Methods for detection and treatment of cancer

Abstract

In alternative embodiments, provided are methods for detecting a cancer, the method comprising: obtaining a sample comprising biological fluids or cells from a subject or human; isolating extracellular vesicles (EVs) having a selected diameter range; determining the presence and concentration of one or at least two proteins, Haptoglobin (HP) and Catalase (CAT), in the selected diameter range from the sample; and correlating the presence of a combination of the one or at least two proteins with a source of the sample, wherein if HP and CAT are individually detected, or if HP and CAT are both detected, then cancer has been positively detected. Also provided are methods for treating, lessoning the symptoms of or decreasing the chance of recurrence of, a cancer comprising administering to an individual in need thereof an antibody (Ab) or an antigen (Ag) binding fragment that specifically binds haptoglobin (HP) and / or a haptoglobin-hemoglobin complex.

Description

[0001] METHODS FOR DETECTION AND TREATMENT OF CANCER

[0002] RELATED APPLICATIONS

[0003] This Patent Convention Treaty (PCT) International Application claims the benefit of priority to U.S. Provisional Patent Application Serial No. (USSN) 63 / 523.872, filed June 28, 2023. The aforementioned application is expressly incorporated herein by reference in their entirety and for all purposes.

[0004] REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0005] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on June 28, 2024, is named “7226. 152961. xml” and is 2,429 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

[0006] TECHNICAL FIELD

[0007] This invention generally relates to biology and cancer diagnosis. In alternative embodiments, provided are methods for detecting Colorectal and / or Lung cancer, the method comprising: obtaining a sample comprising biological fluids or cells from a subject, wherein optionally the subject is a human patient; isolating extracellular vesicles (EVs) having a selected diameter range; determining the presence and concentration of one or at least two proteins, Haptoglobin (HP) and Catalase (CAT), in the selected diameter range from the sample; and optionally correlating the presence of a combination of the at least two proteins with a source of the sample, wherein optionally the source comprises a cancer biopsy or a tissue sample or a control tissue sample, wherein if HP and CAT are individually detected, or if HP and CAT are both detected, then Colorectal and / or Lung cancer have been positively detected.

[0008] BACKGROUND

[0009] Currently, colonoscopy is considered the best and most reliable method for the detection of neoplastic lesions that are at risk of progression to colorectal cancer (CRC). It is recommended as a primary first-line screening test in average and high- risk populations. However, colonoscopy has important and potentially consequential limitations. Importantly, polyps are occasionally missed during the procedure due to challenges with the procedure. Detection rate is highly dependent on quality standards including the colonoscopist skills, technology, and several patient-related factors. There is also a risk of perforation and bleeding. Quintero, E., Gastroenterol Res Pract. 2012 Apr 4; 2012: 846985 doi: 10.1155 / 2012 / 846985. Accordingly, there is an unmet need for methods of testing and diagnosing patients as having colorectal cancer (CRC). There is also an unmet need for better early-stage detection of CRC.

[0010] There is a need for more accurate, faster, and more simple diagnostic screening of CRC in patients. The inventions described herein meet these unsolved challenges and needs.

[0011] Lung cancer is highly treatable when detected at an early stage. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for approximately 85% to 90% of all lung cancer cases. The remaining 10% to 15% of lung cancers are classified as small cell lung cancer (SCLC).

[0012] NSCLC is a broad term that encompasses different subtypes of lung cancer, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Each subtype has distinct characteristics and treatment approaches. Among the NSCLC subtypes, adenocarcinoma is the most prevalent, followed by squamous cell carcinoma.

[0013] Currently, CT (Computed Tomography) scan is considered one of the best methods for early detection of lung cancer. The high sensitivity of CT scans enables the detection of even tiny nodules or lesions, which can be indicative of lung cancer. The level of imaging resolution provided by the cross sectional images can help in differentiating between benign and malignant nodules or tumours. Therefore, CT scan-based lung cancer screening programs have been established for high-risk individuals, such as heavy smokers or those with a history of lung cancer. It's important to note that while CT scans are highly effective in detecting lung cancer, false-positive results can occur, potentially leading to unnecessary' invasive procedures. Additionally, radiation exposure from CT scans should be considered and balanced with the potential benefits. Just like in the case of colorectal cancer. The inventions described herein can help meet these unsolved challenges and needs.

[0014] Extracellular vesicles (EVs) are membranous vesicles secreted from the cells into biological fluids (blood, urine, saliva, CSF, milk, etc). EVs can have different cellular biogenesis, including vesicles generated from the multivesicular body recycling or from plasma membrane budding. In the first case, EVs are referred to as exosomes, while in the second case they are referred to as microvesicles or ectosomes.

[0015] EVs are heterogeneous in size, with a diameter ranging from a few nanometers to micrometres. Given the impossibility of distinguishing exosomes from micro vesicles based on their size, the general nomenclature of small EVs (<200 nm) and large EVs (between 200 nm and 1 um) is widely adopted. Recently, other types of vesicles with a mean diameter below 50-80 nm were descnbed and referred to as exomers, while apoptotic bodies characterized by a mean diameter >1 um were already included in the EVs classification.

[0016] The biological content of EVs reflects the one of the cells of origin, in terms of nucleic acid abundance or presence of specific somatic alterations, proteins, lipids, or metabolites. Blood circulating EVs derive from red blood cells (RBCs), white blood cells (WBCs), platelets (PLTs), and endothelial cells. This evidence has been corroborated by studies involving cell-specific antibodies that were then tested to recognize antigens decorating the surface of EVs. In biofluids from patients with solid tumours, such as colon cancer, breast cancer, prostate cancer, or lung cancer, a fraction of EVs has been demonstrated as originated from tumour cells, then referred to as tumour-derived extracellular vesicles (tEVs).

[0017] SUMMARY

[0018] In alternative embodiments, provided are methods for detecting a cancer or a tumor such as a colorectal (CRC) and / or lung cancer, the method comprising:

[0019] (a) obtaining a sample comprising biological fluids or cells from a subject, wherein optionally the subject is a human patient;

[0020] (b) isolating extracellular vesicles (EVs) having a selected diameter range;

[0021] (c) determining the presence and concentration of one or at least two proteins, Haptoglobin (HP) and Catalase (CAT), in the selected diameter range from the sample, wherein optionally the selected diameter range is between about 50 nm and 500 nm, or between about 100 nm and 400 nm, or between about 150 nm and 300 nm; and

[0022] (d) correlating the presence of a combination of the one or at least two proteins with a source of the sample, wherein optionally the source comprises a cancer biopsy or a tissue sample or a control tissue sample, wherein if HP and CAT are individually detected, or if HP and CAT are both detected, then the cancer or tumor such as a colorectal (CRC) and / or lung cancer have been positively detected.

[0023] In alternative embodiments, provided are methods for treating a cancer or tumor such as a colorectal (CRC) and / or lung cancer, the method comprising:

[0024] (a) detecting whether an individual in need thereof has cancer or tumor such as a colorectal (CRC) and / or lung cancer using a method for detecting cancer or tumor such as a colorectal (CRC) and / or lung cancer as provided herein, for example, using isolated extracellular vesicles (EVs) having a selected diameter range and determining the presence and concentration of one or at least two proteins. Haptoglobin (HP) and Catalase (CAT); and

[0025] (b) treating the individual in need thereof with a drug or therapy to treat or ameliorate the cancer or tumor such as a colorectal (CRC) and / or lung cancer.

[0026] In alternative embodiments, provided are methods for detecting a cancer or a tumor, the method comprising:

[0027] (a) obtaining a sample comprising biological fluids, cells or tissue from a subject, wherein optionally the subject is a human patient or an animal subject, wherein optionally the sample comprises or is derived from a biopsy or a subject tissue sample or a control tissue sample;

[0028] (b) isolating extracellular vesicles (EVs) having a selected diameter range, wherein optionally the selected diameter range of the isolated EVs is between about 50 nm and 500 nm, or between about 100 nm and 400 nm, or between about 150 nm and 300 nm;

[0029] (c) determining the presence of Haptoglobin (HP) and / or Catalase (CAT) in the isolated EVs; and

[0030] (d) measuring the concentration and / or the amount of Haptoglobin (HP) and / or Catalase (CAT) in the sample, and in alternative embodiments:

[0031] - optionally the concentration and / or the amount of Haptoglobin (HP) and / or Catalase (CAT) in the sample is measured by an ELISA (enzyme-linked immunosorbent assay) and / or HPLC (High Performance Liquid Chromatography), and optionally if HP and CAT are individually detected, or if HP and CAT are both detected, then Colorectal and / or Lung cancer have been positively detected or diagnosed, and - optionally if HP and CAT are detected using ELISA (optionally using an ABC AM™ kit) at a level at or above about 580 pg / mL for HP and 10 pg / mL for CAT, or are detected at a level over between about 560 pg / mL to 600 pg / mL for HP and over between about 8 pg / mL to 10 pg / mL for CAT, then the cancer (for example, a colorectal and / or lung cancer) have been positively detected or diagnosed, and

[0032] - optionally if HP alone is detected using an ELISA (optionally using a PROTEINTECH™ kit) at a level at or above about 29,700 pg / mL, or is detected at a level over between about 29,000 pg / mL to 30,000 pg / mL, then the cancer (for example, a colorectal and / or lung cancer) have been positively detected or diagnosed.

[0033] In alternative embodiments, the cancer or tumor is colorectal and / or Lung cancer.

[0034] In alternative embodiments, provided are methods for treating a cancer, the method comprising:

[0035] (a) first detecting whether an individual in need thereof has a cancer or a tumor using a method as provided herein; and

[0036] (b) followed by treating the individual in need thereof with a drug or therapy to treat or ameliorate, or lesson the symptoms of or chance of recurrence of, the tumor or cancer.

[0037] In alternative embodiments;

[0038] - the cancer or tumor is colorectal cancer (CRC) or lung cancer, wherein optionally the lung cancer is non-small cell lung cancer (NSCLC); and optionally the cancer or tumor is a leukemic cell cancer, acute lymphoblastic leukemia (ALL), angioimmunoblastic T cell lymphoma, Burkitt lymphoma, chronic myelogenous leukemia in blast crisis, diffuse large B-cell lymphoma, hairy cell leukemia, myeloma, precursor B lymphoblastic leukemia or lymphoma, follicular lymphoma, diffuse large B- cell lymphoma, mantle cell lymphoma, precursor T lymphoblastic leukemia or lymphoma, non-hematolymphoid sarcoma, or a carcinoma such as a renal cell carcinoma or a metaplastic breast carcinoma, or

[0039] - the cancer or tumor is: an acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, rectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal cancer, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, phary nx cancer, prostate cancer, colorectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and / or urinary bladder cancer.

[0040] In alternative embodiments of methods as provided herein:

[0041] - the treating the individual in need thereof with a drug or therapy to treat or ameliorate, or lesson the symptoms of or chance of recurrence of. the tumor or cancer, comprises administering to an individual in need thereof an antibody (Ab), an antigen (Ag) binding fragment thereof, or a monomeric or dimeric antigen binding protein (ABP) that specifically binds haptoglobin (HP) and / or a haptoglobin-hemoglobin complex;

[0042] - the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds haptoglobin (HP) and / or a haptoglobin-hemoglobin complex, comprises or is fabricated as: an antigen-binding fragment (Fab, or an Ab fragment having just one constant and one variable domain of each of an Ab heavy and light chain), a F(ab')2 (or an Ab digested by pepsin yielding two fragments: a F(ab')2 fragment and a pFc' (pepsin cleavage Fc) fragment), a Fab' (a single chain of a F(ab')2 fragment), a single-chain variable fragment (scFv) (or a fusion protein of a variable region of an Ab heavy and light chain connected together with a linker peptide optionally of about ten to about 25 amino acids in length), a (scFv)2, or a di-scFv or a bi-scFv, or a single peptide chain having two variable heavy and two variable light regions yielding tandem scFv. a minibody (or a fusion protein of a variable region of an Ab heavy and light chain connected together with an alkyl group, optionally a methyl or an ethyl group) a diabody (or an scFv with a linker peptide too short (optionally about five amino acids) for the two variable regions to fold together forcing the scFvs to dimerize), a triabody or a tetrabody (or an scFv with a linker peptide too short (optionally about one or two amino acids) for the two variable regions to fold together forcing the scFvs to trimerize or tetramize), a single-domain antibody (dAB) (or a single variable region of an Ab heavy or Ab light chain), a plurality of complementarity determining region (CDR) fragments, a multispecific antibody formed from two or more antibody fragments, a monoclonal and / or a polyclonal antibody (Ab), and / or a humanized Ab having a non-human (optionally murine) antigen binding fragment or complement determining region (CDR) that specifically binds to human haptoglobulin implanted in a human antibody:

[0043] - the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex is formulated as a pharmaceutical composition, or is formulated for administration in vivo; or is formulated for enteral or parenteral administration, or for oral, intravenous (IV) or intrathecal (IT) administration, or is administered orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially or rectally;

[0044] - the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex is formulated as a nanoparticle, a particle, a micelle or a liposome or lipoplex, a polymersome, a polyplex or a dendrimer; or is formulated as, or contained in, a nanoparticle, a liposome, a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, an emulsion, a lotion, an aerosol, a spray, a lozenge, an aqueous or a sterile or an injectable solution, or an implant; and / or

[0045] - the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex is administered: (a) once a day, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks or once every eight weeks; or (b) at a dosage of between about 1 and 40 mg / Kg, or between about 5 and 20 mg / Kg.

[0046] In alternative embodiments, provided are methods for treating, ameliorating, lessoning the symptoms of or decreasing the chance of recurrence of. a tumor or a cancer, comprising administering to an individual in need thereof an antibody (Ab), an antigen (Ag) binding fragment thereof, or a monomeric or dimeric antigen binding protein (ABP) that specifically binds haptoglobin (HP) and / or a haptoglobin- hemoglobin complex; and in alternative embodiments:

[0047] - the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds haptoglobin (HP) and / or a haptoglobin-hemoglobin complex, comprises or is fabricated as: an antigen-binding fragment (Fab, or an Ab fragment having just one constant and one variable domain of each of an Ab heavy and light chain), a F(ab')2 (or an Ab digested by pepsin yielding two fragments: a F(ab')2 fragment and a pFc' (pepsin cleavage Fc) fragment). a Fab' (a single chain of a F(ab')2 fragment), a single-chain variable fragment (scFv) (or a fusion protein of a variable region of an Ab heavy and light chain connected together with a linker peptide optionally of about ten to about 25 amino acids in length). a (scFv)2, or a di-scFv or a bi-scFv, or a single peptide chain having two variable heavy and two variable light regions yielding tandem scFv, a minibody (or a fusion protein of a variable region of an Ab heavy and light chain connected together with an alkyl group, optionally a methyl or an ethyl group) a diabody (or an scFv with a linker peptide too short (optionally about five amino acids) for the two variable regions to fold together forcing the scFvs to dimerize), a triabody or a tetrabody (or an scFv with a linker peptide too short (optionally about one or two amino acids) for the two variable regions to fold together forcing the scFvs to trimerize or tetramize), a single-domain antibody (dAB) (or a single variable region of an Ab heavy or Ab light chain), a plurality of complementarity determining region (CDR) fragments, or a multispecific antibody formed from two or more antibody fragments;

[0048] - the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex: (a) comprises a murine complement determining region (CDR) that specifically binds to the haptoglobin (HP) or the haptoglobin- hemoglobin complex; or

[0049] (b) is a humanized antibody comprising a murine complement determining region (CDR) that specifically binds to the haptoglobin (HP) or the haptoglobin-hemoglobin complex, wherein optionally the CDR has a sequence comprising SEQ ID NO: 1;

[0050] - the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex is formulated as a pharmaceutical composition, or is formulated for administration in vivo; or is formulated for enteral or parenteral administration, or for oral, intravenous (IV) or intrathecal (IT) administration, or is administered orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially or rectally;

[0051] - the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex is formulated as a nanoparticle, a particle, a micelle or a liposome or lipoplex, a polymersome, a polyplex or a dendrimer; or is formulated as, or contained in, a nanoparticle, a liposome, a tablet, a pill, a capsule, a gel. a geltab, a liquid, a powder, an emulsion, a lotion, an aerosol, a spray, a lozenge, an aqueous or a sterile or an injectable solution, or an implant;

[0052] - the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex is administered: (a) once a day. once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks or once every eight weeks; or (b) at a dosage of between about 1 and 40 mg / Kg, or between about 5 and 20 mg / Kg.; and / or

[0053] - the cancer or tumor is colorectal cancer (CRC) or lung cancer, wherein optionally the lung cancer is non-small cell lung cancer (NSCLC), and optionally th e the cancer or tumor is a leukemic cell cancer, acute lymphoblastic leukemia (ALL), angioimmunoblastic T cell lymphoma, Burkitt lymphoma, chronic myelogenous leukemia in blast crisis, diffuse large B-cell lymphoma, hairy cell leukemia, myeloma, precursor B lymphoblastic leukemia or lymphoma, follicular lymphoma, diffuse large B- cell lymphoma, mantle cell lymphoma, precursor T lymphoblastic leukemia or lymphoma, non-hematolymphoid sarcoma, or a carcinoma such as a renal cell carcinoma or a metaplastic breast carcinoma.

[0054] In alternative embodiments provided are methods for determining the efficacy of a cancer or tumor treatment or therapy, comprising measuring the level of haptoglobin (HP) and / or haptoglobin-hemoglobin complexes in a sample from (or derived from) an individual or subject before beginning the cancer or tumor treatment or therapy, and at least once again after commencing the cancer or tumor treatment or therapy, wherein lower levels of the haptoglobin (HP) and / or haptoglobin-hemoglobin complexes in the individual or subject after commencing the cancer or tumor treatment or therapy as compared to before beginning the cancer or tumor treatment or therapy determines or indicates that the cancer treatment or therapy is effective against the cancer or tumor; and optionally the subject is a human patient or an animal subject, and optionally the sample comprises or is derived from a biopsy or a subject tissue sample or a control tissue sample.

[0055] In alternative embodiments provided are methods for monitoring recurrence of a cancer or tumor, or residual disease, after completion of a cancer or tumor treatment or therapy, comprising measuring the level of haptoglobin (HP) and / or haptoglobin- hemoglobin complexes in a sample from (or derived from) an individual or subject in need thereof:

[0056] (a) before beginning the cancer or tumor treatment or therapy, and / or at least once again after commencing the cancer or tumor treatment or therapy, and

[0057] (b) at least once after completion of the cancer or tumor treatment or therapy, wherein lower levels, or at least not higher levels, of the haptoglobin (HP) and / or haptoglobin-hemoglobin complexes in the individual or subject after completion of the cancer or tumor treatment or therapy as compared to before beginning the cancer or tumor treatment or therapy and / or during the cancer or tumor treatment or therapy determines or indicates that the cancer or tumor has not relapsed or recurred, wherein higher levels of the haptoglobin (HP) and / or haptoglobin-hemoglobin complexes in the individual or subject after completion of the cancer or tumor treatment or therapy as compared to before beginning the cancer or tumor treatment or therapy and / or during the cancer or tumor treatment or therapy determines or indicates a cancer or tumor recurrence, or that the cancer or tumor treatment or therapy was not successfully in substantially eradicating or treating the cancer or tumor.

[0058] In alternative embodiments:

[0059] - the cancer or tumor treatment or therapy comprises chemotherapy, radiation and / or surgery, and optionally the cancer or tumor is colorectal cancer (CRC) or lung cancer, wherein optionally the lung cancer is non-small cell lung cancer (NSCLC); and optionally the cancer or tumor is a leukemic cell cancer, acute lymphoblastic leukemia (ALL), angioimmunoblastic T cell lymphoma, Burkitt lymphoma, chronic myelogenous leukemia in blast crisis, diffuse large B-cell lymphoma, hairy cell leukemia, myeloma, precursor B lymphoblastic leukemia or lymphoma, follicular lymphoma, diffuse large B- cell lymphoma, mantle cell lymphoma, precursor T ly mphoblastic leukemia or lymphoma, non-hematolymphoid sarcoma, or a carcinoma such as a renal cell carcinoma or a metaplastic breast carcinoma, and optionally the subject is a human patient or an animal subject, and optionally the sample comprises or is derived from a biopsy or a subject tissue sample or a control tissue sample.

[0060] The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0061] All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entireties for all purposes.

[0062] DESCRIPTION OF DRAWINGS

[0063] The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

[0064] FIG. 1 graphically illustrates: Haptoglobin and catalase were measured by Mass Spectrometry (MS) in 7 cancer samples (from 4 CRC and 3 NSCLC patients) and compared with 20 control samples (from 10 healthy donors, and 5 diabetes and 5 arthritis patients). The two markers are combined in a composite score (y-axis) as the Iog2 of the sum of m,'z intensities in the MS assay. Statistical significance was assessed by a two-sided / -test and resulted in a / ?- value of L4e’6.

[0065] FIG. 2 graphically illustrates: Haptoglobin and catalase were measured by ELISA (ABCAM™ kits) in 8 cancer samples (from 4 CRC and 4 NSCLC patients) and compared with 6 healthy donor samples. The two markers are combined in a composite score (y-axis) as the sum of catalase concentration (in ng / mL) and 1 / 100 of haptoglobin concentration (in ng / mL). Statistical significance was assessed by a two- sided / -test and resulted in a p-value of 5.6e’4.

[0066] FIG. 3 graphically illustrates: Haptoglobin was measured by ELISA (PROTEINTECH™ kit) in 33 cancer samples (from 15 CRC and 18 NSCLC patients) and compared with 37 control samples (30 healthy donors, 3 diabetes and 4 arthritis patients). Statistical significance was assessed by a two-sided Etest and resulted in a / ?- value of 0.0041.

[0067] Like reference symbols in the various drawings indicate like elements.

[0068] DETAILED DESCRIPTION

[0069] In alternative embodiments, provided are methods for detecting Colorectal and / or Lung cancer, the method comprising: obtaining a sample comprising biological fluids or cells from a subject, wherein optionally the subject is a human patient; isolating extracellular vesicles (EVs) having a selected diameter range; determining the presence and concentration of one or at least two proteins. Haptoglobin (HP) and Catalase (CAT), in the selected diameter range from the sample; and optionally correlating the presence of a combination of the at least two proteins with a source of the sample, wherein optionally the source comprises a cancer biopsy or a tissue sample or a control tissue sample, wherein if HP and CAT are individually detected, or if HP and CAT are both detected, then Colorectal and / or Lung cancer have been positively detected.

[0070] In alternative embodiments, provided are methods of detection of colorectal and non-small cell lung cancer (NSCLC) based on the analysis of the markers carried by the extracellular vesicles present in the serum and plasma of cancer patients. Here we apply SEC technology7to CRC and NSCLC cancer samples (human plasma), to isolate and characterize large EVs from early fractions.

[0071] Our process included a discovery7phase based on mass spectrometry (MS) analysis of EVs isolated from cancer cases versus controls. Subsequently, the protein markers identified to be significantly more or less abundant in cases or controls were selected for scoring using ELISA assay.

[0072] The combination of two protein markers. Haptoglobin (HP) and Catalase (CAT), was identified to be significantly different in cancer cases versus controls (figures 1 & 2) and could provide a method for early detection of colorectal cancer and lung cancer.

[0073] In alternative embodiments, provided are methods directed to therapeutics comprising inhibition of or reducing the levels of circulating HP or complexes involving HP (such as a haptoglobin-hemoglobin complex) for treating, ameliorating, lessoning the symptoms of or decreasing the chance of recurrence of, a cancer or a tumor, including for example colorectal cancer (CRC), or a lung cancer such as a non-small cell lung cancer (NSCLC).

[0074] In alternative embodiments, provided are methods for determining the levels of HP in early treatment samples or fraction to determine the efficacy of the treatment and / or the presence of or the severity of side effects of an anti-cancer therapy (such as radiation and / or surgery) or drug treatment (for example, an anti -tumoral drug; and optionally the levels of HP are determined by a method comprising isolating extracellular vesicles (EVs) having a selected diameter range, and optionally the selected diameter range of the isolated EVs is between about 50 nm and 500 nm, or between about 100 nm and 400 nm, or between about 150 nm and 300 nm.

[0075] Methods as provided herein for the early detection of a cancer of a tumor allow application of a treatment or drug to a patient in need thereof earlier to provide exposure of the cancer or tumor to the treatment or drug, and thus improving the efficacy and prognosis of the treatment or drug.

[0076] In alternative embodiments, methods for determining the levels of HP as provided herein (to determine the efficacy of the treatment and / or the presence of or the severity of side effects of) are used during the course of treatment (including for example surgery, radiation, therapeutics) as a companion diagnostic to provide the health care practitioner more information as to appropriate drugs, therapy regimens and drug dosages.

[0077] In alternative embodiments, methods for determining the levels of HP as provided herein have a prognostic value, allowing physicians to adapt treatment (including for example surgery, radiation, therapeutics) accordingly, for example, whether a more aggressive, or less aggressive, treatment is needed, for example, for a high risk patients or a patient wih an aggressive cancer.

[0078] In alternative embodiments, methods for determining the levels of HP as provided herein, as they can be used to determine the efficacy of an intervention (including for example surgery, radiation, therapeutics) are used to monitor patients during or after the intervention, for example levels of HP are regularly monitored to detect minimal residual disease after a successful intervention, this early detection provides a therapeutic window for efficient interventions that could prevent or delay recurrence. HP can be used also to detect disease recurrence so that relapses could be treated as soon as possible.

[0079] Methods for Treating Cancers and Tumors

[0080] In alternative embodiments, provided are methods for treating, ameliorating, lessoning the symptoms of or decreasing the chance of recurrence of. a tumor or a cancer, comprising administering to an individual in need thereof an antibody (Ab), an antigen (Ag) binding fragment thereof, or a monomeric or dimeric antigen binding protein (ABP) that specifically binds haptoglobin (HP) and / or a haptoglobin- hemoglobin complex, optionally a human haptoglobin (HP) and / or a human haptoglobin-hemoglobin complex.

[0081] In alternative embodiments, the antibody (Ab) that specifically binds to the human haptoglobin (HP) and / or a human haptoglobin-hemoglobin complex is a human Ab (for example, of IgG or IgM isotype) or a humanized Ab, for example, a human antibody having as its antigen binding sequence (for example, as its complement determining sequence, or CDR) a non-human such as a rabbit or a murine (or mouse) amino acid sequence that specifically binds to a human haptoglobin (HP) and / or a human haptoglobin-hemoglobin complex.

[0082] The anti-human haptoglobin antibody, for the example, the murine (or mouse) Ab from which a CDR is extracted to implant in a human Ab superstructure to generate a humanized Ab that specifically binds to a human haptoglobin (HP) and / or a human haptoglobin-hemoglobin complex) can be generated using the human haptoglobin protein, for example, a protein having the sequence (SEQ ID NO: 1):

[0083] 1 msalgavial llwgqlfavd sgndvtdiad dgcpkppeia hgy vehsvry qcknyyklrt 61 egdgvytlnd kkqwinkavg dklpeceadd gcpkppeiah gyvehsvryq cknyyklrte 121 gdgvytlnne kqwinkavgd klpeceavcg kpknpanpvq rilgghldak gsfpwqakmv 181 shhnlttgat lineqwlltt aknlflnhse natakdiapt Itlyvgkkql veiekvvlhp 241 nysqvdigli klkqkvsvne rvmpiclpsk dyaevgrvgy vsgwgmanf kftdhlkyvm 301 Ipvadqdqci rhyegstvpe kktpkspvgv qpilnehtfc agmskyqedt cygdagsafa 361 vhdleedtwy atgilsfdks cavaeygvyv kvtsiqdwvq ktiaen

[0084] In alternative embodiments humanized antibodies used in methods as provided herein comprise CDRs of nonhuman (for example, murine, mouse, rat) antibodies, so as such are chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin. In alternative embodiments, humanized antibodies used in methods as provided herein are human immunoglobulins (recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity', and capacity' to bind human haptoglobulin used in methods as provided herein, some framework sections of the human immunoglobulin are replaced by corresponding nonhuman residues. In alternative embodiments, humanized antibodies used in methods as provided herein may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In alternative embodiments, a humanized antibody heavy or light chain can comprise substantially all of at least one or more variable domains, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the Ab frameworks motifs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin; see for example Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0085] In alternative embodiments human or humanized antibodies are used in methods as provided herein, and they can be an antibody derived from a human or an antibody obtained from a transgenic organism that has been "engineered" to produce specific human antibodies in response to antigenic challenge and can be produced by any method known in the art. In certain techniques, elements of the human heavy and light chain loci are introduced into strains of the organism derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic organism can synthesize human antibodies specific for human antigens, and the organism can be used to produce human antibody-secreting hybridomas. In alternative embodiments a human antibody can also be an antibody wherein the heavy and light chains are encoded by a nucleotide sequence derived from one or more sources of human DNA. A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, or in vitro activated B cells, all of which are known in the art.

[0086] In alternative embodiments any known anti-human haptoglobulin antibody can be used to practice methods as provided herein, and can be used to harvest CDRs for making humanized antibodies that specifically bind to human haptoglobulin, and many anti-human mouse and rabbit haptoglobulin antibodies are known in the art and are commercially available, for example, see Antibodies Online, no. AA 168-319, Catalog No. ABIN7426061, or Catalog No. ABIN2722429. or Catalog No. ABIN7385036, or Catalog No. ABIN7384645, or Catalog No. ABIN5701994, or Catalog No. ABIN6055947, or Catalog No. ABIN6909262, or Catalog No. ABIN6861073, or Catalog No. ABIN6836883.

[0087] In alternative embodiments human or humanized antibodies used in methods as provided herein can comprise introduced mutations (e.g., additions, deletions, and / or substitutions) in a nucleotide sequence encoding the antibody, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that are routinely used to generate amino acid substitutions. In one embodiment, the variable light or heavy chain CDRs derivatives may include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, less than 2 amino acid substitutions, or 1 amino acid substitution relative to the original antibody. In another embodiment, the variable light or heavy chain CDRs derivatives may have conservative amino acid substitutions (e.g. supra) made at one or more predicted non- essential amino acid residues (i.e., amino acid residues which are not critical for the antibody to specifically bind to human CD 19). Mutations can also be introduced randomly along all or part of the variable light or heavy chain CDR coding sequences, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined. In alternative embodiments human or humanized antibodies used in methods as provided herein are encoded by polynucleotides which can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, because a nucleotide sequence of the anti-human haptoglobulin antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0088] A polynucleotide encoding the anti-human haptoglobulin may also be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g.. an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

[0089] In alternative embodiments human or humanized antibodies used in methods as provided herein are produced using a variety of techniques known in the art. including, but not limited to, CDR-grafting (see e.g., European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, Proc. Natl. Acad. Sci., 91 :969-973, chain shuffling (see, e.g., U.S. Pat. No. 5,565,332 and techniques disclosed in for example: U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169: 1119-25 (2002), Caldas et al.. Protein Eng.. 13(5):353-60 (2000), Morea et al.. Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al, Protein Eng., 9(10):895-904 (1996), Couto et al.. Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al.. Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). Framework (FW) residues in the FW regions can be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These FW substitutions are identified by methods well known in the art. e.g., by modeling of the interactions of the CDR and FW residues to identify FW residues important for antigen binding and sequence comparison to identify unusual FW residues at particular positions; see for example, Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al, 1988, Nature, 332:323.

[0090] In alternative embodiments human or humanized antibodies used in methods as provided herein has one or more amino acid residues introduced into it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as "import" residues, which are typically taken from an “import” variable domain. Thus, humanized antibodies comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions from human. Humanization of antibodies is well-known in the art and can essentially be performed following the method of Winter and co-w orkers (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534- 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication No. WO 91 / 09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640). In such humanized chimeric antibodies, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FW residues are substituted by residues from analogous sites in rodent antibodies. Humanization of anti-human haptoglobulin antibodies can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan. 1991. Molecular Immunology 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., Proc. Natl. Acad. Sci., 91 :969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332).

[0091] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity, for example, using a “best- fit” method, where the sequence of the variable domain of a rodent antibody is screened against the entire library7of known human variable-domain sequences. The human sequences which are most closely related to that of the rodent are then screened for the presence of specific residues that may be critical for antigen binding, appropriate structural formation and / or stability of the intended humanized mAb (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). The resulting FW sequences matching the desired criteria are then be used as the human donor FW regions for the humanized antibody. Another method uses a particular FW derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same FW may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA. 89:4285 (1992); Presta et al., J. Immunol., 151 :2623 (1993).

[0092] In alternative embodiments human or humanized antibodies used in methods as provided herein have modified glycosylation, or are agly coslated antibodies (the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for a target antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 . One or more amino acid substitutions can also be made that result in elimination of a glycosylation site present in the Fc region (e.g., Asparagine 297 of IgG). Furthermore, aglycosylated antibodies may be produced in bacterial cells which lack the necessary glycosylation machinery.

[0093] An antibody can also be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC abi 1 i ty of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies as used in methods as provided herein to thereby produce an antibody with altered glycosylation. See, for example, Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17: 176-1, as well as, U.S. Pat. No. 6,946,292; European Patent No: EP 1,176,195; PCT Publications WO 03 / 035835; WO 99 / 54342.

[0094] Antibody formulations for in vivo administration

[0095] In alternative embodiments human or humanized antibodies used in methods as provided herein are formulated with a pharmaceutically acceptable carrier. In alternative embodiments the term “pharmaceutically acceptable” means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may also routinely contain compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, boric, formic, malonic, succinic, and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the antibodies of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

[0096] In alternative embodiments human or humanized antibodies used in methods as provided herein are prepared for storage by mixing the antibody or immunoconjugate having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1999)). in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN, PLURONICS™ or polyethylene glycol (PEG). In alternative embodiments human or humanized antibodies used in methods as provided herein comprise suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.

[0097] In alternative embodiments human or humanized antibodies used in methods as provided herein are formulated in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In alternative embodiments human or humanized antibodies used in methods as provided herein are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0098] In alternative embodiments human or humanized antibodies used in methods as provided herein are formulated suitable for parenteral administration and are in a sterile aqueous or non-aqueous preparation of antibody, which is can be isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or w etting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. In certain embodiments, carrier formulation suitable for various routes of administration can be the same or similar to that described for RITUXAN™. See, Physicians' Desk Reference (Medical Economics Company, Inc., Montvale, N.J., 2005), pp. 958-960 and 1354-1357. In certain embodiments, antibody compositions are formulated for intravenous administration with sodium chloride, sodium citrate dihydrate, polysorbate 80, and sterile water where the pH of the composition is adjusted to approximately 6.5. Those of skill in the art are aware that intravenous injection provides a useful mode of administration due to the thoroughness of the circulation in rapidly distributing antibodies. Intravenous administration, however, is subject to limitation by a vascular barrier comprising endothelial cells of the vasculature and the subendothelial matrix. Still, the vascular barrier is a more notable problem for the uptake of therapeutic antibodies by solid tumors. Lymphomas have relatively high blood flow rates, contributing to effective antibody delivery. Intralymphatic routes of administration, such as subcutaneous or intramuscular injection, or by catheterization of lymphatic vessels, also provide a useful means of treating B cell lymphomas. In certain embodiments, antibodies are self-administered subcutaneously. In such embodiments, the composition is formulated as a lyophilized drug or in a liquid buffer (e.g.. PBS and / or citrate) at about 50 mg / mL.

[0099] In alternative embodiments human or humanized antibodies used in methods as provided herein are entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0100] In alternative embodiments human or humanized antibodies used in methods as provided herein are formulated for in vivo administration in sterile forms, for example, accomplished by filtration through sterile filtration membranes.

[0101] In alternative embodiments human or humanized antibodies used in methods as provided herein are formulated in sustained-release preparations. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylenevinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devized for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S — S bond formation through thiodisulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0102] Exemplary pharmaceutical formulations used in methods as provided herein:

[0103] - a sterile, preservative-free liquid concentrate for intravenous (i.v.) administration of anti-human haptoglobulin antibody, supplied at a concentration of 10 mg / ml in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product can be formulated for i.v. administration using sodium chloride, sodium citrate dihydrate, polysorbate and sterile water for injection. For example, the product can be formulated in 9.0 mg / mL sodium chloride, 7.35 mg / rnL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection. The pH is adjusted to 6.5.

[0104] - a sterile, lyophilized powder in single-use glass vials for subcutaneous (s.c.) injection. The product can be formulated with sucrose, L-histidine hydrochlonde monohydrate, E-histidine and polysorbate 20. For example, each single-use vial can contain 150 mg anti-human haptoglobulin, 123.2 mg sucrose, 6.8 mg L-histidine hydrochloride monohydrate, 4.3 mg L-histidine. and 3 mg polysorbate 20. Reconstitution of the single-use vial with 1.3 ml sterile water for injection yields approximately 1.5 ml solution to deliver 125 mg per 1.25 ml (100 mg / ml) of antibody. - a sterile, preservative-free lyophilized powder for intravenous (i.v.) administration. The product can be formulated with a-trehalose dihydrate, L-histidine HC1, histidine and polysorbate 20 USP. For example, each vial can contain 440 mg anti-human haptoglobulin antibody, 400 mg a, a-trehalose dihydrate, 9.9 mg L- histidine HC1, 6.4 mg L-histidine, and 1.8 mg polysorbate 20, USP. Reconstitution with 20 ml of bacteriostatic water for injection (BWFI), USP, containing 1.1% benzyl alcohol as a preservative, yields a multi-dose solution containing 21 mg / ml antibody at a pH of approximately 6.

[0105] - sterile, lyophilized powder for intravenous infusion in which an anti-human haptoglobulin antibody is formulated with sucrose, polysorbate, monobasic sodium phosphate monohydrate, and dibasic sodium phosphate dihydrate. For example, each single-use vial can contain 100 mg antibody, 500 mg sucrose, 0.5 mg polysorbate 80, 2.2 mg monobasic sodium phosphate monohydrate, and 6.1 mg dibasic sodium phosphate dihydrate. No preservatives are present. Following reconstitution with 10 ml sterile water for injection, USP, the resulting pH is approximately 7.2.

[0106] - sterile, preservative-free solution for subcutaneous administration supplied in a single-use, 1 ml pre-filled syringe. The product can be formulated with sodium chloride, monobasic sodium phosphate dihydrate, dibasic sodium phosphate dihydrate, sodium citrate, citric acid monohydrate, mannitol, polysorbate 80 and water for injection. USP. Sodium hydroxide may be added to adjust pH to about 5.2. For example, each syringe can be formulated to deliver 0.8 ml (40 mg) of drug product. Each 0.8 ml contains 40 mg anti -human haptoglobulin antibody, 4.93 mg sodium chloride, 0.69 mg monobasic sodium phosphate dihydrate, 1.22 mg dibasic sodium phosphate dihydrate, 0.24 mg sodium citrate, 1.04 citric acid monohydrate. 9.6 mg mannitol, 0.8 mg polysorbate 80 and water for injection, USP.

[0107] - sterile, preservative-free, lyophilized powder contained in a single-use vial that is reconstituted with sterile water for injection (SWFI), USP, and administered as a subcutaneous (s.c.) injection. The product can be formulated with sucrose, histidine hydrochloride monohydrate. L-histidine, and polysorbate. For example, a 75 mg vial can contain 129.6 mg or 112.5 mg of an anti-human haptoglobulin antibody, 93. 1 mg sucrose, 1.8 mg L-histidine hydrochloride monohydrate, 1.2 mg L-histidine, and 0.3 mg polysorbate 20. and is designed to deliver 75 mg of the antibody in 0.6 ml after reconstitution with 0.9 ml SWFI, USP. A 150 mg vial can contain 202.5 mg or 175 mg anti-human haptoglobulin antibody, 145.5 mg sucrose, 2.8 mg L-histidine hydrochloride monohydrate, 1.8 mg L-histidine, and 0.5 mg polysorbate 20, and is designed to deliver 150 mg of the antibody in 1.2 ml after reconstitution with 1.4 ml SWFI, USP.

[0108] - sterile, hyophilized product for reconstitution with sterile water for injection. The product can be formulated as single-use vials for intramuscular (IM) injection using mannitol, histidine and glycine. For example, each single-use vial can contain 100 mg anti-human haptoglobulin antibody. 67.5 mg of mannitol. 8.7 mg histidine and 0.3 mg glycine, and is designed to deliver 100 mg antibody in 1.0 ml when reconstituted with 1.0 ml sterile water for injection. As another example, each singleuse vial can contain 50 mg anti-human haptoglobulin antibody, 40.5 mg mannitol, 5.2 mg histidine and 0.2 mg glycine, and is designed to deliver 50 mg of antibody when reconstituted with 0.6 ml sterile water for injection.

[0109] - sterile, preservative-free solution for intramuscular (IM) injection, supplied at a concentration of 100 mg / ml. The product can be formulated in single-use vials with histidine, glycine, and sterile water for injection. For example, each single-use vial can be formulated with 100 mg antibody, 4.7 mg histidine, and 0. 1 mg glycine in a volume of 1.2 ml designed to deliver 100 mg of anti-human haptoglobulin antibody in 1 ml. As another example, each single-use vial can be formulated with 50 mg antibody, 2.7 mg histidine and 0.08 mg glycine in a volume of 0.7 ml or 0.5 ml designed to deliver 50 mg of antibody in 0.5 ml. In alternative embodiments, the pharmaceutical composition is stable at 4°C, or is stable at room temperature.

[0110] In alternative embodiments human or humanized antibodies used in methods as provided herein are dosaged based on a number of factors including the age, sex, species and condition of the subject (e.g., stage of B cell malignancy), the desired degree of cellular depletion, the disease to be treated and / or the particular antibody or antigen-binding fragment being used and can be determined by one of skill in the art. For example, effective amounts of compositions of the invention may be extrapolated from dose-response curves derived in vitro test systems or from animal model (e.g., the cotton rat or monkey) test systems. Models and methods for evaluation of the effects of antibodies are known in the art (Wooldridge et al.. Blood, 89(8): 2994-2998 (1997). Examples of dosing regimens that can be used in methods as provided herein include, but are not limited to, daily, three times weekly (intermittent), weekly, or every 14 days. In certain embodiments, dosing regimens include, but are not limited to, monthly dosing or dosing every 6-8 weeks. Those skilled in the art will appreciate that dosages are generally higher and / or frequency of administration greater for initial treatment as compared with maintenance regimens.

[0111] In alternative embodiments human or humanized antibodies used in methods as provided herein are dosaged in a pharmaceutically acceptable carrier as part of a pharmaceutical composition, and optionally are at least about 0.0005, 0.001, 0.05, 0.075, 0. 1, 0.25, 0.375, 0.5, 1, 2.5, 5, 10, 20. 37.5. or 50 mg / m2 and / or less than about 500, 475. 450, 425, 400, 375, 350. 325, 300. 275, 250, 225. 200, 175. 150, 125, 100, 75, 60, 50, 37.5, 20, 15, 10, 5, 2.5, 1, 0.5, 0.375, 0.1, 0.075 or 0.01 mg / m2. In certain embodiments, the dosage is between about 0.0005 to about 200 mg / m2, between about 0.001 and 150 mg / m2, between about 0.075 and 125 mg / m2, between about 0.375 and 100 mg / m2, between about 2.5 and 75 mg / m2, between about 10 and 75 mg / m2, and between about 20 and 50 mg / m2. In related embodiments, the dosages of antibody used is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5. 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5. 20. 20.5 mg / kg of body weight of a patient. In certain embodiments, the dose of naked antibody used is at least about 1 to 10, 5 to 15, 10 to 20, or 15 to 25 mg / kg of body weight of a patient. In certain embodiments, the dose of antibody used is at least about 1 to 20, 3 to 15, or 5 to 10 mg / kg of body weight of a patient. In other embodiments, the dose of antibody used is at least about 5, 6, 7. 8, 9, or 10 mg / kg of body weight of a patient. In certain embodiments, a single dosage unit of the antibody (optionally in a pharmaceutically acceptable carrier as part of a pharmaceutical composition) can be at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14. 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46. 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70. 72. 74. 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 1 12, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,

[0112] 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,

[0113] 198, 200. 204, 206, 208, 210, 212, 214, 216. 218, 220, 222, 224, 226. 228, 230, 232,

[0114] 234, 236. 238, 240, 242, 244, 246. 248, or 250 micrograms / m2In other embodiments, dose is up to 1 g per single dosage unit.

[0115] In alternative embodiments of methods as provided herein, antibodies are administered at a dose lower than about 375 mg / m2; at a dose lower than about 37.5 mg / m2; at a dose lower than about 0.375 mg / m2; and / or at a dose between about 0.075 mg / m2and about 125 mg / m2. In certain embodiments of methods of the invention, dosage regimens comprise low doses, administered at repeated intervals. For example, in one embodiment, compositions of the invention can be administered at a dose lower than about 375 mg / m2at intervals of approximately even' 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 days.

[0116] In alternative embodiments of methods as provided herein, antibodies are naked antibodies, and a dose of naked antibody used can be at least about 0.1, 0.2, 0.3. 0.4, 0.5, 0.6. 0.7. 0.8, 0.9, 1. 1.5, 2, 2.5, 3. 3.5, 4, 4.5. 5. 5.5, 6, 6.5. 7, 7.5, 8. 8.5. 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5 mg / kg of body weight of a patient. In certain embodiments, the dose of naked antibody used is at least about 1 to 10, 5 to 15, 10 to 20, or 15 to 25 mg / kg of body weight of a patient. In certain embodiments, the dose of naked antibody used is at least about 1 to 20, 3 to 15, or 5 to 10 mg / kg of body weight of a patient. In other embodiments, the dose of naked antibody used is at least about 5, 6, 7, 8, 9, or 10 mg / kg of body w eight of a patient. In certain embodiments, the dose comprises about 375 mg / m2antibody administered weekly for 4 to 8 consecutive weeks. In certain embodiments, the dose is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / kg of body weight of the patient administered weekly for 4 to 8 consecutive weeks.

[0117] Products of manufacture and Kits

[0118] Provided are products of manufacture and kits for practicing methods as provided herein, including for example, anti-human haptoglobulin antibodies, for example, humanized anti-human haptoglobulin antibodies; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein.

[0119] Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and / or Detailed Description sections.

[0120] As used in this specification and the claims, the singular forms “a,” '‘an’’ and “the” include plural referents unless the context clearly dictates otherwise.

[0121] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”. Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (use of the term “about’’) can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[0122] Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of’, “substantially all of’ or “majority of’ encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%. 99% or 99.5%. or more of a referenced amount of a composition.

[0123] The entirety of each patent, patent application, publication and document referenced herein hereby is expressly incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subj ect matter by an examining authority or court.

[0124] Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of', and "consisting of' may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

[0125] The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

[0126] EXAMPLES

[0127] Example 1 : Making and using diagnostic methods as provided herein Methods

[0128] - Samples description.

[0129] Experiments were performed on human K2EDTA plasma samples. Non cancer samples (controls) included 10 healthy donor samples, as well as samples from patients with inflammatory diseases (5 cases of diabetes mellitus type 2, 5 cases of arthritis) purchased with ages matching the cancer samples group (50-75 years old). Stage I solid tumor samples were collected pre-treatment (blood was drawn before surgery / anticancer drugs). 4 cases of colorectal cancer and 4 cases of non-small cell lung cancer were tested.

[0130] - Extracellular vesicles isolation by SEC columns.

[0131] EVs isolation w as performed using qEVsingle / 35 nm Gen 2 isolation columns from IZON, designed for separating EVs from a 150 pL loading volume. Before loading onto the columns, plasma samples were centrifuged at 1,500 x g for 10 minutes to remove any cells and large particles. After the first centrifugation, the supernatant was gently moved to a new7tube and centrifuged again at 10,000 x g for 10 minutes.

[0132] The column was equilibrated at RT and put in an upright position to allow7the buffer to run through it. After that, it was flushed twice with 3 ml of freshly filtered (0.22 pM) PBS.

[0133] 150 pL of the plasma supernatant obtained after the second centrifugation was loaded onto the loading frit of the column and allow ed to run into the column (collected as buffer volume). The column was then topped up with 0.55 ml of filtered PBS (the final buffer volume is 0.7 ml). Once the buffer volume was collected. 170 pL of buffer were repeatedly added to the top of the column and collected as 8 separated fractions.

[0134] - Sample concentration and lysis prior to MS analysis.

[0135] After isolation with qEV column, fraction 3 was concentrated using Merck Amicon Ultra 0.5 ml centrifugal filters unit with a cutoff of 10 kDa. The filters were pre-rinsed to remove traces of glycerine with 100 giL of PBS (centrifugation at 14,000 * g for 10 minutes and reverse spin at 1,000 g for 2 minutes). 300 pL of fraction 3 was then loaded (a pool of 2 isolation columns) and centrifuge at 14,000 x g for 10 minutes. After a reverse spin at 1,000 x g for 2 minutes, the volume was checked. The sample was centrifuged again to reach a final volume of 60 pL (5X concentration).

[0136] The obtained concentrated sample was immediately lysed on ice by adding 60 pL of RIP A™ lysis buffer (89901 Pierce) supplemented with IX HALT PROTEASE INHIBITOR COCKTAIL™ (78438 Thermo Fisher Scientific) and vortexing. The sample was stored at -20°C.

[0137] - Sample prep for MS analysis

[0138] Lysed (RIPA buffer + Protease inhibitors) samples were adjusted to a final concentration of 1% SDS, 100 mM ABC and sonicated at 4°C for 10 min (30 seconds (s) on and 30 s off). Afterward, EVs lysates were then reduced and alkylated through the addition of 10 mM DTT and 22.5 mM iodoacetamide (IAA), with incubation for 30 minutes at 37 °C. Excess IAA was quenched by the addition of DTT.

[0139] Protein digestion was performed using a 1 : 1 ratio of magnetic carboxylate-modified beads (GE Healthcare) as described before (Hughes 2019, doi: 10.1038 / s41596-018- 0082). Briefly, four pL of the prepared bead mix (50 pg / pL) was added to the samples. Afterward, acetonitrile (ACN) was added to a final concentration of 70% (v / v). and samples were mixed at room temperature for 20 min. Subsequently, beads were immobilized by incubation on a magnetic rack for 2 min. The supernatant was discarded, and the pellet was rinsed twice with 70% (v / v) EtOH and 100% ACN. Beads were then resuspended in 40 pL of 50 mM NH4HCO3 supplemented with 1 pg of trypsin and 0.5 pg of Lys-C. After overnight digestion at 37°C, the peptide mixture was collected by incubation on a magnetic rack and subjected to detergent removal using HIPPR™ resin (Thermo Scientific) in accordance with manufacturer's instructions. The supernatant containing purified peptides was transferred into a fresh tube and acidified by adding 1% trifluoroacetic acid. Samples were then desalted by C18 stage-tip, lyophilized, and resuspended in 10 pL of 0. 1% formic acid for LC- MS / MS analysis.

[0140] - LC-MS / MS analysis

[0141] Peptides were separated on an EASY-NLC 1200™ HPLC system (Thermo Scientific) using a 30 cm reversed-phase column (75 pm inner diameter. 1.7 pm bead size, prepacked C 18 column, MSWIL) with a two-component mobile phase (A = 0. 1 % formic acid in water, and B = 0. 1 % formic acid in acetonitrile). The peptides were then eluted using a gradient from 5% to 25% over 52 minutes, followed by 25% to 40% over 8 minutes, and 40% to 98% over 10 minutes at a flow rate of 200 nL / min. The peptides were analyzed on an ORBITRAP FUSION TRIBRID™ mass spectrometer (Thermo Fisher Scientific) in data-dependent mode, with the full scans performed at a resolving power of 120.000 FWHM (mass range: 350-1100 m / z, AGC target value: 4x10e5 ions, maximum injection time: 50 ms). Each full scan was followed by a set of (HCD) MS / MS scans within a 3 sec cycle time, with a collision energy of 30%. Fragment data were acquired in an ion trap (AGC target: 10e4 ions, maximum injection time: 35 ms). The Ion transfer tube temperature was set at 200°C. Dynamic exclusion was enabled and set to 30 sec. with a mass tolerance of 5 ppm. Data were acquired using XCALIBUR (Xcalibur) 4.3™ and TUNE 3.3™ software (Thermo Fisher Scientific). QCLOUD™ (see for example, C. Chiva et al: A cloud-based quality control system for mass spectrometry-based proteomics laboratories. PLOS ONE 13, e0189209 (2018)) was used for all acquisitions to control long-term instrumental performance during the project, using quality control standards. All proteomic data were searched against the in silica digested UNIPROT™ human database (downloaded in March 2023) with major known contaminants and reversed versions of each sequence. PROTEOME DISCOVERER v.2.2.0™ (Thermo Scientific) and MASCOT™ search engine (v.2.6.2™. MATRIXSCIENCE™) were used to identify proteins (precursor mass tolerance: 10 ppm, product mass tolerance: 0.6 Da). Trypsin / P was chosen as the enzy me with 5 missed cleavages. Static modification of carbamidomethyl (C) and variable modification of oxidation (M) and N-terminal protein acetylation were incorporated in the search. FDR was set to < 0.01 for both peptides and proteins. Contaminants were filtered out.

[0142] - Quantification by ELISA of Haptoglobin and Catalase proteins. Haptoglobin (HP) and Catalase (CAT) were measured by ELISA using 2 commercial kits distributed by ABCAM™ (AB219048 and AB277396); alternatively, Haptoglobin (HP) was also measured by ELISA using a commercial kit distributed by PROTEINTECH™ (KE00148ZC1.

[0143] Samples were lysed for 30 minutes on ice with a 0.5 % solution in PBS of IGEPAL CA-630™ (13021 Sigma-Aldrich). A number of embodiments of the invention have been described.

[0144] Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for detecting a cancer or a tumor, the method comprising:(a) obtaining a sample comprising biological fluids, cells or tissue from a subject, wherein optionally the subject is a human patient or an animal subject, wherein optionally the sample comprises or is derived from a biopsy or a subject tissue sample or a control tissue sample;(b) isolating extracellular vesicles (EVs) having a selected diameter range, wherein optionally the selected diameter range of the isolated EVs is between about 50 nm and 500 nm, or between about 100 nm and 400 nm, or between about 150 nm and 300 nm;(c) determining the presence of Haptoglobin (HP) and / or Catalase (CAT) in the isolated EVs; and(d) measuring the concentration and / or the amount of Haptoglobin (HP) and / or Catalase (CAT) in the sample, wherein optionally the concentration and / or the amount of Haptoglobin (HP) and / or Catalase (CAT) in the sample is measured by an ELISA (enzy me-linked immunosorbent assay) and / or HPLC (High Performance Liquid Chromatography), wherein if HP and CAT are individually detected, or if HP and CAT are both detected, then Colorectal and / or Lung cancer have been positively detected or diagnosed, and optionally if HP and CAT are detected using ELISA (optionally using an ABC AM™ kit) at a level at or above about 580 pg / mL for HP and 10 pg / mL for CAT, or are detected at a level over between about 560 pg / mL to 600 pg / mL for HP and over between about 8 pg / mL to 10 pg / mL for CAT, then the cancer (optionally, a colorectal and / or lung cancer) have been positively detected or diagnosed, and and optionally if HP alone is detected using an ELISA (optionally using a PROTEINTECH™ kit) at a level at or above about 29,700 pg / mL, or is detected at a level over between about 29,000 pg / mL to 30,000 pg / mL, then the cancer (optionally, a colorectal and / or lung cancer) have been positively detected or diagnosed.

2. The method of claim 1. wherein the cancer or tumor is colorectal and / or Lung cancer.

3. A method for treating a cancer, the method comprising:(a) first detecting whether an individual in need thereof has a cancer or a tumor using a method as set forth in claim 1 ; and(b) followed by treating the individual in need thereof with a drug or therapy to treat or ameliorate, or lesson the symptoms of or chance of recurrence of, the tumor or cancer.

4. The method of claim 3, wherein the cancer or tumor is colorectal cancer (CRC) or lung cancer, wherein optionally the lung cancer is non-small cell lung cancer (NSCLC).

5. The method of claim 4, wherein the cancer or tumor is a leukemic cell cancer, acute lymphoblastic leukemia (ALL), angioimmunoblastic T cell lymphoma, Burkitt lymphoma, chronic myelogenous leukemia in blast crisis, diffuse large B-cell lymphoma, hairy cell leukemia, myeloma, precursor B lymphoblastic leukemia or lymphoma, follicular lymphoma, diffuse large B- cell lymphoma, mantle cell lymphoma, precursor T lymphoblastic leukemia or lymphoma, non-hematolymphoid sarcoma, or a carcinoma such as a renal cell carcinoma or a metaplastic breast carcinoma.

6. The method of any of claims 3 to 5, wherein the treating the individual in need thereof with a drug or therapy to treat or ameliorate, or lesson the symptoms of or chance of recurrence of, the tumor or cancer, comprises administering to an individual in need thereof an antibody (Ab), an antigen (Ag) binding fragment thereof, or a monomeric or dimeric antigen binding protein (ABP) that specifically binds haptoglobin (HP) and / or a haptoglobin-hemoglobin complex.

7. The method of claim 5. wherein the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP)that specifically binds haptoglobin (HP) and / or a haptoglobin-hemoglobin complex, comprises or is fabricated as: an antigen-binding fragment (Fab, or an Ab fragment having just one constant and one variable domain of each of an Ab heavy and light chain). a F(ab')2 (or an Ab digested by pepsin yielding two fragments: a F(ab')2 fragment and a pFc' (pepsin cleavage Fc) fragment), a Fab' (a single chain of a F(ab')2 fragment). a single-chain variable fragment (scFv) (or a fusion protein of a variable region of an Ab heavy and light chain connected together with a linker peptide optionally of about ten to about 25 amino acids in length), a (scFv)2, or a di-scFv or a bi-scFv, or a single peptide chain having two variable heavy and two variable light regions yielding tandem scFv, a minibody (or a fusion protein of a variable region of an Ab heavy and light chain connected together with an alkyl group, optionally a methyl or an ethyl group) a diabody (or an scFv with a linker peptide too short (optionally about five amino acids) for the two variable regions to fold together forcing the scFvs to dimerize), a triabody or a tetrabody (or an scFv with a linker peptide too short (optionally about one or two amino acids) for the two variable regions to fold together forcing the scFvs to trimerize or tetramize), a single-domain antibody (dAB) (or a single variable region of an Ab heavy or Ab light chain), a plurality of complementarity determining region (CDR) fragments, a multispecific antibody formed from two or more antibody fragments, a monoclonal or a polyclonal antibody (Ab); and / or a humanized Ab having a non-human (optionally murine) antigen binding fragment or complement determining region (CDR) that specifically binds to human haptoglobulin implanted in a human antibody.

8. The method of claim 6 or 7, wherein the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP)that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex is formulated as a pharmaceutical composition, or is formulated for administration in vivo; or is formulated for enteral or parenteral administration, or for oral, intravenous (IV) or intrathecal (IT) administration, or is administered orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially or rectally.

9. The method of claim 8, wherein the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex is formulated as a nanoparticle, a particle, a micelle or a liposome or lipoplex, a polymersome, a polyplex or a dendrimer; or is formulated as, or contained in, a nanoparticle, a liposome, a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, an emulsion, a lotion, an aerosol, a spray, a lozenge, an aqueous or a sterile or an injectable solution, or an implant.

10. The method of any of claim 8 or 9, wherein the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex is administered: (a) once a day. once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every’ six weeks, once every seven weeks or once every eight weeks; or (b) at a dosage of between about 1 and 40 mg / Kg, or between about 5 and 20 mg / Kg.

11. A method for treating, ameliorating, lessoning the symptoms of or decreasing the chance of recurrence of, a tumor or a cancer, comprising administering to an individual in need thereof an antibody (Ab), an antigen (Ag) binding fragment thereof, or a monomeric or dimeric antigen binding protein (ABP) that specifically binds haptoglobin (HP) and / or a haptoglobin-hemoglobin complex.

12. The method of claim 11, wherein the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds haptoglobin (HP) and / or a haptoglobin-hemoglobin complex, comprises or is fabricated as:an antigen-binding fragment (Fab, or an Ab fragment having just one constant and one variable domain of each of an Ab heavy and light chain). a F(ab')2 (or an Ab digested by pepsin yielding two fragments: a F(ab')2 fragment and a pFc' (pepsin cleavage Fc) fragment), a Fab' (a single chain of a F(ab')2 fragment). a single-chain variable fragment (scFv) (or a fusion protein of a variable region of an Ab heavy and light chain connected together with a linker peptide optionally of about ten to about 25 amino acids in length), a (scFv)2, or a di-scFv or a bi-scFv, or a single peptide chain having two variable heavy and two variable light regions yielding tandem scFv, a minibody (or a fusion protein of a variable region of an Ab heavy and light chain connected together with an alkyl group, optionally a methyl or an ethyl group) a diabody (or an scFv with a linker peptide too short (optionally about five amino acids) for the two variable regions to fold together forcing the scFvs to dimerize), a triabody or a tetrabody (or an scFv with a linker peptide too short (optionally about one or two amino acids) for the two variable regions to fold together forcing the scFvs to trimerize or tetramize), a single-domain antibody (dAB) (or a single variable region of an Ab heavy or Ab light chain), a plurality of complementarity determining region (CDR) fragments, a multispecific antibody formed from two or more antibody fragments, a monoclonal and / or a polyclonal antibody (Ab); and / or a humanized Ab having a non-human (optionally murine) antigen binding fragment or complement determining region (CDR) that specifically binds to human haptoglobulin implanted in a human antibody.

13. The method of claim 11 or 12, wherein the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex:(a) comprises a murine complement determining region (CDR) that specifically binds to the haptoglobin (HP) or the haptoglobin-hemoglobin complex; or(b) is a humanized antibody comprising a murine complement determining region (CDR) that specifically binds to the haptoglobin (HP) or the haptoglobin- hemoglobin complex, wherein optionally the CDR has a sequence comprising SEQ ID NO: 1.

14. The method of any of claims 11 to 13, wherein the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin- hemoglobin complex is formulated as a pharmaceutical composition, or is formulated for administration in vivo; or is formulated for enteral or parenteral administration, or for oral, intravenous (IV) or intrathecal (IT) administration, or is administered orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially or rectally.

15. The method of claim 14, wherein the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin-hemoglobin complex is formulated as a nanoparticle, a particle, a micelle or a liposome or lipoplex, a polymersome, a polyplex or a dendrimer; or is formulated as, or contained in, a nanoparticle, a liposome, a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, an emulsion, a lotion, an aerosol, a spray, a lozenge, an aqueous or a sterile or an injectable solution, or an implant.

16. The method of any of claim 14 or 15, wherein the antibody (Ab), antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) that specifically binds the haptoglobin (HP) or the haptoglobin- hemoglobin complex is administered: (a) once a day, once a week, once every two weeks, once every three weeks, once even7four weeks, once every five weeks, once every six weeks, once every seven weeks or once every eight weeks; or (b) at a dosage of between about 1 and 40 mg / Kg. or between about 5 and 20 mg / Kg.

17. The method of claim 11, wherein the cancer or tumor is colorectal cancer (CRC) or lung cancer, wherein optionally the lung cancer is non-small cell lung cancer (NSCLC).

18. The method of claim 11 , wherein the cancer or tumor is a leukemic cell cancer, acute lymphoblastic leukemia (ALL), angioimmunoblastic T cell lymphoma, Burkitt lymphoma, chronic myelogenous leukemia in blast crisis, diffuse large B-cell lymphoma, hairy cell leukemia, myeloma, precursor B lymphoblastic leukemia or lymphoma, follicular lymphoma, diffuse large B- cell lymphoma, mantle cell lymphoma, precursor T lymphoblastic leukemia or lymphoma, non-hematolymphoid sarcoma, or a carcinoma such as a renal cell carcinoma or a metaplastic breast carcinoma, an acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, rectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal cancer, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, colorectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and / or urinary bladder cancer.

19. A method for determining the efficacy of a cancer or tumor treatment or therapy, comprising measuring the level of haptoglobin (HP) and / or haptoglobin- hemoglobin complexes in a sample from (or derived from) an individual or subject before beginning the cancer or tumor treatment or therapy, and at least once again after commencing the cancer or tumor treatment or therapy, wherein lower levels of the haptoglobin (HP) and / or haptoglobin-hemoglobin complexes in the individual or subject after commencing the cancer or tumor treatment or therapy as compared tobefore beginning the cancer or tumor treatment or therapy determines or indicates that the cancer treatment or therapy is effective against the cancer or tumor.

20. The method of claim 19, wherein the subject is a human patient or an animal subject, and optionally the sample comprises or is derived from a biopsy or a subject tissue sample or a control tissue sample.

21. A method for monitoring recurrence of a cancer or tumor, or residual disease, after completion of a cancer or tumor treatment or therapy, comprising measuring the level of haptoglobin (HP) and / or haptoglobin-hemoglobin complexes in a sample from (or derived from) an individual or subject in need thereof:(a) before beginning the cancer or tumor treatment or therapy, and / or at least once again after commencing the cancer or tumor treatment or therapy, and(b) at least once after completion of the cancer or tumor treatment or therapy, wherein lower levels, or at least not higher levels, of the haptoglobin (HP) and / or haptoglobin-hemoglobin complexes in the individual or subject after completion of the cancer or tumor treatment or therapy as compared to before beginning the cancer or tumor treatment or therapy and / or during the cancer or tumor treatment or therapy determines or indicates that the cancer or tumor has not relapsed or recurred, wherein higher levels of the haptoglobin (HP) and / or haptoglobin-hemoglobin complexes in the individual or subject after completion of the cancer or tumor treatment or therapy as compared to before beginning the cancer or tumor treatment or therapy and / or during the cancer or tumor treatment or therapy determines or indicates a cancer or tumor recurrence, or that the cancer or tumor treatment or therapy was not successfully in substantially eradicating or treating the cancer or tumor.

22. The method of any of claims 19 to 21, wherein the cancer or tumor treatment or therapy comprises chemotherapy, radiation and / or surgery.

23. The method of any of claims 19 to 22, wherein the cancer or tumor is colorectal cancer (CRC) or lung cancer, wherein optionally the lung cancer is nonsmall cell lung cancer (NSCLC).

24. The method of any of claims 19 to 23, wherein the cancer or tumor is a leukemic cell cancer, acute lymphoblastic leukemia (ALL), angioimmunoblastic T cell lymphoma, Burkitt lymphoma, chronic myelogenous leukemia in blast crisis, diffuse large B-cell lymphoma, hairy cell leukemia, myeloma, precursor B lymphoblastic leukemia or lymphoma, follicular lymphoma, diffuse large B- cell lymphoma, mantle cell lymphoma, precursor T lymphoblastic leukemia or lymphoma, non-hematolymphoid sarcoma, or a carcinoma such as a renal cell carcinoma or a metaplastic breast carcinoma.

25. The method of any of claims 18 to 23, wherein the subject is a human patient or an animal subject, and optionally the sample comprises or is derived from a biopsy or a subject tissue sample or a control tissue sample.

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