and b) LCDR1, LCDR2, and LCDR3 selected from the group consisting of SEQ ID NOs:26/27/28 and SEQ ID NOs:31/32/33.
In yet other embodiments, an isolated antibody or antigen binding fragment thereof comprises: a) a heavy chain variable region having an amino acid sequence SEQ ID NO: 119 and a light chain variable region having an amino acid sequence of SEQ ID NO:120; or (b) a heavy chain variable region having an amino acid sequence SEQ ID NO:121 and a light chain variable region having an amino acid sequence of SEQ ID NO:122.
Antibodies include monoclonal, polyclonal, human, humanized, chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments, portions, regions, peptides or derivatives thereof, provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis, phage display, or recombinant techniques.
Antibody fragments or portions may lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody such as, for example, Fab, Fab′, F(ab′)2, Fv, scFv, CDR regions, or any portion or peptide sequence of the antibody that is capable of binding antigen or epitope. Antibody fragments may be produced from intact antibodies using methods well known in the art, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). See e.g., Wahl et al., 24 J. Nucl. Med. 316-25 (1983). Portions of antibodies may be made by any of the above methods, or may be made by expressing a portion of the recombinant molecule. For example, the CDR region(s) of a recombinant antibody may be isolated and subcloned into the appropriate expression vector. See, e.g. U.S. Pat. No. 6,680,053.
Polyclonal antibodies can be prepared by any known method. Polyclonal antibodies are raised by immunizing an animal (e.g. a rabbit, goat, rat, mouse, donkey, etc) by multiple subcutaneous or intraperitoneal injections of the relevant antigen (a purified peptide fragment, full-length recombinant protein, fusion protein, etc) optionally conjugated to keyhole limpet hemocyanin (KLH), serum albumin, etc. diluted in sterile saline and combined with an adjuvant (e.g. Complete or Incomplete Freund's Adjuvant) to form a stable emulsion. The polyclonal antibody is then recovered from blood, ascites and the like, of an animal so immunized. Collected blood is clotted, and the serum decanted, clarified by centrifugation, and assayed for antibody titer. The polyclonal antibodies can be purified from serum or ascites according to standard methods in the art including affinity chromatography, ion-exchange chromatography, gel electrophoresis, dialysis, etc.
The monoclonal antibodies of the present disclosure can be prepared by hybridoma fusion techniques. (see, Kohler and Milstein, Nature, 256:495-97 (1975); Brown et al., J. Immunol., 127 (2):539-46 (1981); and Yeh et al., Int. J. Cancer, 29:269-75 (1982)). These techniques involve the injection of an immunogen (e.g., purified antigen or cells or cellular extracts carrying the antigen) into an animal (e.g., a mouse) so as to elicit a desired immune response (i.e., production of antibodies) in that animal. The immunogenic composition is injected, for example, into a mouse, and after a sufficient time the mouse is sacrificed and somatic antibody-producing lymphocytes are obtained. Antibody-producing cells may be derived from the lymph nodes, spleens and peripheral blood of primed animals. Spleen cells are preferred. Mouse lymphocytes give a higher percentage of stable fusions with the mouse myelomas described below. The use of rat, rabbit and frog somatic cells is also possible. The spleen cell chromosomes encoding desired immunoglobulins are immortalized by fusing the spleen cells with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol (PEG). Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques; for example, the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md.
The resulting cells, which include the desired hybridomas, are then grown in a selective medium, such as HAT medium, in which unfused parental myeloma or lymphocyte cells eventually die. Only the hybridoma cells survive and can be grown under limiting dilution conditions to obtain isolated clones. The supernatants of the hybridomas are screened for the presence of antibody of the desired specificity, e.g., by immunoassay techniques using the antigen that has been used for immunization. Positive clones can then be subcloned under limiting dilution conditions, and the monoclonal antibody produced can be isolated. Various conventional methods exist for isolation and purification of the monoclonal antibodies so as to free them from other proteins and other contaminants. Commonly used methods for purifying monoclonal antibodies include ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography (see, e.g., Zola et al., in Monoclonal Hybridoma Antibodies: Techniques and Applications, Hurell (ed.) pp. 51-52 (CRC Press 1982)). Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites fluid) using techniques known in the art (See, generally, Fink et al., supra, at page 123, FIG. 6-1).
Generally, the individual cell line may be propagated in vitro, for example in laboratory culture vessels, and the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration or centrifugation. Alternatively, the yield of monoclonal antibody can be enhanced by injecting a sample of the hybridoma into a histocompatible animal of the type used to provide the somatic and myeloma cells for the original fusion.
Alternatively, monoclonal antibodies can also be made using recombinant DNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cell, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. Also, recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described (McCafferty et al., 1990, Nature, 348:552-554; and Marks et al., 1991, J. Mol. Biol., 222:581-597).
The polynucleotide(s) encoding a monoclonal antibody can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted 1) for those regions of, for example, a human antibody to generate a chimeric antibody or 2) for a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.
Human or humanized antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated (See, e.g., U.S. Pat. No. 5,750,373). Human antibodies can also be made in transgenic mice containing human immunoglobulin loci that are capable upon immunization of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Examples of methods used to generate humanized antibodies are described in U.S. Pat. No. 5,225,539.
Antibodies can be prepared and selected from a phage library, as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-314, Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al., 1991, J. Mol. Biol., 222:581). Techniques for the generation and use of antibody phage libraries are also described in U.S. Pat. Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404 (each of which is incorporated by reference in its entirety). Affinity maturation strategies and chain shuffling strategies (Marks et al., 1992, Bio/Technology 10:779-783, incorporated by reference in its entirety) are known in the art and may be employed to generate high affinity human antibodies.
In embodiments, nucleic acids coding for a heavy chain and light chain variable domains of an antibody of the specificity as described herein are shown in FIGS. 19 and 20. A nucleic acid coding for a heavy chain variable domain of an antibody as described herein comprises a nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:24. A nucleic acid coding for a light chain of an antibody as described herein comprises a nucleic acid sequence of SEQ ID NO:25 or SEQ ID NO:30.
Screening of antibodies for desired binding specificity is conducted according to standard methods. In embodiments, an antibody specifically binds to a 100 kDa glycoprotein that has a colorectal cancer membrane bound and a soluble form, has a UV absorbance peak at about 228 nm, an isoelectric point of about 3.5 to 4, a sialic acid content of about 20%. In embodiments, the antibodies are also screened for binding to the 100 kDa glycoprotein, and for not substantially binding to 180 kDa CEA and/or ACT. In embodiments, antibodies are isolated that bind to a cancer cell extract obtained by a method comprising a) contacting colorectal and/or liver cancer cells with an acid to form an extract; b) separating components of the extract by molecular weight to form a first fraction having components with a molecular weight of about 60 kDa or greater; c)isolating components of the first fraction that have a molecular weight of about 75 kDa or greater and less than about 200 kDa, and d) isolating a 100 kDa fraction.
In some embodiments, the antibodies are further screened for the ability to bind to tissue samples of cancers as compared to normal tissue of the same tissue type. In embodiments, the antibodies are further screened for binding to colon or lung tumor cells and for lack of binding to a non diseased tissue such as colon, or lung tissue. In yet other embodiments, the antibodies are optionally screened for binding to a 100 kDa glycoprotein that has an amino acid sequence of SEQ ID NO:1.
It should be understood that the present invention encompasses the antibodies described herein and any fragments thereof containing the active binding region of the antibody, such as Fab, F(ab)2 and Fv fragments. Such fragments can be produced from the antibodies using techniques well established in the art (see, e.g., Rousseaux et al., in Methods Enzymol., 121:663-69, Academic Press (1986)).
In addition, embodiments of the present disclosure encompass antibodies that are capable of binding to the same epitope as the antibodies and/or competing with the antibody for binding at that site. These include antibodies having the same epitope specificity as the antibodies that specifically bind to a 100 kDa glycoprotein that has a sequence of SEQ ID NO:1 as described herein, and does not substantially bind to other linear epitopes of CEACAM5 as described herein. For example, class, isotype and other variants of the antibody of the invention may be constructed using recombinant class-switching and fusion techniques known in the art (see, e.g., Thammana et al., Eur. J. Immunol., 13:614 (1983); Spira et al., J. Immunol. Meth., 74:307-15 (1984); Neuberger et al., Nature, 312:604-08 (1984); and Oi et al., supra)). Thus, chimeric antibodies or other recombinant antibodies (e.g., antibody fused to a second protein such as a lymphokine) having the same binding specificity as the antibodies fall within the scope of this invention.
Also included within the scope of the invention are anti-idiotypic antibodies of the antibodies that specifically bind to a 100 kDa glycoprotein that has a colorectal cancer membrane bound and a soluble form, has a UV absorbance peak at about 228 nm, an isoelectric point of about 3.5 to 4, a sialic acid content of about 20%, and does not substantially bind to 180 kDa CEA and/or ACT. These anti-idiotypic antibodies can be produced using the antibodies as immunogen and are useful for diagnostic purposes in detecting humoral response to tumors and in therapeutic applications, e.g., in a vaccine, to induce an anti-tumor response in patients (See, e.g., Nepom et al., Cancer And Metastasis Reviews, 6:487-501 (1987); and Lee et al., Proc. Nat'l. Acad. Sci. (USA), 82:6286-90 (1985)).
Human monoclonal antibodies may be made by using the 100 kDa antigen of the disclosure, to sensitize human lymphocytes to the antigen in vitro followed by EBV-transformation or hybridization of the antigen-sensitized lymphocytes with mouse or human lymphocytes as described by Borrebaeck et al. (Proc. Nat'l. Acad. Sci. (USA), 85:3995-99 (1988)). Therefore, human monoclonal antibodies or chimeric antibodies that bind the 100 kDa antigen are also included within the scope of the present invention.
Methods
The disclosure provides methods of screening, diagnosis, and treatment monitoring of patients having a cancer such as colon cancer or lung cancer.
In embodiments, a method involves combining one or more antibodies that specifically bind to a 100 kDa glycoprotein that has a colorectal cancer membrane bound and a soluble form, has a UV absorbance peak at about 228 nm, an isoelectric point of about 3.5 to 4, a sialic acid content of about 20%, and does not substantially bind to 180 kDa CEA and/or ACT in a human tissue or fluid sample; and detecting the binding of the antibody to the 100 kDa glycoprotein antigen in the sample. In embodiments, the presence or amount of the 100 kDa glycoprotein detected is indicative of the presence of or risk of cancer as compared to control sample without cancer. In embodiments, the 100 kDa glycoprotein is increased in the sample as compared to a control sample from a subject not having cancer. In embodiments, subjects having about 6.5 units/ml or greater of the 100 kDa glycoprotein are at increased risk for colorectal cancer or tubular adenomas.
In embodiments, the sample from the subject is a blood or serum sample and the immunoassay is able to detect the risk of or presence of stage 1 colon carcinoma and/or tubular adenomas. In embodiments, the assay detects these conditions with a sensitivity and/or a specificity of at least 70%, 80%, 90%, 95%, and any number in between 70-100%.
In one aspect, an immunoassay comprises one or more of any of the antibodies as described herein. In embodiments, an immunoassay for determining whether a subject is at risk of colorectal cancer comprises: a) combining an antibody or antigen binding fragment thereof with a human tissue or fluid sample from the subject; wherein the antibody or antigen binding fragment specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, but does not bind to glycosylated 180 kDa CEA and one or more linear peptides consisting of a linear peptide of 15 amino acids from amino acids 1-60 of SEQ ID NO:2, a linear peptide of amino acids 111 to 125 of SEQ ID NO:3, and/or a linear peptide of 15 amino acids from amino acids 150-701 of SEQ ID NO:4; and b) determining the amount of the antigen in the human tissue or fluid by determining the amount of the antibody antigen complex. In am embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a capture antibody attached to a solid substrate. In embodiments, the antibody is detectably labelled. In embodiments, one or more capture antibodies can be utilized.
In other embodiments, an immunoassay further comprises a second antibody that specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, but does not bind to glycosylated 180 kDa CEA and one or more linear peptides consisting of a linear peptide of 15 amino acids from amino acids 1-60 of SEQ ID NO:2, a linear peptide of amino acids 110 to 125 of SEQ ID NO:3, and/or a linear peptide of 15 amino acids from amino acids 150-701 of SEQ ID NO:4; and b) determining the amount of the antigen in the human tissue or fluid by determining the amount of the antibody antigen complex. In am embodiments, the second antibody is a monoclonal antibody. In other embodiments, the second antibody is a capture antibody attached to a solid substrate. In embodiments, the antibody is detectably labelled.
In embodiments, the pair of antibodies in an immunoassay bind to an epitope in the N terminal domain of CEACAM5 (SEQ ID NO:5) and do not bind to epitopes in the other domains of CEACAM5 as described herein.
In embodiments, an immunoassay further comprises an additional antibody that specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1. In embodiments the additional antibody is detectably labelled. In embodiments, the additional antibody is a polyclonal antibody.
In embodiments, detectable labels include, without limitation, a radionuclide, an enzyme, a fluorescent agent and a chromophore.
In embodiments, a method of the disclosure involves a screening method for subjects. A subject may visit a health care facility, for example, as a part of routine checkup, and a sample is obtained from the subject. The sample is then screened for the presence of a 100 kDa glycoprotein in accord with methods as described herein. If an increase of the 100 kDa glycoprotein as compared to control is seen in the sample or if the presence or amount of the antigen exceeds a predetermined cutoff value, the health care worker directs the subject to further cancer screening.
In a specific example, a subject comes in for an annual physical and a blood sample is taken. The blood sample is tested in accord with the kits and methods as described herein for the presence or amount of the 100 kDa glycoprotein. If an amount of the 100 kDa antigen detected is about 6.5 units/ml or greater as compared to control in the sample, the health care worker directs the subject to further cancer screening, such as a colonoscopy rather than waiting for the recommended time for conducting such routine screening. In addition, the health care worker may direct the subject to more specific diagnostic methods such as a biopsy, CT scan, MRI, PET scan, alone or in conjunction with other biomarker tests.
In embodiments, the immunoassay methods as described herein identifies with 95% sensitivity the Stage I (Earliest stage histologically confirmed) of Colorectal Cancer. In other embodiments, the immunoassay also identifies over 60% of the Tubular Adenoma polyps which if not removed will most likely become cancer. In embodiments, the antibodies in the immunoassay are selected or modified in order to provide an immunoassay for detecting tubular adenoma or stage I colorectal cancer with a sensitivity of at least 70% to 100% including every number in between and a specificity of at least 70%400%, including every number in between.
In embodiments, a method comprises monitoring treatment of a patient with cancer for efficacy by a) combining an antibody or antigen binding fragment thereof with a human tissue or fluid sample from the subject; wherein the antibody or antigen binding fragment specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, but does not bind to glycosylated 180 kDa CEA and one or more linear peptides consisting of a linear peptide of 15 amino acids from amino acids 1-60 of SEQ ID NO:2, a linear peptide of amino acids 111 to 125 of SEQ ID NO:3, and/or a linear peptide of 15 amino acids from amino acids 150-701 of SEQ ID NO:4; and b) determining the amount of the antigen in the human tissue or fluid by determining the amount of the antibody antigen complex. In embodiments, a decrease in the presence or amount of the antigen detected as compared to a value at the beginning of treatment is indicative of the efficacy of treatment of cancer. In embodiments, the cancer is selected from the group consisting of colon cancer and lung cancer. In embodiments, a treatment is identified as efficacious if it results in a decrease in the amount of the 100 kDa antigen of at least 20% or more. In embodiments, the value at the beginning of treatment is about 6.5 units/ml or greater.
In embodiments, an immunoassay kit is provided for use in the methods described herein. In some embodiments, an immunoassay kit comprises: a) a first and/or second antibody that specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, but does not bind to glycosylated 180 kDa CEA and one or more linear peptides consisting of a linear peptide of 15 amino acids from amino acids 1-60 of SEQ ID NO:2, a linear peptide of amino acids 110 to 125 of SEQ ID NO:3, and/or a linear peptide of 15 amino acids from amino acids 150-701 of SEQ ID NO:4; b) an additional antibody that specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, labeled with a signal generating element; and c) a calibrator.
In embodiments, the antibody or antigen binding fragment binds to a conformational epitope. In some embodiments, the antibody or antigen binding fragment specifically binds to and/or competes for binding to an epitope comprising amino acids 61-69 (SEQ ID NO:6), 73-77 (SEQ ID NO:7), or 69-82 KGERVDGNRQIIGY (SEQ ID NO:8), and 96-107(SEQ ID NO:9) of SEQ ID NO:1. In other embodiments, the antibody or antigen binding fragment specifically binds to an epitope comprising amino acids 61-69(SEQ ID NO:6), 78-98(SEQ ID NO:10), 96-107(SEQ ID NO:9), and 127-139(SEQ ID NO:11) of SEQ ID NO:1.
In embodiments, the antibody or antigen binding fragment does not substantially bind to epitopes in one or all other domains of CEACAM5 selected from the group consisting of amino acids 146-237 (A1 Ig-2), amino acids 238-322 (B1Ig-3), amino acids 324-415 (A2 Ig-4), amino acids 416-498 (B2 Ig-5), amino acids 502-593 (A3 Ig-6) amino acids 594-677 (B3 Ig-7). The amino acid numbers will be shifted one number from SEQ ID NO:1 because of the deletion of amino acid 320. In embodiments, the first and/or second antibody are monoclonal antibodies having an epitope specificity as described herein and the additional antibody is a polyclonal antibody that bind to a number of different epitopes. In embodiments, one or more monoclonal antibodies having different epitope specificity are used as capture antibodies.
In embodiments, the first and second antibody can comprise the CDRs and/or the heavy chain or light chain variable domain sequences as described herein.
In yet other embodiments, a method further comprises displaying the presence or amount of the 100 kDa glycoprotein in the sample on a detectable reader. In yet other embodiments, a method further comprises communicating the presence or amount of the 100 kDa glycoprotein in the sample and/or the presence or absence of cancer to a health care worker. In embodiments, the cancer is selected from the group consisting of as colon cancer and lung cancer. In embodiments, the sample is from a biopsy, a serum, or a blood sample. In embodiments, a detectably labeled antibody is administered in vivo to identify the cancer. In embodiments, the antibody is a human or humanized antibody comprising the heavy and/or light chain CDRS as described herein
In embodiments, a detectable reader is able to convert the signal from the detectably labeled antibody into a numerical value. The detectable reader employed depends on the label employed in the immunoassay. For example, for fluorescent labels or antibody labeled with an enzyme that converts a substrate labeled with fluorescent label, a detectable reader is a spectrofluorometer. In other embodiments, the detectable reader detects a radioactive label. In embodiments the detectable reader comprises a display and/or a printer. The reader may include software that automatically takes into account background and uses a standard curve based on an appropriate calibrator to provide a numerical value. In embodiments, the numerical value is communicated to a health care worker via a network including a wireless network.
The methods and kits of the disclosure are useful as point of care assays. The methods and assays as described herein can be used in conjunction with other cancer screening methods such as colonoscopy, lung imaging, biopsy, PET scans, magnetic resonance imaging, ct scans, and assay for other biomarkers associated with cancer.
In embodiments, one or more antibodies are selected to provide an immunoassay that can detect the presence or absence of a 100 kDa glycoprotein as described herein in a tissue or serum sample from a patient having cancer with a specificity and/or sensitivity of at least 70%400%, including every number in between. In embodiments, the first antibody and/or second antibody is a monoclonal antibody. In embodiments, the additional antibody is a polyclonal antibody.
In embodiments, at least two antibodies are selected in order to form an antibody pair that specifically binds to a 100 kDa glycoprotein in a human tissue or bodily fluid sample in an immunoassay. In embodiments, antibodies that bind to the 100 kDa glycoprotein may bind to the same epitope or different epitopes on the protein.
Immunohistiological techniques involve contacting a biological specimen such as a tumor tissue specimen with the antibodies of the invention and then detecting the presence on the specimen of the antibodies complexed to their antigen. The formation of such antibody-antigen complexes with the specimen indicates the presence of tumor cells in the tissue. Detection of the antibodies on the specimen can be accomplished using techniques known in the art, such as the immunoperoxidase staining technique, the avidin-biotin (ABC) technique or immunofluorescence techniques (see, e.g., Ciocca et al., Meth. Enzymol., 121:562-79 (1986); Hellstrom et al., Cancer Research, 46:3917-23 (1986); and Kimball (ed.), Introduction To Immunology (2nd Ed.), pp. 113-117, Macmillan Publ. Co. (1986)).
Serologic diagnostic techniques involve the detection and quantitation of tumor-associated antigens that have been secreted or “shed” into the serum or other biological fluids of patients thought to be suffering from carcinoma. Such antigens can be detected in the body fluids using techniques known in the art such as radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA) wherein an antibody reactive with the “shed” antigen is used to detect the presence of the antigen in a fluid sample (see, e.g., Uotila et al., J. Immunol. Methods, 42:11 (1981) and Allum et al., “Monoclonal Antibodies in the Diagnosis and Treatment of Malignant Conditions” Surg. Ann., 18:41-64, 48-51 (1986)). These assays, using the antibodies disclosed herein, therefore can be used for the detection in biological fluids of the antigens with which the antibodies react and thus provide for the detection of various carcinomas in human patients. Thus, it is apparent from the foregoing that the antibodies of the invention can be used in most assays involving antigen-antibody reactions. These assays include, but are not limited to, standard RIA techniques, both liquid and solid phase, as well as ELISA assays, immunofluorescence techniques, and other immunocytochemical assays (see, e.g., Sikora et al. (eds.), Monoclonal Antibodies, pp. 32-52, Blackwell Scientific Publications, (1984)).
The antibodies of the disclosure are also useful for in vivo diagnostic applications for the detection of human tumors. One such approach involves the detection of tumors in vivo by tumor imaging techniques using the antibodies labeled with an appropriate imaging reagent that produces detectable signal. Imaging reagents and procedures for labeling antibodies with such reagents are well known (see, e.g., Wensel and Meares, Radio Immunoimaging and Radioimmunotherapy, Esevier, N.Y. (1983); Colcher et al., Meth. Enzymol., 121:802-16 (1986)). The labeled antibodies may be detected by a technique such as radionuclear scanning (see, e.g., Bradwell et al. in Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. (eds.), pp. 65-85, Academic Press (1985)).
In some embodiments, the antibodies are useful as a therapeutic agent, especially if linked to a toxic molecule. Antibodies that have been screened for binding to cancer cell tissue as described herein are useful in a method for treating a cancer comprising administering an antibody that specifically binds to a 100 kDa glycoprotein that has a colorectal cancer membrane bound and a soluble form, has a UV absorbance peak at about 228 nm, an isoelectric point of about 3.5 to 4, a sialic acid content of about 20%, has a sequence of SEQ ID NO:1, and does not substantially bind to 180 kDA CEA and/or ACT in a subject suspected of having cancer. In embodiments, the antibodies are human or humanized antibodies comprising the heavy chain CDRs and/or light chain CDRS described herein. In embodiments, the cancer is selected from colon cancer and lung cancer. In embodiments, the cancer is colon cancer.
For diagnostic purposes, antibodies can be attached to a solid surface. Solid surfaces, include without limitation, membranes, multiwell plates, chromatography media, glass slides, latex beads, magnetic beads, microarray chips, capillary tubes and chips. Antibodies can also be labeled with a signal generating element. Signal generating elements include dyes, enzymes, radioactive labels, and chemiluminescent reagents.
In some embodiments, a cutoff value for classifying unknown samples as having the disease or not having the disease is identified. In embodiments, a cutoff value is determined by diluting each of plurality of samples from subjects known to have cancer and detecting the amount of the 100 kDa glycoprotein as compared to a similar set of dilutions from samples subject known not to have cancer. In embodiments, a cutoff is selected that discriminates between bodily fluid samples such as serum samples that are indicative of the presence of cancer as compared to samples known not to have cancer. In embodiments, a cutoff value for the presence of colorectal cancer or tubular adenomas in a serum sample is at least about 6.5 units/ml.
Kits
The disclosure also provides kits for diagnosis of cancer, such as colon or lung cancer. The kit comprises at least one antibody that specifically binds to a 100 kDa glycoprotein that has a colorectal cancer membrane bound and a soluble form, has a UV absorbance peak at about 228 nm, an isoelectric point of about 3.5 to 4, a sialic acid content of about 20%, and does not substantially bind to 180 kDa CEA and/or ACT. In embodiments, the antibody is labeled with a signal generating element.
In yet other embodiments, the kit comprises a) a first and/or second antibody that specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, but does not bind to glycosylated 180 kDa CEACAM5 and one or more linear peptides consisting of a linear peptide of 15 amino acids from amino acids 1-60 of SEQ ID NO:2, a linear peptide of amino acids 111 to 125 of SEQ ID NO:3, and/or a linear peptide of 15 amino acids from amino acids 150-701 of SEQ ID NO:4; b) an additional antibody that specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, labeled with a signal generating element; and c) a calibrator. The antibodies may bind to the same epitope or different epitopes. One of the antibodies is optionally attached to a solid surface. Solid surfaces include membranes, beads, magnetic beads, microwell plates, slides, and micorarrays. The other antibody is optionally labeled with a detectable label or signal generating element. Detectable labels or signal generating elements include biotin, fluorescent dyes, chemiluminescent tags, radioactive tags, and enzymes.
The kit may also optionally comprise a calibrator or standard as a positive or negative control. The standard may comprise a cancer cell extract obtained by a method comprising a) contacting colorectal and/or liver tumor cells with an acid to form an extract; b) separating components of the extract by molecular weight to form a first fraction having components of about 60 kDa or greater; c)isolating components of the first fraction that have a molecular weight of about 75 kDa or greater and less than about 200 kDa; and d) isolating a protein of 100 kDa molecular weight. In embodiments, a calibrator is a 100 kDa glycoprotein having an amino acid sequence of SEQ ID NO:1. In embodiments, the tumor cell is from colorectal cancer. In other embodiments, the kit comprises a negative control such as bovine serum albumin, ACT, and/or 180 kDA CEA.
In embodiments, a kit comprises instructions for conducting an immunoassay to detect the presence of a 100 kDa glycoprotein. The instructions provide a standard curve and instructions for diluting the positive and negative control. The instructions provide a cutoff value of 6.5 units/ml. If the antigen is detected at a level of 6.5 units/ml. or greater the sample is identified as positive for an increased risk of colorectal or lung cancer.
Also included with the disclosure are point of care kits and assay formats. In such assays, the biological sample is blood, urine, saliva, and/or tears and testing is conducted in a doctor's office, emergency room, hospital room, outpatient center, and the like. In embodiments, an antibody as described herein is attached to a solid substrate such as a multiwell plate, dipstick, or membrane based test strip. When the antibody is contacted with the patient sample, if the antigen is detected in the patient sample, the solid substrate changes color in at least one location which can be read visually. In an alternative embodiment, the antibody may be incorporated in a meter that draws a blood sample and then if the antigen is detected gives a detectable signal that is read by the meter.
In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the scope of this invention in any manner.
Example 1
A perchloric acid tumor extract prepared from liver and colon cancer tissues was used to prepare polyclonal and monoclonal antibodies. The HAI test referred to as the Tennagen assay was developed solely using a polyclonal antibody and was used to diagnose colon cancer. An ELISA test using two specific monoclonal antibodies as capture antibodies and the polyclonal antibody as a signal generating antibody was developed later. A cocktail of the two specific monoclonal antibodies in the ELISA Assay repeatedly have a sensitivity of 95+% to Early Stage Colorectal carcinoma and a specificity of 94+% when serum samples from patients not known to have this condition are analyzed.
Immunohistochemical studies have shown the two monoclonal antibodies attach to the cell membranes of adenocarcinoma of colorectal cancer but do not attach to normal tissue. No attachment to cells in bladder or breast tissue is seen.
Materials and Methods
Immunization
Lots of several Tennessee Antigen (CA11-19), used in the immunization of goats for the production of polyclonal antibodies, were used to immunize the Balb/C mice for the production of the mouse hybridomas. The Tennessee Antigen (CA11-19) lots were perchloric acid extracts from adenocarcinoma tissue with purification steps including Sepharose 4B and Sephadex G200 chromatography as used for all antigen approved for immunization of goats and mice after the initial characterization of the antigen.
A large number of monoclonal antibodies were cloned from the hybridomas generated from three of the immunized mice. The hybridomas and monoclonals were initially screened using the approved Tennessee Antigen coated RBCs, a component of the TennaGenTM Assay. The hybridoma and monoclonal supernates were mixed with the Tennessee Antigen coated RBCs and hemagglutation titer was determined. Hybridoma and monoclonal cell culture supernates containing antibodies to Tennessee Antigen (CA11-19) resulted in the smooth mat agglutination. The HA assay was used as the screening test for screening large numbers of hybridomas and later monoclonal antibody production in cell culture supernates.
Using the Limiting Dilution technique, 23 monoclonal antibodies were produced from the best hybridomas. With extensive evaluation of the 23 clones, 7 clones were selected for further evaluation in the ELISA assay. Immunohistochemical studies confirmed the monoclonals would bind to adenocarcinoma of colon and lung. Two monoclonals also had positive staining with squamous cell and adenocarcinoma of the Lung. The negative controls were bladder and breast tissue. The final selection for the capture antibodies was a monoclonal cocktail utilizing 5E5-1 and 5A1-1 following the second blind serum panel from the National Cancer Institute. Summary of NCI evaluation using a Double Blinded Serum Panel supplied by the NCI through Mayo Clinic for the CA11-19 Mono-Poly ELISA Sandwich Assay is shown.
ELISA Assay
The ELISA uses a high binding microtiter plate coated with a cocktail of two mouse monoclonal antibodies, 5E5-1 & 5A1-1, the anti-Tennessee Antigen polyclonal antibody conjugated to alkaline phosphatase for use as the detection antibody and Tennessee Antigen lots approved for use as calibrators are calibrators in the CA11-19 ELISA Kit. The ELISA uses neat serum and not perchloric acid extracts.
The protocol for an ELISA is as follows:
Coating: each well of a high binding 96 well Easy Wash EIA/RIA plate was coated two hours at room temperature ( ̃21° C.) followed by overnight at refrigeration (4-8° C.) with 125 uL of optimized by titration the solution of monoclonal cocktail (Capture Antibodies) in ELISA Coating Buffer. This equates to approximately 1 μg of antibody-per-well.
Blocking: after 3×300 μL washes with PBS T 20 Wash Buffer, the plates were blocked with 300 μL-per-well of 1.2% SEA Block in ELISA Coating Buffer for 2 hrs. At room temperature. Microtiter plate is further blocked with 2% Sucrose, and 4% Polyvinylpyrrolidone Buffer for 45 minutes.
Storage: Plates were dried by inverting on plastic drying racks for 24 hours at room temperature, then placed in foil pouches along with a dessicant packet and humidity indicator, and then stored at 40° C.
Sample: Plates, Calibrators and Sample Buffer were moved to room temperature and allowed to equilibrate. 25 μL of calibrators, and controls were added to the 2 microtiter wells of plates along with 25 μl of unknown samples followed by 75 uL of Sample Diluting Buffer. Plates were sealed, placed in ziplock bag and incubated at 37 degree C. overnight for 2 hrs.
CA11-19 plates were washed 6×300 μL with distilled water and then 100 uL of CA11-19 Polyclonal alkaline phosphatase conjugate was added to each well, Plates were sealed, placed in ziplock bag and left at 37 degree C. for 2 hrs.
CA11-19 plates were washed 6×300 μL with Distilled water and 100 uL CA11-19 Substrate was added to each well. Plates were placed at 37 degree C. for 30 min.
Measurement: The absorbance of each well at 405 nm was measured immediately using a standard plate reader.
Quantification: The absorbance of the standards was plotted using a point to point curve and the absorbance of the controls and unknowns are read against the curve.
The calibrators for the ELISA test are manufactured following SOP/Procedure using Tennessee Antigen (CA11-19) lots approved for use as a calibrator. The unitage is based on the original unitage of a vault reference standard and manufacturing allows for the dilution of antigen lot to obtain the standard required unitage. The Scientific advisory board at the FDA meeting made the statement that Tumor Markers were not pure enough to have a purified uniform standard. The World Health Organization made a similar statement concluding that the Tumor Marker Tests should be reported at “units/ml” and not in a weight such as ng or pc until the protein was completely 100% pure for a reference standard. Many tumor markers such as AFP, B-HCG, CA15-3, CA19-9, CA125, CA27-29, and PAP are reported as U/ml while CEA and PSA are reported in ng/ml.
Immunohistochemistry
The various monoclonal (cloned hybridomas) antibodies and goat antibody created for the TennaGen Assay were further characterized using Immunohistochemisty studies at two different independent facilities, Summa Medical, TX and U of Hawaii, HI. The monoclonal 5E5-1 and 5A1-1 stained adenocarcinoma of the colon and lung as well as squamous cell carcinoma of the lung just as the goat antisera; however, the signal from the 5E5-1 antibody was stronger.
Results
This test configuration was used to measure the CA11-19 levels in 422 patients. 222 samples were collected from GI Associates, TN) and 200 samples were purchased commercially from Equitech Bio, TX. The serum from these patients was assayed in the CA11-19 ELISA Assay. Results are summarized below in FIG. 1 and Tables 1, 2 and 3. With the use of the monoclonal cocktail of 5E5-1 and 5A1-1 along with improvements in other ELISA reagents, the sensitivity has been greater than 90% with specificity between 84 to 92% depending on the clinical status of the patient.







TABLE 1







All cancerous patients histologically confirmed categorized by cancer stage using 


a 6.5 units/mL cut-off to determine true positive, true negative, 


false positive, and false negative rates. Serum levels













Number
True
True
False
False


Colon Cancer/Histological confirmed
Samples
Positive
Negative
Positive
Negative















Stage I
21
21(100%)
0
0
0


Stage II
23
23(100%)
0
0
0


Stage III
17
17(100%)
0
0
0


Stage IV
11
7(64%)
0
0
4(36%)


Unknown Stage (pathology report not-provided)
9
 9(100%)
0
0
0
















TABLE 2







Polyps considered to be precancerous-Tubular Adenoma, Adenoma 


malignancy and Villous Adenoma. Serum levels












DIAGNOSIS
Number
True
True
False
False


POLYPS
Samples
Positive
Negative
Positive
Negative















Tubular 
53
17(32%) 
0
0
36(68%)


Adenoma







Adenoma 
1
1(100%)
0
0
0


Malignancy







Villous 
1
1(100%)
0
0
0


Adenoma







Hyperplastic
26
0
21(81%)
5(19%)
0


Polyp-not 
13
0
12(92%)
1(8%) 
0


classified







No Pathology





















TABLE 3







All non-cancerous patients categorized by condition using a 6.5 units/mL


cut-off to determine true positive, true negative, false positive, and false negative


rates. Serum levels












Condition
NumberSamples
TruePositive
TrueNegative
FalsePositive
FalseNegative















GI Bleeding
36
0
27(75%)
9(25%)
0


Crohns Disease
10
0
 8(80%)
2(20%)
0


Diverticulitis
70
0
62(89%)
8(11%)
0


Ulcerative
5
0
 5(100%)
0
0


Colitis







Hemorrhoids
24
0
22(92%)
2(8%) 
0


Change in
20
0
13(65%)
7(35%)
0


Bowel Habits







Normal GI
73
0
66(90%)
7(10%)
0


Misc GI Disease
6
0
 5(83%)
1(17%)
0


Undiagnosed
2
0
0
0
0









FIG. 1 shows a summary of all data from the 422 patient clinical study. Panel A shows the combined data for all non-cancerous and cancerous patients. The box covers the middle 50% of the data while the line is the median. The whiskers cover the middle 90% of the data (from 5% to 95%). Panel B is a receiver operator characteristic (ROC) curve comparing the cancerous vs. non-cancerous samples.
The box & whiskers plot (FIG. 1A) shows that the CA11-19 levels in non-cancerous tissues are lower overall than in cancerous patients. The box and whiskers plot comparing the levels of antigen detected in samples from patients known to have cancer as compared to control samples, establishes a cutoff value for 6.5 units/ml as indicative of the presence of colorectal cancer based on the lower limit of the whisker for the middle 90% of the data. When this data is plotted as a ROC curve (FIG. 1B), there is an apparent shift to the upper left quadrant consistent with a positive correlation between CA11-19 and cancer. The sensitivity and specificity of this test was 95% and 84% respectively when a cut-off of 6.5 units/mL is used. When the non-cancerous patients (Table 3) and cancerous patients (Table 1) are separated out by condition and the 6.5 units/mL cut-off is applied, the conditions giving the highest true positive, false positive, true negative, and false negative rate are seen. The highest false negative rate was seen in tubular adenomas (Table 2). Colon cancer from stages I-III gave 100% detection while stage IV was 64%.
Harvested monoclonal antibody supernates from 5E5-1 and 5A1-1 were purified and used in a cocktail as the capture antibody on high binding microtiter plates for further clinical evaluations. Two different Blind Serum Panels were assayed using two different lots of coated microtiter plates. The new lot used the 2012 cocktail as the capture antibody while the second lot used microtiter plates manufactured in 2009-2010 previously used in the evaluation conducted in 2009-2010. The serum panels were assembled at EDP Biotech and delivered to Dr. B. F. Overholt's office at Gastrointestinal Associates (GIA) where they were blind coded for assay. The CA11-19 (shown as CA1-18) assays were performed and results were taken to GIA where the codes were identified. The results are shown in Table 4 and 5.







TABLE 4







(Serum Panel A)











Disease Present
Disease Absent
Total Number













CA1-18 Elevated
27
6
33


CA1-18 Normal
5
33
38


TOTAL
32
39
71
















TABLE 5







(Serum Panel B)











Disease Present
Disease Absent
Total Number













CA1-18 Elevated
21
11
32


CA1-18 Normal
1
29
30


TOTAL
22
40
62









Table 4 above compares Colorectal cancer and Normals confirmed by colonoscopy. Serum Panel A (Jul. 23, 2012) shows a Sensitivity of 84.4% and Specificity of 78.9%. Polyps and Lung Cancer not included in calculation. The CA11-19 Lot 2012 showed a sensitivity of 95.5% with specificity of 80.0 percent seen in Serum Panel B using the colorectal and benign GI data. Polyps and Lung Cancer not included in calculation. When data from Serum Panel A and B are combined, a sensitivity of 92.3% and specificity of 85% is seen in Stage I Colorectal cancer as compared to Benign GI Disease.
Discussion
These results show that an ELISA assay with two capture monoclonal antibodies and a detectably labeled polyclonal antibody prepared against the Tennessee antigen repeatedly have a sensitivity of 95+% to Early Stage Colorectal carcinoma and a specificity of 94+% as measured in serum.
Immunohistochemical studies have shown the two monoclonal antibodies attach to the cell membrane of adenocarcinoma of colorectal cancer but do not attach to normal tissue. No attachment to cells in bladder or breast tissue is seen.
Example 2
The identity of the antigen that is being detected by the antibody assays described above has been difficult to determine. The perchloric acid carcinoma cell extract CA11-19 contains a number of different proteins. Further purification of the perchloric acid extract has identified a fraction of about 60-65 kDa and a fraction of about 100 kDa. However, the 60-65 kDa fraction, when used to immunize animals, has not provided any antibodies that specifically detect early stage colorectal carcinoma.
Isolation and Characterization of Antigens Present in the CA11-19 Preparation.
Human adenocarcinoma tumor tissue from colon and liver were extracted. The purification procedure involved a perchloric acid extraction with further purification steps including Sepharose 4B® and Sephadex G200® chromatography. Following SOP guidelines, SDS gel, Western Blot, immunoelectrophoresis and antigen content/protein determinations were used for all antigen lots. Over 100 different tumor tissues were extracted into individual Tennessee Antigen (CA11-19) extract lots.
Amino Acid analysis, UV absorbance at 228 and Molecular Weight determinations have been repeatedly made of different antigen lots and compared to CEA. The results are shown in Table 6.













TENNESSEE ANTIGEN(CA11-



TEST
19)
180 kDa CEA







Molecular Weight
100,000 Daltons
200,000 Daltons


UV Absorbance
Peak @ 228 nm
Peak @ 280 nm


AMINO ACID STUDIES




Lysine content
GREATER (7.6 moles %)
Less (2.9 moles %)


Glutamic acid, Glycine, Alanine
More than CEA
Less Than TNAG


Aspartic acid, Serine, Valine,
Less than CEA
Less than TN AG


isoleucine




Leucine
Less than CEA
More than TN AG


CARBOHYDRATE ANALYSIS
More than CEA's
Less than TN AG


Sialic Acid
19.88 moles % (average)
3.6 moles % (CEA WHO)


SDS Polyacrylamide
Band at
No single band at



100,000 Daltons
100,000 Daltons


Isoelectric Focusing
3.5-4.0 range
2.4, 3.0, 4.5-6.4 crude CEA prep




2.0-3.0 in purified prep


Immunoelectrophoresis/
No cross reactivity with



Hemagglutination-inhibition
Gold's original CEA,




Go's CEA, WHO-CEA,




Egan CEA, or Roche CEA









Similar characterizations were done comparing the 100 kDa antigen to the characteristics of alpha 1 anti-chymotrypsin (ACT), The results are shown in Table 7.










COMPARISON OF TENNESSEE ANTIGEN, ACT, and CEA











CA1-18
ACT
CEA













Molecular Weight
100 kDa
65 kDa
180 kDa


UV Peak
228
395
280


Isoelectric Point
3.5-4
5-5.3
3.5-4


Globulin Region
Beta
Alpha
Beta


Cancer Site
GI-Lung
Prostate, 
All Solid




Gastric
Tumors


Cancer Stage
EARLY
Advanced 
Advanced









SDS PAGE and Western Blot

A large number of perchloric acid extracts of tumor and normal tissue were removed from −20° C. storage and sterile filtered. All the Tennessee Antigen and Normal tissue extracts were perchloric acid extract with no further purification. The Tennessee Antigen perchloric acid extract was more stable than the Tennessee Antigen Lots that had been further purified for use as immunogen and characterization work. These samples were screened for the presence of CA11-19 by SDS-PAGE separation (FIG. 2A) and then goat anti-CA11-19 polyclonal Conjugate Western in two different batches (FIG. 2B).
A subset of samples showed clean single band reactivity in the 55-76 kDa region while others showed a generic reactivity where the entire protein pool showed a streak in the Western. The only immunizing antigen (#19) available showed good Western reactivity as did another sample (#28) which was deemed reactive enough to be used for amino acid analysis. Selected samples showing a clean single band reactivity were rerun on SDS-PAGE with the goal of excising the Western reactive region for protein identification. 11 gel slices were removed and given to Protein Discovery. They digested the samples, sequenced the released peptides via mass spectrometry, and then probed available databases to determine the protein make-up of the gel slice. Interestingly, ACT was detected 3× more often than any other protein with albumin coming in second.
Discussion
The isolated fraction from colorectal and/or liver tumors has components with a molecular weight of 55 to 75 kDa and a component with a 100 kDa. (FIG. 2A) The western blot shows reactivity with a 60 kDa and larger molecular weight proteins. (FIG. 2B). The identity of these bands was studied.
Very early data from two independent institutions showed that the Tennessee Antigen used as the immunogen for antibody production had a molecular weight close to 100 kDa. Two points important to the characterization studies: 1) the immunologically active portion of Tennessee Antigen has 100,000 molecular weight. Only the purified 100,000 MW material used as the immunogen has produced antibodies (monoclonal and polyclonal) that can measure a difference between Early Stage adenocarcinoma of colorectal and lung cancer serum with greater than 90% accuracy. The 60 kDa fraction used as the immunogen in rabbit, goat and mouse was not successful in producing a usable antibody. Further use of the 60 kDa material as the immunogen for antibody production in chicken and mouse also did not produce a useable antibody for the detection of colorectal and lung cancer when used as the capture antibody in a ELISA. The second significant point in the Tennessee Antigen Characterization is the documentation that Tennessee Antigen has a major UV peak at 228 and a small minor peak at 280 which is very different from Alpha 1 antichymotrypsin (ACT)-UV peak 395, and Alpha 1 antitrypsin-UV peak 296. Previous physical characterizations distinguish this band of material from 180 kDa CEA as well.
A total of 23 different monoclonal antibodies to Tennessee Antigen were produced, initially studied to be potentially useful and are stored in cryotanks for further evaluation for their diagnostic and therapeutic utility. Further immunohistochemical studies would identify the potential value of the additional 21 monoclonals to Tennessee Antigen. Experiments to further characterize the 100 kDa antigen involve amino acid sequencing of purified material and epitope mapping of the monoclonal antibodies.
Example 3
Separation of the 100 kDa material and the 60-65 kDa material Separation of high molecular weight material from low molecular weight material in adenocarcinoma tumor cell extracts in preparation for amino acid sequencing of large molecular weight fraction
Materials and Methods
Tumor Cell Material
Two different tumor cell specimens were obtained from adenocarcinoma from colorectal tissue. One tumor cell specimen is a stage II adenocarcinoma identified as 167B-80. A second tumor cell specimen is also an adenocarcinoma stage II identified as 173-81. Control colorectal and tubular adenoma serum specimens are identified as HF numbers.
Tumor Cell Extraction
Tumor cell extracts from each tumor cell specimen were prepared by mincing into very small pieces and extracting the material with 1.4% perchloric acid.
Column Chromatography
Aqueous Perchloric acid extracts of each tumor cell extract were separated by column chromatography on Sepharose 4B. Fractions were collected and each fraction was tested for absorbance at 280 and in the ELISA assay. Fractions containing peak absorbance at 280 and ELISA activity were pooled and were further separated using Superdex 200 chromatography. Fractions were collected from Superdex 200 column and tested for absorbance at 280 and in the ELISA. Fractions were run on SDS polyacrylamide gel.
ELISA
The ELISA uses a high binding microtiter plate coated with a cocktail of two mouse monoclonal antibodies, 5E5-1 and 5A1-1, the anti-Tennessee Antigen polyclonal antibody conjugated to alkaline phosphatase for use as the detection antibody. Large molecular weight and low molecular weight fractions from the column chromatography were tested in the ELISA assay for reactivity with the antibodies that can detect serum samples from patients having cancer.
The protocol for an ELISA is as follows:
Coating: each well of a high binding 96 well Easy Wash EIA/RIA plate was coated two hours at room temperature ( ̃21° C.) followed by overnight at refrigeration (4-8° C.) with 125 uL of optimized by titration the solution of monoclonal cocktail (Capture Antibodies) in ELISA Coating Buffer. This equates to approximately 1 μg of antibody-per-well.
Blocking: after 3×300 μL washes with PBS T 20 Wash Buffer, the plates were blocked with 300 μL-per-well of 1.2% SEA Block in ELISA Coating Buffer for 2 hrs. at room temperature. Microtiter plate is further blocked with 2% Sucrose, and 4% Polyvinylpyrrolidone Buffer for 45 minutes.
Storage: Plates were dried by inverting on plastic drying racks for 24 hours at room temperature, then placed in foil pouches along with a dessicant packet and humidity indicator, and then stored at 40° C.
Sample: Plates, Calibrators and Sample Buffer were moved to room temperature and allowed to equilibrate. 25 μL of calibrators, and controls were added to the 2 microtiter wells of plates along with 25 μl of unknown samples followed by 75 uL of Sample Diluting Buffer. Plates were sealed, placed in ziplock bag and incubated at 37 degree C. overnight for 2 hrs.
CA11-19 plates were washed 6×300 μL with distilled water and then 100 uL of CA11-19 Polyclonal alkaline phosphatase conjugate was added to each well, Plates were sealed, placed in ziplock bag and left at 37 degree C. for 2 hrs.
CA11-19 plates were washed 6×300 μL with Distilled water and 100 uL CA11-19 Substrate was added to each well. Plates were placed at 37 degree C. for 30 min.
Measurement: The absorbance of each well at 405 nm was measured immediately using a standard plate reader.
Quantification: The absorbance of the standards was plotted using a point to point curve and the absorbance of the controls and unknowns are read against the curve.
The calibrators for the ELISA test were manufactured following SOP/Procedure using Tennessee Antigen (CA11-19) lots approved for use as a calibrator. The unitage was based on the original unitage of a vault reference standard and manufacturing allows for the dilution of antigen lot to obtain the standard required unitage.
The polyclonal antibody does not substantially bind to pools of human serum, 180 kDa CEA preparations or CEACAM 6 (NCA).
Results
The results in FIG. 3 show that for each tumor cell extract bands of protein as determined by absorbance at 280 are found in fractions F9 to F13. The peak of the ELISA activity of the fractions indicate antigenic activity is predominantly found in fractions F10 to F12. Fractions F9 to F12 were pooled. The pooled fractions from each of the tumor specimens were then run on Superdex 200 column. (data not shown) A broad band of protein is found in fraction F7 to F13. Each fraction was tested in the ELISA and fractions showing peak activity were pooled and run on an SDS gel. A band was found at around 100 kDa (data not shown).
The band at 100 kDa will be analyzed further. The band will be cut out and analyzed for amino acid sequence.
Discussion
Previous studies have identified an antigen derived from tumor cell extracts detectable in a CA11-19 ELISA assay that is predictive of the presence of colorectal cancer in blood samples from patients. Characterization of the tumor cell extract revealed inactive protein fractions at 50-60 kDa and an active fraction at 100 kDa. In order to further characterize the 100 kDa fraction, separation and removal of the material having a molecular weight of 50-60 kDa was conducted. Separation of the lower molecular weight material from the higher molecular weight material was accomplished using column chromatography. Fractions that contained predominantly the 100 kDa material and that were active in the ELISA assay are being analyzed for amino acid sequence. This analysis should provide the identity of the antigen.
Example 4
N Glycan Profiling of CA11-19 Antigen
The sample preparation methods for N-glycan profiling by MALDI-MS are summarized in FIG. 4.
SDS-PAGE
The 100 kDa CA11-19 material is referred to as 168B antigen and was prepared as described in Example 3. The sample was denatured by adding equal amounts of 2×SDS sample buffer and incubated at 100° C. for 5 min. The mixture then divided into small portions and loaded on each lane. The samples were separated in 7.5% SDS-PAGE gel. After electrophoresis, the resolved proteins were stained with Coomassie Brilliant Blue (CBB). (FIG. 5)
In-Gel PNGase F Digestion and Extraction of Released N-Glycans
In gel PNGase F digestion was performed according to the method of (Kuster et al Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high performance liquid chromatography. Anal. Biochem., (1997) 250, 82-101). Briefly, target glycoprotein bands were excised from CBB-stained gel sheet and the gel bands were further cut into smaller pieces. The gel pieces were then destained alternately with 50 mM ammonium bicarbonate (AmBic) and 100% acetonitrile until the color turned clear. Then proteins in gel were reduced with dithiothreitol (DTT), followed by carboxyamidomethylation with iodoacetamide (IAM). DTT and IAM in gel were washed out alternately with 50 mM AmBic and 100% acetonitrile. Dehydrated gel pieces were reswelled with PNGase F solution (PNGase F in 50 mM AmBic) on ice for 45 min initially, and then the digestion was carried out at 37° C. overnight. Released N-glycans were extracted from the gels by stepwise extraction increasing the proportion of acetonitrile in 5% formic acid. The extracts were dried.
N-Glycan Preparation
Released N-glycans were purified from peptides by passage through a Sep-Pak C18 cartridge. The glycan fraction was eluted with 5% acetic acid and dried by lyophilization. The fractions were dried by lyophilization then permethylated based on the method of Anumula and Taylor, (Anumula et al., A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates. Anal Biochem, 1992. 203(1): p. 101-108.), prior to purification again with a C18 cartridge. The permethylated N-glycans were then analyzed with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).
MALDI-TOF Analysis
MALDI/TOF-MS of the permethylated N-linked glycans was performed in the reflector positive ion mode using α-dihyroxybenzoic acid (DHBA, 20 mg/mL solution in 50% methanol) as a matrix. The spectrum was obtained by using AB Sciex TOF/TOF 5800 MADLI mass spectrometry system.
Results:
Separation of CA11-19 168B by SDS-PAGE
CA11-19 168B was separated by SDS-PAGE for in-gel digestion. After electrophoresis, the resolved proteins were stained with CBB and images of gels are shown in FIG. 5. FIG. 5A shows localization of target glycoprotein 100 KD band and an additional band at 45 KD observed in the CA11-19 168B. FIG. 5B shows pictures of actual gels used for your sample analysis. Since the 100 KD bands were not clearly visible to observe in FIG. 5B, we cut the region that corresponds to the molecular weight of 100 KD and were excised for N-glycan profiling. We also performed the N-glycan analysis on the 45 KD band with the same procedures as a reference.
N-Glycan Profiling by MALDI/TOF-MS
The permethylated N-glycans from samples provided were profiled by MALDI/TOF MS analysis. The MALDI-full mass spectra of each sample bands are shown in FIG. 6A-B.
In-gel digestion and N-glycan analysis from 100 KD bands reveals peaks that may correspond to several glycan structures (m/z 1416.6, m/z 1661.7, and m/z 2039.9). However, the intensity of peaks is almost same as the noise level because the trace amount of sample concentration (FIG. 6A). Although we can clearly detect glycan associated with the sample and they are biantennary structures with and without galactose, it is possible that we are not detecting all the glycans present due to lack of sufficient sensitivity from small amount of sample obtained for 100 kD band.
The primary structures that were detected in 45 kDa bands using MALDI/TOF-MS were complex type N-glycans such as GlcNAc1Man3GlcNAc2 (m/z 1416.6) and GlcNAc2Man3GlcNAc2 (m/z 1661.7). The complex type N-glycans with fucose were also observed in 45 kDa band (FIG. 6B). It should be noted that the relative peak intensities for these glycans were much higher than that of the 100 KD band.
A summary of all results following N-glycan profiling is shown in Table 8.







TABLE 8







Summary of N-glycans from the CA 11-19 168B detected by MALDI-MS. Gal(○),


Galactose; Man(●), Mannose; Fuc(▾), Fucose; GlcNAc (▪), N-acetylglucosamine;










Suggested structure
Observed m/z :
45 Kda
100 Kda









1171.5
✓
✓


 






1416.6
✓
✓


 






1590.7

✓


 






1661.7
✓
✓


 






1794.7

✓


 






1865.8

✓


 






1906.8

✓


 






2039.8
✓
✓


 






2069.8

✓


 






2110.9

✓


 






2284.9
✓
✓


 






2489.0

✓









Discussion

N-glycan analysis from 100 KD bands indicates that peaks correspond to several glycan structures as shown in Table 8. In the SDS-PAGE (FIG. 5), we could definitely see the difference between the concentration of 45 KDa bands and 100 KDa. Consequently, we were able to observe much stronger signals from the 45 KD band as compared to the 100 KD band.
Example 5
O Glycan Profiling of CA11-19
The sample preparation methods for O-glycan profiling by MALDI-MS are summarized in FIG. 7.
SDS-PAGE
Sample material CA11-19BSTK and CA11-19 168BNC were prepared as described in Example 3. Sample STK differed from NC by being concentrated 5 fold. The samples were denatured by adding equal amounts of 2×SDS sample buffer and incubated at 100° C. for 5 min. Then the mixture was divided into small portions and loaded on each lane. The samples were separated in 7.5% SDS-PAGE gel. After electrophoresis, the resolved proteins were stained with Coomassie Brilliant Blue (CBB).
In-Gel O-Glycan Release by Reductive β-Elimination
In gel reductive β-elimination was performed according to the method of Dell, A et al (Dell, A. et al. Mass spectrometry of Carbohydrate-Containing Biopolymers. Methods Enzymol., (1994) 230, 108-132.) Briefly, target glycoprotein bands were excised from CBB-stained gel sheet and the gel bands were further cut into smaller pieces. The gel pieces were then destained alternately with 50 mM ammonium bicarbonate (AmBic) and 100% acetonitrile until the color turned clear. And then, gel pieces were washed with ethyl acetate and were washed with acetonitrile. O-glycans were released from gels by β-elimination. The gels were rehydrated by adding 100 mM sodium hydroxide and 1M sodium borohydrate. The samples were incubated for 18 h at 45° C. The sample was placed on ice and neutralized with 10% acetic acid. The neutralized samples were loaded onto AG-50WX8 cation exchange column to desalt. Ten percent acetic acid in methanol was added to samples to remove the borate.
O-Glycan Preparation
Released O-glycans were purified from peptides by passage through a Sep-Pak C18 cartridge. The glycan fraction was eluted with 5% acetic acid and dried by lyophilization. The fractions were dried by lyophilization then permethylated based on the method of Anumula and Taylor (cited supra), prior to purification again with a C18 cartridge. The permethylated O-glycan were then analyzed with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).
MALDI-TOF Analysis
MALDI/TOF-MS of the permethylated N-linked glycans was performed in the reflector positive ion mode using α-dihyroxybenzoic acid (DHBA, 20 mg/mL solution in 50% methanol) as a matrix. The spectrum was obtained by using AB Sciex TOF/TOF 5800 MADLI mass spectrometry system.
Nanospray Ionization FTMS
NSI-MSMS analysis was determined using a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source. Permethylated O-linked glycans were dissolved in 1 mM NaOH in 50% methanol then infused directly into the instrument at a constant flow rate of 0.5 μL/min. A full FTMS spectrum was collected at 30 000 resolution with 3 microscans. The capillary temperature was set at 210° C. and MS analysis was performed in the positive ion mode. For total ion mapping (automated MS/MS analysis), m/z range, 500 to 2000 was scanned with ITMS mode in successive 2 mass unit windows.
Results:
Separation of CA11-19 168BSTK and CA11-19 168BNC by SDS-PAGE
CA11-19 168BNC and CA11-19 168BSTK were separated by SDS-PAGE for in-gel digestion. See FIG. 8. After electrophoresis, the gels were stained with CBB. FIG. 8 shows the localization of target glycoprotein 100 KDa of CA11-19 168BNC and CA11-19 168BSTK. Since the 100 kDa band of CA 11-19 168BSTK was not clearly visible to observe in FIG. 8, the gel was cut the region that corresponds to the molecular weight of 100 KDa and gel slices excised for O-glycan profiling.
O-Glycan Profiling by MALDI/TOF-MS
Permethylated O-glycans from the sample were analyzed by MALDI/TOF MS as well as NSI-MS and total ion mapping.
The MALDI-TOF-full mass spectra of 100 KDa sample bands in both samples are not shown in this report because MALDI-TOF analysis did not reveal major glycan signals but instead presented mostly non-glycan signals. The samples were further analyzed with a LTQ Orbitrap XL mass spectrometer. The full FTMS scan of the sample show similar results as MALDI-TOF analysis with some O-glycan signals. NSI-FTMS spectra of O-glycan from the 100 KDa band from the both samples are shown in FIG. 9. The upper spectrum presents O-glycans of CA11-19 168BNC(A) and the lower spectrum shows O-glycans of CA11-19 168BSTK(B). The major O-glycan structures are the core 2 structure (m/z 779) and the core3 with fucose (m/z 953). In the SDS-PAGE (FIG. 8), we could definitely see the difference of sample concentration between CA11-19 168BNC and CA11-19 168BSTK. Consequentially, we did not observe the strong O-glycan signals from sample CA11-19 168BNC in sample CA11-19 168BSTK.
To confirm the O-glycan structures, total ion mapping (TIM) was carried out by NSI LTQ Orbitrap XL MS. Using TIM scan method, we were able to confirm several O-glycans which is summarized in Table 9. Both analyses showed a similar trend. CA11-19 168BNC sample shows clearer MSMS spectra than CA 11-19 168BSTK sample because of the sample concentration.
The MSMS spectra of the permethylated O-glycan at m/z 936, m/z 953, and m/z 1157 derived from the O-glycan spectrum of CA 11-19 168BNC (FIG. 9 upper spectrum) are shown in FIG. 10 as examples. As explained in FIG. 10A, MSMS profile of the molecular ion at m/z 936 (NeuAc-HexNAc-HexNAc), the diagnostic b ion is shown at m/z 398, together with the complementary y ions at m/z 561, m/z 659, and m/z 677. The MSMS profile of the molecular ion at m/z 953 (Hex-DHex-HexNAc2) and m/z 1157 (Hex2-DHex-HexNAc2) are interpreted in FIGS. 10B and 10C.
The MSMS profile of CA 11-19 168BNC and CA 11-19 168BSTK showed the same patterns and we present spectra from CA 11-19 168BNC in this report.







TABLE 9







Summary of O-glycans from the CA11-19 168B glycoproteins











Structural assignment
Theoretical m/z
Observed m/z
NC
STK


















534.29
534.29
✓
—


 






575.32
575.32
✓
✓


 






691.36
691.37
✓
—


 






779.41
779.42
✓
✓


 






895.46
895.47
✓
✓


 






936.49
936.50
✓
✓


 






953.51
953.50
✓
✓


 






983.51
983.52
✓
✓


 






1140.59
1140.60
✓
—


 






1157.60
1157.61
✓
✓


 






1187.61
1187.63
✓
—









Discussion

Our results indicate that the major O-linked glycans from CA11-19 168BNC and CA11-19 168BSTK are the core 2 structure (m/z 779) and the core3 with fucose (m/z 953). Since the amount of CA11-19 168BSTK glycoprotein was smaller than CA11-19 168BNC, the full ms of CA11-19 168BNC showed more O-glycan signals. It is being noted that the assigned glycan structures in the current report are based on common biosynthetic pathways.
Example 6
Amino Acid Sequence of 100 kDa Material
The 100 kDa (168B0 material was isolated and analyzed for amino acid sequence.
Amino Acid Sequence Analysis
One sample (in solution) was received. The proteins were reduced, alkylated, and digested with trypsin using the FASP protocol. The sample was resuspended in 2% acetonitrile/0.1% formic acid and analysis by LC-ESI-MS/MS. A 120 min. gradient was used and MS analysis was performed with an LTQ Orbitrap Velos mass spectrometer. MS spectra were searched using the MASCOT algorithm and the results generated an Excel spreadsheet. Searches using the tryptic peptides were conducted against a Human uniprot database. The protein was identified as having a sequence of CEACAM5 isofrom 2 (SEQ ID NO:1).
Peptide Mass Fingerprint of Antigen 168B
In order to characterize the antigen 168B, we submitted the sample to trypsin, chymotrypsin and ASP-N proteolysis followed by LC-LTQ Orbitrap MS/MS analysis. For the characterization of the antigen, a nano-LC chromatography was processed using a Ultimate 3000 (Dionex) system in line with a LTQ Orbitrap XL mass spectrometer (Thermo).
Sample Preparation:
5 μl of the antigen (2.5 μM) was mixed with 20 μl of ammonium bicarbonate (25 mM, pH 8.3). After mixing 2.5 μl of DTT (500 mM) is added to the solution. The mixture was then incubated 1 hour at 55° C. After incubation, 2.5 μl of iodioacetamide (1M) is added before 1 hour of incubation at room temperature in a dark room. After incubation, the solution is diluted 1/5 by adding 120 μl of the buffer used for the proteolysis.
145 μl of the reduced/alkyled antigen was mixed with 2 μl of trypsin (Roche Diagnostic) at ratio 1/20. The proteolytic mixture was incubated overnight at 37° C.
145 μl of the reduced/alkyled antigen was mixed with 2 μl of chymotrypsin (Roche Diagnostic) at ratio 1/20. The proteolytic mixture was incubated overnight at 30° C.
145 μl of the reduced/alkyled antigen was mixed with 2 μl of ASP-N(Roche Diagnostic) at ratio 1/20. The proteolytic mixture was incubated overnight at 30° C.
After proteolysis, 10 μl of the peptide generated by proteolysis was loaded onto a nano-liquid chromatography system (Ultimate 3000, Dionex).
A 95/05/0.1 H2O/ACN/HCOOH v/v/v
B 20/80/0.1 H2O/ACN/HCOOH v/v/v
gradient 5-40% B in 35 minutes
injected volume 10 μl
precolumn 300-μm ID×5-mm C4 PepMapTM
precolumn flow rate 30 μl/min
column 75-μm ID×5-cm C4 PepMapTM
column flow rate 200 nl/min
The LTQ orbitrap MS analysis has been performed with the following parameters:
















needle voltage
1.8 V



capillary voltage
  5 V



μscan MS
1



μscan MS2
1



MS range m/z
300-1700



MS/MS strategy
MS + 6CID MS/MS



Min. signal required
500



Ion isolation window
3 m/z units



Normalized collision energy
35%



Default charge state
2



Activation Q
0.25



Activation time
30



Dynamic exclusion
ON



Dynamic exclusion params
RC 1, RD 30s, ED 30s



Charge state screening
ON



Charge state rejection
ON



Charge state reject. Params
+1 and unassigned rejected










Validation Filters

Peptide Delta CN≧0.1

Peptide Xcorrvs Charge state≧1.5 (+1), 2.00(+2), 2.50(+3), 3.00(≧+4)

Peptide Probability≦0.001

Peptide Matches=1

Protein Number of Different Peptides≧2

Protein Exclude this reference: Keratin

Results

The sequence of CEACAM5 isoform 2 was used to analyze the mass spec data of the tryptic peptides. 18 tryptic peptides were identified in the sequence of 168B, covering 29.8%:











Identified Tryptic peptides of 168B
















Signif-
SEQ



Peptide
MH+
Position
icance
ID NO
















MESPSAPPHR
1108.52
 1-10
94.9
40


 



WCIPWQR
988.48
11-18
95.7
41


 



LLLTASLLTF
1987.12
18-35
96.8
42



WNPPTTAK






 



VDGNR
560.27
73-77
95.9
43


 



QIIGYVIGTQ
2177.13
78-98
92.4
44



QATPGPAYSG







R






 



SDLVNEEATG
1465.69
127-139
95.8
45



QFR






 



VYPELPKPSI
2328.2
140-160
94.6
46



SSNNSKPVED







K






 



LQLSNGNR
901.48
191-198
99.2
47


 



TLTLFNVTR
1064.6
199-207
97.1
48


 



NDTASYK
798.36
208-214
98.9
49


 



CETQNPVSAR
1104.51
215-224
93.6
50


 



LQLSNDNR
959.49
368-375
98.5
51


 



TLTLLSVTR
1003.61
376-384
92.2
52


 



NSGLYTCQAN
1866.81
470-487
97.2
53



NSASGHSR






 



AYVCGIQNSV
1481.71
567-580
92.5
54



SANR






 



INGIPQQHTQ
1807.02
628-643
95.3
55



VLFIAK






 



ITPNNNGTYA
2113.01
644-663
95.1
56



CFVSNLATGR






 



NNSIVK
674.38
664-669
96.5
57









The sequence of CEACAM5 isoform 2 was used to analyze the mass spec data of the chymotryptic peptides. 35 chymotryptic peptides were identified in the sequence of B168, covering 65.3%:











Identified Chymotrypsin peptides of 168B














Signif-
SEQ


Peptide
MH+
Position
icance
ID NO














ESPSAPPHRW
1163.55
 2-11
92.5
58


 


ESTPFNVAEGKEVL
2209.5 
39-58
94.4
59


LLVHNL






 


HLFGYSWYK
1199.56
61-69
96.5
60


 


KGERVDGNRQIIGY
1604.85
69-82
90.3
61


 


VIGTQQATPGPAY
1302.66
83-95
94.5
62


 


SGREIIYPNASL
1319.69
 96-107
96.6
63


 


IQNIIQNDTGF
1262.63
109-119
97
64


 


VNEEATGQF
 994.44
130-138
92.3
65


 


TCEPETQDATY
1257.49
166-176
93.5
66


 


VNNQSLPVSPRL
1323.73
180-191
94
67


 


NVTRNDTASY
1140.52
204-213
96.1
68


 


TISPLNTSYRSGEN
1877.93
241-257
98.1
69


LNL






 


SCHAASNPPAQY
1245.53
258-269
94.3
70


 


IPNITVNNSGSY
1278.63
286-297
93.6
71


 


TCQAHNSDTGL
1146.48
298-308
90.5
72


 


NRTTVTTITVY
1268.68
309-319
92.8
73


 


ITSNNSNPVEDEDA
1787.82
326-342
96.5
74


VAL






 


TCEPEIQNTTY
1298.55
343-353
97.6
75


 


SVTRNDVGPYECGI
1993.92
381-398
92.6
76


QNEL






 


SVDHSDPVIL
1081.55
399-408
93.3
77


 


GPDDPTISPSY
1148.51
413-423
94.5
78


 


YRPGVNLSLSCHAA
2331.09
426-447
96.3
79


SNPPAQYS






 


IDENIQQHTQEL
1467.7 
450-461
91.5
80


 


ISNITEKNSGL
1175.62
463-473
92.5
81


 


VKTITVSAELPKPS
2296.25
490-511
93.8
82


ISSNNSKP






 


TCEPEAQNTTY
1256.5 
521-531
94.5
83


 


VNGQSLPVSPRL
1266.71
535-546
96.2
84


 


NVTRNDARAY
1179.58
559-568
98.1
85


 


VCGIQNSVSANRSD
1859.92
569-586
95.6
86


PVTL






 


GPDTPIISPPDSSY
1445.67
591-604
95.6
87


 


SCHSASNPSPQY
1277.52
613-624
90.5
88


 


RINGIPQQHTQVL
1503.83
627-639
90.6
89


 


IAKITPNNNGTY
1305.67
641-652
92.2
90


 


SGTSPGLSAGAT
1004.46
676-687
94.8
91


 


ATVGIMIGVLVGVA
1298.75
686-699
96.2
92









9 peptides were identified by Asp N proteolysis in the sequence of B168, covering 16.8%:











Identified ASP-N peptides of 168B














Signif-
SEQ


Peptide
MH+
Position
icance
ID NO














DTGFYTLHVIKS
1380.7158
116-127
94.5
 93


 


DAVAFTCEPETQ
1310.5569
161-172
96.2
 94


 


DTASYKCETQNPV
2012.9454
209-226
92.5
 95


SARRS






 


DSVILNVLYGP
1189.6463
227-237
94.7
 96


 


DNRTLTLLSVTRN
1502.8285
373-385
92  
 97


 


DVGPYECGIQNEL
1622.7366
386-400
98.6
 98


SV






 


DPVILNVLYGP
1199.667
404-414
93.3
 99


 


DARAYVCGIQNSV
1910.9137
564-581
95.7
100


SANRS






 


DTPIISPP
839.4509
593-600
96.1
101









Based on the results obtained, we designed overlap mapping of the trypsin, chymotrypsin and ASP-N peptides (FIG. 14). Combining the peptides of Trypsin, Chymotrypsin and ASP-N proteolysis, 85.6% of the sequence is covered. The polypeptide is identified as CEACAM5 isoform 2 as shown in FIG. 14 (SEQ ID NO:1)

Example 7
Antibody Characterization: Mass Spectroscopy of Antibodies Cross-Linked with Antigen Sample Preparation
Samples provided included Antibody 1 5E5-1: 0.2 mg/ml; 2 ml; glycine buffer; Antibody 2 5A1-1: 1.2 mg/ml; 2 ml; glycine buffer; and Antigen 168B: 0.8 mg/ml; 100 μl; PBS pH 7.4. Antibody 5E5 and Antibody 5A1-1 have been shown to identify a biomarker for colorectal cancer in serum from patients. The antigen is identified as 168B and is a partially glycosylated form of CEACAM5 as described herein.
Three mAb/Ag interactions samples were prepared with the following concentrations:
A Antigen: 168B Antibody: 5E5-1 Mix 168B/5E5-1
5 microliters of the antigen (4 micromolar) were mixed with 5 microliters of antibody (2 micromolar) to form a mixture of 10 microliter (2 micromolar antigen and 1 micromolar antibody)
B Antigen: 168B Antibody: 5A1-1 Mix 168B/5A1-1
5 microliters of the antigen (4 micromolar) were mixed with 5 microliters of antibody (2 micromolar) to form a mixture of 10 microliter (2 micromolar antigen and 1 micromolar antibody)
C Antigen: 168B Antibody: 5A1-1/5E5-1 Mix 168B/5A1-1/5E5-1
5 microliters of the antigen (4 micromolar) were mixed with 2 microliters of each antibody (2 micromolar) to form a mixture of 10 microliter (2 micromolar antigen/1 micromolar antibody5A1-1/1 micromolar antibody 5E5-1)
1 μl of the mixture obtained was mixed with 1 μl of a matrix composed of a re-crystallized sinapinic acid matrix (10 mg/ml) in acetonitrile/water (1:1, v/v), TFA 0.1% (K200 MALDI Kit). After mixing, 1 μl of each sample was spotted on the MALDI plate (SCOUT 384). After crystallization at room temperature, the plate was introduced in the MALDI mass spectrometer and analyzed immediately. The analysis has been repeated in triplicate.
Cross-Link Experiments
The mixtures prepared above (9 μl left for each mixture) was submitted to cross-linking using CovalX's K200 MALDI MS analysis kit. 9 μl of the mixture was mixed with 1 μl of K200 Stabilizer reagent (2 mg/ml) and incubated at room temperature. CovalX uses a mixture of amine based crosslinking agents to stabilize the interactions between the antibody and the antigen. After the incubation time (180 minutes), the samples were prepared for MALDI analysis. The samples were analyzed by High-Mass MALDI analysis immediately after crystallization.
High-Mass MALDI MS Analysis
The MALDI ToF MS analysis has been performed using CovalX's HM3 interaction module with a standard nitrogen laser and focusing on different mass ranges from 0 to 2000 kDa. For the analysis, the following parameters have been applied:
Mass Spectrometer:
Linear and Positive mode
Ion Source 1: 20 kV
Ion Source 2: 17 kV
Lens: 12 kV
Pulse Ion Extraction: 400 ns
HM3:
Gain Voltage: 3.14 kV
Acceleration Voltage: 20 kV
To calibrate the instrument, an external calibration with clusters of Insulin, BSA and IgG has been applied. For each sample, 3 spots were analyzed (300 laser shots per spots). The presented spectrum corresponds to the sum of 300 laser shots. The MS data were analyzed using CovalX's Complex Tracker analysis software version 2.0.

Results

Analysis of the mix A: 5E5-1/168B, showed that the antigen and the antibody were detected with observed MH+=92.879 kDa (168B-antigen) and MH+=158.498 kDa. (5E5-1 antibody)(FIG. 11A). The cross-linking experiment was completed after 180 minutes incubation time with the crosslinking reagent K200. After cross-linking, we detected two additional peaks with MH+=258.945 kDa [5E5-1•B168] and MH+=354.164 kDa [5E5-1•2B168] (FIG. 11B). Using Complex Tracker software, we overlaid the control and cross-link spectra. The overlay confirmed the detection of two non covalent complexes [5E5-1•B168] and [5E5-1•2B168] (FIG. 11C).
Analysis of the Mix B: 5A1-1/168B, showed that the antigen and the antibody were detected with MH+=92.743 kDa (168B) and MH+=159.125 kDa. (5A1-1) (data not shown). The cross-linking experiment was completed after 180 minutes incubation time with the crosslinking reagent K200. After cross-linking, we detected two additional peaks with MH+=259.155 kDa [5A1-1•B168] and MH+=355.025 kDa [5A1-1•2B168] (data not shown). Using Complex Tracker software, we overlaid the control and cross-link spectra. The overlay confirmed the detection of two non covalent complexes [5A1-1•B168] (antibody binding with a single antigen) and [5A1-1•2B168] (antibody binding to two antigens)(data not shown).
Analysis of the Mix C: 5E5-1/5A1-1/168B showed that the antigen and the antibody were detected with MH+=92.910 kDa (168B) and MH+=158.988 kDa (5E5, 5A1-1). (FIG. 12A). The cross-linking experiment was completed after 180 minutes incubation time with the crosslinking reagent K200. After cross-linking, we detected two additional peaks with MH+=260.155 kDa and MH+=356.012 kDa (FIG. 12B). Using Complex Tracker software, we overlaid the control and cross-link spectra. The overlay confirmed the detection of two non covalent complexes [5E5-1•B168] and [5E5-1•2B168]. (FIG. 12C).
Discussion
The sandwich assay indicated that the simultaneous binding of the monoclonal antibodies 5E5-1 and 5A1-1 on the antigen 168 B was not observed. The analysis indicated that the epitope of these two monoclonal antibodies on the antigen 168B are located in the same close region of the protein.
Example 8
Epitope Mapping of Antibody 5E5-1
A competition assay with peptides derived from the antigen was conducted to determine if the epitope was linear.
Pepsin Proteolysis
In order to determine the nature of the epitope, we performed a proteolysis of 168B antigen with immobilized pepsin. 10 μl of the antigen with a concentration of 2.5 μM were mixed with immobilized pepsin 2.5 μM and incubated at room temperature for 30 minutes. After the incubation time the sample was centrifuged and the supernatant was pipetted. The completion of the proteolysis was controlled by High-Mass MALDI mass spectrometry in linear mode and reflectron mode. The pepsin proteolysis was optimized in order to obtain large amounts of peptides in the 1000-3500 Da range.
Pepsin Peptides/Antibody/Antigen Mixing and Incubation
5 μl of the antigen peptides generated by proteolysis was concentrated 10× using speed vac and mixed with 5 μl of 5E5-1 antibody (4 μM) and incubated at 37° for 2 hours.
Antigen Peptides/Antigen/Antibody Mixing
After incubation of the antibodies with the antigen peptides, 5 μl of the mixture is mixed with 5 μl of the intact antigen (4 μM). After mixing, the mixture contains the following proteins: 168B peptides 5E5-1 Antibody/168B antigen peptides Mix 5E5-1 antibody/168B peptides/168B antigen.
Interaction Analysis
For the competition assay, the antibody/antigen interaction analysis was performed with the same protocol as described in Example 6.
Results
For this experiment, the antigen and the antibody were detected with MH+=92.879 kDa and MH+=158.498 kDa. (FIG. 13A). The cross-linking experiment was completed after 180 minutes incubation time with the crosslinking reagent K200. After cross-linking, we detected two additional peaks with MH+=258.945 kDa and MH+=354.164 kDa. Using Complex Tracker software, we overlaid the control and cross-link spectra. (FIG. 13B) The overlay confirmed the detection of two non covalent complexes [5E5-1•B168] and [5E5-1•2B168] (FIG. 13C).
After competition, we did not detect inhibition of the binding of the antibody 5E5-1 on the antigen 168B. After competition, we observed two non covalent complexes: [5E5-1•B168] and [5E5-1•2B168] (FIG. 13).
Discussion
The competition assay indicated that peptides of the antigen are not inhibiting the binding Antibody/Antigen. The epitope 5E5-1 on 168B is not linear. The epitope is based on the conformation of the antigen.
Characterization of the Molecular Interfaces B168/5E5-1
In order to determine the epitope of 5E5-1 antibody on 168B antigen with high resolution the antibody/antigen complexes are incubated with deuterated cross-linkers and subjected to multienzymatic cleavage. After enrichment of the cross-linked peptides, the samples are analyzed by high resolution mass spectrometry (nLC-Orbitrap MS) and the data generated are analyzed using XQuest and Stavrox software.
For this analysis, a nLC in combination with Orbitrap mass spectrometry have been used as described above. 5 μl of the antigen sample (concentration 4 μM) was mixed with 5 μl of the antibody sample (Concentration 2 μM) in order to obtain an antibody/antigen mix with final concentration 1 μM/2 μM. The mixture was incubated at 37° C. for 180 minutes. In a first step, 1 mg of d0 cross-linker was mixed with 1 mg of dl 2 cross-linker. The 2 mg prepared were mixed with 1 ml of DMF in order to obtain a 2 mg/ml solution of DSS do/d12. 10 μl of the antibody/antigen mix prepared previously were mixed with 1 μl of the solution of cross-linker d0/d12 prepared (2 mg/ml). The solution is incubated 180 minutes at room temperature in order to achieve the cross-linking reaction.
In order to facilitate the proteolysis, it is necessary to reduce the disulfide bond present in this protein. The cross-linked sample was mixed with 20 μl of ammonium bicarbonate (25 mM, pH 8.3). After mixing 2.5 μl of DTT (500 mM) is added to the solution. The mixture is then incubated 1 hour at 55° C. After incubation, 2.5 μl of iodioacetamide (1M) is added before 1 hour of incubation at room temperature in a dark room. After incubation, the solution is diluted 1/5 by adding 120 μl of the buffer used for the proteolysis.
145 μl of the reduced/alkyled cross-linked sample was mixed with 2 μl of trypsin (Roche). The proteolytic mixture was incubated overnight at 37° C.
For this proteolysis, the buffer is Tris-HCL 100 mM, CaCl2 10 mM, pH7.8. 145 μl of the reduced/alkyled cross-linked complex were mixed with 2 μl of α-chymotrypsin 200 μM and incubated overnight at 30° C.
145 μl of the reduced/alkyled cross-linked sample was mixed with 2 μl of ASP-N (Roche). The proteolytic mixture was incubated overnight at 37° C.
Results
After cross-linking, the peptides generated by multi-enzymatic proteolysis are covering 81% of the total antigen sequence. After trypsin digestion of the antibody/antigen cross-linked complex with deuterated d0 d12, the nLC-orbitrap MS/MS analysis allowed to detect 32 peptides having monolinks. These monolinked peptides have been detected with both Xquest and Stavrox software.
After trypsin digestion of the antibody/antigen cross-linked complex with deuterated d0 d12, the nLC-orbitrap MS/MS analysis allowed to detect 4 cross-linked peptides between the antigen 168B and the antibody 5E5-1 heavy chain. These cross-linked peptides have been detected with both Xquest and Stavrox software.









Trypsin Cross-link Identified: 168B/5E5-1



Protein 1/Protein 2



QIIGYVIGTQQATPGPAYSGR-LSGTAGVHSQVQL-a19-b2



 



Position Protein 1/Protein2



168B 78-98 5E5-1 HC 11-24



(1-4 of heavy chain variable domain)



 



Protein 1/Protein 2



QIIGYVIGTQQATPGPAYSGR-LSGTAGVHSQVQL-a21-b2



 



Position Protein 1/Protein2



168B 78-98 5E5-1 HC 11-24



(1-4 of heavy chain variable domain)



 



Protein 1/Protein 2



SDLVNEEATGQFR-VQLQQSGAD-a1-b6



 



Position Protein 1/Protein2



168B 127-139 5E5-1 HC 21-29 



(1-9 of heavy chain variable domain)



 



Protein 1/Protein 2



SDLVNEEATGQFR-VQLQQSGAD-a13-b6



 



Position Protein 1/Protein2



168B 127-139 5E5-1 HC 21-29



(1-9 of heavy chain variable domain)






After chymotrypsin digestion of the antibody/antigen cross-linked complex with deuterated d0 d12, the nLC-orbitrap MS/MS analysis allowed to detect 4 cross-linked peptides between 168 B antigen and 5E5-1 antibody Heavy Chain and light chain. These cross-linked peptides have been detected with both Xquest and Stavrox software.










Chymotrypsin Cross-link Identified: 168B/5E5-1



Protein 1/Protein 2



HLFGYSWYK-QLLGLLLLCFQGTRC-a6-b14



 



Position Protein 1/Protein2



168B 61-69 5E5-1 LC 6-20



 



Protein 1/Protein 2



HLFGYSWYK-IQMTQITSS-a9-b8



 



Position Protein 1/Protein2



168B 61-69 5E5-1 LC 22-30



(2-9 of light chain variable domain)



 



Protein 1/Protein 2



SGREIIYPNASL-GVHSQVQLQQS-a1-b4



 



Position Protein 1/Protein2



168B 96-107 5E5-1 HC 16-26



(1-7 of heavy chain variable domain)



 



Protein 1/Protein 2



SGREIIYPNASL-GVHSQVQLQQS-a3-b11



 



Position Protein 1/Protein2



168B 96-107 5E5-1 HC 16-26



(1-7 of heavy chain variable domain)






After ASP-N digestion of the antibody/antigen cross-linked complex we did not detect any crosslinked peptides between 168B and 5E5-1.

Discussion

Using chemical cross-linking, High-Mass MALDI mass spectrometry and nLC-Orbitrap mass spectrometry we were able to characterize the interaction interface between the antigen 168B and the monoclonal antibody 5E5-1. Our analysis indicates that the epitope of this monoclonal antibody includes the following amino acids on 168B: 66; 69; 96; 98; 127; 139. On the antibody the paratope includes the following amino acids: Heavy chain: 6 Light Chain: 9. These results are illustrated in FIG. 15.
Example 9
A competition assay with peptides derived from the antigen was conducted to determine if the epitope was linear for antibody 5A1-1.
Pepsin Proteolysis
In order to determine the nature of the epitope, we performed a proteolysis of 168B antigen with immobilized pepsin. 10 μl of the antigen with a concentration of 2.5 μM were mixed with immobilized pepsin 2.5 μM and incubated at room temperature for 30 minutes. After the incubation time the sample was centrifuged and the supernatant was pipetted. The completion of the proteolysis was controlled by High-Mass MALDI mass spectrometry in linear mode and reflectron mode. The pepsin proteolysis was optimized in order to obtain large amounts of peptides in the 1000-3500 Da range.
Pepsin Peptides/Antibody/Antigen Mixing and Incubation
5 μl of the antigen peptides generated by proteolysis was concentrated 10× using speed vac and mixed with 5 μl of 5A1-1 antibody (4 μM) and incubated at 37° for 2 hours.
Antigen Peptides/Antigen/Antibody Mixing
After incubation of the antibodies with the antigen peptides, 5 μl of the mixture is mixed with 5 μl of the intact antigen (4 μM). After mixing, the mixture contains the following proteins: 168B peptides 5A1-1 Antibody/168B antigen peptides Mix 5A1-1 antibody/168B peptides/168B antigen.
Interaction Analysis
For the competition assay, the antibody/antigen interaction analysis was performed with the same protocol as described in Example 6.
Results
For this experiment, the antigen and the antibody were detected with MH+=92.879 kDa and MH+=158.498 kDa. (data not shown). The cross-linking experiment was completed after 180 minutes incubation time with the crosslinking reagent K200. After cross-linking, we detected two additional peaks with MH+=258.945 kDa and MH+=354.164 kDa. Using Complex Tracker software, we overlaid the control and cross-link spectra. (data not shown) The overlay confirmed the detection of two non covalent complexes [5A1-1•B168] and [5A1-1•2B168] (data not shown).
After competition, we did not detect inhibition of the binding of the antibody 5A1-1 on the antigen 168B. After competition, we observed two non covalent complexes: [5A1-1•B168] and [5A1-1•2B168] (data not shown). This indicates the epitope is not linear.
Characterization of the Molecular Interfaces B168/5A1-1
In order to determine the epitope of 5A1-1 antibody on 168B antigen with high resolution the antibody/antigen complexes are incubated with deuterated cross-linkers and subjected to multienzymatic cleavage. After enrichment of the cross-linked peptides, the samples are analyzed by high resolution mass spectrometry (nLC-Orbitrap MS) and the data generated are analyzed using XQuest and Stavrox software.
For this analysis, a nLC in combination with Orbitrap mass spectrometry have been used as described above. 5 μl of the antigen sample (concentration 4 μM) was mixed with 5 μl of the antibody sample (Concentration 2 μM) in order to obtain an antibody/antigen mix with final concentration 1 μM/2 μM. The mixture was incubated at 37° C. for 180 minutes. In a first step, 1 mg of d0 cross-linker was mixed with 1 mg of d12 cross-linker. The 2 mg prepared were mixed with 1 ml of DMF in order to obtain a 2 mg/ml solution of DSS do/d12. 10 μl of the antibody/antigen mix prepared previously were mixed with 1 μl of the solution of cross-linker d0/d12 prepared (2 mg/ml). The solution is incubated 180 minutes at room temperature in order to achieve the cross-linking reaction.
In order to facilitate the proteolysis, it is necessary to reduce the disulfide bond present in this protein. The cross-linked sample was mixed with 20 μl of ammonium bicarbonate (25 mM, pH 8.3). After mixing 2.5 μl of DTT (500 mM) is added to the solution. The mixture is then incubated 1 hour at 55° C. After incubation, 2.5 μl of iodioacetamide (1M) is added before 1 hour of incubation at room temperature in a dark room. After incubation, the solution is diluted 1/5 by adding 120 μl of the buffer used for the proteolysis.
145 μl of the reduced/alkyled cross-linked sample was mixed with 2 μl of trypsin (Roche). The proteolytic mixture was incubated overnight at 37° C.
For this proteolysis, the buffer is Tris-HCL 100 mM, CaCl2 10 mM, pH7.8. 145 μl of the reduced/alkyled cross-linked complex were mixed with 2 μl of α-chymotrypsin 200 μM and incubated overnight at 30° C.
145 μl of the reduced/alkyled cross-linked sample was mixed with 2 μl of ASP-N (Roche). The proteolytic mixture was incubated overnight at 37° C.
Results
After cross-linking, the peptides generated by multi-enzymatic proteolysis are covering 81% of the total antigen sequence. After trypsin digestion of the antibody/antigen cross-linked complex with deuterated d0 d12, the nLC-orbitrap MS/MS analysis allowed to detect 35 peptides having monolinks. These monolinked peptides have been detected with both Xquest and Stavrox software.
After trypsin digestion of the antibody/antigen cross-linked complex with deuterated d0 d12, the nLC-orbitrap MS/MS analysis allowed to detect 4 cross-linked peptides between the antigen 168B and the antibody 5A1-1 heavy chain. These cross-linked peptides have been detected with both Xquest and Stavrox software.









Trypsin Cross-link Identified: 168B/5A1-1



Protein 1/Protein 2



VDGNR-VKPGASVKL-a5-b2



 



Position Protein 1/Protein2



168B 73-77 5A1-1 HC 11-19



 



Protein 1/Protein 2



VDGNR-VKPGASVKL-a5-b2



 



Position Protein 1/Protein2



168B 73-77 5A1-1 HC 11-19






After chymotrypsin digestion of the antibody/antigen cross-linked complex with deuterated d0 d12, the nLC-orbitrap MS/MS analysis allowed to detect 10 cross-linked peptides between 168 B antigen and 5A1-1 antibody Heavy Chain and light chain. These cross-linked peptides have been detected with both Xquest and Stavrox software.










Chymotrypsin Cross-link Identified: 168B/5A1-1



Protein 1/Protein 2



HLFGYSWYK-VGDRVTITCRA-a6-b4



 



Position Protein 1/Protein2



168B 61-69 5A1-1 LC 15-25



 



Protein 1/Protein 2



HLFGYSWYK-VGDRVTITCRA-a9-b10



 



Position Protein 1/Protein2



168B 61-69 5A1-1 LC 15-25



 



Protein 1/Protein 2



KGERVDGNRQIIGY-TCRASQSISSYLN-a1-b2



 



Position Protein 1/Protein2



168B 69-82 5A1-1 LC 22-34



 



Protein 1/Protein 2



KGERVDGNRQIIGY-TCRASQSISSYLN-a4-b10



 



Position Protein 1/Protein2



168B 69-82 5A1-1 LC 22-34



 



Protein 1/Protein 2



KGERVDGNRQIIGY-VKPGASVKLS-a9-b2



 



Position Protein 1/Protein2



168B 69-82 5A1-1 HC 11-19



 



Protein 1/Protein 2



KGERVDGNRQIIGY-VKPGASVKLS-a9-b6



 



Position Protein 1/Protein2



168B 69-82 5A1-1 HC 11-19



 



Protein 1/Protein 2



SGREIIYPNASL-ASVKLSC-a1-b2



 



Position Protein 1/Protein2



168B 96-107 5A1-1 HC 16-22



 



Protein 1/Protein 2



SGREIIYPNASL-ASVKLSC-a3-b4



 



Position Protein 1/Protein2



168B 96-107 5A1-1 HC 16-22



 



Protein 1/Protein 2



SGREIIYPNASL-CKASGYTFTNYWINWVKP-a11-b2



 



Position Protein 1/Protein2



168B 96-107 5A1-1 HC 21-39



 



Protein 1/Protein 2



SGREIIYPNASL-CKASGYTFTNYWINWVKP-a11-b17



 



Position Protein 1/Protein2



168B 96-107 5A1-1 HC 21-39






After ASP-N digestion of the antibody/antigen cross-linked complex we did not detect any crosslinked peptides between 168B and 5A1-1.

Discussion

Using chemical cross-linking, High-Mass MALDI mass spectrometry and nLC-Orbitrap mass spectrometry we were able to characterize the interaction interface between the antigen 168B and the monoclonal antibody 5A1-1. Our analysis indicates that the epitope of this monoclonal antibody includes the following amino acids on 168B: 66; 69; 72; 77; 96; 98; 106. On the antibody the paratope includes the following amino acids: Heavy chain: 15; 19; 21; 23; 25; 40 and CDRH1. Light Chain: 18; 24; 31 and CDRL1. These results are illustrated in FIG. 16.
It is apparent that many modifications and variations of this invention as set forth above may be made without departing from the spirit and scope. The specific embodiments described are given by way of example only, and the invention is limited only by the terms of the appended claims.

## Claims

1. An immunoassay for determining whether a subject is at risk of cancer, the immunoassay comprising:
a) combining an antibody or antigen binding fragment thereof with a sample from the subject; wherein the antibody or antigen binding fragment thereof specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, but does not substantially bind to glycosylated 180 kDa CEA and to one or more linear peptides selected from the group consisting of 15 amino acids from amino acids of SEQ ID NO:2, amino acids of SEQ ID NO:3, and 15 amino acids from amino acids of SEQ ID NO:4, and wherein said antibody or antigen binding fragment binds to an epitope comprised within amino acids of SEQ ID NO:6, 7, 8, 9, 10 or 11; and
b) determining the amount of the antigen in the sample by determining the amount of the antibody antigen complex.

2. The immunoassay of claim 1, wherein the antibody or antigen binding fragment thereof is a monoclonal antibody or antigen binding fragment thereof.

3. The immunoassay of claim 2, wherein the antibody or antigen binding fragment is a capture antibody or antigen binding fragment thereof.

4. The immunoassay of claim 1, further comprising a second antibody or antigen binding fragment thereof, wherein the second antibody or antigen binding fragment thereof specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, but does not substantially bind to glycosylated 180 kDa CEA and to one or more linear peptides selected from the group consisting of 15 amino acids from amino acids of SEQ ID NO:2, amino acids of SEQ ID NO:3, and 15 amino acids from amino acids of SEQ ID NO:4, and wherein said antibody or antigen binding fragment binds to an epitope comprised within amino acids of SEQ ID NO:6, 7, 8, 9, 10 or 11.

5. The immunoassay of claim 4, wherein the second antibody or antigen binding fragment thereof is a monoclonal antibody or antigen binding fragment thereof.

6. The immunoassay of claim 5, wherein the second antibody or antigen binding fragment thereof is a capture antibody or antigen binding fragment thereof.

7. The immunoassay of claim 1, further comprising an additional antibody or antigen binding fragment thereof that specifically binds to the 100 kDa glycoprotein comprising SEQ ID NO:1.

8. The immunoassay of claim 7, wherein said additional antibody or antigen binding fragment thereof is labeled with a label selected from the group consisting of a radionuclide, an enzyme, a fluorescent agent and a chromophore.

9. The immunoassay of claim 8, wherein the additional antibody or antigen binding fragment thereof is a polyclonal antibody or antigen binding fragment thereof.

10. The immunoassay of claim 1, further comprising identifying the subject as at risk for cancer if the amount of the antigen in the sample is about 6.5 units/ml or greater.

11. The immunoassay of claim 1, wherein the sample is a human tissue or fluid.

12. An immunoassay for determining whether a subject is at risk of cancer, the immunoassay comprising:
a) combining an antibody or antigen binding fragment thereof with a sample from the subject wherein the antibody or antigen binding fragment thereof specifically binds to a 100 kDa glycoprotein comprising SEQ ID NO:1, but does not substantially bind to glycosylated 180 kDa CEA and to one or more linear peptides selected from the group consisting of 15 amino acids from amino acids of SEQ ID NO:2, amino acids of SEQ ID NO:3, and 15 amino acids from amino acids of SEQ ID NO:4, wherein the antibody or antigen binding fragment thereof comprises:
heavy chain CDRs (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region having the sequence of SEQ ID NO:119; and
light chain CDRs (LCDR1, LCDR2 and LCDR3) from a light chain variable region having the sequence of SEQ ID NO:120; and

b) determining the amount of the antigen in the sample by determining the amount of the antibody antigen complex.

13. The immunoassay of claim 12, wherein the antibody or antigen binding fragment thereof comprises the heavy and light chain variable regions.