methods to determine the level or state of activation of an oncogenic fusion protein, to optimize th

专利摘要:
  METHODS FOR DETERMINING THE LEVEL OR STATUS OF ACTIVATION OF AN ONCOGENIC FUSION PROTEIN, TO OPTIMIZE THERAPY AND / OR TO REDUCE TOXICITY IN A SUBJECT HAVING CANCER, TO SELECT AN SUITABLE ANTI-CANCER MEDICATION FOR THE TREATMENT OF A TREATMENT FOR A TREATMENT OF A TREATMENT FOR A TREATMENT OF A TREATMENT FOR A TREATMENT OF A CHEMISTRY. FROM A CANCER TO TREATMENT WITH ANTI-CANCER MEDICINE, TO PREDICT THE RESPONSE OF A SUBJECT HAVING CANCER TO TREATMENT WITH ANTI-CANCER MEDICINE, AND, TO DETERMINE WITHOUT A SUBJECT HAVING CANCER IS RESISTANT TO TREATMENT WITH ANTI-CANCER MEDICATION.The present invention provides antibody-based arrangements for detecting the activation state and / or total amount of one or a plurality of oncogenic fusion proteins in a biological sample, such as whole blood or tumor tissue, and methods of using these. In certain examples, the activation state and or the total amount of oncogenic fusion protein (s) present in a sample can be measured in combination with one or a plurality of signal transduction molecules. The compositions and methods of the present invention have the advantages of specificity associated with enzyme linked immunosorbent assays, sensitivity associated with signal amplification, and high yield multiplexing associated with microarrays.
公开号:BR112012009296A2
申请号:R112012009296-0
申请日:2010-10-20
公开日:2021-02-02
发明作者:Sharat Singn；Xinjun Liu
申请人:Prometheus Laboratories Inc.；
IPC主号:

专利说明:

, “METHODS FOR DETERMINING THE LEVEL OR STATE OF; ACTIVATION OF AN ONCOGENIC FUSION PROTEIN, TO OPTIMIZE THERAPY AND / OR TO REDUCE TOXICITY IN A SUBJECT HAVING CANCER, TO SELECT A MEDICINE — SUITABLE FOR ANY CANCER TREATMENT, TO IDENTIFY AN ANCINATORY TREATMENT RESPONSE -CANCER, TO i PREDICT THE RESPONSE OF A SUBJECT HAVING CANCER TO J TREATMENT WITH ANTI-CANCER MEDICINE, AND, FOR DETERMINING IF A SUBJECT HAVING CANCER IS RESISTANT TO TREATMENT WITH ANTI-CANCER MEDICINE ”
CROSS REFERENCE TO RELATED REQUESTS This application claims the priority of provisional application US 61 / 253,393, filed on October 20, 2009, provisional application US 61 / 305,084, filed on February 16, 2010, provisional application US 61 / 327,487, filed on April 23, 2010, and provisional application US 61 / 383,037, filed on September 15, 2010, the disclosures of which are incorporated herein by reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION Fusion proteins, also known as chimeric proteins, are proteins created by joining two or more genes that originally encode separate proteins. The translation of this fusion gene results in a simple polypeptide with functional properties derived from each of the original proteins. Mutant — chimeric proteins occur when a large-scale mutation, typically a chromosomal translocation, creates an unprecedented coding sequence that contains parts of the coding sequences for two different genes. Naturally occurring fusion proteins are important in cancer, where
”The BCR-ABL fusion protein is a well-known example - of an oncogenic fusion protein.
It can be considered as the main oncogenic regulator of chronic myelogenous leukemia (CML), but it is also associated with acute lymphoblastic leukemia (ALL). In fact, the brand - cytogenetics of CML is the Philadelphia (Ph) chromosome, which results in the formation of the BCR-ABL fusion gene that encodes a 210 kDa protein.
In fact, the resulting BCR-ABL fusion protein is an active tyrosine kinase that is important for the pathogenesis of CML.
Although imatinib (Gleevec ”) is currently the initial therapy for patients recently diagnosed with CML, about 20-25% of patients do not achieve lasting complete cytogenetic responses.
Studies have shown that reactivation of BCR-ABL signaling in the presence of continued treatment with imatinib is the main cause of resistance.
In most patients, reactivation results from mutations in the BCR-ABL kinase domain that hinder the binding of imatinib and lead to the induction of drug resistance.
As such, measurement of S activity of BCR-ABL is useful in predicting response to therapy with tyrosine kinase inhibitors 2, such as imatinib, as well as in identifying patients who develop resistance to such inhibitors.
To date, the methods available to detect BCR-ABL activity depend on the measurement of phosphorylated CRKL (PpCRKL), a BCR-ABL substrate.
For example, La Rosee et al. (Haematologica, 93: 765-9 (2008)) describe a Western blot analysis of total leukocyte lysates to determine the level of pCRKL as a substitute for BCR-ABL activity (see also, Hochhaus et al. —Leukemia, 16: 2190-6 (2002); White et al., J.
Clin.
Oncol, 25: 4445-51 (2007)). Similarly, Khorashad et al. (Haematologica, 94: 8 1-4 (2009)) Does S describe a method based on flow cytometry to measure the level of Ss pCRKL to assess BCR-ABL activity (see also, Hamilton et al., Leukemia, 20: 1035-9 (2006)). However, these methods do not have the
7 specificity and sensitivity that is required to determine the presence or - level of BCR-ABL activity in a sample, as they are simple antibody assays that depend on the detection of phosphorylation of a substitute protein. Thus, specific and sensitive methods are necessary to detect the activity of BCR-ABL, as well as the activity of other oncogenic fusion proteins, for diagnostic, prognostic and therapeutic purposes. The present invention satisfies this need and provides related advantages as well. ; SUMMARY OF THE INVENTION The present invention provides antibody-based arrangements for detecting the activation state and / or total amount of one or a plurality of oncogenic fusion proteins in a biological sample, such as whole blood (for example, a lysate prepared at from isolated rare circulating cells or leukocytes) or tumor tissue (for example, a fine needle aspirate) and methods of using these. In certain examples, the activation state and / or total amount of oncogenic fusion protein (s) present in a sample can be measured in combination with one or a plurality of signal transduction molecules. The compositions and methods of the present invention have the specificity advantages associated with enzyme linked immunosorbent assays, sensitivity associated with signal amplification and high throughput multiplexing associated with microarrays.
In one aspect, the present invention provides a method for determining the level or state of activation of a fusion protein - oncogenic, the method comprising: (a) contacting a cell extract with a first binding fraction specific to a first domain of a first full-size protein, under suitable conditions to transform the first full-size protein present in the cell extract into a úÚÚ | - | NN——
"complex comprising the first full size protein and the first 'binding fraction, wherein the first domain of the first full size protein does not have a corresponding oncogenic fusion protein, which comprises a second domain different from the first - size protein total fused to a first domain of a second protein of different total size; (b) removing the complex from step (a), from the: cell extract, to form a cell extract devoid of the first full-size protein; (oc) placing the cell extract of step (b) in contact with a second fraction of specific binding for the second domain other than the first full-size protein, under conditions suitable for transforming the oncogenic fusion protein present in the cell extract into a complex comprising the oncogenic fusion protein and the second mother-binding fraction i (d) determine the level or state of activation of the complex of step (c), determining by means of hence the level or state of activation of the oncogenic fusion protein.
In particular embodiments, determining the level or state of activation of an oncogenic fusion protein includes measuring a level of expression (eg, concentration) and / or activation (eg, phosphorylation) of an oncogenic fusion protein (eg, in a cell extract).
In preferred embodiments, steps (c) and (d) of the method of - the present invention comprise an enzyme linked immunosorbent assay (ELISA), a flow cytometry assay, a label selection assay, or a detection detection assay. double proximity in the manner described here.
In a particular modality of the detection test of
NT is the level or state of activation of an oncogenic fusion protein, the method. comprising: (a) incubating a cell extract with a serial dilution of capture antibodies specific for the oncogenic fusion protein to 5 - form a plurality of captured oncogenic fusion proteins, where the capture antibodies are limited on a solid support , wherein the oncogenic fusion protein comprises a first domain that: corresponds to a first protein and a second domain that corresponds to a different second protein, and in which the capture antibodies are - specific to the first domain of the fusion protein; (b) incubating the plurality of captured oncogenic fusion proteins with at least two types of detection antibodies specific for the second domain of the oncogenic fusion protein, to form a plurality of detectable captured oncogenic fusion proteins, wherein the detection antibodies comprise : (1) a plurality of antibodies independent of the state of. activation marked with a facilitation fraction, and (2) a plurality of activation state-dependent antibodies marked with a first element of a signal amplification pair, where the facilitation fraction generates an oxidizing agent that channels and reacts with the first element of the signal amplification pair; (c) incubating the plurality of detectable captured oncogenic fusion proteins with a second element of the amplification pair of 25. signal, to generate an amplified signal, and (d) detecting the amplified signal generated from the first and second elements of the signal amplification pair. ex In another particular modality of the detection test of HUH
'level or state of activation of an oncogenic fusion protein, the method - comprising:
(a) incubate a cell extract with a serial dilution of capture antibodies specific for the oncogenic fusion protein to
- forming a plurality of captured oncogenic fusion proteins, where the capture antibodies are limited to a solid support, where the oncogenic fusion protein comprises a first domain that i corresponds to a first protein and a second domain that corresponds to
'to a second different protein, and in which the capture antibodies are - specific to the first domain of the fusion protein;
(b) incubating the plurality of captured oncogenic fusion proteins with at least two types of detection antibodies to form a plurality of detectable captured oncogenic fusion proteins, wherein the detection antibodies comprise:
(1) a plurality of antibodies independent of the activation state labeled with a facilitation fraction, in which the antibodies. independent of the activation state are specific to the first domain of the fusion protein, and
(2) a plurality of activation state dependent antibodies labeled with a first element of a signal amplification pair, wherein the activation state dependent antibodies are specific to the second domain of the fusion protein,
wherein the facilitation fraction generates an oxidizing agent that channels and reacts with the first element of the signal amplification pair;
(c) incubating the plurality of captured detectable oncogenic fusion proteins with a second element of the signal amplification pair, to generate an amplified signal, and (d) detecting the amplified signal generated from the first and
: In another aspect, the present invention provides a method "to optimize therapy and / or reduce toxicity in a subject with cancer, and who receives a period of therapy for the treatment of cancer, the method comprising: (a) isolating cancer cells after administration of an anti-cancer drug; (b) lyse isolated cells to produce a cell extract;: (c) measure a level of expression and / or activation of a protein: oncogenic fusion in the cell extract, using an assay described herein, and (d) comparing the measured level of expression and / or activation of the oncogenic fusion protein with a level of expression and / or activation of the oncogenic fusion protein, measured at an earlier time, during the period of therapy, and (e) determining a subsequent dose of the therapy period - for the subject, or whether a different therapy period can be administered to the subject based on the comparison of step (d).
. In certain embodiments, one or more signal transduction molecules present in the cell extract are detected, in addition to one or more oncogenic fusion proteins. Examples of - signal transduction molecules include, without limitation, receptor tyrosine kinases, non-receptor tyrosine kinases, components of the tyrosine kinase signaling cascade, and / or substrates for one or more oncogenic fusion proteins (for example, BCR- GLA). In some examples, signal transduction molecules are detected using the methods described here, with the exception that, - depending on the assay, both the two antibodies (i.e., the capture antibody and the detection antibody) and the three antibodies (i.e., the capture antibody and both detection antibodies) are directed to the same protein. In other examples, signal transduction molecules are
In particular modalities, one or more of the signal transduction molecules: present in the cell extract are detected together with one or more oncogenic fusion proteins, using the assays (for example, immunoassays) described here.
In certain embodiments, the present invention also provides kits for performing the double proximity detection assays described herein, comprising: (a) a serial dilution of one or a plurality of capture antibodies maintained on a solid support, in which: capture antibodies are specific to one or more analytes of interest (for example, oncogenic fusion proteins or signal transduction molecules); and (b) a plurality (e.g., at least two types) of detection antibodies for each analyte of interest. The kits can optionally further comprise other reagents such as, for example, the first and second elements of the signal amplification pair.
Other objects, features and advantages of this. invention will be evident to those skilled in the art from the description and figures below.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows an embodiment of the assay format of the present invention, which depends on the co-location of two additional enzyme-linked detection antibodies for subsequent channel formation events for each target oncogenic fusion protein bound.
Figure 2 shows schematically the application of the arrangements —devention for drug selection throughout the cancer treatment period.
Figures 3 A-D show various modalities of the assay format of the present invention to detect expression and levels of activation of oncogenic fusion proteins such as BCR-ABL.
aa es between the IR and RD ea e ni ae o
: Figure 4 shows the BCR-ABL signal in K562 cells after - removal of free BCR.
Figure 5 shows the BCR signal in K562 cells after removal of free BCR.
Figure 6 shows the detection of total and phosphorylated levels of BCR-ABL in K562 cells.
Figure 7 shows the level of phosphorylation ("BCR-ABL phospho") and the total amount ("BCR-ABL total") of BCR-ABL detected in human chronic myelogenous leukemia p K562 cells with or without a decrease in —BCR free using beads coupled to the BCR C-terminal antibody.
Figure 8 shows the phosphorylated BCR-ABL signal in K562 cells with or without free BCR removal using beads coupled to the BCR C-terminal antibody. In particular, Figure 8A provides a microarray comparison of the phosphorylated BCR-ABL signal detected in K562 cell lysates, with or without the removal of full-size BCR ("Untreated accounts" = Untreated BCR versus "Treated accounts "= BCR. Removed with beads that contains an antibody specific to the C-terminus of full-size BCR conjugated to them). Figure 8B provides a graphical representation of the microarray data with relative fluorescence units (RFU) as a function of the cell number.
Figure 9 shows the total BCR-ABL signal in K562 cells with or without the removal of free BCR, using beads coupled to the C-terminal BCR antibody. In particular, Figure 9A provides a microarray comparison of the total BCR-ABL signal detected in K562 cell lysates - with or without full-size BCR removal ("Untreated accounts" = Unremoved BCR versus "Treated accounts" = BCR removed with beads that contains an antibody specific for the C terminus of full size BCR conjugated to them). Figure 9B provides a graphical representation of the a a ee e and ra ee a and a microarray data with relative fluorescence units (RFU) as -: a function of the cell number. Figure 10 shows the removal of full-size free BCR from an extract of K562 cells, after putting the cell extract in contact with beads coupled to the B-terminal C-antibody. In particular, Figure 10A provides a microarray comparison of the total BCR signal detected in K562 cell lysates with or without the removal of full size BCR ("Untreated accounts" = Untreated BCR versus "Treated accounts" = BCR removed with beads containing a specific antibody —for full-length BCR C termination conjugated to them). Figure 10B provides a graphical representation of the microarray data with relative fluorescence units (RFU) as a function of the cell number. Figure 11 shows that the full-size free BCR and ABL proteins, but not the BCR-ABL fusion protein, are present in white blood cells (WBCs).
Figure 12 shows that the full-size free BCR present in WBCs inhibited the phosphorus BCR-ABL signal in K562 cell extracts, when such K562 cell extracts were greater with the WBC extracts.
Figure 13 shows the total BCR-ABL signal in larger K562 cells with WBC extracts after removal of free BCR, using beads coupled to the BCR C-terminal antibody. In particular, Figure 13A shows that the free BCR signal was saturated when the K562 cell extracts were larger with the WBC extracts. After treatment with beads - coupled to the C-terminal BCR antibody, the free BCR was removed. Figure 13B shows that the BCR-ABL signal was not altered with or without treatment with beads in the same experiment.
Figure 14 shows that the BCR-ABL inhibitor, imatinib
'but not the expression (i.e., total levels) of the BCR-ABL protein in cells. K562.
Figure 15 shows that the BCR-ABL inhibitor, nilotinib (Tasigna ), Dose-dependent inhibited the activation (ie, phosphorylation), but not the expression (ie, total levels), of BCR-ABL protein in K562 cells. .
Figure 16 shows that the BCR-ABL inhibitor, dasatinib (Sprycel ), Dose-dependent inhibited the activation (ie, phosphorylation), but not the expression (ie, total levels), of BCR-ABL protein in cells K562.
Figure 17 shows that CRKL is both present and activated (i.e., phosphorylated) in K562 cells.
Figure 18 shows that CRKL is present in human A431 squamous cell carcinoma cells and is activated (i.e., phosphorylated) by treating EGF.
Figure 19 shows that CRKL is present in T47D human ductal breast epithelial tumor cells, but is not activated (i.e., phosphorylated) upon treatment with EGF.
Figure 20 shows that CRKL is present in T47D 20 cells. and is activated (ie, phosphorylated) at low levels by treatment with heregulin (HRG).
Figure 21 shows that CRKL is present in human MCF-7 breast adenocarcinoma cells and is activated (i.e., phosphorylated) at low levels by treatment with heregulin (HRG).
Figure 22 illustrates the presence of activated (i.e., phosphorylated) CRKL in white blood cells (WBCs) from different donors.
Figure 23 illustrates that JAK2 is activated (i.e., phosphorylated) in K562 cells and A431 cells. MOTHER
Figure 24 illustrates that phosphorylated BCR-ABL can be detected and measured in cell lysates prepared from K562 cells isolated from blood, using magnetic anti-CDA45 beads. Figure 25 illustrates that total BCR-ABL levels have not - been altered when an antibody targeting the full-length BCR C-termination (where the C-terminal domain is not present in BCR-ABL) has been recognized on the same device of the same piece, as an antibody directed to the N-terminal region of BCR-ABL. Figure 26 illustrates that the natural free BCR signal detected with a specific N-terminal BCR antibody was reduced when an antibody directed to the natural BCR C terminations was recognized on the same device on the same piece. DETAILED DESCRIPTION OF THE INVENTION
1. Introduction Haematological malignancies are the types of cancer that affect blood, bone marrow and lymph nodes. Because the three are intimately connected through the immune system, a disease that affects one of the three will often affect the others as well. For example, although lymphoma is technically a disease of the lymph nodes, it often disperses to the bone marrow and blood. Chromosomal translocations, which create fusion proteins with novel coding sequences that contain parts of the coding sequences for two different genes, are a common cause of these diseases, but are a less common cause of solid tumors. As such, it is important to identify the presence and / or activity of - oncogenic fusion proteins associated with hematological malignancies, in order to provide the appropriate prognosis and treatment for patients with these types of cancer. For example, the BCR-ABL fusion protein is associated with chronic myelogenic leukemia (CML), as well as lymphoblastic leukemia | U OF
: acute (ALL). In particular, the BCR-ABL protein is an active tyrosine kinase that is important for the pathogenesis of cancer. Although imatinib (Gleevec ”) is currently the initial therapy for patients recently diagnosed with CML, about 20-25% of patients do not achieve lasting complete cytogenetic responses. Studies have shown that reactivation of BCR-ABL kinase activity, in the presence of continued treatment with imatinib, is the main cause of resistance. As such, measuring BCR-ABL activity is useful in predicting response to therapy with tyrosine kinase inhibitors, such as imatinib, as well as in identifying patients who develop resistance to such inhibitors.
The present invention provides methods for detecting the activation state and / or the total amount of one or a plurality of fusion proteins (alone or in combination with one or a plurality of signal transduction molecules) in isolated cells using a system of antibody-based array assay. Cell extracts prepared from isolated leukocytes, circulating cells, or other cell types are particularly used in the methods described herein. In some embodiments, the high-throughput multiplex assays of the present invention can detect the activation state of one or more oncogenic fusion proteins, and / or signal transduction molecules, at the single cellular level. In fact, signal transduction molecules, such as EGFR, can be detected with a sensitivity of about 100 zeptomols and a linear dynamic range of about 100 zeptomols to about 100 fentomols. As such, single cell detection of the activation state of one or more fusion proteins - oncogenic, and / or signal transduction molecules, facilitates the prognosis and diagnosis of cancer, as well as the determination of targeted and personalized therapies.
Figure 1 illustrates an exemplary P—— detection test
| oncogenic, such as BCR-ABL, is linked to a capture antibody and two detection antibodies (i.e., an antibody independent of the activation state and an antibody dependent on the activation state). Capture antibody 1 binds to the BCR portion of the fusion protein regardless of its activation state.
Although the antibody independent of the activation state 2 binds to the ABL portion of the fusion protein independent of its activation state, the antibody dependent on the activation state 3 binds to the ABL portion of the fusion protein dependent on its activation state (for example, example, the activation-dependent antibody will bind only to the activated form of BCR-ABL with a phosphorylated residue). The antibody independent of the activation state is marked with a facilitation fraction 4 ("Enzyme A"), and the antibody dependent on the activation state is marked with a first element of a signal amplification pair 5 ("Enzyme B") . The binding of both detection antibodies to the ABL portion of the fusion protein introduces the facilitation fraction in sufficient proximity to the first element of the signal amplification pair, in such a way that a signal generated by the facilitation fraction can be channeled into the first element signal amplification pair, resulting in the generation of a detectable and / or amplifiable signal.
Various methods for channeling by proximity are described herein and are also known in the art including, but not limited to, FRET, FRET with time-resolved fluorescence, LOCI, etc.
An advantage of channeling by proximity, as used in the methods of the present invention, is that a simple detectable signal is generated only for those analytes (for example, fusion proteins or signal transduction molecules) that have bound to all three antibodies, resulting in better assay specificity, less foundation and simplified detection.
As explained in more detail here, to assess potential anti-cancer therapies for an individual patient, cells
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7 varied doses.
The stimulation of the growth factor can then be. performed for about five minutes (for example, about 1-5 minutes) or for several hours (for example, about 1-6 hours). Differential activation of signaling pathways with and without anti-cancer drugs can assist in the selection of an appropriate cancer therapy, at an appropriate dose for each individual patient.
The cells can also be isolated from a patient during treatment with an anti-cancer drug, and stimulated with one or more growth factors to determine whether a change in therapy can be implemented.
As such, figure 2 shows that the methods of the present invention advantageously assist the physician in providing the correct anti-cancer medication, in the correct dose, at the correct time, to each patient. YOU.
Definitions
As used herein, the following terms have the meanings assigned to them, unless otherwise specified.
The term "cancer" includes any element of a class of diseases characterized by the uncontrolled growth of abnormal cells.
The term includes all known cancers and neoplastic conditions, whether they are characterized as malignant, benign, soft-tissue or solid, and cancers of all stages and degrees, including pre- and post-20: metastatic cancers.
Non-limiting examples of different types of cancer include hematological malignancies (eg, leukemia, lymphoma), osteogenic sarcoma (eg, Ewing's sarcoma), soft tissue sarcomas (eg, protuberant dermatofibrosarcoma (DFSP), rhabdomyosarcoma), others soft tissue malignancies, carcinomas - thyroid papillaries, prostate cancer, gastric cancer (for example, stomach), breast cancer, lung cancer (for example, non-small cell lung cancer), digestive and gastrointestinal cancers (for example, example, colorectal cancer, gastrointestinal stromal tumors, tumors N——
h bile duct cancer and small intestine cancer), esophageal cancer, "gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, ovarian cancer, kidney cancer (eg kidney cell carcinoma), nervous system cancer central, skin cancer, choriocarcinomas and cancers - head and neck. As used here, a "tumor" comprises one or more cancer cells.
A "hematological malignancy" includes any type of cancer that affects the blood, bone marrow and / or lymph nodes. Examples of haematological malignancies include, but are not limited to, leukemia, lymphoma, and multiple myeloma. Non-limiting examples of different types of leukemia include chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenic leukemia (AML) and large granular lymphocytic leukemia. Subtypes of CML include, for example, chronic monocytic leukemia. ALL subtypes include, for example, precursor of acute B-cell lymphoblastic leukemia, acute B-cell pro-cell lymphoblastic leukemia, precursor of acute T-cell lymphoblastic leukemia and acute biphenotypic leukemia. CLL subtypes include, for example, B-cell pro-lymphocytic leukemia. AML subtypes include, for example, acute pro-myelocytic leukemia, acute myeloblastic leukemia and acute megakarioblastic leukemia. Examples of different types of lymphoma include, but are not limited to, Hodgkin's lymphoma (four subtypes) and non-Hodgkin's lymphoma, such as, for example, small lymphocytic lymphoma (SLL), large and diffuse B-cell lymphoma (DLBCL), lymphoma follicular (FL), Mantle cell lymphoma (MCL), hairy cell leukemia (HCL), marginal zone lymphoma (MZL), Burkitt's lymphoma (BL), post-transplant lymphoproliferative disorder (PTLD), pro-lymphocytic leukemia T-cell (T-PLL), B-cell pro-lymphocytic leukemia (B-PLL), Waldenstrom macroglobulinemia (also known as lymphoplasmacytic lymphoma) and TT and aaa DES DA Ia LA N ——— õ “& h
À The term "analyte" includes any molecule of interest, - typically a macromolecule such as a polypeptide, whose presence, quantity and / or identity are determined. In certain examples, the analyte is a cellular component of a cancer cell, preferably an oncogenic fusion protein or a signal transduction molecule.
The term "transform" or "transformation" includes a physical and / or chemical change of an analyte or sample to extract the analyte, or change or modify the analyte in the manner defined herein. As used herein, an extraction, a manipulation, a chemical precipitation, an ELISA, a complexation, an immuno-extraction, a physical or chemical modification of the analyte or sample to measure an analyte level or concentration or activation state are all a transformation. In other words, as long as the analyte or sample is not identical before and after the transformation stage, the change or modification is a transformation.
As used herein, the term "serial dilutions" is intended to include a series of concentrations descending from a particular sample (for example, cell lysate) or reagent (for example, antibody). A serial dilution is typically produced by a process of mixing a measured amount of an initial concentration of a sample, or reagent, with a diluent (for example, dilution buffer) to create a lower concentration of the sample or reagent, and repeat the process sometimes sufficient to obtain the desired number of serial dilutions. The sample or reagent can be serially diluted at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, or 1,000 times to produce a dilution in; series comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 , 30, 35, 40, 45, or 50 concentrations descending from the sample or reagent. For example, a serial dilution, comprising a 2-fold serial dilution of a capture antibody reagent in
FE is the amount of the initial capture antibody concentration with one: equal amount of dilution buffer to create a capture antibody concentration of 0.5 mg / mL, and repeat the process to obtain capture antibody concentrations of 0, 25 mg / ml, 0.125 mg / ml, 0.0625 mg / ml, 0.0325 mg / ml, etc.
The term "upper dynamic range", as used herein, refers to the ability of an assay to detect a specific analyte in just one cell or in many thousands of cells. For example, the immunoassays described here have a higher dynamic range as a result of detecting — advantageously a particular oncogenic fusion protein, or signal transduction molecule of interest, in about 1-10,000 cells (for example, about 1.5, 10 , 25, 50, 75, 100, 250, 500, 750, 1,000, 2,500, 5,000, 7,500 or
10,000 cells) using a serial dilution of capture antibody concentrations.
The term "fusion protein" or "chimeric protein" includes a protein created by joining two or more genes that originally encode separate proteins. Such gene fusions are typically generated when a chromosomal translocation replaces the terminal exons of a gene with intact exons from a second gene. This creates a unique gene that can be transcribed, joined and translated to produce a functional fusion protein. In particular embodiments, the fusion protein is an oncogenic fusion protein, that is, a fusion protein involved in oncogenesis. Examples of oncogenic fusion proteins include, but are not limited to, BCR-ABL, DEK-CAN, E2A-PBX1, RARa-PML, IREL- —URG, CBFB-MYHII, AMLI-MTG8, EWS-FLI, LYT-10- Lime, HRX-ENL, HRX-AF4, NPM-ALK, IGH-MYC, RUNX1I-ETO, TEL-TRKC, TEL-AML1, MLL-AFA4, TCR-RBTN2, COLIA1-PDGF, E2A-HLF, PAX3-FKHR, ETV6- NTRK3, RET-PTC, TMRSS-ERG and TPR-MET.
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'The term "signal transduction molecule" or "signal transducer" includes proteins and other molecules that carry out the process in which a cell converts an extracellular signal or stimulus into a response, which typically involves ordered sequences of biochemical reactions within the - cell Examples of signal transduction molecules include, but are not limited to, receptor tyrosine kinases such as EGFR (eg, EGFR / HER-1 / ErbB1, HER-2 / NewErbB 2, HER-3 / ErbB3, HER-4 / ErbB4), VEGFR-I / FLT-1, VEGFR-2 / FLK-1 / KDR, VEGFR-3 / FLT-, FLT-3 / FLK-2, PDGFR (e.g. PDGFRA, PDGFRB), c-Met , c-KIT / SCFR, INSR (insulin receptor), IGF-IR, IGF-IIR, IRR (insulin receptor related receptor), CSF-IR, FGFR1-4, HGFR 1-2, CCKA4, TRK AC, MET, RON, EPHA 1-8, EPHB 1-6, AXL, MER, TYRO3, TIE 1-2, TEK, RYK, DDR 1-2, RET, c-ROS, V-cadherin, LTK (z tyrosine kinase leukocyte), ALK (anaplastic lymphoma kinase), ROR 1-2, MUSK, AATYK —15 1-3, RTK 106, and truncated forms of the tir receptor osine kinases such as' p95ErbB2; non-tyrosine receptor kinases such as Src, Frk, Btk, Csk, Abl, Zap70, Fes / Fps, Fak, Jak, Ack, and LIMK; components of the tyrosine kinase signaling cascade such as Akt, MAPK / ERK, MEK, RAF, PLA2, MEKK, JINKK, INK, p38, Shc (p66), PI3K, Ras (for example, K-Ras, —N-Ras , H-Ras), Rho, Racl, Cde42, PLC, PKC, p70 S6 kinase, p53, cyclin D1, STATI, STAT3, PIP2, PIP3, PDK, mTOR, BAD, p21, p27, ROCK, IP3, TSP-1 , NOS, PTEN, RSK 1-3, INK, c-Jun, Rb, CREB, Ki67 and paxiline; nuclear hormone receptors such as estrogen receptor (ER), progesterone receptor (PR), androgen receptor, glucocorticoid receptor, mineralocorticoid receptor, vitamin A receptor, vitamin D receptor, retinoid receptor, thyroid hormone receptor and orphan recipients; nuclear receptor co-activators and repressors and combinations thereof.
CT ASSES Pa
The term "sample", as used herein, includes any biological specimen obtained from a patient. Samples include, without limitation, whole blood, plasma, serum, ductal lavage fluid, breast aspirate, lymph (for example, disseminated tumor cells from the lymph node), - bone marrow aspirate, saliva, urine, excrement (ie, sputum, sputum, bronchial lavage fluid, tears, fine needle aspiration (eg, collected by random periareolar fine needle aspiration), any other body fluid, a tissue sample (eg tumor tissue) such as a biopsy of a tumor (for example, needle biopsy) or a lymph node (for example, sentinel node biopsy), and cell extracts thereof. In some embodiments, the sample is whole blood or a fractional component thereof, such as plasma, serum, red blood cells, leukocytes such as peripheral blood mononuclear cells and / or rare non-circulating cells. In particular embodiments, the sample is obtained by isolating leukocytes or circulating cells from a solid tumor from whole blood, 'or a cellular fraction thereof, using any technique known in the art. In other embodiments, the sample is a sample of tumor tissue inserted in paraffin and fixed with formalin (FFPE), for example, from a solid tumor.
As used herein, the term "circulating cells" includes
20. extratumor cells that have undergone both metastasis and micrometastasis from a solid tumor. Examples of circulating cells include, but are not limited to, circulating tumor cells, cancer stem cells and / or cells that are migratory to the tumor (eg, circulating endothelial progenitor cells, circulating endothelial cells, pro-angiogenic myeloid cells - circulating, dendritic cells current, etc.).
A "biopsy" refers to the process of removing a tissue sample for assessment of diagnosis or prognosis, and to the tissue specimen itself. Any biopsy technique known in the technology can be NV ————
i biopsy applied will generally depend on the type of tissue evaluated and the size and type of the tumor (ie, solid or suspended (ie, blood or ascites)), among other factors. Representative biopsy techniques include excisional biopsy, incisional biopsy, needle biopsy (eg, needle heart biopsy, fine needle aspiration biopsy, etc.), surgical biopsy and bone marrow biopsy. Biopsy techniques are discussed, for example, in Harrison's Principles of Internal Medicine, Kasper, et al., Eds., 16th ed., 2005, chapter 70, and elsewhere V. Those skilled in the art will understand that biopsy techniques can be performed to identify cancerous and / or precancerous cells in a given tissue sample.
The term "subject", or "patient", or "individual", typically includes humans, but can also include other animals such as, for example, other primates, rodents, canines, felines, horses, sheep, similar porcinosis.
'An "array" or "microarray" comprises a distinct set and / or serial dilution of capture antibodies immobilized or maintained on a solid support such as, for example, glass (for example, a glass slide), plastic, chips , pins, filters, beads (for example, - magnetic beads, polystyrene beads, etc.), paper, membrane (for example, nylon, nitrocellulose, polyvinylidene fluoride (PVDF), etc.), bundles of fibers, or any another suitable substrate. Capture antibodies are generally immobilized or maintained on the solid support through covalent or non-covalent interactions (eg, ionic bonds, hydrophobic interactions, hydrogen bonds, Van der Waals forces, dipole-dipole bonds). In certain examples, capture antibodies comprise capture labels that interact with capture agents attached to the solid support. The arrangements used in the assays of the present invention typically comprise a plurality of different UU N antibodies ———— Ã & "&
Capture and / or concentrations of capture antibody that are coupled to the surface of a solid support in different known / addressable locations. The term "capture antibody" is intended to include an immobilized antibody that is specific to (i.e., binds, is bound by, or - forms a complex with) one or more analytes of interest in a sample, such as a cellular extract of leukocytes or rare circulating cells. In preferred embodiments, the capture antibody is maintained on a solid support in an array. Capture antibodies suitable for immobilizing any of a variety of oncogenic fusion proteins, or signal transduction molecules, on a solid support are available from Upstate (Temecula, CA), Biosource (Camarillo, CA), Cell Signaling Technologies (Danvers, MA), R&D Systems (Minneapolis, MN), Lab Vision (Fremont, CA), Santa Cruz Biotechnology (Santa Cruz, CA), Sigma (St.
“Louis, MO), and BD Biosciences (San Jose, CA). . The term "detection antibody", as used herein, includes an antibody comprising a detectable tag that is specific to (i.e., binds, is bound by, or forms a complex with) one or more analytes of interest in a sample. The term also includes an antibody that is specific for one or more analytes of interest, where the antibody can be linked by another species that comprises a detectable tag. Examples of detectable tags include, but are not limited to, biotin / streptavidin tags, “nucleic acid tags (e.g., oligonucleotide), chemically reactive tags, fluorescent tags, enzyme tags, radioactive tags and combinations thereof. Detection antibodies suitable for detecting the activation state and / or total amount of any of a variety of oncogenic fusion proteins, or signal transduction molecules, are available from Upstate (Temecula, CA), Biosource (Camarillo , CA), Cell Signaling Technologies
'CA), Santa Cruz Biotechnology (Santa Cruz, CA), Sigma (St. Louis, MO), and - BD Biosciences (San Jose, CA). As a non-limiting example, phospho-specific antibodies against various phosphorylated forms of signal transduction molecules, such as EGFR, c-KIT, c-Src, FLK-1, PDGFRA, —PDGFRB, Akt, MAPK, PTEN, Raf , and MEK, are available from Santa Cruz Biotechnology.
The term "activation-state-dependent antibody" includes a detection antibody that is specific for (i.e., binds to, is linked to, or forms a complex with) a particular activation state of one or more —analites of interest in a sample. In preferred embodiments, the activation-dependent antibody detects the phosphorylation, ubiquitination, and / or complexation state of one or more analytes, such as one or more oncogenic fusion proteins or signal transduction molecules. In some embodiments, phosphorylation of the ABL kinase domain of the BCR-ABL fusion protein is detected using an activation-dependent antibody. In other embodiments, the phosphorylation of elements of the EGFR family of the receptor tyrosine kinases, and / or the formation of heterodimeric complexes between elements of the EGFR family is detected using antibodies dependent on the activation state.
Non-limiting examples of activation states of oncogenic fusion proteins that are suitable for detection with antibodies dependent on the activation state include phosphorylated forms of BCR-ABL, DEK-CAN, E2A-PBX1, RARo-PML, IREL-URG, CBF / 5- MYHI11, AML1 -MTG8, EWS-FLI, LYT-10-Cald, HRX-ENL, HRX-AFA4, NPM-ALK, IGH-MYC, RUNXI-ETO, TEL-TRKC, TEL-AML1I, MLL-AF4 , TCR-RBTN2, COLIAI-PDGF, E2A-HLF, PAX3-FKHR, ETV6-NTRK3, RET-PTC, TMRSS-ERG and TPR-MET. Examples of activation states (listed in parentheses) of signal transduction molecules that
1 activation include, but are not limited to, EGFR (EGFRvIII, phosphorylated (p-) - EGFR, EGFR: Shc, ubiquitinated (u-) EGFR, p-EGFRvIII); ErbB2 (truncated p95 (Tr) -ErbB2, p-ErbB2, p95: Tr-p-ErbB2, HER-2: Shc, ErbB2: PI3K, ErbB2: EGFR, ErbB2: ErbB3, ErbB2: ErbB4); ErbB3 (p-ErbB3, ErbB3: PI3K, - p-ErbB3: PIK, ErbB3: Shc); ErbB4 (p-ErbB4, ErbB4: Sho); c-Met (pc-Met or c-Met / HGF complex), ER (p-ER (S118, S167); IGF-IR (p-IGF-IR, IGF-IRIRS, IRS: PIBK, p-IRS, IGF -IR: PBKO; INSR (p-INSR); KIT (p-KIT); FLT3 (p-FLT3); HGFRI (p-HGFRID); HGFR2 (p-HGFR2); RET (p-RET); PDGFRa (p -PDGFRa); PDGFRP (p-PDGFRP); VEGFRI (p-VEGFRI, VEGFRLPLCg, VEGFRISrc); VEGFR2 (p-VEGFR2, VEGFR2: PLCy, VEGFR2: Src, VEGFR2: VEGFR2: VEGFR2: VEGFR2: hepar sulfate); (p-VEGFR3); FGFR1 (p-FGFR1); FGFR2 (p-FGFR2); FGFR3 (p-FGFR3); FGFR4 (p-FGFR4); Tiel (p-Tiel); Tie2 (p-Tie2); EphA ( p-EphA);. EphB (p-EphB); NFKB and / or IKB (p-IK (S32), p-NFKB (S536), p-P65: IKBa); 15th Akt (p-Akt (T308, S473 )); PTEN (p-PTEN); Bad (p-Bad (SI 12, S136), Bad: '14-3-3); mTor (p-mTor (S2448)); p70S6K (p-p70S6K (T229, T389)); Mek (p-Mek (S217, S221)); Erk (p-Erk (T202, Y204)); Rsk-1 (p-Rsk-1 (T357, S $ 363)); Ink (p-Jnk (T183, Y185)); P38 (p-P38 (T180, Y182)); Stat3 (p-Stat-3 (Y705, S727)); Fak (p-Fak (Y576)); Rb (p-Rb (S249 , T252, S780)); Ki67; p53 (p-p53 (S392, S20)); CREB (p-CREB (SI 33)); c-Jun (p-c-Jun (S63)); cSrce (p-cSre (Y416)); and paxiline (p-paxiline (Y118)).
The term "activation state independent antibody" includes a detection antibody that is specific for (i.e., binds, is bound by, or forms a complex with) one or more analytes of interest in a sample, regardless of their activation state. For example, the antibody independent of the activation state can detect both phosphorylated and non-phosphorylated forms of one or more analytes, such as one or more oncogenic fusion proteins or signal transduction molecules.
The term "nucleic acid" or "polynucleotide" includes D deoxyribonucleotides or ribonucleotides, and polymers thereof, both in the form of single strand and double strand such as, for example, DNA and RNA. Nucleic acids include nucleic acids that contain known —nucleotide analogs, or modified main part residues or bonds, which are synthetic, naturally occurring and non-naturally occurring, and which have similar binding properties to the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O- methyl ribonucleotides and peptide nucleic acids (PNAs). Unless specifically limited, the term includes nucleic acids that contain known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants of this and complementary sequences, as well as the sequence explicitly indicated.
The term "oligonucleotide" refers to a single-stranded oligomer or polymer of RNA, DNA, RNA / DNA hybrid and / or a mimetic thereof. In certain examples, oligonucleotides are composed of naturally occurring (ie, unmodified) nitrogenous bases, sugars and nucleoside bonds (main part). In certain other examples, oligonucleotides comprise modified nitrogenous bases, sugars and / or linkages between nucleosides. As used herein, the term "imperfect matching pattern" or "imperfect matching region" refers to a portion of an oligonucleotide that does not show 100% complementarity with its complementary sequence. An oligonucleotide can have at least one, two, three, four, five, six, or more regions of imperfect pairing. At
'by 1, 2, 3, 4,5,6,7,8,9, 10, 11, 12, or more nucleotides. Imperfectly matched motifs or regions may comprise a single nucleotide or may comprise two, three, four, five, or more nucleotides. The phrase "restrictive hybridization conditions" refers to - conditions under which an oligonucleotide will hybridize in its complementary sequence, but not in other sequences. The restrictive conditions are sequence dependent and will be different in different circumstances. Longer strings hybridize specifically at higher temperatures. An extensive roadmap for hybridizing nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). In general, restrictive conditions are selected to be about 5-10 ºC lower than the. thermal melting (Tm) for the specific sequence at a defined ionic concentration pH. T ,, is the temperature (in defined ionic concentration, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the equilibrium target sequence (as the target sequences are present in excess, in T ,, 50 % of the probes are occupied in equilibrium). Restrictive conditions can also be achieved with the 20th addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive sign has at least twice the background hybridization, preferably 10 times the background hybridization.
The terms "substantially identical" or "identity - substantial", in the context of two or more nucleic acids, refer to two or more sequences or subsequences that are the same, or have a specific percentage of nucleotides that are the same (ie is at least about 60%, preferably at least about 65%, 70%, 75%, 80%,
j compared and aligned for maximum correspondence with a comparison window, or determined region, measured using a comparison sequence algorithm or by manual alignment and visual inspection. This definition, when indicated by the context, also refers analogously to the complement of a sequence. Preferably, the substantial identity exists in a region that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides in size. The term "tyrosine kinase inhibitor" includes any of a variety of therapeutic agents or medicaments, which act as selective or non-selective inhibitors of receptor and / or non-receptor tyrosine kinases. Without being bound by any particular theory, tyrosine kinase inhibitors generally inhibit the tyrosine kinase target by binding to the enzyme's ATP binding site. Examples of tyrosine kinase inhibitors include, but are not limited to, imatinib (Gleevec *; STIS71), nilotinib (Tasigna ”), dasatinib (Sprycel ), Bosutinib (SKI-606), gefitinib (ressa”), sunitinib (Sutentº ; SU11248), erlotinib (Tarceva ”; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SUS416), vatalanib (PTK787 / ZK222584), sorafenib (BAY 43-9006), leflunomom ! 10O1), vandetanib (Zactima'M; ZD6474), derivatives thereof, analogs thereof and combinations thereof. Additional tyrosine kinase inhibitors suitable for use in the present invention are described in, for example, U.S. patents 5,618,829, 5,639,757, 5,728,868, 5,804,396, 6,100,254,
6,127,374, 6,245,759, 6,306,874, 6,313,138, 6,316,444, 6,329,380, 6,344,459,
6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424,
6,740,665, 6,794,393, 6,875,767, 6,927,293 and 6,958,340. Those skilled in the art will know of other tyrosine kinase inhibitors suitable for use in the present invention. In certain examples, the tyrosine kinase inhibitor is administered in a pharmaceutically acceptable form including, without
, aluminum, calcium, lithium, magnesium, potassium, sodium or zinc; an ammonium salt - such as a tertiary amine or quaternary ammonium salt, and an acidic salt such as a succinate, tartrate, bitartrate, dihydrochloride, salicylate, hemisuccinate, citrate, isocitrate, malate, maleate, mesylate, hydrochloride salt - hydrobromide, phosphate, acetate, carbamate, sulfate, nitrate, format, lactate, gluconate, glucuronate, pyruvate, oxalacetate, fumarate, propionate, aspartate, glutamate or benzoate.
The term "incubate" is used interchangeably with "put in touch" and "expose" and does not imply any specific period or temperature requirements, unless otherwise indicated.
The terms "complete cytogenetic response", "complete cytogenetic remission", "CCyR" and "CCgR" include the clinically accepted criteria, defined to be in the absence of cells positive for chromosome - Philadelphia in metaphase, among a population of at least 20 cells in —metaphase, in the manner determined by chromosomal banding of Î ± cells isolated from bone marrow. In certain instances, when the metaphase of cells isolated from bone marrow cannot be obtained or evaluated by chromosomal banding, the term can be defined as the presence of <1% positive nuclei for BCR-ABL, from those of at least 200 nuclei classified in the manner determined by fluorescent in situ hybridization of interphase (FISH) blood cells. Interphase FISH can be performed, for example, with BCR-ABL extra signal, double color, double fusion, or in situ hybridization probes. For further descriptions of these terms, see, for example, O'Brien et al., N. Engl. J. Med., 348: 994-1004 - (2003); Hughes et al., N. Eng.U. Med., 349: 1423-1432 (2003); and Bacarani et al., J. Clin. Oncol, 27: 6041-6051 (2009).
A "major molecular response", "major molecular remission" or "MMR" is achieved when the level of a fusion protein
"with respect to one or more levels of control protein, such as full size BCR and / or full size GLA.
In certain embodiments, a major molecular response is achieved when the ratio of oncogenic fusion protein levels (eg, BCR-ABL levels) to control protein levels (eg, BCR or ABL levels), during therapy with anti-cancer medication (eg, tyrosine kinase inhibitor), is reduced by at least about 2-3 logs with respect to the ratio of the same proteins prior to therapy with anti-cancer medication.
Unlike the definitions of main molecular response that depend on the detection of transcribed levels of mRNA, the antibody-based double proximity detection assays of the present invention advantageously provide the ability to detect a cancer cell (for example, CML) in the medium of about 100,000 cells from a healthy donor, thereby enabling the determination of a major molecular response by measuring and comparing protein levels of BCR-ABL, BCR, and / or ABL.
In other modalities, a major molecular response is a clinical classification, defined as a reduction of at least 3-logs below a standardized baseline value on a logarithmic scale (base 10), in the ratio of BCR mRNA transcripts. -ABL for both GLA and BCR mRNA transcripts, expressed as a percentage of the levels of ABL or BCR mRNA transcripts.
In certain examples, the level of mRNA transcripts is determined using real-time quantitative RT-PCR (qCPR) methods. In additional modalities, a major molecular response is the condition when the ratio of BCR-ABL to ABL mRNA transcripts, —BCRor control is determined by PCRq to be a value defined as <0.1% on the international scale (IS). IS is anchored on the profile reduction scale established by the laboratories participating in the International Randomized Study of Interferon versus STIS71 (IRIS) clinical study.
See, for
"al., Sangue, 108: 28-37 (2006), Brand et al., Sangue, 112: 3330-3338 (2008); and
- Baccarani et al., J.
Clin.
Oncol, 27: 6041-6051 (2009). A "complete molecular response", "complete molecular remission" or "CMR" is achieved when the level of an oncogenic fusion protein, such as BCR-ABL, decreases by at least about 3-4 logs with respect to one or more more levels of control protein, such as full size BCR and / or full size GLA.
In certain embodiments, a complete molecular response is achieved when the ratio of oncogenic fusion protein levels (eg, BCR-GLA levels) to protein control levels (eg, BCR or GLA levels), during medication anti-cancer therapy, is reduced by at least about 3-4 logs with respect to the ratio of the same proteins prior to therapy with anti-cancer medication (eg, tyrosine kinase inhibitor). Unlike the definitions of - complete molecular response, which depend on the detection of transcribed levels of mRNA, the antibody-based double proximity detection assays of the present invention advantageously provide the ability to detect a cancer cell (for example, CML) in the through approximately 1,000,000 cells from a healthy donor, thereby enabling the determination of a complete molecular response by measuring and comparing protein levels of —BCR-ABL, BCR, and / or ABL.
In other modalities, a complete molecular response is a clinical classification in which the BCR-ABL mRNA transcripts are not detectable by PCRqg, and / or nest PCR, in at least two consecutive samples of blood of adequate quality, in order to ensure the ability to detect a 4.0-4.5-log drop in BCR-ABL mRNA levels.
In certain examples, a complete molecular response can be defined as a reduction of at least 4.5-logs below a standardized baseline value on a logarithmic scale in the BCR-ABL ratio for ABL, BCR or mRNA mRNA transcripts. control, expressed n (2006); Muller et al., Leukemia., 23: 1957-1963 (2009); and Baccarani et al., J. r Clin.
Oncol., 27: 6041-6051 (2009). The term "period of therapy" includes any therapeutic approach taken to relieve or prevent one or more symptoms associated with cancer, such as a hematological malignancy (for example, leukemia, lymphoma, etc.). The term includes administering any compound, medication, procedure and / or regimen used to improve the health of an individual with cancer, and includes any of the therapeutic agents described herein.
Those skilled in the art will understand that both the period of therapy and the dose of the current therapy period can be changed (for example, increased or decreased), based on the expression and / or activation levels of one or more oncogenic fusion proteins and / or signal transduction molecules, determined using the methods of the present invention. . II.
Description of modalities
The present invention provides antibody-based arrangements for detecting the activation state, and / or the total amount of one or more oncogenic fusion proteins and / or signal transduction molecules in a biological sample, such as an extract or cell lysate .
The present invention also provides methods of using such arrangements to facilitate cancer prognosis and diagnosis, the prediction or identification of resistance to drug treatment, and the determination of targeted and personalized therapies.
In particular embodiments, the compositions and methods of the present invention advantageously identify patients who are resistant to therapy with a tyrosine kinase inhibitor, such as imatinib, due to mutations in the - target protein kinase (e.g., BCR-ABL), non-suitability therapeutic regimen, and / or administration of a dose of subideal medication.
In a particular embodiment, the present invention provides assays, such as, for example, immunoassays, for real-time detection
BCR-ABL, substrates of these and / or other signal transduction molecules in a biological sample, such as an extract or cell lysate. As such, the present invention advantageously provides benefits to patients with hematological malignancies, such as CML, who receive one or more targeted therapies, selecting and monitoring them for the duration of the therapy and assessing whether they can be changed to a targeted therapy. alternative such as, for example, nilotinib (Tasigna ), to efficiently inhibit the target molecule (eg, BCR-ABL) with minimal toxicity.
In certain embodiments, the present invention provides a method with a higher sensitivity range for detecting oncogenic fusion proteins, such as BCR-ABL. Current immunoassays to detect BCR-ABL proteins in cell extracts can detect, in general, - a BCR-ABL positive leukemic cell in 10-100,000 normal cells, equivalent to a detection sensitivity of 10-0.001% (see, for example, Jilani et al., Leuk. Res., 32: 936-943 (2008); Weerkamp et al., Leukemia, 23: 1 106-1117 (2009); Raponi et al., Haematologica, 94: 1767-1770 (2009); and US patent publication 2006/0172345). The proximity assays described here advantageously exhibit greater sensitivity in about 1: 100,000-10,000,000 cells (ie, a leukemic cell for about 100,000-10,000,000 normal cells; equivalent to a detection sensitivity of about 0.001- 0.00001%) or about 1: 1,000,000-10,000,000 cells (that is, a leukemic cell for about
1,000,000-10,000,000 normal cells; equivalent to a detection sensitivity of about 0.0001-0.00001%) and include, for example, about 1: 100,000 cells, 1: 200,000 cells, 1: 300,000 cells, 1: 400,000 cells, 1: 500,000 cells, 1: 600,000 cells, 1: 700,000 cells, 1: 800,000 cells, 1: 900,000 cells, 1: 1,000,000 cells, 1: 2,000,000 cells, 1: 3,000,000
1: 7,000,000 cells, 1: 8,000,000 cells, 1: 9,000,000 cells, 1: 10,000,000 cells, 1: 100,000-500,000 cells, 1: 100,000-1,000,000 cells, 1: 500,000-
1,000,000 cells, 1: 100,000-5,000,000 cells, 1: 500,000-10,000,000 cells, 1: 2,000,000-10,000,000 cells, 1: 5,000,000-10,000,000 cells, 1: 1,000,000 -7,500,000 cells, 1: 1,000,000-5,000,000 cells, and any other ranges here. The sensitivity of the methods described here can be comparable to or exceed that of standard BCR-ABL assays based on nucleic acid (eg, PCRq or nest PCR), which is in the detection range of 1 leukemic cell to 10,000-1,000. 000 normal cells. For additional descriptions of the sensitivity range of the BCR-ABL assays based on nucleic acid, see, for example, Press et al., Blood, 107: 4250-4256 (2006) and Radish JP, Blood, 114: 3376-3381 (2009).
In particular embodiments, the antibody-based proximity assays of the present invention advantageously enable a greater degree of sensitivity and / or specificity in detecting the presence, level, and / or activation status of oncogenic fusion proteins, such as BCR- ABL, compared to current immunoassays and nucleic acid-based assays to detect BCR-ABL, thereby providing a more accurate determination of response indicators, for example, such as a 20 response: complete cytogenetics, a major molecular response, a complete molecular response and combinations of these. As a non-limiting example, current nucleic acid assays are not sensitive enough to detect very small amounts of BCR-ABL transcripts in a patient sample, such that a determination of a complete molecular response using current nucleic acid assays, does not necessarily mean that the patient is cured and no longer has BCR-ABL-mediated disease (eg, CML).
In one aspect, the present invention provides a method for determining the level or state of activation of an oncogenic fusion protein, the method comprising: (a) contacting a cell extract with a first specific binding fraction for a first domain of a first full-size protein, in conditions suitable for transforming the first full-size protein present in the cell extract into a complex comprising the first full-size protein and the first binding fraction, wherein the first domain of the first size protein total does not have a corresponding oncogenic fusion protein, which comprises a second domain different from the first full size protein fused to a first domain of a second different total size protein; . (b) removing the complex from step (a), from the extract! 15 cell, to form a cell extract devoid of the first full-size protein; (c) placing the cell extract from step (b) in contact with a second fraction of specific binding for the second domain different from the first full-size protein, under conditions suitable for transforming the oncogenic fusion protein present in the cell extract into a complex which comprises the oncogenic fusion protein and the second binding fraction; and (d) determining the level or state of activation of the complex of step (c), thereby determining the level or state of activation of the oncogenic fusion protein.
In one embodiment, the cell extract comprises an extract of cells isolated from a sample. In certain examples, the sample is selected from whole blood, serum, plasma, fine needle aspirated breast, lymph, saliva and combinations thereof. In another modality, the sample is obtained from a cancer patient. In some instances, cancer can be caused by the formation of an oncogenic fusion protein due to a chromosomal translocation in the cancer cells. - Examples of such cancers include, but are not limited to, hematological malignancy, osteogenic sarcoma, soft tissue sarcoma and combinations thereof. In particular modalities, hematological malignancy is leukemia or lymphoma. In a preferred embodiment, leukemia is chronic myelogenous leukemia (CML). In another embodiment, isolated cells, from which the cell extract or lysate is prepared, may comprise circulating tumor cells, leukocytes, or combinations thereof. In certain embodiments, isolated cells are stimulated in vitro with growth factors. In some instances, the isolated cells are - incubated with an anti-cancer drug before stimulation with growth factor. In other examples, isolated cells are lysed after growth factor stimulation to produce the cell extract.
In some embodiments, the cell extract is prepared from samples of bone marrow or whole blood collected fresh or frozen. As a non-limiting example, a whole blood sample treated with anticoagulants (for example, EDTA, heparin and / or acid-citrate-dextrose (ACD)) is first separated into a plasma or serum fraction and a cell fraction. The cell fraction can be processed by hypotonic lysis of red blood cells with ammonium chloride, and / or centrifugation by Ficoll-HyPaque density gradient, to isolate - leukocytes from a blood sample. Isolated cells, present in the cell fraction, can be lysed to thereby transform the isolated cells into a cell extract by any technique known in the art, such as those described in Raponi et al., LeukRes, 32: 923-43
(2008); Weerkamp et al., Leukemia, 23: 1106-1117 (2009) and in U.S. patents
6,610,498 and 6,686,165. In some instances, isolated leukocytes can be treated with one or more permeable protease inhibitors in the cell prior to lysis. Cell permeable protease inhibitors include, but are not limited to, diisopropyl fluorophosphate (DFP), 4- (2-aminoethyl) benzenesulfonyl fluoride (AEBSF), phenylmethanesulfonyl fluoride (PMSF) and mixtures thereof. As a non-limiting example, isolated leukocytes are incubated for 10-30 minutes on ice in a buffer containing 20mM AEBSF and PMSF ImMM in PBS. The cells are gently centrifuged for 5 minutes, at 520g, at 4 ºC, to separate the supernatant and the isolated leukocytes. The treatment of isolated leukocytes with protease inhibitors is described, for example, in Weerkamp et al., Leukemia, 23: 1106-. 1117 (2009).
In some instances, isolated leukocytes can be incubated for up to 30 minutes on ice in RIPA lysis buffer that contains one or more protease inhibitors. The RIPA buffer essentially comprises or consists of 50 mM Tris HCl, pH7.5, 150 mM NaCl, 1% NP40 and 0.1% sodium dodecyl sulfate. In other certain examples, the RIPA buffer does not contain sodium deoxycholate. After incubation, the cell mixture is centrifuged at> 18,000g for 1-10 minutes, at 4 ° C, to separate the cell extract and cell debris. The cell extract is collected. For additional descriptions of cell lysis protocols, see, for example, Raponi et al., Haematologica, 94: 1767-1770 (2009); Weerkamp et al., Leukemia, 23: 1106-1117 (2009); and U.S. patent publication US 2006/0172345. In some embodiments, plasma is prepared from samples of fresh peripheral whole blood collected and treated with one or more anticoagulants (eg EDTA, heparin or ACD), as described limiting, blood samples can be separated in a fraction of plasma or serum, and a cellular fraction. Plasma can be stored at -70-80 ºC until tested, or tested in 96 hours of blood sample collection.
In certain embodiments, the oncogenic fusion protein is - selected from the group consisting of BCR-ABL, DEK-CAN, E2A-PBXI1, RARo-PML, IREL-URG, CBFB-MYH11, AMLI-MTG8, EWS-FLI, LYT - 10-Cal, HRX-ENL, HRX-AF4, NPM-ALK, IGH-MYC, RUNX1I-ETO, TEL-TRKC, TEL-AML1, MLL-AF4, TCR-RBTN2, COLIA1-PDGF, E2A-HLF, PAX3 -FKHR, ETV6-NTRK3, RET-PIC, TMRSS-ERG, TPR-MET and combinations thereof. In particular modalities, the oncogenic fusion protein is BCR-ABL. In certain examples, the first full size protein is BCR, the first domain of the first full size protein comprises the BCR terminal carboxyl region (BCR-C), and the second. different domain from the first full size protein comprises the 215 amino terminal region of BCR (BCR-N). In other certain examples, the second protein of different total size is ABL, the first domain of the second protein of different total size comprises the terminal carboxyl region of ABL (ABL-C), and the second different domain of the second protein of different total size comprises the amino terminal region of ABL (ABL- * N). In an alternative embodiment, the first full size protein is GLA and the second different full size protein is BCR.
In some embodiments, the activation state is selected from the group consisting of a phosphorylation state, a state of ubiquitination, a state of complexation and combinations thereof. In a preferred embodiment, the oncogenic fusion protein is BCR-ABL and the activation state is a phosphorylation state.
In other embodiments, the test methods of the present invention comprise further determining the level or state of particulars, to one or more signal transduction molecules comprising a BCR-ABL substrate such as, for example, CRKL, JAK2, STATS, Srco , FAK, c-ABL, c-CBL, SHC, SHP-2, VAV, BAP-1I and combinations thereof.
In a particular embodiment, the first binding moiety comprises a first antibody. In some examples, the first antibody is affixed to a solid support. Non-limiting examples of solid supports include glass, plastic, chips, pins, filters, beads, paper, membrane, bundles of fibers and combinations thereof. In preferred embodiments, the first antibody is affixed to a bead (for example, magnetic bead, polystyrene bead, etc.), and the bead functions as a diminishing label to remove the first full-size protein from the cell extract.
"In another particular embodiment, the second binding fraction comprises a second antibody. In some instances, the first antibody is affixed to a solid support. Non-limiting examples of solid support include glass, plastic, chips, pins, filters , beads, paper, membrane, bundles of fibers and combinations thereof In a preferred embodiment, the second antibody is maintained on a solid support, such as a - membrane (for example, nylon, nitrocellulose, PVDF, etc.), in another direction, the second antibody is affixed to a bead (for example, magnetic bead, polystyrene bead, etc.), where the bead can optionally contain a dye such as a fluorophore (for example, a colored bead) In examples where a plurality of beads - is used, each bead may contain an independently selected dye, such as a fluorophore (for example, a red or infrared fluorophore) of different intensities or with different excitation and / or emission spectra.
In preferred embodiments, steps (c) and (d) comprise a double proximity detection assay (also known as a collaborative proximity immunoassay ("COPIA")), in the manner described herein. In other embodiments, steps (c) and (d) comprise an enzyme-linked immunosorbent assay (ELISA), a flow cytometry assay, or a label selection assay as described herein.
In modalities where steps (c) and (d) comprise a double proximity detection assay, step (c) may additionally comprise: (c) placing the cell extract of step (b) in contact with a third bond fraction and a fourth binding fraction under suitable conditions to transform the oncogenic fusion protein, present in the cell extract, into a complex comprising the fusion protein - oncogenic and the second, third and fourth binding fractions: 15 where the third binding fraction is marked with a facilitation fraction and is specific for (for example, it specifically binds to) one or more epitopes present in the following domains or sequences: (1) the first domain of the second protein of different total size; (11) the second domain is different from the first full-size protein; or (iii) the fusion site between the second different domain of the first full size protein and the first domain of the second different full size protein, where the fourth binding moiety is marked with a first element of an amplification pair signal, and is specific to the first domain of the second protein of different total size, and in which the facilitation fraction generates an oxidizing agent that channels and reacts with the first element of the signal amplification pair.
In modalities where steps (c) and (d) comprise a double proximity detection test, step (d) can additionally comprise:
(d) incubating the complex of step (c ') with a second element of the signal amplification pair to generate an amplified signal, and (d ") detecting the amplified signal generated from the first and second elements of the amplification pair of signal.
In certain embodiments, the cell extract of step (b) can be brought into contact with a serial dilution of the second binding fraction to form a plurality of complexes comprising the oncogenic fusion protein and the second binding fraction. In some embodiments, the third and fourth linker fractions may comprise the third and fourth antibodies, respectively. In some examples, the third and fourth antibodies are both antibodies independent of the activation state. In such examples, the amplified signal generated from the first and second elements of the signal amplification pair is correlated with the total amount of the oncogenic fusion protein. In other examples, the third l15 antibody is an antibody independent of the activation state and the fourth antibody is an antibody dependent on the activation state. In such examples, the amplified signal generated from the first and second elements of the signal amplification pair are correlated with the amount of activated oncogenic fusion protein (e.g., phosphorylated).
In other embodiments, the third link fraction can be directly marked with the facilitation fraction. In yet other modalities, the fourth link fraction is marked directly with the first element of the signal amplification pair. In alternative embodiments, the fourth linkage fraction is marked with the first element of the —part of signal amplification by means of a link between a first element of a conjugated binding pair in the second detection antibody, and a second element of the conjugated binding pair in the first element of the signal amplification pair. In these modalities, the first element of the pair of
In additional modalities, the facilitation fraction is glucose oxidase.
In certain examples, glucose oxidase and the third binding moiety are conjugated to a sulfhydryl-activated dextran molecule.
In such examples, the sulfhydryl-activated dextran molecule has a weight
- molecular of about 500kDa.
In other examples, the oxidizing agent is hydrogen peroxide (HO). In such examples, the first element of the signal amplification pair is a peroxidase such as, for example, horseradish peroxidase (HRP), and / or the second element of the signal amplification pair is a tyramide reagent such as, for example, example, biotin-tiramide.
In particular embodiments, the amplified signal is generated by oxidation of biotin-tiramide peroxidase to produce an activated tiramide.
In certain examples, the activated tiramide is detected directly.
In other certain examples, activated tiramide is detected by adding a reagent of
- signal detection.
Non-limiting examples of E 15 detection reagent include a streptavidin-labeled fluorophore, and a combination of a streptavidin-labeled peroxidase and a chromogenic reagent, such as, for example, 3,3 ', 5,5'-tetramethylbenzidine (TMB). In certain embodiments, the test methods of the present invention further comprise:
(e) placing the cell extract in contact with a fifth specific binding fraction for a second domain of the second protein of different total size, under conditions suitable to transform the second protein of different total size present in the cell extract into a complex comprising the second protein of different total size and the
- fifth binding fraction, in which the second domain of the second protein of different total size is not present in the oncogenic fusion protein; and
(D remove the complex from step (e), from the cell extract, to form a cell extract devoid of the second full-length protein Fr Aiferente; —ó OO OD »O in which step (e) is performed before, during, or after step (a). In a particular embodiment, the fifth binding fraction comprises a fifth antibody. In some examples, the fifth antibody is affixed to a solid support. Non-limiting examples of solid support - include glass, plastic, chips, pins, filters, beads, paper, membrane, bundles of fibers and combinations thereof. In preferred embodiments, the fifth antibody is affixed to a bead (for example, magnetic bead, polystyrene bead, etc.), and the bead functions as a decrease label to remove the second protein of a different total size from the cell extract.
Figure 3A illustrates an exemplary proximity assay (300) to detect the presence (total level) and / or activation state (phosphorylation level) of an oncogenic fusion protein, such as BCR-ABL (310). The oncogenic fusion protein encoded by the chimeric BCR- - ABL gene varies in size, depending on the break point in the BCR gene. The BCR-ABL fusion gene is generated by the reciprocal translocation of the ABL gene | located on the long arm of chromosome 9, and the BCR gene located on the long arm of chromosome 22, resulting in the oncogenic fusion gene on chromosome 22q9-, also referred to as the Philadelphia chromosome. See, for example, Kurzock et al., N. Engl. J. Med. 319: 990-8 (1988); Rosenberg et al.,
20. Adv. Hn Virus Res. 35: 39-81 (1988). Depending on the chromosomal breakpoints that translate the ABL gene to the N termination of the BCR gene, different sizes of the BCR-ABL fusion genes are formed. It is determined that the break points in the ABL gene are distributed over approximately 200kb between exons 1b and a2, and the break points of the gene —BCR are grouped in the three regions; the major break point (M-BCR) between exons 13-15 (b2-b4); the smallest break point (m-BCR) between alternative exons 1 and 2 (el and e2); and the micro break point (mu-BCR) at intron 19 (el9). Thus, depending on the rearrangement of the BCR-ABL gene, exclusive BCL-ABL proteins are formed. See, for example, Konopka et al., Cell 37: 1035-42 (1984); van Dongen, IIM, Leukemia 19: 1292-5 (2005). The p210 BCR-ABL protein is generated from b3a2 (el4a2) and / or b2a2 (el3a2) gene transcripts, which are detected in more than 95% - of CML cases and a subset of ALL. This protein contains 1,790 amino acids and is composed of a BCR oligomerization domain (OLI) at the N-terminus, followed by the BCR S / T kinase domain, a BCR amino acid sequence insert that is not present in normal BCR, followed by SH3, SH2 and Y kinase domains of GLA domains, as well as the C-terminal proline-rich GLA domain. The p190 BCR-ABL fusion protein is encoded by the Ela2 fusion gene transcript, which is mainly associated with Ph positive ALL. The rare cases of CML are due to a translocation of the p190 type BCR-ABL gene, and - in these, the disease tends to have a prominent monocytic component,: 15 which resembles chronic myelomonocytic leukemia (CMML). The p230 BCR-ABL protein is formed from the el9a2 gene transcript that is associated with neutrophilic CML, classical CML and AML. See, for example, Konopka et al., Cell 37: 1035-42 (1984); van Dongen, JJM, Leukemia 19: 1292-5 (2005). The exceptional cases of CML have been described with the
20. BCR break points outside the three regions of defined grouping, or with unusual break points in GLA (see, for example, Melo, Baillieres Clin. Haematol., 10: 203-22 (1997)). The proximity assay described here is capable of detecting both the total and activated levels of a BCR-ABL protein encoded by a chimeric BCR-ABL gene, with a breakpoint anywhere in the BCR gene.
As shown in figure 3A, the full-size BCR protein can first be removed from a patient sample using a specific decrease label for the BCR terminal carboxyl region of how this is an account, in which an antibody specific to the region full size BCR terminal carboxyl is affixed (321b). Once the full-size BCR is removed from the sample, a capture antibody specific for the amino terminal region of BCR (BCR-N) is used to capture the BCR-ABL fusion protein. Since BCR-ABL is captured by binding between BCR-N and the specific capture antibody for BCR-N, the total concentration of BCR-ABL is determined (340), and activated BCR-ABL (350) is also determined is measured. In some embodiments, the facilitation fraction (for example, GO) is coupled to an antibody specific for the GLA terminal carboxyl region (ABL-C), and the first element of the signal amplification pair (for example, HRP) is coupled to a specific antibody for ABL-C in an epitope different from that recognized by the antibody coupled to the facilitation fraction. In certain examples, the antibody coupled to the signal amplification pair is an antibody independent of the activation state that binds to ABL-C, regardless of its activation state, and thereby measures the total concentration of BCR-ABL ( 340). In other certain examples, the antibody coupled to the signal amplification pair is an activation-dependent antibody that binds to phospho-ABL-C (for example, pY245, pY412), and thereby measures the concentration of BCR- GLA activated (350).
20. In an alternative embodiment, the facilitation fraction (eg, GO) is coupled to a specific antibody to BCR-N in an epitope other than that recognized by the BCR-ABL capture antibody, and the first element of the amplification pair signal (for example, HRP) is coupled to an antibody specific to ABL-C. The antibody coupled to the signal amplification pair can be an antibody independent of the activation state, or an antibody dependent on the activation state in the manner described herein. In some embodiments, the patient's full-size ABL protein before capturing and detecting the expression and / or activation of BCR-ABL using a specific decrease label for the full-length ABL amino terminal region (330). A non-limiting example of a decrease label like this is an account in which an antibody specific for the full-length GLA amino terminal region is affixed (321a). In these modalities, once BCR-ABL is captured by binding between ABL-C and a specific capture antibody for ABL-C, the total concentration of BCR-ABL is determined (360), and the activated BCR-ABL ( 370) is also measured. In some embodiments, the facilitation fraction (for example, GO) is coupled to an antibody specific for ABL-C in an epitope different from that recognized by the capture antibody BCR-ABL, and the first element of the signal amplification stop (for example, example, HRP) is coupled to a specific antibody to BCR-N. In certain examples, the antibody coupled to the facilitation fraction is an antibody independent of the activation state that binds to ABL-C, regardless of its activation state,: and thereby measures the total concentration of BCR-ABL (360 ). In other certain examples, the antibody coupled to the facilitation fraction is an activation-dependent antibody that binds to phospho-ABL-C (eg, pY 245, pY412) and thereby measures the concentration of activated BCR-ABL (370).
In modalities where the facilitation fraction is GO and the first element of the signal amplification pair is HRP, the binding of both the GO-coupled antibody and the HRP-coupled antibody to the BCR-ABL fusion protein brings the GO fraction close enough with the HRP fraction, in such a way that a signal generated by the GO fraction (ie, H7O,) can —channel into the HRP fraction, resulting in the generation of a detectable and / or amplifiable signal. An advantage of channeling by proximity, as used in the methods described here, is that a simple detectable signal that is correlated with the levels of total or activated BCR-ABL protein is capture, antibody coupled to the facilitation fraction, and antibody coupled to the + signal amplification pair), resulting in less specificity of the assay, less foundation and simplified detection.
An alternative modality (400) for detecting the presence - (total level) and / or activation state (phosphorylation level) of BCR-ABL (410) using binding antibodies is shown in figure 3B. Again, the beads (421a and / or 421b) are used to remove full-size BCR and / or GLA proteins, for example, by their carboxyl and amino terminations, respectively. Next, by capturing the N-terminal portion of BCR-ABL using a specific binding fraction immobilized on a solid support, the total concentration of BCR-ABL is determined (440), and the activated BCR-ABL (450) is also determined. measure. Total BCR-ABL (460) and activated protein (470) can also be determined by capturing ABL-C, either using a - binding antibody to measure total BCR-ABL levels, or using a specific antibody for ABL phosphorylation to measure the levels of activated BCR-GLA.
In particular, Figure 3B (440) illustrates that the total amount of BCR-ABL present in a biological sample, such as serum, can be detected by placing the captured analyte in contact with (1) a first detection antibody (i.e., binding antibody), specific for the site or melting point between the BCR amino terminal region (BCR-N) and the ABL terminal carboxyl region (ABL-O), and (ii) a second ABL specific detection antibody -Ç. Both the first and second detection antibodies bind to BCR-ABL regardless of their activation state. The first detection antibody (i.e., binding antibody) is labeled with glucose oxidase (GO), and the second detection antibody is labeled with horseradish peroxidase (HRP). The binding of both the first and the second detection antibody to the BCR-ABL fusion protein brings the fraction i and “the signal generated by the GO fraction (ie, H; O7) can channel the HRP fraction, ——— - resulting in generation one detectable and / or amplifiable signal.
Figure 3B (450) further illustrates that the amount of activated BCR-ABL present in a biological sample, such as serum, can be detected by placing the captured analyte in contact with (1) a first detection antibody (ie, binding antibody), specific for the site or melting point between BCR-N and ABL-C, and (li) a second detection antibody specific for an activated (e.g., phosphorylated) form of BCR-ABL. detection antibody binds to BCR-ABL, regardless of its activation state, while the second detection antibody binds to an activation site (eg, phosphorylation) present in the ABL-C domain of the fusion protein.
The first detection antibody (ie, binding antibody) is labeled with glucose oxidase (GO), and the second - detection antibody is labeled with horseradish peroxidase (HRP). At the 15th connection, both the first and the second detection antibody with the BCR-ABL fusion protein brings the GO fraction in sufficient proximity to the HRP fraction, in such a way that a signal generated by the GO fraction (H2O>,) can be channeled into the fraction HRP, resulting in the generation of a detectable and / or amplifiable signal.
In embodiments where steps (c) and (d) comprise an - ELISA, the ELISA may comprise a sandwich ELISA.
Any suitable antibody pair can be used for the capture and detection of antibodies in a sandwich ELISA.
Those skilled in the art will know and understand how to select an appropriate antibody pair for the assay.
In general, two antibodies that are selected bind to the target of interest, for example, —BCR-ABL, in different epitopes, in such a way that the binding of the first antibody (capture) does not interfere with the second (detection) antibody. In preferred embodiments, the first antibody (capture) binds to the second domain other than the first full-size protein (for example, BCR-
; second protein of different total size (eg ABL-C). In - certain embodiments, the detection antibody will be conjugated to an enzyme, for example, horseradish peroxidase (HRP) or alkaline phosphatase (AP), to assist in the detection of the complex.
In other embodiments, an S - secondary antibody conjugated to an enzyme (for example, HRP or AP), which binds to the detection antibody, can be used in the assay.
In general, the complex will then be detected using a luminescent substrate, for example, Ultra LITE'TM (NAG Research Laboratories); SensoLyte &(AnaSpec); maximum sensitivity substrate SuperSignal ELISA Femto (Thermo Scientific); - SuperSignal ELISA Pico chemiluminescent substrate (Thermo Scientific); and CPSD (disodium phosphate 3- (4-methoxypyrospiro (1,2-dioxetane-3,2 '- (5'-chloro) tricycle [3.3.1.13,7] decan) -4-yl) phenyl; Tropix, Inc ). The CPSD substrate can be found in chemiluminescent detection systems, such as - for example, the ELISA-Light "M (Applied Biosystems) system. In one preferred embodiment, the BCR-ABL sandwich ELISA comprises the use of an anti-BCR-N antibody as the capture antibody, where the capture antibody is maintained on a solid support such as a microplate well, and an anti-ABL-C antibody conjugated to HRP as the detection antibody, where the detection antibody can comprise an antibody independent of the activation state or an antibody dependent on the activation state (for example, phospho-specific) Figure 3C illustrates exemplary sandwich ELISA modalities (500) to detect the presence (total level) and / or activation status (phosphorylation level) of BCR-ABL (510) The beads (521a and / or 521b) are used to remove the full-size protein BCR and / or ABL, for example, by its carboxyl terminations and N, respectively.
Then, using ELISA and capturing the N-termination portion of BCR-ABL that uses a specific binding fraction, the total concentration of BCR-ABL is
'Total GLA (560) and activated protein (570) can also be determined. capturing ABL-C, and then placing the captured BCR-ABL fusion protein in contact with a specific detection antibody for BCR-N, or a detection antibody specific for the phosphorylation site in ABL-C, respectively, using ELISA .
In embodiments where steps (c) and (d) comprise a flow cytometry assay (FCM), the FCM assay may comprise a fluorescence activated cell separation assay (FACS). Flow cytometry is a technique for counting and examining microscopic particles, such as cells, suspending them in a fluid stream and passing them through an electronic detection device. Flow cytometry allows simultaneous multiparametric analysis of the physical and / or chemical characteristics of up to thousands of particles per second. In flow cytometry, - a beam of light (usually laser light) of a single wavelength is - 15 directed to a hydrodynamically focused stream of fluid. Numerous detectors are focused on the point where the current passes through the light beam: one in line with the light beam (forward scattering or FSC) and several perpendicular to it (lateral scattering (SSC) and one or more fluorescent detectors) . Each suspended particle of about 0.2 to - about 150 micrometers that passes through the beam spreads the beam, and the fluorescent chemicals found in the particle or attached to the particle can be excited by emitting light over a longer wavelength. than that of the light source. This combination of scattered and fluorescent light is captured by the detectors and, analyzing fluctuations in the - luminosity in each detector (one for each peak of fluorescent emission), it is then possible to direct various types of information regarding the physical and chemical structure of each individual particle. . FSC is related to cell volume and SSC depends on the internal complexity of the particle. Some are
'light scattering for measurement, while other flow cytometers. they form images of each individual particle fluorescence, scattered light and transmitted light.
FACS is a specialized type of flow cytometry.
S - Provides a method for distributing a heterogeneous mixture of particles in two or more containers, one particle at a time, based on the specific light scattering and fluorescent characteristics of each particle.
It is a scientific instrument used, since it is provided fast, objective and quantitative that records fluorescence signals from individual particles, as well as - physical separation of particles of particular interest.
The particle suspension is held in the center of a limited, rapidly flowing liquid stream.
The flow is arranged in such a way that there is a great separation between the particles with respect to their diameter.
A mechanism of. vibration causes the particle stream to break into droplets - 15 individual.
The system is adjusted so that there is a low probability of more than one particle per droplet.
Just before the current breaks into droplets, the flow passes through a fluorescence measurement station, where the fluorescent character of interest for each particle is measured.
An electric charge ring is placed only at the point where the current breaks into droplets.
A charge is placed on the ring based on the fluorescence intensity immediately before the measurement, and the opposite charge is captured in the droplet, as it has been broken from the current.
The charged droplets then fail to reach an electrostatic deflection system that deflects the droplets in a container based on their charge.
In - some systems, the charge is applied directly to the current, and droplet separation maintains the charge from the same signal as the current.
The current then becomes neutral after the droplets are separated.
A FACS assay can be performed using a FACSCAalibur flow cytometer, available from BD
S1
: In certain modalities, a FACS assay is performed using - two antibodies that bind to the target of interest, for example, BCR-ABL, in different epitopes, in such a way that the binding of the first antibody (capture) does not interfere with the second (detection) antibody. In —preferred modalities, the first antibody (capture) binds to the second domain other than the first full-size protein (for example, BCR-N), and the second (detection) antibody binds to the first domain of the second size protein different total (for example, GLA-C). In certain embodiments, the capture antibodies are affixed to the beads, such as - polystyrene beads, and the beads are stained internally with fluorophores.
In some embodiments, the detection antibody is conjugated to a fluorophore or an enzyme to aid in the detection of the complex.
The detection antibody may comprise an antibody independent of the - activation state or an antibody dependent on the activation state (for example, | 15. phospho-specific). In other embodiments, a fluorophore, enzyme, or other detection fraction that binds to the detection antibody, can be used in the assay.
In general, the complex can then be detected in the manner described above.
In modalities where steps (c) and (d) comprise a label selection assay, the label selection assay can comprise a Luminexº assay ”. Luminexº assays are available, for example, from Invitrogen Corporation (Carlsbad, CA). In some examples, the label selection assay comprises a Luminexº multiplex assay format.
In general, two antibodies that are selected bind to the target - of interest, for example, BCR-ABL, in different epitopes, in such a way that the binding of the first antibody (capture) does not interfere with the second (detection) antibody. In preferred embodiments, the first antibody (capture) binds to the second domain different from the first protein of bind to the first domain of the second protein of different total size (e.g., ABL-C). In certain embodiments, the capture antibodies are affixed to the polystyrene beads, and the beads are stained internally with red and infrared fluorophores of different intensities. In some embodiments, the detection antibody is conjugated to a fluorophore or an enzyme to aid in the detection of the complex. The detection antibody can comprise an antibody independent of the activation state or an antibody dependent on the activation state (e.g., phospho-specific). In other embodiments, a fluorophore, enzyme, or other detection fraction, which binds to the detection antibody, can be used in the assay. In general, the complex can then be detected, for example, by using a detection system such as a Luminex & 100TM or 2007M detection system, in which the accounts can be read in a single file, by - double laser, for classification and quantification of each analyte.
215 Figure 3D illustrates exemplary flow cytometry and label selection modalities (600) to detect the presence (total level) and / or activation state (phosphorylation level) of BCR-ABL (610). The beads (621a and / or 621b) are used to remove full-size BCR and / or GLA proteins, for example, by their carboxyl and N terminations, respectively. Next, a specific account to capture the portion of the N termination of BCR-ABL that uses a specific capture fraction, and a specific account for the termination C of ABL, are used. The total protein (640) is determined by counting molecules with the two specific labels or colored beads. Similarly, the amount of activated protein can - be determined using a bead with a capture antibody specific for the phosphorylated portion of ABL and the N-terminus of BCR-ABL. Counting these two specific colored beads or labels, the amount of activated BCR-GLA is measured (650). Total BCR-GLA (660) and activated protein (670)
To a specific capture account, and placing the captured BCR-ABL * fusion protein in contact with a specific account for BCR-N, or a specific account for the phosphorylated portion of ABL. Counting these two specific colored beads or labels, the amount of total BCR-ABL - (660) or activated (670) protein is measured.
Non-limiting examples of antibodies suitable for use in methods for measuring levels of total and / or activated BCR-ABL protein, illustrated in Figures 3A-3D, include those shown in Table 1 below.
Table 1. Exemplary antibodies for the BCR-GLA assays of the present invention. Target Ab Clone Epitope Supplier Ber AF5129 N-terminal R&D sc-48422 H-5 N-terminal Santa Cruz 1684 EPS35Y C-terminal Epitomics Abl AFS414 C-terminal R&D 4G10 phospho-tyrosine Millipore õ ab62189 pY245 Abcam PABO0397 pY245 Novus - ab47315 pY4 ab55284 pY412 Abcam NB100-92665 pY412 Novus Additional examples of antibodies that bind to both tumor-specific BCR-ABL fusion proteins and non-oncogenic natural full-size BCR or ABL proteins include, but are not limited to, the specific 7C6 monoclonal antibody of the BCR b2 epitope that recognizes b2a2 p210 BCR-ABL, b3a2 p210 BCR-ABL, p160 BCR and p130 BCR proteins described in Dhut et al., Oncogene, 3: 561-6 (1988), the SHE domain specific 8E9 antibody that recognizes ela2 p190 BCR-ABL, b2a2, b3a2, and pl45 GLA proteins described in US patents 5,369,008 and
6,610,498, the BCR amino termination specific antibody described in US patent 6,107,457, and the mouse monoclonal% SC-23 specific antibody of the ABL carboxyl termination (24-1 1) from Santa Cruz. Examples of antibodies connection points suitable for. binding at the site or melting point of the chimeric BCR-ABL protein includes, but is not limited to, the mouse monoclonal antibody t3908 specific for the binding of BCR-ABL b2a2 (L99H4), available from Cell Signaling Technology, Inc. (Danvers, MA ), the isolated antibodies specific to the p210 BCR-ABL fusion protein described in US patent publication 2005021430], the BCR-ABL b3a2-specific polyclonal antibody b3a2 described in van Denderen et al., Leukemia, 6: 1107 -12 (1992), and the BCR-ABL ela2-specific monoclonal antibody ela2 (ER-FP1) described in van —Denderen etal., Leukemia, 8: 1503-9 (1994). BCR-ABL binding antibodies can be used for the detection of BCR-ABL fusion proteins that are associated, but without limitation, with Ph positive leukemias.
In certain embodiments, the methods of the present invention. provide cell extract preparation from bone marrow samples - 15 (for example, bone marrow aspirate), or whole blood, freshly collected or frozen, recovering or isolating cells of interest such as white blood cells (for example, chronic myelogenous leukemia cells (CML)), for example, using a magnetic capture bead with anti-CD45 antibodies and / or anti-CD15 antibodies, without any “washing step after cell recovery or isolation.
The cell extract thus obtained can be analyzed with respect to the level of expression and / or activation of one or more oncogenic fusion proteins, such as BCR-ABL, substrates thereof, pathways thereof or combinations thereof.
Without being tied to any particular theory, eliminating the need for any of the washing steps after cell isolation is advantageous, due to the cells of interest that can be recovered from blood or bone marrow samples without changing the concentration intracellularity of an anti-cancer drug, such as a tyrosine kinase inhibitor.
As presented
: washing, as described herein, is contrary to accepted practice in the technique of - washing cells after isolation (for example, cells linked to the wash count) and provides cell extracts from recovered cells without substantial dilution of an anti-cancer drug , such as a tyrosine kinase inhibitor (eg, Gleevec ”, Tasigna”, Sprycelº, etc.) within cells.
In alternative embodiments, the methods of the present invention provide simultaneous detection of the total amount, and / or activation state of an oncogenic fusion protein (for example, BCR-ABL), in combination with one or both natural full-size proteins which contains sequences or domains found in the oncogenic fusion protein (for example, full-size BCR and / or ABL). In a particular modality, the present method allows the detection and / or measurement of both total levels of BCR-ABL, as well as the total levels of BCR and / or ABL of. total natural size in a biological sample, such as a sample of - 15 blood or bone marrow aspirate. In certain embodiments, the levels of natural protein (for example, BCR and / or full-length GLA) are determined along with the levels of oncogenic fusion protein (for example, BCR-ABL) in a multiplexed manner in the same piece. In these modalities, the full-size protein can be isolated - advantageously together with the oncogenic fusion protein, in such a way that the levels of these molecules are determined in the same piece.
As shown in example 10 below, these alternative modalities for the methods of the present invention can be used to detect and / or measure the total levels of BCR-GLA, as well as the total natural size BCR or GLA levels, and a ratio of total BCR-GLA levels to the full-size BCR or GLA levels can be calculated. In some examples, the ratio of BCR-GLA levels to BCR or GLA levels is calculated for
S for example, a major molecular response (MMR), a complete molecular response (CMR), a complete cytogenetic response (CCyR), and combinations thereof. In other examples, these alternative modalities for the methods of the present invention can be used to monitor changes in BCR-ABL expression with respect to a control, such as full-size BCR or ABL (for example, by calculating a ratio from total BCR-GLA levels to full-size BCR or GLA levels) as a therapy function (eg, tyrosine kinase inhibitor therapy).
The methods of the present invention are used particularly to determine the activation state (for example, phosphorylation) of one or more oncogenic fusion proteins, such as BCR-ABL, in patients at risk of developing, suspected of having, or diagnosed with a . cancer, such as a hematological malignancy (eg, leukemia, - 15 lymphoma, etc.). In certain examples, the methods of the present invention assist, assist or facilitate in the diagnosis of cancer in a subject, by measuring levels of activated (for example, phosphorylated) fusion protein (for example, phosphoryl BCR-ABL levels) for determine whether the subject expresses an activated form of the oncogenic fusion protein (for example, a BCR-ABL positive patient). In other embodiments, the methods of the present invention are performed on a subject already determined to express an activated form of the oncogenic fusion protein to optimize therapy, reduce toxicity, and / or monitor the efficiency of the therapeutic treatment. In a particular aspect of these modalities, the level - of activated BCR-ABL protein can be determined in a BCR-ABL positive patient, during the period of therapy (for example, while the patient is in therapy with an anti-cancer drug, such as such as Gleevecº, Tasigna ”, Sprycelº, etc.), to optimize therapy, reduce toxicity, and / or
Both levels of total and activated oncogenic fusion protein (eg "phosphorylated) (eg, BCR-ABL) are measured according to the antibody-based assays of the present invention, and a ratio of the protein levels of total activated oncogenic fusion (for example, ratio of phosphorus-total BCR-ABL protein levels) can be calculated and used to assess the duration of therapy for a subject, for example, by comparing the ratio of phosphate oncogenic fusion protein levels / total with the same ratio calculated for the subject in an initial period (for example, in an initial period, although in therapy with anti-cancer medication or at a point of time before therapy with anti-cancer medication). modalities, the ratio of the levels of activated oncogenic fusion protein to total (for example, the ratio of phosphorus to total BCR-ABL protein levels) can be calculated with respect to the levels of one or more control proteins such as, for example , one or both s the natural full-size proteins that contain sequences or domains found in the oncogenic fusion protein (for example, BCR and / or ABL for BCR-ABL fusion protein). In preferred embodiments, the total level of the control protein is unaffected or substantially changed by anti-cancer drug therapy. The methods of the present invention are also particularly
20. used to determine the activation state (for example, phosphorylation) of one or more signal transduction molecules, in one or multiple pathways associated with an oncogenic fusion protein, such as BCR-ABL, in patients at risk of developing, suspected of having, or diagnosed with, cancer such as haematological malignancy (eg - leukemia, lymphoma, etc.). Exemplary signal transduction molecules include BCR-ABL substrates such as, for example, CRKL, JAK2, STATS, Src, FAK, c-ABL, c-CBL, SHC, SHP-2, VAV, BAP-1 and combinations of the same. In certain examples, the methods of this
: subject measuring levels of activated oncogenic fusion protein (eg, phosphorylated) (eg, phosphorylated BCR-ABL levels), and activated signal transduction molecule levels (eg, phosphorylated) (eg, levels CRKL phospho, phospho-JAK2, phospho-STATS, etc.), to determine whether the subject expresses an activated form of the oncogenic fusion protein (for example, a BCR-ABL positive patient) and / or an activated form of a or more signal transduction molecules in the pathway.
In other embodiments, the methods of the present invention are carried out on a subject already determined to express an activated form of the fusion protein - homogeneous to optimize therapy, reduce toxicity, and / or monitor the efficiency of therapeutic treatment.
In a particular aspect of these modalities, the levels of activated BCR-ABL protein and one or more components of the signal transduction pathway (for example, CRKL, JAIK2,. STATS, Src, FAK, etc.) can be determined in a patient positive - 15 for BCR-ABL, during the therapy period (for example, while the patient is in therapy with anti-cancer medication, such as Gleevecº , Tasigna ”, Sprycelº, etc.), to optimize the therapy, reduce the toxicity, and / or monitor the efficiency of therapeutic treatment.
In some embodiments, both the levels of total and activated oncogenic fusion protein (eg, phosphorylated) (eg, BCR-ABL) are measured according to the antibody-based assays of the present invention, and a ratio of the levels of activated oncogenic fusion protein to total (for example, ratio of phosphorus-to-total BCR-ABL protein levels) can be calculated (for example, with respect to the levels of one or more control proteins) and used to assess the period of therapy for a subject, for example, by comparing the ratio of phospho / total oncogenic fusion protein levels to the same ratio, calculated for the subject at an early stage (for example, at an early stage although in therapy with anti-cancer medication or in one mode, both component levels of the signal transduction pathway
- total how much activated is measured and a ratio of component levels of the total to activated signal transduction path (for example, ratio of CRKL, JAK2, or phospho / total STATS protein levels) can be calculated (for example, with respect to at the levels of one or more control proteins) and used to evaluate the period of therapy of a subject, for example, comparing the ratio of the levels of the component of the phospho / total signal transduction pathway with the same ratio calculated for the subject in a early stage (for example, at an early stage although on anti-drug therapy
cancer therapy, or in a period of time before therapy with anti-cancer medicine). In certain examples, the level of expression of an oncogenic fusion protein (for example, BCR-ABL) can be correlated or related to the level of activation (for example,
- phosphorylation) of the signal transduction components downstream, such as 2 15 CRKLJAK2, STATS, Ste, FAK, etc.
In a particular aspect, the present invention provides a method for optimizing therapy, and / or reducing toxicity in a subject with cancer, and who receives a period of therapy for the treatment of cancer, the method comprising:
(a) isolating cancer cells after administration of an anti-cancer drug (for example, one or more tyrosine kinase inhibitors such as Gleevec *, Tasigna ”, Sprycelº, etc.);
(b) lyse the isolated cells to produce a cell extract; (c) measuring an expression and / or activation level (for example, —phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay described herein; and (d) comparing the measured level of expression and / or activation of the oncogenic fusion protein with a level of expression and / or activation of the ds o a and o
The oncogenic fusion protein, measured at an earlier time, during the. therapy period; and (e) determining a subsequent dose of the therapy period for the subject, or whether a different therapy period can be administered to the subject based on the comparison of step (d).
In particular modalities, both the level of expression and the level of activation of the oncogenic fusion protein are measured in the cell extract, for example, by performing one of the proximity tests described here. In certain preferred embodiments, the oncogenic fusion protein comprises BCR-ABL. In other certain preferred embodiments, the subject expresses an activated form of the oncogenic fusion protein. In a particularly preferred embodiment, the subject is positive for BCR-ABL (for example, the subject was determined to have detectable levels - of phospho-BCR-ABL prior to administration of the anti-cancer drug).
2115 In some embodiments, both levels of total and activated oncogenic fusion protein (eg, phosphorylated) (eg, BCR-ABL) are measured in the cell extract according to the antibody-based assays of the present invention, and a ratio of activated oncogenic fusion protein levels to total (eg ratio of phosphorus-total BCR-ABL protein levels) can be calculated and used to assess the period of therapy for a subject, for example, by comparing the ratio of the levels of phospho / total oncogenic fusion protein with the same ratio calculated for the subject at an early stage (for example, at an early stage, although in anti-cancer drug therapy, or in a period of time before anti-cancer drug therapy -cancer). As illustrated in example 6 below, the levels of the phosphorus / total oncogenic fusion protein ratio (eg, BCR-ABL) in the inhibition of the anti-cancer drug are correlated with the percentage inhibition of the fusion protein signal.
: GLA) under treatment with anti-cancer medication (see, for example, figures. 14C, 15C, and 16C). In other embodiments, the ratio of levels of activated oncogenic fusion protein to total (for example, ratio of phosphorus-to-total BCR-ABL protein levels) can be calculated with respect to the levels of one or more control proteins such as, for example , one or both of the natural full-length proteins that contain sequences or domains found in the oncogenic fusion protein (for example, BCR and / or ABL for BCR-ABL). In preferred embodiments, the total level of the control protein is unaffected or substantially changed by anti-cancer drug therapy.
In certain aspects of the methods described here to optimize therapy, less than about 50% inhibition of activation (eg, phosphorylation) of levels of oncogenic fusion protein (eg, BCR-ABL) in a subject, indicates a need of increasing the subsequent dose of the - therapy period, or administering a different therapy period (eg. 15 changing the current therapy period by changing to a different anti-cancer drug) in order to prevent or reduce the risk of cancer recurrence on the subject. As a non-limiting example, in examples where the subject is in therapy with Gleevec , less than about 50% inhibition of the level of phosphorylation of BCR-ABL fusion protein indicates a need to increase the dose - subsequent to Gleevec ”, or change the therapy from the subject to Tasigna ”. In certain instances, less than about 49%, 48%, 47% a, 46%, 45%, 44%, 43 Yo, 42 Yo, 41%, 40%, 35%, 30%, 25%, 20% , 15%, 10%, or 5% activation inhibition (eg, phosphorylation) of oncogenic fusion protein levels (eg, BCR-ABL) in a subject, indicates a need to increase the dose - subsequent period of therapy, or change the current therapy period. In some embodiments, the percentage of inhibition of activation of the levels of oncogenic fusion protein can be determined by calculating a ratio of the levels of activated oncogenic fusion protein to total (for example, ratio of levels of oncogenic fusion protein).
: more control proteins (eg, BCR and / or ABL full size to BCR-ABL), and comparing the ratio of levels of phospho / total oncogenic fusion protein with the same ratio calculated for the subject at an early stage (for example, at an early stage, although in therapy with anti-cancer medicine, or in a period of time before therapy with anti-cancer medicine). Those skilled in the art will experience lower and higher suitable doses, in which the period of current therapy can be adjusted in such a way that therapy with the drug is optimized, for example, a subsequent dose that is at least about 1.5, 2 , 2,5, 3, 3,5, 4, 4,5, 5, 5,5, 6, 6,5, 7, 7,5, 8, 8,5, 9, 9,5,10,15 , 20,25,30,35,40,45, 50 or 100 times higher than the current dose.
In other certain aspects of methods to optimize therapy, less than about 50% inhibition of activation (eg, phosphorylation) of the levels of oncogenic fusion protein (eg, BCR-ABL) in one. subject, indicates a lack of compliance by the subject with the period of therapy (for example, the subject is not taking the anti-cancer medication regularly i or in the manner instructed by a doctor), and / or the existence of possible side effects or associated toxicity with the therapy period. In these modalities, it is recommended that the current therapy period be carefully monitored (for example, by a doctor or other healthcare professional) for compliance, or that a different therapy period be administered (for example, the therapy period change to a different anti-cancer drug) in order to increase compliance and / or prevent or reduce the risk of side effects. In certain instances, less than about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42% 41%, 40%, 35%, 30%, 25%, 20%, 15 %, 10%, or 5% inhibition of activation of levels of oncogenic fusion protein in a subject, indicates a failure of the subject to comply with the period of therapy, and / or the existence of possible side effects or toxicity associated with the period of therapy.
] oncogenic fusion protein can be determined by calculating a ratio of - activated oncogenic fusion protein levels to total (for example, ratio of phosphorus / total BCR-ABL protein levels), optionally with respect to the levels of one or more control proteins (for example, BCR and / or S-GLA - total size for BCR-GLA), and comparing the ratio of phosphorus / total oncogenic fusion protein levels to the same ratio calculated for the subject at an early stage (for example, at an early stage, albeit in anti-cancer drug therapy, or in a period of time before anti-cancer drug therapy).
In additional aspects of the methods described here to optimize therapy, more than about 80% inhibition of activation (eg, phosphorylation) of levels of oncogenic fusion protein (eg, BCR-ABL) in a subject, indicates that the subject is in therapy. correct and with the correct dose. In certain instances, more than about 81%, 82 2 15%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% inhibition of activation (eg, phosphorylation) of oncogenic fusion protein levels (eg, BCR-ABL) in a subject, indicates that the subject is in therapy as the correct anti-cancer drug and in the correct dose In some modalities, the percentage of inhibition of activation of the levels of oncogenic fusion protein can be determined by calculating a ratio of the levels of activated oncogenic fusion protein to total (for example, ratio of phosphorus / total BCR-ABL protein levels), optionally with respect to the levels of one or more control proteins (for example, BCR and / or ABL size - total to BCR-ABL), and then comparing the ratio of phospho / total oncogenic fusion protein levels with the same ratio calculated for the subject at an early stage (for example, at an early stage, albeit on med therapy anti-cancer treatment, or a period of time before therapy i In a related aspect, the present invention provides a: method for optimizing therapy, and / or reducing toxicity in a subject with cancer, and who receives a period of therapy for the treatment of cancer, the method comprising: (a) isolating cancer cells after administration of an anti-cancer drug (for example, one or more tyrosine kinase inhibitors such as Gleevecº , Tasigna ”, Sprycel” , etc.); (b) lyse the isolated cells to produce a cell extract; (c) measuring a level of expression and / or activation (eg, phosphorylation) of an oncogenic fusion protein, and one or more signal transduction molecules in its pathway, in the cell extract using an assay described herein; and (d) comparing the measured level of expression and / or activation of the oncogenic fusion protein, and signal transduction molecules, with a —level of expression and / or activation of the oncogenic fusion protein, and signal transduction molecules , measured at an initial time during the therapy period; and (e) determining a subsequent dose of the therapy period for the subject, or whether a different therapy period can be administered to the “subject” based on the comparison of step (d).
In particular modalities, both the level of expression and the level of activation of the oncogenic fusion protein and one or more signal transduction molecules are measured in the cell extract, for example, by performing one of the proximity assays described here. In certain preferred embodiments, the oncogenic fusion protein comprises BCR-ABL. In other certain preferred embodiments, signal transduction molecules include BCR-ABL substrates such as, for example, CRKL, JAK2, STATS, Src, FAK, c-ABL, c-CBL, SHC, SHP-2, VAV, BAP -I e is an activated form of the oncogenic fusion protein.
In one mode. particularly preferred, the subject is positive for BCR-ABL (for example, the subject was determined to have detectable levels of phospho-BCR-
GLA before the administration of the anti-cancer medication). In some embodiments, both the levels of total and activated oncogenic fusion protein (for example, phosphorylated) (for example, BCR-ABL) and the component levels of the signal transduction pathway (for example, CRKL, JAK2, STATS) , are measured in the cell extract according to the antibody-based assays of the present invention, and a ratio of activated oncogenic fusion protein levels to total (e.g. ratio of phosphorus / total BCR-ABL protein levels), and a ratio of component levels of the total to activated signal transduction pathway (eg ratio of CRKL, JAK2, or phospho / total STATS protein levels), can be. calculated and used to assess the period of therapy for a subject, for example. 15 example, comparing the ratio of phospho / total oncogenic fusion protein and the levels of the signal transduction path component with the same ratio, calculated for the subject at an early stage (for example, at an early stage, although in therapy with anti-cancer medicine, or in a period of time before anti-cancer medicine therapy). In other embodiments, the ratio of activated oncogenic fusion protein levels to total (for example, ratio of phosphorus-to-total BCR-ABL protein levels) and ratio of activated signal transduction molecule levels to total (for example, ratio of CRKL, JAK2, or phosphorus / total STATS protein levels) can be calculated with respect to the levels of one or more control proteins such as, for example, one or both natural full-size proteins that contain sequences or domains found in the oncogenic fusion protein (for example, BCR and / or ABL for BCR-ABL). In preferred embodiments, the total level of the control protein is unaffected or
Ú In certain aspects of the methods described here to optimize "therapy, less than about 50% activation inhibition (eg, phosphorylation) of one, two, three, four, five, six, or more levels of fusion protein oncogenic (for example, BCR-ABL), and / or component levels of the signal transduction pathway (for example, CRKL, JAK2, STATS), in a subject, indicates a need to increase the subsequent dose for the period of therapy or to administer a different therapy period (for example, changing the current therapy period by changing to a different anti-cancer medication) in order to prevent or reduce the risk of cancer recurrence in the subject.
As a non-limiting example, in examples where the subject is on Gleevecº therapy, less than about 50% inhibition of the level of phosphorylation of BCR-ABL fusion protein, and / or a component of the signal transduction pathway, such as such as CRKL, JAK2, and / or STATS, indicates a need to increase the subsequent dose of Gleevec * or to change the subject's therapy to Tasigna ”. In certain instances, less than about 49 K%, 48%, 47 Y%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 35%, 30%, 25%, 20 %), 15%, 10%, or 5% activation inhibition (for example, phosphorylation) of one, two, three, four, five, six, or more levels of oncogenic fusion protein (for example, BCR-ABL ), and / or component levels of the signal transduction pathway (for example, CRKL, JAK2, STATS), in a subject, indicates a need to increase the subsequent dose of the therapy period, or to change the current therapy period.
In some examples, the percentage of inhibition of the levels of activation of oncogenic fusion protein and / or signal transduction molecule can be determined by calculating a ratio of the levels of activated oncogenic fusion protein to total (for example, ratio of protein levels Phosphorus / total BCR-ABL), and / or a ratio of component levels of the total to activated signal transduction pathway (for example, ratio of CRKL protein levels, JAK2, or phosphorus / total STATS),
For example, full size BCR and or GLA for BCR-GLA), and comparing the calculated phospho / total ratio with the same ratio calculated for the subject in an early stage (for example, in an early stage, although in therapy with anti-cancer drug, or in a period of time before anti-cancer drug therapy). Those skilled in the art will experience higher and lower doses, in which the period of current therapy can be adjusted, in such a way that therapy with the drug is optimized, for example, a subsequent dose that is at least about 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, 15, 20, 25, 30, 35, 40, 45, 50 or 100 times greater or less than the current dose.
In other certain aspects of methods to optimize therapy, less than about 50% activation inhibition (eg, phosphorylation) of one, two, three, four, five, six, or more levels of 'oncogenic ( for example, BCR-ABL), and / or component levels of the 2 15 signal transduction pathway (for example, CRKL, JAK2, STATS), in a subject, indicates a subject's lack of compliance with the therapy period (for example, example, the subject is not taking the anti-cancer medication regularly, or in the manner instructed by the doctor), and / or the existence of possible side effects or toxicity associated with the period of therapy.
In these modalities, it is recommended that the current therapy period be carefully monitored (for example, by a doctor or other healthcare professional) for compliance, or that a different therapy period be administered (for example, the current therapy period either by switching to a different anti-cancer drug) in order to —increase compliance and / or prevent or reduce the risk of side effects.
In certain instances, less than about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% inhibition of activation of one, two, three, four, five, six, or more levels of TF -— Oncogenic fusion protein (eg; BER-ABE), and / or levels in ----
'component of the signal transduction pathway (eg, CRKL, JAK2, - STATS), in a subject, indicates a subject's lack of compliance with the therapy period and / or the existence of possible side effects or toxicity associated with the period of therapy. In some embodiments, the - percentage of inhibition of activation of the levels of oncogenic fusion protein - and / or signal transduction molecule can be determined by calculating a ratio of the levels of activated oncogenic fusion protein to total (for example, ratio of the levels phosphorus / total BCR-ABL protein), and / or a ratio of component levels of the total to activated signal transduction pathway (for example, ratio of CRKL, JAK2, or phosphorus / total STATS protein levels), optionally with relation to the levels of one or more control proteins (for example, BCR and / or ABL of full size for BCR-ABL), and comparing the calculated phosphorus / total ratio with the same ratio calculated for the subject «at an early stage ( for example, at an early stage, although in therapy 2 15 with anti-cancer medication, or in a period of time before therapy with anti-cancer medication).
In additional aspects of the methods described here to optimize therapy, more than about 80% inhibition of activation (eg, phosphorylation) of one, two, three, four, five, six, or more levels of 20. oncogenic fusion protein (eg, BCR-ABL), and / or component levels of the signal transduction pathway (eg, CRKL, JAK2, STATS), in a subject, indicates that the subject is on the correct therapy and dose correct. In certain instances, more than about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 Y%, 96%, 97% 98%, 99%, or 100% activation inhibition (eg, phosphorylation) of one, two, three, four, five, six, or more levels of oncogenic fusion protein ( for example, BCR-ABL), and / or component levels of the signal transduction pathway (for example, CRKL, JAK2, STATS), in a subject,
Correct dose. In some modalities, the percentage of inhibition of the levels of. activation of oncogenic fusion protein and / or signal transduction molecule can be determined by calculating a ratio of the levels of activated oncogenic fusion protein to total (for example, ratio of phosphorous / total protein —BCR-ABL) and / or a ratio of component levels of the total to activated signal transduction pathway (for example, ratio of CRKL, JAK2, or phospho / total STATS protein levels), optionally to the levels of one or more control proteins (for example, Full-size BCR and / or GLA to BCR-GLA), and then comparing the calculated phospho / total ratio with the same ratio calculated for the subject in an early stage (for example, in an early stage, albeit in anti-drug therapy -cancer, or a period of time before anti-cancer drug therapy).
In other aspects of the methods described here to optimize the. 15 therapy, the activation of an alternative signal transduction pathway indicates a need to change or adjust the current therapy period (for example, switching to a different anti-cancer drug). As a non-limiting example, in examples where the subject is in therapy with Gleevecº, the activation (for example, phosphorylation) of an alternative signal transduction pathway, such as Src, indicates a need to switch the subject's therapy to Sprycelº or Tasigna ”.
In another aspect, the present invention provides a method for selecting an anti-cancer drug suitable for the treatment of a cancer, the method comprising: (a) isolating cancer cells after administration of an anti-cancer drug, or before incubation with an anti-cancer medication; (b) lyse the isolated cells to produce a cell extract;
A) (c) determining an expression and / or activation level (for example, phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay described herein; and (d) determining whether the anti-cancer drug is suitable or S - inappropriate for the treatment of cancer, by comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference expression and / or activation profile generated in the absence of the anti-cancer medication.
In a preferred embodiment, the method for selecting an appropriate anti-cancer drug for the treatment of cancer comprises: (a) isolating cancer cells after administration of an anti-cancer drug, or before incubation with an anti-cancer drug cancer; (b) lyse the isolated cells to produce a cell extract; (c) determining an expression and / or activation level (for example, phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay comprising a serial dilution of capture antibodies specific for the oncogenic fusion protein, at which —The capture antibodies are limited to a solid support; (d) comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference expression and / or activation profile generated in the absence of the anti-cancer drug; and (e) indicate whether the anti-cancer drug is suitable for the treatment of cancer when the level of expression and / or activation detected for the oncogenic fusion protein is altered (for example, substantially lower), compared with the profile of reference expression and / or activation. a ne o a a ea O o
In some embodiments, the methods of the present invention. they can be used to assist or assist in the selection of an anti-cancer medication suitable for the treatment of a cancer such as, for example, a hematological malignancy. In other embodiments, the methods of the present invention can be used to improve the selection of an anti-cancer drug suitable for the treatment of a cancer such as, for example, a hematological malignancy. In certain embodiments, the method additionally or alternatively comprises the step of indicating whether the anti-cancer drug is unsuitable for the treatment of cancer when the level of expression and / or activation detected for the oncogenic fusion protein is not changed (for example, is not substantially altered), compared to the reference expression and / or activation profile. In additional modalities, one or more signal transduction molecules present in the cell extract are detected, in addition to one or more oncogenic fusion proteins, and the anti-cancer drug is determined to be suitable or inappropriate based on this "molecular profile".
In yet another aspect, the present invention provides a method for identifying a cancer's response to treatment with an anti-cancer drug, the method comprising: (a) isolating cells from a cancer after administration of an anti-cancer drug, or before incubation with an anti-cancer medication; (b) lyse the isolated cells to produce a cell extract; (c) determining an expression and / or activation level (e.g., phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay described herein; and (d) identifying the cancer as responsive or unresponsive to treatment with the anti-cancer drug, comparing the level of expression TT - «(or activation detected for the oncogenic fusion protein with a perfitde ———
T2] expression and / or reference activation generated in the absence of the drug. anti-cancer.
In a preferred embodiment, the method for identifying a cancer's response to treatment with an anti-cancer drug comprises: (a) isolating cancer cells after administration of an anti-cancer drug, or before incubation with an anti-cancer drug - cancer; (b) lyse the isolated cells to produce a cell extract; (c) determining an expression and / or activation level (for example, phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay comprising a serial dilution of capture antibodies specific for the oncogenic fusion protein, at which capture antibodies are limited to a solid support; (d) comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference expression and / or activation profile generated in the absence of the anti-cancer drug; and (e) indicate whether the cancer is responsive to treatment with the anti-cancer drug when the level of expression and / or activation detected for the oncogenic fusion protein is altered (for example, substantially decreased), compared to the expression profile and / or reference activation.
In some embodiments, the methods of the present invention can be used to assist or assist in the identification of a cancer response, such as, for example, a hematological malignancy, for treatment with an anti-cancer drug. In other embodiments, the methods of the present invention can be used to improve the identification of the response of a cancer such as, for example, a malignancy
BB k modalities, the method additionally or alternatively comprises a. step of indicating whether the cancer is unresponsive to treatment with the anti-cancer drug, when the level of expression and / or activation detected for the oncogenic fusion protein is not altered (for example, it is not substantially decreased), compared to the profile expression and / or reference activation. In additional modalities, one or more signal transduction molecules present in the cell extract are detected, in addition to one or more oncogenic fusion proteins, and the cancer is identified as responsive or unresponsive to treatment based on this "molecular profile." In yet another aspect, the present invention provides a method for predicting the response of a subject with cancer to treatment with an anti-cancer drug, the method comprising: (a) isolating cancer cells after administration of an anti-cancer drug cancer, or before incubation with an anti-cancer drug; (b) lyse the isolated cells to produce a cell extract; (oc) determining an expression and / or activation level (for example, phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay described herein; and (d) predict the likelihood that the subject will respond to treatment with the anti-cancer drug, comparing the level of expression and / or activation detected for the oncogenic fusion protein with a generated expression and / or reference profile in the absence of the drug —anti-cancer.
In a preferred embodiment, the method for predicting the response of a subject with cancer to treatment with an anti-cancer drug comprises:
i (a) isolate cancer cells after administering one. anti-cancer medicine, or before incubation with an anti-cancer medicine; (b) lyse the isolated cells to produce a cell extract; (c) determining an expression and / or activation level (for example, phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay comprising a serial dilution of capture antibodies specific for the oncogenic fusion protein, at which capture antibodies are limited to a solid support; (d) comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference expression and / or activation profile generated in the absence of the anti-cancer drug; and (e) indicate whether the subject is likely to respond to treatment with the anti-cancer drug when the level of expression and / or activation detected for the oncogenic fusion protein is changed (for example, substantially decreased), compared to the expression profile and / or reference activation.
In some embodiments, the methods of the present invention can be used to assist or assist in predicting the likelihood that a subject will respond to treatment with an anti-cancer drug for a cancer such as, for example, a hematological malignancy. In other embodiments, the methods of the present invention can be used to improve the prediction of a subject's likelihood of responding to treatment with an anti-cancer drug for a cancer such as, for example, a hematological malignancy. In certain embodiments, the method further comprises or alternatively, the stage of indicating whether the subject is unlikely to respond to treatment with the anti-cancer medication, when the level of expression and / or activation detected for the tad: tornãot protein.
It is substantially reduced), compared to the expression and / or activation profile - of reference. In additional modalities, one or more signal transduction molecules present in the cell extract are detected, in addition to one or more oncogenic fusion proteins, and the probability that the subject S - will respond to treatment is predicted based on this "molecular profile" .
In a further aspect, the present invention provides a method for determining whether a subject with cancer is resistant to treatment with an anti-cancer drug, the method comprising: (a) isolating cells from a cancer after administration of an anti-cancer drug. cancer, or before incubation with an anti-cancer drug; (b) lyse the isolated cells to produce a cell extract; (c) determining an expression and / or activation level (e.g., phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay described herein; and (d) determine whether the subject is resistant or sensitive to treatment with the anti-cancer drug, by comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference profile and / or activation generated in the absence of the drug —anti-cancer, or in the presence of the anti-cancer drug at an early stage.
In a preferred embodiment, the method for determining whether a subject with cancer is resistant to treatment with an anti-cancer drug comprises: (a) isolating cancer cells after administration of an anti-cancer drug, or before incubation with a anti-cancer medication; (b) lyse the isolated cells to produce a cell extract;
'(c) determining a level of expression and / or activation (for example, phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay comprising a serial dilution of capture antibodies specific for the oncogenic fusion protein, where —the capture antibodies are limited to a solid support; (d) comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference expression and / or activation profile generated in the absence of the anti-cancer drug, or in the presence of the anti-cancer drug in a stage initial; and (e) indicate whether the subject is resistant to treatment with the anti-cancer drug when the level of expression and / or activation detected for the oncogenic fusion protein has not been changed (for example, it is not substantially decreased), compared to the profile expression and / or reference activation.
In some embodiments, the methods of the present invention can be used to assist or assist in identifying a subject with cancer, who is resistant to treatment with an anti-cancer drug, or in determining the likelihood of a subject with cancer being resistant to treatment. with an anti-cancer medication, in which the subject has a - cancer such as, for example, a hematological malignancy. In other embodiments, the methods of the present invention can be used to improve the identification of a subject with cancer, who is resistant to treatment with an anti-cancer drug, or in determining the likelihood that a subject with cancer is resistant to treatment with an - anti-cancer medication, in which the subject has a cancer such as, for example, a hematological malignancy.
In certain modalities, the method additionally or alternatively comprises the step of indicating whether the subject is sensitive to the treatment "cancer;
TI fa (for example, phosphorylation) detected for the oncogenic fusion protein is - altered (for example, substantially decreased), compared to the reference expression or activation profile. Non-limiting examples of reasons why a subject with cancer may be resistant to treatment with an S - anti-cancer drug, include the presence of one or more mutations in the oncogenic fusion protein of interest (for example, BCR-ABL) , non-compliance with the therapeutic regimen, and / or administration of a dose of subideal medication. With respect to a subideal drug dose of the anti-cancer drug, the method may additionally comprise the step of increasing the next dose or subsequent dose of the anti-cancer drug administered to the subject. In additional modalities, one or more signal transduction molecules present in the cell extract are detected, in addition to one or more oncogenic fusion proteins, and the subject is identified as resistant or sensitive to treatment based on this "molecular profile." In particular modalities, the level of expression (for example, total) and / or the level of activation (for example, phosphorylation) of the oncogenic fusion protein, or of the signal transduction molecule, is considered to be "altered" in the presence of an anti-cancer medication when it is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
20. 50%, 55%, 60%, 65%, 70%, 75 Y%, 80%, 85%, 90%, or 95% more or less expressed or activated than in the absence of the anti-cancer medication. In one embodiment, the level of expression (eg, total) and / or activation level (eg, phosphorylation) of the oncogenic fusion protein, or signal transduction molecule, is "substantially - decreased" in the presence of an anti-cancer drug when it is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% less expressed or activated than in the absence of the anti-cancer medication. In additional modalities, it is considered that the level of expression (by to-total ivet-of activation to-phosphorylation) -d:
'oncogenic fusion or signal transduction molecule, is. "substantially decreased" in the presence of an anti-cancer drug (1) when there is a change from the high or strong expression and / or activation of the oncogenic fusion protein, or the signal transduction molecule, without the anti-cancer drug, for medium, weak, low, or very weak expression and / or activation of the oncogenic fusion protein, or the signal transduction molecule, with the anti-cancer drug, or (2) when there is a change from the expression and / or medium activation of the oncogenic fusion protein, or signal transduction molecule, without the anti-cancer medication, for the expression, and / or weak, low, or very weak activation of the oncogenic fusion protein, or the signal transduction, with the anti-cancer medication.
In some embodiments, the expression level and / or activation level of the oncogenic fusion protein, or signal transduction molecule, is expressed as a relative fluorescence unit (RFU) value that corresponds to the signal strength for an analyte of particular interest, which is determined using, for example, a proximity assay such as the collaborative proximity immunoassay (COPIA) described herein.
In other modalities, the level of expression and / or the level of activation of the protein - of oncogenic fusion, or of the signal transduction molecule, is expressed as MIT OE, TA TAN, A + "or" + ", which corresponds to the increasing the signal strength for a particular analyte of interest that is determined using, for example, a proximity assay such as COPIA.
In some examples, an undetectable or minimally detectable level of expression or activation of a particular analyte of interest, which is determined using, for example, a proximity assay such as COPIA, can be expressed as "-" or "t". In other examples, a low level of expression or activation of a particular analyte of interest, which is determined
'expressed as "+". In yet other examples, a level of expression or. moderate activation of a particular analyte of interest, which is determined using, for example, a proximity assay such as COPIA, can be expressed as "++". In still other examples, a high level of expression or activation of a particular analyte of interest, which is determined using, for example, a proximity assay such as COPIA, can be expressed as "+++". In additional examples, a very high level of expression or activation of a particular analyte of interest, which is determined using, for example, a proximity assay such as COPIA, can be expressed as "++++", In yet other modalities, the level of expression and / or the level of activation of the oncogenic fusion protein, or the molecule signal transduction is quantified by calibrating or normalizing the RFU value, which is determined using, for example, a proximity test such as COPIA, against a standard curve generated by the particular analyte of interest. In certain examples, a computational unit (CU) value can be calculated based on the standard curve. In other examples, the CU value can be expressed as "-", "4", "+", "++", "+++" or "+++", according to the previous description for intensity of signal.
In certain embodiments, the level of expression or activation of a particular analyte of interest, when expressed as "-", "4", "+", "++", "+++" or "+++", can correspond to a level of expression or activation that is at least about 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, 15, 20, 25, 30, 35, 40, 45, 50, or 100 times greater or less (for example, about 1.5 -3.2-3.2-4.2-5.2-10.2-20, 2-50, 3-5, 3-10, 3-20, 3-50, 4-5, 4-10 , 4-20, 4-50, 5-10, 5-15, 5-20, or 5-50 times greater or less) than a reference expression level or activation level, for example, when compared to a negative control, such as an IgG control,
: when compared to a positive control, such as a pan-CK control, - when compared to a level of expression or activation determined in the presence of an anti-cancer drug, and / or when compared to a level of expression or activation determined in the absence of an anti-cancer drug In some instances, the correlation is specific for analyte. As a non-limiting example, a "+" expression or activation level, determined using, for example, a proximity test such as COPIA, can correspond to a 2-fold increase in expression or activation for an analyte and an increase of 5 times for another analyte, - when compared to a reference expression or activation level.
In particular modalities, both the levels of total and activated oncogenic fusion protein (for example, phosphorylated) (for example, 3 BCR-ABL), and that of the signal transduction molecule, are measured in the “cell extract according to the assays the antibody base of the present invention, and a ratio of levels of activated oncogenic fusion protein levels to total (e.g. ratio of phosphorous / total BCR-ABL protein levels), or a ratio of activated signal transduction molecule levels for total (for example, ratio of CRKL, JAK2, or phosphorus / total STATS protein levels), can be calculated and then compared to the same ratio, —calculated based on the expression and activation patterns generated in the absence of the anti-drug -cancer.
In some embodiments, the level of expression or activation of the reference oncogenic fusion protein, or signal transduction molecule, determined in step (c) is obtained from a normal cell, such as an individual's non-cancerous cell. healthy person who does not have cancer, such as hematological malignancy. In other certain embodiments, the level of expression or activation of the reference oncogenic fusion protein, or signal transduction molecule, determined in step (c) is obtained from a cell
: tumor from a sample (for example, cell extract) from a patient - with a cancer, such as leukemia or lymphoma.
In some embodiments, the level of expression or activation of the reference oncogenic fusion protein, or signal transduction molecule, determined in step (c) is obtained from a cell (for example, a tumor cell obtained from a sample) that is not treated with the anti-cancer medication. In particular modalities, the cell that is not treated with the anti-cancer drug is obtained from the same sample as the isolated cell (for example, a test cell to be investigated), used to produce the cell extract, is obtained. In certain instances, the presence of a lower level of expression or activation of the oncogenic fusion protein, or the signal transduction molecule, compared to the level of expression or is the reference activation indicates that the anti-cancer drug is suitable for . cancer treatment (for example, the tumor is more likely to respond to the anti-cancer drug). In other certain examples, the presence of an identical, similar, or greater level of expression or activation of the oncogenic fusion protein, or the signal transduction molecule, compared to the reference level or activation level, indicates that the drug anti-cancer is unsuitable for the treatment of - cancer (for example, the tumor is less likely to respond to the anti-cancer drug).
In alternative modalities, the level of expression or activation of the reference oncogenic fusion protein, or signal transduction molecule, determined in step (c) is obtained from a cell sensitive to the anti-cancer drug, which is treated with the anti-cancer medicine. In such embodiments, the presence of an identical, similar or lower level of expression or activation of the oncogenic fusion protein, or of the signal transduction molecule, compared to the level of expression or activation of ference -— indi "E ad j
M cancer treatment (for example, the tumor is more likely to respond to the anti-cancer drug). In other certain alternative modalities, the level of expression or activation of the reference oncogenic fusion protein, or signal transduction molecule, - determined in step (c) is obtained from a cell resistant to the anti-cancer drug, which is treated with the anti-cancer drug. In such modalities, the presence of an identical, similar or higher level of expression or activation of the oncogenic fusion protein, or of the signal transduction molecule, compared to the reference level of expression or activation, indicates that the anti- cancer is unsuitable for cancer treatment (for example, the tumor is less likely to respond to the anti-cancer drug). 'In certain embodiments, a higher level of expression or - activation of the oncogenic fusion protein, or signal transduction molecule, determined in step (c) is considered to be present in a cell extract when the level of expression or activation is at least about 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, 15, 20, 25, 30.35, 40, 45, 50, or 100 times greater (for example, about 1.5-3, 2-3, 2-4, 2- 5, 2- 10, 2-20, 2-50, 3-5, 3-10, 3-20, 3-50, 4-5, 4-10, 4-20, 4-50, 5-10, 5-15, 5-20, —or5-50 times higher) than the reference expression or activation level of the analyte that corresponds to a cell (for example, a cancer cell obtained from a patient sample) not treated with the drug anti-cancer, in a cell sensitive to the anti-cancer drug, treated with the anti-cancer drug, or in a cell resistant to the anti-cancer drug, treated with the anti-cancer drug.
In other embodiments, a lower level of expression or activation of the oncogenic fusion protein, or signal transduction molecule, determined in step (c) is considered to be present in an extract
K 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 , 15, 20, 25, 30, 35, "40, 45, 50, or 100 times smaller (e.g., about 1.5-3, 2-3, 2-4, 2-5, 2- 10, 2-20, 2-50, 3-5, 3-10, 3-20, 3-50, 4-5, 4-10, 4-20, 4-50, 5-10, 5-15, 5- 20, or 5-50 times lower) than the reference expression level or activation of the analyte that corresponds to a cell (for example, a cancer cell obtained from a patient sample) not treated with the anti-cancer drug, in a cell sensitive to the anti-cancer drug treated with the anti-cancer drug, or in a cell resistant to the anti-cancer drug treated with the anti-cancer drug. THE.
Antibody arrays In one aspect, the present invention provides an array with a higher dynamic range comprising a plurality of serial dilution of capture antibodies specific for one or more analytes in a cell extract, where capture antibodies are limited in a solid support.
In some embodiments, the cell extract is prepared from a sample of whole blood, urine, sputum, bronchial lavage fluid, tear, breast aspirate, lymph, saliva, and / or fine needle aspirate (FNA) sample.
As a non-limiting example, a whole blood sample is first separated into a fraction of plasma or serum, and a cell fraction (i.e., cell precipitate). The cell fraction typically contains red blood cells, white blood cells (leukocytes), and / or circulating cells from a solid tumor, such as circulating tumor cells (CTCs), circulating endothelial cells (CECs), circulating endothelial progenitor cells (CEPCs) ), cancer stem cells - (CSCs) and combinations of these.
The isolated cells present in the cell fraction can be lysed, thereby transforming the isolated cells into a cell extract, by any technique known in the art.
In some examples, the cell extract comprises an extract typically isolated from a patient sample using one or more separation methods including, for example, immunomagnetic separation (see, for example, Racila et al., Proc. Natl. Acad. Set USA, 95: 4589-4594 (1998); Bilkenroth et al., Int. J. Cancer, 92: 577-582 (2001)), microfluidic separation S (see, for example, Mohamed et al., IEEE Trans. Nanobiosci ., 3: 251-256 (2004); Lin et al, Abstract No. 5147, 977 AACR Annual Meeting, Washington, DC (2006)), FACS (see, for example, Mancuso et al., Blood, 97: 3658 -3661 (2001)), density gradient centrifugation (see, for example, Baker et al., Clin. Cancer Res., 13: 4865-4871 (2003)), and reduction methods (see, for example, Meye et al., Int. J. Oncol, 21: 521-530 (2002)).
In other examples, the cell extract comprises an extract of leukocytes, such as granulocytes (polymorphonuclear leukocytes), which include, for example, neutrophils, basophils and eosinophils, agranulocytes (leukocytes - mononuclear) that include, for example, blood mononuclear cells peripheral, such as lymphocytes and monocytes, and macrophages and mixtures of these. Leukocytes can be isolated from whole blood using any separation method known in the art including, for example, Ficoll-HyPaque density gradient centrifugation, hypotonic red blood cell lysis, and the use of density gradient media, such as Lymphoprep "M and - Polymorphprep"! M (Axis-Shield; Oslo, Norway).
In certain embodiments, isolated leukocytes, circulating cells or other cells (for example, cells obtained from a solid tumor by means of fine needle aspiration) can be stimulated in vitro with one or more growth factors before, during and / or after incubation with one or more anti-cancer drugs of interest. Stimulatory growth factors include, but are not limited to, epidermal growth factor (EGF), heregulin (HRG), TGF-oa, PIGF, angiopoietin (Ang), NRG1, PGF, TNF-a, VEGF, PDGF, IGF, FGF , HGF, cytokines and the like. In other cases; intas-isotad A Hisadas; toad
'stimulation of the growth factor and / or treatment with anti-cancer medication, to produce the cell extract (eg, cell lysate) using any technique known in the technology. Preferably, cell lysis is initiated between about 1-360 minutes after stimulation of the growth factor, and more preferably at two different time intervals: (1) about 1-5 minutes after stimulation of the growth factor; and (2) between about 30-180 minutes after stimulation of the growth factor. Alternatively, the cell lysate can be stored at -80 ° C until use. The protocols for the isolation, stimulation and lysis of circulating cells are described in PCT publication WO 2008/036802, which is incorporated herein by reference to its Integral, for all purposes. Protocols for the preparation of tumor cell extracts from tissue, biopsy or primary cultures are described in PCT publication WO 2009/108637, which is hereby "incorporated by reference in its entirety, for all purposes.
In certain embodiments, the anti-cancer drug comprises an anti-signaling agent (i.e., a cytostatic drug), such as a monoclonal antibody or a tyrosine kinase inhibitor, an anti-proliferative agent; a chemotherapeutic agent (i.e., a cytotoxic drug), a hormonal therapeutic agent, a radiotherapeutic agent, a vaccine, and / or any other compound with the ability to reduce or cancel the uncontrolled growth of abnormal cells, such as cancer cells. In some embodiments, the isolated cells are treated with one or more anti-signaling agents, anti-proliferative agents and / or hormonal therapeutic agents, in combination with at least one chemotherapeutic agent.
Examples of anti-signaling agents include, without limitation, monoclonal antibodies such as trastuzumab (Herceptin ), Alemtuzumab (Campath ), Bevacizumab (Avastin "), cetuximab (Erbitux ), DEI Er pani b-EVectibixaM) rituximab- ( Laugh and
And tositumomab (BEXXAR9), tyrosine kinase inhibitors such as mesylate - from imatinib (Gleevec ), Nilotinib (Tasigna ”), dasatinib (Sprycel”), bosutinib (SKI-606), gefitinib (Iressa ”), sunitinib (Sutent ), erlotinib (Tarceva ”), lapatinib (GW-572016; Tykerb ), canertinib (CI 1033), semaxinib (SUS416), vatalanib (PT 787 / ZK222584), sorafenib (BAY 43-9006; Nexavar”), leflunomide ( SOUTH 01) and vandetanib (ZACTEVIATM; ZD6474), and combinations thereof.
Exemplary anti-proliferative agents include mTOR inhibitors such as sirolimus (rapamycin), temsirolimus (CCI-779) and —everolimus (RADOO0I1), Akt inhibitors such as 1L6-hydroxymethyl-chiro-inositol-2- (R) -2- O-methyl-3-O-octadecyl-sn-glycerocarbonate, 9-methoxy-2-methyl acetate — ellipticin, 1,3-dihydro-1- (1 - ((4- (6-phenyl-IH- imidazo [4,5- 'glquinoxalin-7-yl) phenyl) methyl) -4-piperidinyl) -2H-benzimidazol-2-one, —10- (4 "- - (N-diethylamino) butyl) -2-chlorophenoxazine , 3-formileromone thiosemicarbazone (Cu complex (IDCl), API-2, a l5-mer peptide derivative of amino acids 10-24 of the proto-oncogene TCL1 (Hiromura et al., J. Biol. Chem., 279: 53407- 53418 (2004), KP372-1, and the compounds described in Kozikowski et al., J. Am. Chem. Soc, 125: 1144-1145 (2003) and Kau et al., Cancer Cell, 4: 463-476 ( 2003) and combinations thereof.
Non-limiting examples of chemotherapeutic agents include platinum-based drugs (eg, oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin, satraplatin, etc.), alkylating agents (eg, cyclophosphamide, ifosfamide, corambucil, busulfan, melfalan, mecloretamine, uramustine, thiotepa, nitrosureas, etc.), anti-metabolites (for example, 5-fluorouracil, azathioprine, 6-mercaptopurine, methotrexate, leucovorin, capecitabine, cytarabine, floxuridine, fludarabine, gemcethexine) (Gemzar) (Gemzar) ALDVITA ), Raltitrexed, etc.), plant alkaloids (e.g. vincristine, vinblastine, vinorelbine,
7 topoisomerase inhibitors (eg, irinotecan, topotecan, ansacrine, - etoposide (VP16), etoposide phosphate, teniposide, etc.), anti-tumor antibiotics (eg, doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), salts — pharmaceutically acceptable of these, stereoisomers thereof, derivatives thereof, analogs thereof and combinations thereof.
Examples of hormonal therapeutic agents include, without limitation, aromatase inhibitors (for example, aminoglutetimide, anastrozole (Arimidexº), letrozole (Femara ”), vorozole, exemestano (Aromasin ), 4-androsteno-3,6,17-triona ( 6-0XO), 1,4,6-androstatrien-3,17-dione (ATD), formestane (Lentaron ), Etc.), selective estrogen receptor modulators (eg, bazedoxifene, clomiphene, fulvestrant, fasofoxifene , raloxifene, tamoxifen, toremifene, etc.), steroids (for example, dexamethasone), finasteride, and —gonadotropin-releasing hormone (GnRH) agonists such as goserelin, pharmaceutically acceptable salts thereof, stereoisomers of these, derivatives thereof, analogs thereof and combinations thereof.
Non-limiting examples of cancer vaccines include ANYARA from Active Biotech, DCVax-LB from Northwest Biotherapeutics, EP- — 2101 from TDM Pharma, GV1001 from Pharmexa, 1IO-2055 from Idera Pharmaceuticals, INGN 225 from Introgen Therapeutics and Stimuvax from Biomira Merck.
Examples of radiotherapeutic agents include, but are not limited to, radionuclides such as “Sc,“ Cu, Cu, Sr, 6y, Ty, Y, Rh, Mag, "mm, Wing, Pm, Sm, 1667, VLy," Re, Re , 2AtE 2PBi, - optionally conjugated with antibodies directed against tumor antigens.
In particular embodiments, the one or more analytes present in the cell extract comprise one or a plurality of proteins of
It is signal transduction molecules. Non-limiting examples of proteins - oncogenic fusion and signal transduction molecules of interest are described above. In some embodiments, each serial dilution of capture - antibodies comprises a series of decreasing concentrations of capture antibody. In certain examples, the capture antibodies are serially diluted at least 2 times (for example, 2, 5, 10, 20, 50, 100, 500, or 1,000 times) to produce a serial dilution comprising an adjusted number (for example, example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more) of - decreasing concentrations of capture antibody, which are identified in the array. Preferably, at least 2, 3, 4, 5, or 6 replicates of each dilution of capture antibody are identified in the array. : In other modalities, the solid support comprises glass. (for example, a glass slide), plastic, chips, pins, filters, beads, 15th paper, membrane (for example, nylon, nitrocellulose, polyvinylidene fluoride (PVDF), etc.), bundles of fibers, or any other suitable substrate. In a preferred embodiment, capture antibodies are limited (for example, through covalent or non-covalent interactions) on glass slides coated with a nitrocellulose polymer such as, 20. for example, FASTº slides, which are commercially available from Whatman Inc. (Florham Park, NJ).
As a non-limiting example, an addressable microarray of the present invention may comprise a serial dilution of the capture antibody to determine the activation status of BCR-ABL in a cell extract, in which the capture antibody is directed to the BCR domain of the BCR-ABL fusion protein and detection antibodies (for example, both activation-dependent antibodies and activation-independent antibodies) are targeted to the
. how much the antibodies independent of the activation state are directed: to the BCR domain of the BCR-ABL fusion protein, and the antibody dependent on the activation state is directed to the ABL domain. The arrangements may additionally comprise a plurality of different - capture antibodies directed to the additional fusion proteins, and / or signal transduction molecules in a series of descending concentrations (i.e., serial dilutions), in which the capture antibodies they are coupled to the surface of the solid support in different addressable locations. Those skilled in the art will understand that the arrangement can be any configuration that allows for discrete signaling for each of the activated oncogenic fusion proteins, and / or signal transduction molecules to be detected. For example, the arrangement may be a line or a network 'of distinct regions (for example, points or spots) on the surface of the. support, where each region contains a different capture antibody or capture agent (that is, it binds to the capture label present on the capture antibody). The array can be configured for use in methods where the activation states of a plurality of oncogenic fusion proteins and / or signal transduction molecules are detected in a simple multiplex assay. In various embodiments, the plurality comprises at least 2, 3, 4,, 5,6,7,8,9,10,15,20,25, 30,35, 40, 45, 50, or more oncogenic fusion proteins and / or signal transduction molecules. In particular embodiments, the plurality comprises the BCR-ABL fusion protein in combination with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 , 45, 50, or more additional oncogenic fusion proteins and / or signal transduction molecules.
B. Double proximity detection assays In particular aspects, the assays of the present invention to detect the activation state of a particular analyte of interest in a
"Circulating a solid tumor, are a high yield multiplex proximity assay (ie, three-antibodies) with a higher dynamic range. As a non-limiting example, in situations where the analyte is a simple protein (eg, EGFR ), the three antibodies used in the proximity assay can comprise: (1) a capture antibody specific for the analyte; (2) a detection antibody specific for an activated form of the analyte (i.e., state-dependent antibody) ; and (3) a detection antibody that detects the total amount of the analyte (i.e., antibody independent of the activation state) .The antibody dependent on the activation state is capable of detecting, for example, phosphorylation, ubiquitination, and / or complex state of the analyte The antibody independent of the activation state is generally capable of detecting both activated and non-activated forms of the analyte.
. As another non-limiting example, in situations where the “analyte is a fusion protein (for example, BCR-ABL), which contains a first domain that corresponds to a protein (for example, BCR) that is fused to a second domain which corresponds to another protein (for example, ABL), the three antibodies used in the proximity assay can comprise: (1) a capture antibody specific to the first domain - fusion protein; (2) a detection antibody specific to an activated form of the second domain of the fusion protein (i.e., antibody dependent on the activation state); and (3) a detection antibody that detects the total amount of the fusion protein specifically binding to the second domain of the fusion protein, regardless of its activation state (i.e., - antibody independent of the activation state). The activation-dependent antibody is capable of detecting, for example, phosphorylation, ubiquitination, and / or complexation state of the fusion protein. The antibody independent of the activation state is in general capable of detecting both r In a preferred aspect, the present invention provides one. a method for performing a high performance multiplex immunoassay with a higher dynamic range, the method comprising: (a) incubating a cell extract with one or a plurality of serial dilution of capture antibodies, specific for one or more fusion proteins, to form a plurality of captured fusion proteins, wherein the capture antibodies are limited to a solid support, where each fusion protein comprises a first domain that corresponds to a first protein and a second domain that corresponds to a different second protein, and wherein the capture antibodies are specific to the first domain of the fusion proteins; (b) incubating the plurality of fusion proteins captured with k detection antibodies, specific for the second domain of the fusion proteins, to form a plurality of detectable captured fusion proteins, wherein the detection antibodies comprise: (1) a plurality of activation state-independent antibodies marked with a facilitation fraction, and (2) a plurality of activation-state antibodies marked with a first element of a signal amplification pair, where the facilitation fraction generates a oxidizing agent that channels and reacts with the first element of the signal amplification pair; (c) incubating the plurality of captured detectable fusion proteins with a second element of the signal amplification pair, to generate an amplified signal, and (d) detecting the amplified signal generated from the first and second elements of the amplification pair of signal.
In an alternative aspect, the method for performing a high-throughput multiplex immunoassay with a higher dynamic range FA == comprehendIrte: DO mm
- (a) incubate a cell extract with one or a plurality of. serial dilution of capture antibodies, specific for one or more fusion proteins, to form a plurality of captured fusion proteins, wherein the capture antibodies are limited to a solid support, where each fusion protein comprises a first domain that corresponds to a first protein and a second domain that corresponds to a different second protein, and in which the capture antibodies are specific to the first domain of the fusion proteins; (b) incubating the plurality of captured fusion proteins with detection antibodies to form a plurality of detectable captured fusion proteins, wherein the detection antibodies comprise: (1) a plurality of antibodies independent of the activation state marked with a facilitation fraction, in which the antibodies. independent of the activation state are specific to the first domain —fusion proteins, and (2) a plurality of activation-state-dependent antibodies labeled with a first element of a signal amplification pair, in which the state-dependent antibodies activation are specific to the second domain of fusion proteins, in which the facilitation fraction generates an oxidizing agent that channels and reacts with the first element of the signal amplification pair; (c) incubating the plurality of captured detectable fusion proteins with a second element of the signal amplification pair, to generate an amplified signal, and (d) detecting the amplified signal generated from the first and second elements of the amplification pair of signal.
In another alternative aspect, the method for performing a high-throughput multiplex immunoassay with a higher dynamic range FA - = comprtete: DDD
. (a) incubating a cell extract with one or a plurality of. serial dilution of capture antibodies, specific for one or more fusion proteins, to form a plurality of captured fusion proteins, wherein the capture antibodies are limited to a solid support, where each fusion protein comprises a first domain that corresponds to a first protein and a second domain that corresponds to a different second protein, and in which the capture antibodies are specific to the first domain of the fusion proteins; (b) incubating the plurality of fusion proteins captured with detection antibodies to form a plurality of detectable captured fusion proteins, wherein the detection antibodies comprise: (1) a plurality of activation-independent antibodies marked with a facilitation fraction, where antibodies' independent of the activation state are specific for the sequence, site, —fusion point between the first and second domains of the fusion proteins (i.e., binding antibodies), and (2) a plurality of activation state-dependent antibodies labeled with a first element of a signal amplification pair, where activation-state-dependent antibodies are specific —for the second domain of fusion proteins, where the facilitation fraction generates an agent oxidation channel that channels and reacts with the first element of the signal amplification pair; (oc) incubate the plurality of captured fusion proteins detectable with a second element of the signal amplification pair, to — generate an amplified signal, and (d) detect the amplified signal generated from the first and second elements of the amplification pair of signal.
In certain embodiments, one or more transduction molecules or fusion proteins. Examples of signal transduction molecules of interest. are described above and include, without limitation, receptor tyrosine kinases, non-receptor tyrosine kinases and / or components of the tyrosine kinase signaling cascade. The signal transduction molecules can - be detected using the methods described here, with the exception that all three antibodies (ie, the capture antibody and both detection antibodies) are directed to the same protein, or using any method known to those skilled in the art. In addition, signal transduction molecules can be detected using the simple detection assays (i.e., two antibodies) described in PCT publication WO 2008/036802, incorporated herein by reference in their entirety for all purposes. In particular embodiments, one or more of the signal transduction molecules present in the cell extract are detected together with one or more fusion proteins using the immunoassays and arrays described herein. In some examples, the cell extract is incubated with capture antibodies already limited on a solid support. In other examples, the cell extract is first incubated with capture antibodies in solution, and then placed in contact with a solid support to immobilize the captured analytes, for example, by means of capture labels present in the capture antibodies that interact with agents capture devices attached to the solid support.
In some embodiments, the detection antibodies are incubated with analytes that are bound to the capture antibodies in solution or bound to a solid support. In certain examples, the cell extract comprising a plurality of analytes is first incubated with the detection antibodies in solution, and then placed in contact with capture antibodies in solution or limited on a solid support. In other certain examples, the cell extract comprising a plurality of
. detection in solution, and then placed in contact with a solid support - to immobilize the antibody-analyte complexes, for example, by means of capture labels present in the capture antibodies or detection antibodies that interact with capture agents bound on the solid support . - Before the detection step, immobilized complexes can be washed to remove uncomplexed antibodies, washed complexes can be released sequentially from the support surface, and channeling by proximity to each of the analytes to be tested can be detected by a suitable method, as described herein.
In embodiments where the surface of the support comprises capture agents limited in the array, the incubation step may comprise placing the cell extract comprising a plurality of: analytes in solution in contact with the capture antibodies and antibodies from. detection, using an excess of all three antibodies to direct the reaction to completion. In a variation of the method, the resulting antibody-analyte complexes are affixed to a solid phase and washed to remove unbound antibodies. As shown in figure 2 of PCT publication WO 2008/036802, which is incorporated herein by reference in its entirety, for all purposes, capture antibody 1 may comprise capture label 10. The complexes are affixed to a solid phase 12 by means of a capture agent 11, which is adhered to the solid phase and binds to the capture label, thereby immobilizing the complex. The immobilized complex is washed with a suitable buffer, and then released from the solid phase by the addition of a release agent 13. The release agent - can work by any mechanism that results in the release of the washed complex. In one embodiment, the capture label comprises a cleavable site that is recognized and cleaved by the release agent. In another modality, represented in figure 2, the release agent competes with the
9%. The capture can be a first oligonucleotide that hybridizes to a - partially complementary - oligonucleotide (i.e., the capture label) affixed to the capture antibody, and the release agent can be an oligonucleotide that is completely complementary to the capture agent, - resulting in ribbon displacement and release of the washed complex from the solid phase. Other examples of suitable capture labels / capture agents / release agents that can be used include, but are not limited to, 2,4-dinitrophenol (DNP) / anti-DNP antibody / 2,4-DNP lysine, T2 / anti antibody -T3 / 13, ouabain / anti-digoxin / digoxin antibody and —detiobiotin / streptavidin / biotin (see, for example, Ishikawa et al., J. Clin.
Lab Anal, 12: 98-107 (1998)).
After the washed complex is released from the solid phase, it is either: (1) placed in contact with a support surface comprising capture molecules limited in the arrangement that specifically binds capture labels to the capture antibody, and (2) dissociated, and the dissociated detection antibodies are brought into contact with a support surface comprising capture agents that specifically bind capture labels to the detection antibodies. Figure 2 of PCT publication WO 2008/036802 represents the modality in which the washed complex is dissociated, and the dissociated detection antibodies are brought into contact with the support surface 14. The support surface comprises a plurality of capture molecules limited in an "addressable" or "location code" arrangement. Each distinct region of the array comprises a unique capture agent 9 that binds - specifically to the capture label 8 present in the antibody, regardless of the activation state of detection 2, or to the antibody dependent on the activation state 3, limiting and organizing by means of addition the labeled detection antibodies in the array. In a preferred embodiment, the agents of
«Specifically on each other. Addressable arrays comprising and oligonucleotide capture molecules are also known in the art (see, for example, Keramas et al., Lab Chip, 4: 152-158 (2004); Delrio-Lafreniere et al, Diag. Microbiol. Infect. Dis., 48: 23-31 (2004)).
The presence of the detection antibodies in each distinct region of the array can be detected directly or indirectly with a fraction, such as a facilitation fraction or a first element of a signal amplification pair. Examples of fractions that can be detected directly include fluorophores, chromophores, colloidal gold, colored latex, et. In one embodiment, both fractions are selected independently with fluorophores. Any pair of fluorophores that provide a distinguishable reading, while in close proximity to one another, can be used such as, for example, Cy3 / Cy5, Cy5 / phycohertrin and the like.
. Alternatively, if a targetable oligonucleotide array is used, both fractions can have the same fluorophore distributed in different location codes. Confocal laser scanning microscopy can be used to detect fluorophore fractions that are adhered to the array. In assays where complexes are released from the array prior to detection, such as in ribbon displacement assays, suitable methods for detecting fluorophore fractions include capillary flow confocal laser-induced fluorescence, nano-HPLC, micro-capillary electrophoresis, etc.
In some embodiments, antibodies independent of the activation state additionally comprise a detectable fraction. In such examples, the amount of the detectable fraction is correlated with the amount of one or more of the analytes in the cell extract. Examples of detectable fractions include, but are not limited to, fluorescent tags, chemically reactive tags, enzyme tags, radioactive tags and the like. Preferably, the detectable fraction is a fluorophore such as a fluorescein dye (FITC), Oregon Green'M; rhodamine, Texas red,. tetrarodamine isothiocinate (TRITCO), a CyDye "M fluorine (for example, Cy2, Cy3, Cy5) and the like. The detectable fraction can be coupled directly or indirectly to the antibodies regardless of the activation state, using the" methods well known in the art.
In certain examples, antibodies independent of the activation state are directly marked with the facilitation fraction. The facilitation fraction can be coupled to antibodies independent of the activation state using methods well known in the art. A facilitation fraction suitable for use in the present invention includes any molecule capable of generating an oxidizing agent that channels (ie, is directed to) and reacts with (that is, binds, is bound by, or forms a complex with ): another molecule in proximity (that is, spatially close or close): with the facilitation fraction. Examples of fractions of facilitation include, without limitation, enzymes such as glucose oxidase (GO) or any other enzyme that catalyzes an oxidation / reduction reaction involving molecular oxygen (Ox) as an electron acceptor, and photosynthesizers such as blue of methylene, cane rose, porphyrins, squarate dyes, phthalocyanines and the like. Non-limiting examples of oxidizing agents include hydrogen peroxide (H2O), a simple oxygen, and any other compound that transfers oxygen atoms or gains electrons in an oxidation / reduction reaction. Preferably, in the presence of a suitable substrate (for example, glucose, light, etc.), the facilitation fraction (for example, glucose oxidase, photosensitizer, etc.) generates an oxidizing agent (for example, - hydrogen peroxide ( HO), simple oxygen, etc.) that channels and reacts with the first element of the signal amplification pair (for example, horseradish peroxidase (HRP), hapten protected by a protective group, an enzyme inactivated by thioether binding to a enzyme inhibitor, etc. The two fractions are in close proximity to each other.
The preparation of sulfhydryl modified dextran molecules and their use in the preparation of conjugates between an antibody and a facilitation moiety, such as glucose oxidase (GO), is described in PCT publication WO 2009/108637, which is incorporated herein by reference in its integral, with all the purposes.
In other certain examples, antibodies independent of the activation state are indirectly marked with the facilitation fraction, by hybridizing between a ligand oligonucleotide conjugated to the antibodies independent of the activation state, and a complementary ligand oligonucleotide conjugated to the facilitation fraction . Linking oligonucleotides can be coupled to the facilitation moiety or to antibodies independent of the activation state using methods well known in the art. In some embodiments, the ligand-conjugated oligonucleotide in the facilitation fraction exhibits 100% complementarity with the conjugate-ligand oligonucleotide in antibodies independent of the activation state. In other embodiments, the oligonucleotide linker pair comprises at least one, two, three, four, five, six, or more regions of imperfect matching, for example, by hybridization under conditions of restrictive hybridization. Those skilled in the art will understand that antibodies independent of the activation state, specific for different analytes, can be either conjugated to the same ligand oligonucleotide or to different ligand oligonucleotides.
The size of the ligand oligonucleotides that are conjugated to the facilitation fraction or to the antibodies independent of the - activation state can vary. In general, the linker sequence can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides in size. Typically, random nucleic acid sequences are generated by coupling. As a non-limiting example, a library of distinct contiguous ones: a spacer domain, signature domain and conjugation domain. Preferably, the ligand oligonucleotides are determined by efficient coupling without impairing the function of the facilitation moiety or antibodies independent of the activation state, in which they are conjugated.
The sequences of ligand oligonucleotides can be determined to prevent or minimize any secondary structure formation in a variety of test conditions. Melting temperatures are typically carefully monitored for each segment in the binder to allow participation in all test procedures. In general, the melting temperature range of the segment of the ligand sequence is not greater than 5 ºC. Computer algorithms (for example, OLIGO 6.0) to determine the melting temperature, secondary structure, and loop-like structure, in defined ionic concentrations, can be used to analyze each of the three different domains in each ligand. The combined complete sequences can also be analyzed with respect to their structural characterization and their comparability with other conjugated ligand oligonucleotide sequences, for example, if they hybridize under conditions of restrictive hybridization to a complementary ligand oligonucleotide.
The spacer region of the ligand oligonucleotide provides adequate separation of the conjugation domain from the oligonucleotide crosslinking site. The conjugation domain works to bind molecules labeled with a complementary sequence of ligand oligonucleotides in the —conjugation domain through nucleic acid hybridization. Nucleic acid-mediated hybridization can be performed both before and after the formation of the antibody-analyte complex (ie, antigen), providing a more flexible assay format. Unlike many methods that bind to antibodies or other molecules, they have minimal impact on the specific affinity of antibodies in their target analyte or in the function of conjugated molecules. In some embodiments, the signature sequence domain of the ligand oligonucleotide can be used in a complex of multiplexed protein assays. The multiple antibodies can be conjugated to ligand oligonucleotides with different signature sequences. In multiplex immunoassays, sequences of reporter oligonucleotides labeled with appropriate probes can be used to detect cross-hybridization between antibodies and their antigens in the multiplex assay format.
Linking oligonucleotides can be conjugated to antibodies or other molecules using several different methods. For example, linker oligonucleotides can be synthesized with a thiol group at either the 5 'or 3' end. The thiol group can be deprotected using reducing agents (for example, TCEP-HC1) and the resulting binders can be purified using a rotating desalination column. The resulting unprotected linker oligonucleotides can be conjugated to the primary amines of antibodies, or other types of proteins, using heterobifunctional crosslinkers such as SMCC. Alternatively, the 5'-phosphate groups on oligonucleotides can be treated with water soluble carbodiimide EDC to form phosphate esters, and subsequently be coupled to the amine-containing molecules. In certain examples, the diol in the 3'-ribose residue can be oxidized to groups - aldehyde and then conjugated to the amine groups of antibodies, or other types of proteins, using reductive amination. In other certain examples, the linker oligonucleotide can be synthesized with a biotin modification at both the 3 'and 5' end and conjugated to molecules labeled with
Linking oligonucleotides can be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al., J. Am. Chem. Soc, 109: 7845 (1987); Scaringe et al., Nucl. Acids Res., 18: 5433 (1990); Wincott et al., Nucl. Acids Res., 23: 2677-2684 (1995); and Wincott et al., Methods Mol Bio., 74:59 (1997). In general, oligonucleotide synthesis uses common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 'end and phosphoramidites at the 3' end. Suitable reagents for oligonucleotide synthesis, methods for deprotecting nucleic acid, and methods for purifying nucleic acid are known to those skilled in the art.
The preparation and use of antibodies conjugated to the oligonucleotide for simultaneous detection of total and phosphorylated analytes is described in PCT publication WO 2008/036802, which is incorporated herein by: reference in its entirety, for all purposes.
In certain examples, antibodies dependent on the activation state are labeled directly with the first element of the signal amplification pair. The element of the signal amplification pair can be coupled to the activation-dependent antibodies using methods well known in the art. In other certain examples, the antibodies are dependent. of the activation state are indirectly marked with the first element of the signal amplification pair, by means of a connection between a first element of a binding pair conjugated to the antibodies dependent on the activation state and a second element of the binding pair conjugated to the first element of the signal amplification pair. The elements of the binding pair (e.g., biotin / streptavidin) can be coupled to the element of the signal amplification pair, or to the antibodies dependent on the activation state, using methods well known in the art. Examples of wild amplification pair (HRP) elements, catalase, chloroperoxidase, cytochrome, peroxidase, eosinophil peroxidase, glutathione peroxidase, lactoperoxidase, myeloperoxidase, thyroid peroxidase, deiodinase and the like. Other examples of signal amplifying pair elements include haptens protected by a protecting group and enzymes inactivated by thioether binding to an enzyme inhibitor.
Capture antibodies, antibodies independent of the activation state, and antibodies dependent on the activation state are typically selected to minimize competition between them with respect to the binding of the analyte (that is, all antibodies can simultaneously bind to their - proteins fusion molecules or corresponding signal transduction molecules). In an example of proximity channeling, the facilitation fraction is glucose oxidase (GO) and the first element in the 3rd signal amplification pair is horseradish peroxidase (HRP). When the GO is brought into contact with a substrate such as glucose, it generates an oxidizing agent (ie, hydrogen peroxide (EH2O,)). If HRP is channeling by proximity to the GO, the H, O, generated by the GO is channeled and complex with the HRP to form a complex HRP-H, O ,; that, in the presence of the second element of the signal amplification pair (for example, a chemiluminescent substrate, such as luminol or isoluminol, or a fluorogenic substrate such as tiramide (for example, biotin-tiramide), homovanilic acid, or 4- hydroxyphenyl acetic acid), generates an amplified signal. Methods of using GO and HRP in a proximity assay are described, for example, in Langry et al., U.S. Dept. of Energy Report No. UCRL-ID-136797 (1999). When biotin-tiramide is used as the - second element of the signal amplification pair, the complex HRP-H, O, oxidizes the tiramide to generate a reactive tiramide radical that covalently bonds close to the nucleophilic residues. Activated tiramide is either detected directly or detected by adding a streptavidin reagent or a combination of a streptavidin-labeled peroxidase and a chromogenic reagent.
Examples of fluorophores suitable for use in the present invention include, but are not limited to, an Alexa Fluor dye (e.g., Alexa Fluor 555), fluorescein, fluorescein isothiocyanate (FITC), Oregon Green'M; rhodamine, Texas red, tetrarodamine isothiocyanate (TRITC), a CyDye "M fluorine (eg, Cy2, Cy3, Cy5) and the like.
The streptavidin label can be attached directly or indirectly to the fluorophore or peroxidase using methods well known in the art.
Non-limiting examples of reagent — chromogenic suitable for use in the present invention include 3.3 ', 5.5 "- tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB), 2,2'-azino-bis ( 3-ethylbenzothiazoline-6-sulfonic) (ABTS), 4-chloro-1-naptol (4CN) and / or 'porphyrinogen. In another example of proximity channeling, the facilitation fraction is a photosensitizer, and the first element The signal amplification pair is a large molecule labeled with multiple haptens that are protected by protective groups, which prevent the binding of the haptens to a specific binding partner (for example, ligand, antibody, etc.). of the signal amplification pair may be a dextran molecule labeled with protected biotin, coumarin and / or fluorescein molecules.
Suitable protecting groups include, but are not limited to, phenoxy, analino, olefin, thioether, and selenoether protecting groups.
Additional photosensitizers and hapten-protected molecules, suitable for use in the proximity assays of the present invention, are described in U.S. patent 5,807,675. When the photosensitizer is excited by light, it generates an oxidizing agent (ie, simple oxygen). If the hapten molecules are in channeling by proximity to the photosensitizer, the simple oxygen generated by the haptens & ibilizadoré Hizad> to produce carbonyl groups (ketones or aldehydes) and sulfinic acid, releasing the protective groups of the haptens. The unprotected haptens are then available to specifically bind to the second element of the signal amplification pair (for example, a specific de-binding partner that can generate a detectable signal). For example, when the hapten is biotin, the specific binding partner may be an enzyme-labeled streptavidin. Exemplary enzymes include alkaline phosphatase, / 3-galactosidase, HRP, etc. After washing to remove unbound reagents, the detectable signal can be generated by adding a detectable substrate (e.g., fluorescent, chemiluminescent, chromogenic, etc.) to the enzyme, and be detected using suitable methods and instrumentation known in the art. Alternatively, the detectable signal 'can be amplified using tyramide signal amplification, and the activated - tyramide can be detected either directly or detected by adding a signal detection reagent, in the manner described above.
In yet another example of proximity channeling, the facilitation fraction is a photosensitizer, and the first element of the signal amplification pair is an enzyme-inhibitor complex. The enzyme and the - inhibitor (for example, phosphonic acid-labeled dextran) are linked together by a cleavable linker (for example, thioether). When the photosensitizer is excited by light, it generates an oxidizing agent (ie, simple oxygen). If the enzyme-inhibitor complex is channeling by proximity to the photosensitizer, the simple oxygen generated by the photosensitizer is channeled and reacts with the cleavable ligand, releasing the enzyme inhibitor, thereby activating the enzyme. An enzyme substrate is added to generate a detectable signal or, alternatively, an amplification reagent is added to generate an amplified signal.
In an additional example of proximity channeling, the facilitation fraction is HRP, the first element of the signal amplification pair is a protected hapten or an enzyme-inhibitor complex in the manner described above, and the protecting groups comprise p-alkoxy phenol. A - addition of phenylenediamine and H5O; it generates a reactive phenylenediimine that channels into the protected hapten or enzyme-inhibitor complex, and reacts with p-alkoxy phenol protecting groups to produce exposed haptens or a reactive enzyme. The amplified signal is generated and detected in the manner described above (see, for example, U.S. patent 5,532,138 and 5,445,944).
Those skilled in the art will understand that binding partners, other than antibodies, can be used to immobilize and / or detect one or more analytes of a cell extract according to the proximity assays - (i.e., three-antibodies) described herein . Non-limiting examples of such binding partners include analyte linkers or receptors, substrates of the —analyte, binding domains (e.g., PTB, SH2, etc.), aptamers and the like.
An exemplary protocol for performing the proximity assays described herein is provided in example 1. In another embodiment, the present invention provides kits for performing the previously described proximity assays comprising: (a) a serial dilution of a plurality of antibodies of captures limited on a solid support and (b) a plurality of detection antibodies (for example, antibodies independent of the activation state and antibodies dependent on the activation state). In some examples, the kits may additionally contain instructions for methods of using the kit to detect the activation states of one or a plurality of fusion proteins, and / or signal transduction molecules. The kits can also contain any of the additional reagents described in the invention, such as, for example, first and second elements of the signal amplification pair, tyramide signal amplification reagents, substrates for the facilitation fraction, wash buffers, etc.
IV. Construction of antibody arrays In certain respects, the present invention provides antibody-based arrays to detect the activation state of one or a plurality of fusion proteins, in a cell extract, using a serial dilution of capture antibodies limited in a solid support. The arrangements used in the assays of the present invention typically comprise a plurality of one or more different capture antibodies, in a range of concentrations of capture antibody that are coupled to the surface of a solid support in different addressable locations. p The solid support can comprise any substrate suitable for immobilizing proteins. Examples of solid supports include, but are not limited to, glass (for example, a glass slide), plastic, chips, pins, filters, beads (for example, magnetic beads, polystyrene beads, etc.), paper, membranes, bundles of fibers, gels, metal, ceramics and the like. Membranes, such as nylon (Biotrans "M, ICN Biomedicals, Inc. (Costa Mesa, CA); Zeta-Probe”, Bio-Rad Laboratories (Hercules, CA)), —nitrocellulose (Protranº, Whatman Inc. (Florham Park, NN)) and PVDF (ImmobilonTM, Millipore Corp. (Billerica, MA)), are suitable for use as solid supports in the arrangements of the present invention. nitrocellulose, for example, FAST slides, which are available - commercially from Whatman Inc. (Florham Park, NJ).
Particular aspects of the solid support that are desirable include the ability to bind to large amounts of capture antibodies, the ability to bind capture antibodies with TT denaturation - minimal; -and-8 inability to bind to other proteins. Another aspect | “—D—— Ow is that the solid support can exhibit minimal" capillarity "when antibody solutions containing capture antibodies are applied to the support. A solid support with minimal capillarity allows small aliquots of capture antibody solution applied to the support to result in - small, defined patches of immobilized capture antibody.
Capture antibodies are typically limited directly or indirectly (for example, by means of capture labels) to the solid support through covalent or non-covalent interactions (for example, ionic bonds, hydrophobic interactions, hydrogen bonds, Van der forces Waals, dipole-dipole connections). In some embodiments, the capture antibodies are affixed covalently to the solid support using a homobifunctional or heterobifunctional crosslinker, using standard crosslinking methods and conditions. Suitable crosslinkers are available: commercially from suppliers such as, for example, Pierce Biotechnology
15. (Rockford, IL).
Methods for generating the arrays of the present invention include, but are not limited to, any technique used to construct arrays of protein or nucleic acid. In some embodiments, the capture antibodies are identified in an array using a microidentifier, which are typically robotic printers equipped with split pins, blind pins or prints. Robotic systems suitable for printing the antibody arrangements described here include the PixSys 5000 robot (Cartesian Technologies; Irvine, CA) with split pins ChipMaker2 (TeleChem International; Sunnyvale, CA), as well as other robotic printers - available from BioRobics (Wobum, MA) and Packard Instrument Co. (Meriden, CT). Preferably, at least 2, 3, 4, 5, or 6 replicates of each dilution of capture antibody are identified in the array.
Another method for generating antibody arrays from the capture antibody dilution in each position of the selected array, by placing a capillary dispenser on a solid support in conditions efficient to pull a defined volume of liquid in the support, in which this process is repeated using dilutions of capture antibody selected in - each array position selected to create a complete array. The method can be practiced to form a plurality of such arrangements, where the solution deposition step is applied to a selected position on each of a plurality of solid supports, in each repeated cycle. A further description of such a method can be found, for example, in U.S. patent 5,807,522. In certain examples, devices for printing on paper can be used to generate the antibody arrays of the present invention. í For example, the desired capture antibody dilution can be loaded W into the print head of a domestic inkjet printer and printed on a suitable solid support (see, for example, Silzel et al., Clin. Chem., 44: 2036-2043 (1998)). In some embodiments, the arrangement generated on the solid support has a density of at least about 5 points / cm ”, and preferably at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, —110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600 , 650, 700, 750, 800, 850, 900, 950, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000 or
9,000, or 10,000 points / cm ”. In certain examples, the dots on the solid support represent - each a different capture antibody. In other certain examples, multiple points on the solid support represent the same capture antibody, for example, as a serial dilution comprising a series of decreasing concentrations of capture antibody. AND
Additional examples of methods for preparing and constructing antibody arrays on solid supports are described in U.S. patents.
6,197,599, 6,777,239, 6,780,582, 6,897,073, 7,179,638 and 7,192,720, US patent publications 20060115810, 20060263837, 20060292680 and 20070054326; and Varnum et al., Methods Mol. Biol., 264: 161-172 (2004).
Methods for examining the antibody arrays are known in the art and include, without limitation, any technique used to examine the protein or nucleic acid arrays. Microarray examiners suitable for use in the present invention are available from PerkinElmer (Boston, MA), Agilent Technologies (Palo Alto, CA), Applied Precision (Issaquah, WA), GSI Lumonics Inc. (Billerica, MA), and Axon Instruments (Union City, CA). As a non-limiting example, a: GSI ScanArray 3000 for fluorescence detection can be used with. ImaGene software for quantification.
V. Drug Selection and Optimization for Cancer Therapy In certain respects, the present invention provides methods for the selection of appropriate therapies for infraring or interrupting one or more unregulated signaling pathways. In other certain respects, the present invention provides methods for optimizing therapy and / or reducing toxicity in a subject with cancer who is receiving a period of therapy for the treatment of cancer. Thus, the present invention can be used to facilitate the determination of personalized therapies, based on the particular molecular signature provided by the collection of activated oncogenic fusion proteins and / or signal transduction proteins in a given patient's cancer or tumor.
Thus, in a particular aspect, the present invention provides a method for optimizing therapy and / or reducing toxicity in a
RSA subject with cancer, and who receives a period of therapy for the treatment of cancer, the method comprising: (a) isolating cancer cells after administration of an anti-cancer drug (for example, one or more tyrosine inhibitors - kinasetal such as Gleevecº, Tasigna ”, Sprycelº , etc.); (b) lyse the isolated cells to produce a cell extract; (c) measuring a level of expression and / or activation (for example, phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay described herein; and (d) comparing the measured level of expression and / or activation of the oncogenic fusion protein with a level of expression and / or activation of the oncogenic fusion protein, measured at an earlier time, during the IS period of therapy; and . (e) determining a subsequent dose of the therapy period — for the subject, or whether a different therapy period can be administered to the subject based on the comparison of step (d).
In particular modalities, both the levels of total and activated oncogenic fusion protein (for example, phosphorylated) (for example, BCR-ABL) are measured in the cell extract, according to the antibody-based assays of the present invention, and a ratio of activated oncogenic fusion protein levels to total (eg ratio of phosphorus-total BCR-ABL protein levels) can be calculated and used to assess the period of therapy for a subject, for example, by comparing the ratio of the levels of phospho / total oncogenic fusion protein with the same ratio - calculated for the subject at an early stage (for example, at an early stage, although in anti-cancer drug therapy, or in a period of time before drug therapy anti-cancer).
In a related aspect, the present invention provides a TT ——— method to para-optimize therapy and / or to reduce toxicity in a subject with ÚÚ | - | cancer, and receiving a period of therapy for the treatment of cancer, the method comprising: (a) isolating cancer cells after administration of an anti-cancer drug (for example, one or more tyrosine inhibitors - quinasetalcom as Gleevec *, Tasigna ”, Sprycelº, etc.); (b) lyse the isolated cells to produce a cell extract; (c) measure an expression and / or activation level (for example, phosphorylation) of an oncogenic fusion protein and one or more signal transduction molecules in its pathway in the cell extract, using an assay here —described; ) to compare the measured level of expression and / or activation of the oncogenic fusion protein and signal transduction molecules with a - level of expression and / or activation of the oncogenic fusion protein and molecules: of signal transduction, measured at an initial moment during the therapy period (e) determining a subsequent dose of the therapy period for the subject, or whether a different therapy period can be administered to the subject based on the comparison of step (d). In particular embodiments, both the levels of total and activated oncogenic fusion protein (for example, phosphorylated) (for example, BCR-ABL), and the component levels of the signal transduction pathway (for example, CRKL, JAK2, STATS ), are measured in the cell extract, according to the antibody-based assays of the present invention, and a ratio of activated oncogenic fusion protein levels to total (eg ratio of phosphorus / total BCR-ABL protein levels) and a ratio of component levels of the total to activated signal transduction pathway (for example, ratio of CRKL, JAK2, or phospho / total STATS protein levels) can be calculated and used to assess the therapy period for a TT - - subject; for example, comparing the ratio of fusion protein levels —UúÚúÚúÚúÚú | phosphorus / total oncogenic, and the component of the signal transduction pathway, with the same ratio calculated for the subject in an early stage (for example, in an early stage, although in therapy with anti-cancer medication, or in a period of time before therapy with anti-cancer medication).
In another aspect, the present invention provides a method for selecting an anti-cancer drug suitable for the treatment of a cancer, the method comprising: (a) isolating cancer cells after administration of an anti-cancer drug, or before incubation with an anti-cancer medication; (b) lyse the isolated cells to produce a cell extract; '(oc) determining an expression and / or activation level (eg, phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay described herein; and (d) determine whether the anti-cancer drug is suitable or inappropriate for the treatment of cancer, by comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference expression and / or activation profile generated in the absence of anti-cancer medication.
In a preferred embodiment, the method for selecting an appropriate anti-cancer drug for the treatment of cancer comprises: (a) isolating cancer cells after administration of an anti-cancer drug, or before incubation with an anti-cancer drug - cancer; (b) lyse the isolated cells to produce a cell extract; (c) determining a level of expression and / or activation (for
Cell PE using an assay comprising a serial dilution of capture antibodies specific for the oncogenic fusion protein, wherein the capture antibodies are limited to a solid support; (d) compare the level of expression and / or activation detected for S the oncogenic fusion protein with a reference expression and / or activation profile generated in the absence of the anti-cancer drug; and (e) indicate whether the anti-cancer drug is suitable for the treatment of cancer when the level of expression and / or activation detected for the oncogenic fusion protein is changed (for example, - substantially decreased), compared to the expression profile and / or reference activation. In certain examples, the preferred embodiment can. additionally understand, that is, as step (O, or alternatively understand, that is, as step (e), the step of indicating whether the drug —anti-cancer is unsuitable for the treatment of cancer when the level of expression and / or activation detected for the oncogenic fusion protein is not altered (for example, it is not substantially decreased), compared to the expression profile and / or reference activation. In other examples, one or more signal transduction molecules present in the cell extract are detected, in addition to one or more oncogenic fusion proteins, and the anti-cancer drug is determined to be suitable or inappropriate based on this “molecular profile.” In particular modalities, both the levels of total and activated oncogenic fusion protein (for example, phosphorylated) (eg, BCR-ABL) are measured in the cell extract, according to the antibody-based assays of the present invention, and a ratio of the levels of activated oncogenic fusion protein to total ratio (eg ratio of phosphorus-to-total BCR-ABL protein levels) can be calculated and used to determine TT see anti-cancer drug is suitable or inappropriate for the treatment of
PE cancer, for example, comparing the ratio of phosphorus / total oncogenic fusion protein levels with the same ratio calculated based on the reference expression and activation profiles, which were generated in the absence of the anti-cancer drug.
In another aspect, the present invention provides a method for identifying a cancer's response to treatment with an anti-cancer drug, the method comprising: (a) isolating cancer cells after administration of an anti-cancer drug, or before incubation with an anti-cancer medication; (b) lyse the isolated cells to produce a cell extract; (c) determining a level of expression and / or activation (for example, phosphorylation) of an oncogenic fusion protein in the extract: cell, using an assay described herein; and (d) identify the cancer as responsive or not responsive to treatment with the anti-cancer drug, comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference expression and / or activation profile generated in the absence of anti-cancer medication.
In a preferred embodiment, the method for identifying a cancer's response to treatment with an anti-cancer drug comprises: (a) isolating cancer cells after administration of an anti-cancer drug, or before incubation with an anti-cancer drug - cancer; (b) lyse the isolated cells to produce a cell extract; (c) determining a level of expression and / or activation (for example, phosphorylation) of an oncogenic fusion protein in the TT-cell extract, -using an assay comprising a serial dilution of | N ————
capture antibodies specific for the oncogenic fusion protein, where the capture antibodies are limited on a solid support; (d) compare the level of expression and / or activation detected for the oncogenic fusion protein with a reference and / or activation profile of reference generated in the absence of the anti-cancer drug; and (e) indicate whether the cancer is responsive to treatment with the anti-cancer drug when the level of expression and / or activation detected for the oncogenic fusion protein is altered (for example, substantially decreased), compared to the expression profile and / or reference activation. In certain examples, the preferred embodiment may further comprise, i.e., as step (1), or comprise. alternatively, that is, as step (e), the step of indicating whether the cancer is not: responsive to treatment with the anti-cancer drug when the level of expression and / or activation detected for the oncogenic fusion protein is not changed ( for example, is not substantially decreased), compared to the reference expression and / or activation profile. In other examples, one or more signal transduction molecules present in the cell extract are detected, in addition to one or more oncogenic fusion proteins, and the cancer is identified as responsive or unresponsive to treatment based on this "molecular profile".
In particular embodiments, both the levels of total and activated oncogenic fusion protein (eg, phosphorylated) (eg, BCR-ABL) are measured in the cell extract, according to the —sensitive antibody base of the present invention, and a ratio of activated oncogenic fusion protein levels to total (for example, ratio of phosphorus-to-total BCR-ABL protein levels) can be calculated and used to identify whether the cancer is responsive or not responsive to treatment with the TT drug --- —-Anti-eâneer, for example, comparing the ratio of N-fusion protein levels—————
phosphorus / total oncogenic with the same ratio calculated based on the reference expression and activation profiles, which were generated in the absence of the anti-cancer medication.
In yet another aspect, the present invention provides a - method for predicting the response of a subject with cancer to treatment with an anti-cancer drug, the method comprising: (a) isolating cells from a cancer after administration of an anti-cancer drug -cancer, or before incubation with an anti-cancer medication; (b) lyse the isolated cells to produce a cell extract; (c) determining an expression and / or activation level (eg, phosphorylation) of an oncogenic fusion protein in the extract. cell, using an assay described herein; and . (d) predict the likelihood that the subject will respond to treatment with the anti-cancer drug, comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference expression and / or activation profile, generated in the absence of the anti-cancer medication.
In a preferred embodiment, the method for predicting the response of a subject with cancer to treatment with an anti-cancer drug comprises: (a) isolating cancer cells after administration of an anti-cancer drug, or before incubation with an anti-cancer drug; (b) lyse the isolated cells to produce a cell extract; (c) determining an expression and / or activation level (eg, phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay comprising a serial dilution of POE rr NE —————
capture antibodies specific for the oncogenic fusion protein, where the capture antibodies are limited on a solid support; (d) compare the level of expression and / or activation detected for the oncogenic fusion protein with a reference and / or activation profile of - reference, generated in the absence of the anti-cancer drug; and (e) indicate whether the subject is likely to respond to treatment with the anti-cancer drug when the level of expression and / or activation detected for the oncogenic fusion protein is changed (for example, substantially decreased), compared to the expression profile - and / or reference activation. In certain examples, the preferred embodiment may further comprise, that is, as step (f), or comprise. alternatively, that is, as step (e), the step of indicating whether the subject: likely will not respond to treatment with the anti-cancer drug, —when the level of expression and / or activation detected for the oncogenic fusion protein is not changed (for example, it is not substantially decreased), compared to the reference expression and / or activation profile. In other examples, one or more signal transduction molecules present in the cell extract are detected, in addition to one or more fusion proteins 20. oncogenic, and the probability that the subject will respond to treatment is predicted based on this "molecular profile".
In particular modalities, both the levels of total and activated oncogenic fusion protein (for example, phosphorylated) (for example, BCR-ABL) are measured in the cell extract, according to the antibody-based assays of the present invention, and a ratio of activated oncogenic fusion protein levels to total (for example, ratio of phosphorus-total BCR-ABL protein levels) can be calculated and used to predict whether the subject will be likely to respond to treatment with TT Anti Radiation -cancer, for example, comparing the ratio of UU levels NTE—— ""
phospho / total oncogenic fusion protein with the same ratio calculated based on the reference expression and activation profiles, which were generated in the absence of the anti-cancer drug.
In a further aspect, the present invention provides a - method for determining whether a subject with cancer is resistant to treatment with an anti-cancer drug, the method comprising: (a) isolating cells from a cancer after administration of an anti-cancer drug cancer, or before incubation with an anti-cancer drug; (b) lyse the isolated cells to produce a cell extract; (c) determining an expression and / or activation level (eg, phosphorylation) of an oncogenic fusion protein in the extract. cell, using an assay described herein; and: (d) determine whether the subject is resistant or sensitive to treatment with the anti-cancer drug, by comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference expression and / or activation profile, generated in the absence of the anti-cancer drug or in the presence of the anti-cancer drug at an early stage.
In a preferred embodiment, the method for determining whether a subject with cancer is resistant to treatment with an anti-cancer drug comprises: (a) isolating cancer cells after administration of an anti-cancer drug, or before incubation with a anti-cancer medication; (b) lyse the isolated cells to produce a cell extract; (c) determining an expression and / or activation level (eg, phosphorylation) of an oncogenic fusion protein in the cell extract, using an assay comprising a serial dilution of FAN
NE specific capture antibodies for the oncogenic fusion protein, where the capture antibodies are limited on a solid support; (d) comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference and / or activation profile of reference, generated in the absence of the anti-cancer drug or in the presence of the anti-cancer drug in a early stage; and (e) indicate whether the subject is resistant to treatment with the anti-cancer drug when the level of expression and / or activation detected for the oncogenic fusion protein is not altered (for example, it is not - substantially decreased), compared to expression profile and / or reference activation. In certain examples, the preferred embodiment can. additionally understand, that is, as a step (Yes, or understand. alternatively, that is, as a step (e), the step of indicating whether the subject is sensitive to treatment with the anti-cancer medication when the level of expression and / or activation detected for the oncogenic fusion protein is altered (for example, substantially decreased), compared to the expression profile and / or reference activation. In other examples, one or more signal transduction molecules present in the cell extract are detected, in addition to of one or more oncogenic fusion proteins, and the subject is identified as resistant or sensitive to treatment based on this "molecular profile". In particular modalities, both the levels of total and activated oncogenic fusion protein (for example, phosphorylated) ( for example, BCR-ABL) are measured in the cell extract, according to the antibody-based assays of the present invention, and a ratio of activated oncogenic fusion protein levels to total (eg (ratio of phosphorus-to-total BCR-ABL protein levels) can be calculated and used to determine HUH
7 Ú It is cancer, for example, comparing the ratio of the levels of fusion protein: phosphorus / total oncogenic with the same ratio calculated based on the Na expression and reference activation profiles, which were generated in the absence of. anti-cancer medication or in the presence of the anti-cancer medication at an early stage.
In some embodiments, the methods of the present invention may further comprise sending or reporting the results of step (d) to a physician, for example, an oncologist or general practitioner. In other embodiments, the methods of the present invention may comprise - additionally recording or storing the results of step (d) in a computer database or other machine or device suitable for storing information, for example, in a laboratory. In some embodiments, the methods of the present invention may further comprise the step of obtaining a sample from a subject with cancer, from which cells, such as cancer cells, are isolated. The sample can be obtained from a subject either before treatment with an anti-cancer drug (for example, before incubation with an anti-cancer drug) or after administration of an anti-cancer drug (for example, at any time throughout cancer treatment). Suitable samples include, but are not limited to, whole blood, plasma, serum, ductal lavage fluid, breast aspirate, lymph (eg, disseminated tumor cells from the lymph node), bone marrow aspirate, saliva, urine, excrement (ie , feces), sputum, bronchial lavage fluid, tears, fine needle aspiration (for example, collected by random periareolar fine needle aspiration), any other body fluid, a tissue sample (for example, tumor tissue), such as a biopsy of a tumor (for example, needle biopsy) or a lymph node (for example, sentinel lymph node biopsy), and cell extracts thereof. In Tr ———> some -modalities, the sample is whole blood or a fractional component TTITTFAI IO O aaa N—— 0 "-
: * - of this, such as plasma, serum, red blood cells, leukocytes such as "peripheral blood mononuclear cells, and / or rare circulating Fa cells. In particular modalities, the sample is obtained by isolating" leukocytes or circulating cells from a solid tumor from whole blood, or a cellular fraction thereof, using any technique known in the art. If the isolated cells are obtained from a subject who has not received treatment with an anti-cancer drug, the isolated cells can be incubated in vitro, under appropriate conditions, with one or a cocktail of anti-cancer drugs that target one or more of the analytes. to be detected in step (o. In certain modalities, cancer is a hematological malignancy (for example, leukemia, lymphoma), osteogenic sarcoma (for example, Ewing's sarcoma), soft tissue sarcoma (for example, DFSP,. rhabdomyosarcoma), another soft tissue malignancy, papillary thyroid carcinoma, or prostate cancer. In particular modalities, cancer is caused by the formation of an oncogenic fusion protein due to a chromosomal translocation in the cancer cells or tumor. , isolated cells are stimulated in vitro with growth factors in the manner described here. In others - modalities, the anti-cancer drug may comprise one or more of the therapeutic agents techniques described herein including, but not limited to, monoclonal antibodies, tyrosine kinase inhibitors, chemotherapeutic agents, hormonal therapeutic agents, radiotherapeutic agents and vaccines. In some embodiments, the activation state detected for the oncogenic fusion protein present in the cell extract can be, for example, a phosphorylation state, a state of ubiquitination, a state of complexation, or combinations thereof. In other embodiments, the solid support may comprise, for example, glass, plastic, chips, pins, TT filters, beads, paper, membrane, bundles of fibers and combinations thereof. úÚ HUH
: “- In yet other modalities, the capture antibodies are limited to the solid support in a targetable arrangement. mp In certain embodiments, the assay in step (c) comprises: P (1) incubating (for example, contacting) the cell extract with dilution in a series of capture antibodies to form a plurality of captured oncogenic fusion proteins (for example , to transform the oncogenic fusion proteins, present in the cell extract, into captured oncogenic fusion protein complexes comprising the oncogenic fusion proteins and capture antibodies);
(11) incubating (e.g., contacting) the plurality of oncogenic fusion proteins captured with detection antibodies, which comprise antibodies independent of the activation state and antibodies dependent on the specific activation state for the same or different
And domain of the oncogenic fusion protein, to form a plurality of detectable captured oncogenic fusion proteins (for example, to transform the captured oncogenic fusion protein complexes into detectable captured oncogenic fusion protein complexes comprising the captured and oncogenic fusion proteins detection antibodies),
where antibodies independent of the activation state are marked with a facilitation fraction, antibodies dependent on the activation state are marked with a first element of a signal amplification pair, and the facilitation fraction generates an oxidizing agent that channels and reacts with the first element of the signal amplification pair;
(iii) incubate (for example, put in contact) the plurality of detectable captured analytes with a second element of the signal amplification pair to generate an amplified signal, and are [RCC —————————— A
: It is (iv) to detect the amplified signal generated from the first and second second elements of the signal amplification pair. - Antibodies independent of the activation state can be. tagged directly with the facilitation fraction or indirectly tagged with the facilitation fraction, for example, by hybridizing between an oligonucleotide conjugated to the antibodies independent of the activation state and a complementary oligonucleotide conjugated to the facilitation fraction.
Similarly, antibodies dependent on the activation state can be directly labeled with the first element of the signal amplification pair or indirectly marked with the first element of the signal amplification pair, for example, by means of a link between a first element of a binding pair conjugated to the activation state-dependent antibodies and a second element of the binding pair. in conjunction with the first element of the signal amplification pair.
In certain examples, the first element of the binding pair is biotin, and the second element of the binding pair is an avidin, such as streptavidin or neutravidine.
In some modalities, the facilitation fraction may be, for example, glucose oxidase (GO). In certain examples, GO and antibodies independent of the activation state can be conjugated to a sulfhydryl-activated dextran molecule.
The sulfhydryl-activated dextran molecule typically has a molecular weight of about 500kDa (for example, about 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750kDa). In other embodiments, the oxidizing agent can be, for example, hydrogen peroxide (HO). In yet other embodiments, the first element of the signal amplification pair can be, for example, a peroxidase such as horseradish peroxidase (HRP). In additional embodiments, the second element of the TT signal amplification pair -may be, for example, a tiramide reagent (eg biotin-tiramide) ÉÚúÚ N ——— >>>>
: * - Preferably, the amplified signal is generated by oxidation of biotin-tiramide peroxidase to produce an activated tiramide (for example, to transform the biotin-tiramide into an activated tiramide). Activated tiramide - can be detected directly or detected indirectly, for example, with the addition of a signal detection reagent. Non-limiting examples of signal detection reagents include streptavidin-labeled fluorophore, and combinations of streptavidin-labeled peroxidases and chromogenic reagents such as, for example, 3,3 ', 5,5'-tetramethylbenzidine (TMB).
In certain examples, HRP and activation-dependent antibodies can be conjugated to a sulfhydryl-activated dextran molecule. The sulfhydryl-activated dextran molecule typically has a molecular weight of about 70kDa (e.g., about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100kDa ).
SAW. Production of antibodies The generation and selection of antibodies not yet commercially available to analyze the activation states of oncogenic fusion proteins, or signal transduction molecules, in a biological sample such as an extract or cell lysate, according to the invention, can be accomplished in several ways. For example, one of them is to express and / or purify a polypeptide of interest (i.e., antigen) using the protein expression and purification methods known in the art, while another way is to synthesize the polypeptide of interest using peptide synthesis methods solid phase compounds known in the art. See, for example, —Guideto Protein Purification, Murray P. Deutcher, ed., Meth. Enzymol., Vol. 182 (1990); Solid Phase Peptide Synthesis, Greg B. Fields, ed., Meth. Enzymol., Vol. 289 (1997); Kiso et al., Chem. Pharm. Bull., 38: 1192-99 (1990); Mostafavi et al., Biomed. Pept. Proteins Nucleic Acids, 1: 255-60, FOOT
: “Purified or synthesized polypeptide can then be injected, for example, [into mice or rabbits, to generate polyclonal antibodies or monoclonal Fat.
Those skilled in the art will recognize that many procedures - available for the production of antibodies, for example, in the manner - describe Antibodies, A Laboratory Manual, Harlow and Lane, Eds., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1988). Those skilled in the art will also understand that binding fragments or Fab fragments, which mimic (for example, maintain the functional binding regions of) antibodies, can also be prepared from genetic information by various procedures.
See, for example, Antibody Engineering: A Practical Approach, Borrebaeck, Ed., Oxford University Press, Oxford (1995); and Huse et al., J.
Immunol, 149: 3914-3920 (1992). 'In addition, several publications have reported the use of technology. phage display to produce and select libraries of polypeptide paraseligara a selected target antigen (see, for example, Cwirla et al., Proc.
Natl.
Acad.
Sci.
USA, 87: 6378-6382 (1990); Devlin et al., Science, 249: 404-406 (1990); Scott et al., Science, 249: 386-388 (1990); and Ladner et al., U.S. patent 5,571,698). A basic concept of phage display methods is the establishment of a physical association between a polypeptide encoded by a phage DNA and a target antigen.
This physical association is provided by the phage particle, which exhibits a polypeptide as part of a capsid that encompasses the phage genome, which encodes the polypeptide.
The establishment of a physical association between the polypeptides and their genetic material allows the selection of a simultaneous mass of a very high number of phages that carry different polypeptides.
The phage that exhibits a polypeptide with affinity for a target antigen binds to the target antigen, and these phages are enriched by selecting affinity for the target antigen.
The identity of the polypeptides displayed from these phages can be TT —— - - determined from their respective genomes.
Using these methods, a Ú |
'* - polypeptide identified as having a binding affinity with a desired target Ni antigen can then be mass-synthesized by aa means. conventional (see, for example, U.S. patent 6,057,098). : The antibodies that are generated by these methods can then be selected by first separating by affinity and specificity with the purified polypeptide antigen of interest and, if required, comparing the results of the affinity and specificity of the antibodies with other desired polypeptide antigens that are excluded from the connection. The separation procedure may involve immobilizing the purified polypeptide antigens in wells separate from the microtiter plates. The solution containing a potential antibody, or group of antibodies, is then placed in the respective microtiter wells and incubated for about 30 minutes to 2 hours. The microtiter wells are then washed and one. labeled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase, if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 minutes and then washed. The substrate is added to the wells and a colored reaction will appear where the antibody to the immobilized polypeptide antigen is present.
The antibodies thus identified can then be further analyzed for affinity and specificity. In the development of immunoassays for a target protein, the purified target protein acts as a standard that measures the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because of the binding affinity of - various antibodies may differ, for example, certain combinations of antibody may interfere with each other sterically, the performance of an antibody assay may be a more important measure than the absolute affinity and specificity of that antibody.
Macaws NM
: É Those skilled in the art will understand that many approaches [can be obtained in the production of antibodies or binding fragments, and in UN separation and selection by affinity and specificity with various polypeptides. of interest, but these approaches do not change the scope of the present invention.
A. Polyclonal antibodies Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of interest and an adjuvant. They can be used to conjugate the —polypeptide of interest to a carrier protein, which is immunogenic in the species to be immunized such as, for example, Californian keyhole hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using an agent Non-limiting examples of bifunctional or derivative agents include —maleimidobenzoyl sulfosuccinimide ester (conjugation by means of cysteine residues), N-hydroxysuccinimide (conjugation by means of lysine residues), glutaraldehyde, succinic anhydride, SOC », and RIN-C = NR, where R and R, are different alkyl groups. Animals are immunized against the polypeptide of interest —or an immunogenic conjugate, or a derivative thereof, combining, for example, 100 ug (for rabbits) or 5 ug (for mice) of the antigen, or conjugated to 3 volumes of Freund's complete adjuvant, and injecting the solution intradermally at multiple sites. After one month, the animals are stimulated with about 1/5 to 1/10 of the original amount of polypeptide, - or conjugated in incomplete Freund's adjuvant, by subcutaneous injection at multiple sites. Seven to fourteen days later, the animals are bled and the serum is assayed for antibody titration. The animals are stimulated in a typical way until the titration reaches a plateau. Preferably, the animal is stimulated NN ———— “At
D 'different immunogenic protein and / or by means of a different cross-linking reagent "can be used. Conjugates can also be prepared in a recombinant cell culture as fusion proteins. In certain examples, À aggregating agents, such as alum, can be used to improve the immune response.
B. Monoclonal antibodies Monoclonal antibodies are generally obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that comprise the population are identical, except —for possible naturally occurring mutations that may be present in smaller amounts. Thus, the modifier '"monoclonal" indicates the characteristic of the antibody as not being a mixture of antibodies: discrete. For example, monoclonal antibodies can be prepared. using the hybridoma method described by Kohler et al., Nature, 256: 495 (1975), or by any recombinant DNA method known in the art (see, for example, U.S. patent 4,816,567).
In the hybridoma method, a mouse or other appropriate host animal (e.g., hamster) is immunized, in the manner described above, to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the polypeptide of interest used for immunization. Alternatively, lymphocytes are immunized in vitro. The immunized lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (see, for example, Goding, Monoclonal - Antibodies: Principles and Practice, Academic Press, pp. 59- 103 (1986)). The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of non-CBT parental myeloma cells ——————
: The hypoxanthine guanine phosphoribosyl transferase (HGPRT) enzyme, the DO culture medium for hybridoma cells will typically include hypoxanthine, ass aminopterin and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable, high-level production of antibody by the cells producing the selected antibody, and / or are sensitive to a medium, such as the HAT medium. Examples of such preferred myeloma cell lines for the production of human monoclonal antibodies include, but are not limited to, murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumor cells (available from Salk Institute Cell Distribution Center; San Diego, CA), SP-. 2 or X63-Ag8-653 (available from the American Type Culture Collection; Rockville, MD), and human myeloma or mouse-human heteromyeloma cell lines (see, for example, Kozbor, JJ. Immunol, 133: 3001 ( 1984); and Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).
The culture medium in which the hybridoma cells are grown can be assayed for the production of monoclonal antibodies directed against the polypeptide of interest. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined using, for example, the Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).
After identifying the hybridoma cells that produce HUH
. Ú can be subcloned by limiting dilution procedures and grown: by standard methods (see, for example, Goding, Monoclonal Antibodies: SEND Principles and Practice, Academic Press, pp. 59-103 (1986)). Suitable culture media for this purpose include, for example, the D-MEM or RPMI1640 medium. In addition, hybridoma cells can be grown in vivo as tumor ascites in an animal. Monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid or serum, by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or chromatography by affinity. The DNA encoding the monoclonal antibodies can be isolated and sequenced easily using conventional procedures (for example, using oligonucleotide probes that are capable of binding. Specifically to the genes encoding the heavy and light chains of the murine antibodies). Hybridoma cells function as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells, such as β cells. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce the antibody, to induce the synthesis of monoclonal antibodies in the recombinant host cells. See, for example, Skerra et al., Curr. Opin. Immunol., 5: 256-262 (1993); and Pluckthun, Immunol Rev., 130: 151-188 (1992). DNA can also be modified, for example, by replacing the coding sequence for human heavy and light chain constant domains in place of murine homologous sequences (see, for example, the U.S. patent
4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 (1984)), or covalently joining in the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin peptide. PE err and
; i In an additional embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody libraries for phage expression, generated using the techniques described in, - for example, McCafferty et al., Nature, 348: 552 -554 (1990); Clackson et al., - Nature, 352: 624-628 (1991); and Marks et al., J.
Mol.
Biol., 222: 581-597 (1991). The production of high-affinity human monoclonal antibodies (nM range) by shuffling chains is described in Marks et al.
BioTechnology, 10: 779-783 (1992). The use of combinatorial infection and in vivo recombination as a strategy to build very large phage libraries is described in Waterhouse et al., Nuc.
Acids Res., 21: 2265-2266 (1993). Thus, these techniques are viable alternatives in the traditional monoclonal antibody hybridoma methods for the generation of antibodies. monoclonal. , Ç.
Humanized Antibodies Methods for humanizing non-human antibodies are known in the art.
Preferably, a humanized antibody has one or more amino acid residues introduced from a non-human source.
These non-human amino acid residues are often referred to as "import" residues, which are typically obtained from a "import" variable domain. Humanization can be performed by essentially replacing the sequences of the hypervariable region of a non-human antibody with the corresponding sequences of a human antibody.
See, for example, Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); and Verhoeyen et al., Science, 239: 1534-1536 (1988). Accordingly, such "humanized" antibodies are chimeric antibodies (see, for example, U.S. patent 4,816,567), in which substantially less than one intact human variable domain has been replaced by the corresponding sequence from a non-human species.
In TT -Hpractic; -humanized antibodies are typically human antibodies in UN N ———
y i that some residues from hypervariable region, and possibly some residues: from region of structure (FR), are replaced by residues from NA analogous sites: rodent antibodies.
, The choice of human variable domains, both light and heavy, to be used in the preparation of humanized antibodies described here is an important consideration to reduce antigenicity. According to the so-called "most suitable" method, the variable domain sequence of a rodent antibody is selected against the entire known human variable domain sequence library. The human sequence that is closest to that of the rodent is then accepted as the human FR for the humanized antibody (see, for example, Sims et al., J. Immunol, 151: 2296 (1993); and Chothia et al., JM Biol, 196: 901 (1987)). Another method uses a particular RF derived from the consensus sequence of all antibodies. humans from a particular subgroup of heavy and light chains. The same FR can be used for several different humanized antibodies (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al., J. Immunol., 151: 2623 (1993)). It is also important that the antibodies are humanized with high affinity retention with the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analyzing parental sequences and various conceptual humanized products, using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. The inspection of these displays allows the analysis of the probable role of the residues in the functioning of the Tr—— candidate immunoglobulin sequence, that is, the analysis of residues that TTITFO Sc No
. 'influences the candidate immunoglobulin's ability to bind to its antigen. í In this way, RF residues can be selected and combined from the recipient and import sequences, in such a way that the characteristics of the desired antibody, such as greater affinity with the target antigen (s) ), are achieved. In general, residues from the hypervariable region are directly and specifically involved in the influence of antigen binding. Various forms of humanized antibodies are contemplated in accordance with the present invention. For example, the humanized antibody can be an antibody fragment, such as a Fab fragment. Alternatively, the humanized antibody can be an intact antibody, such as an intact IgA, IZG or IeM antibody. . D. Human antibodies - As an alternative to humanization, human antibodies can be generated. In some modalities, transgenic animals (for example, mice) that can be produced are capable, through immunization, of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been reported that the homozygous deletion of the antibody's heavy chain binding region (JH) gene in chimeric and mutant germline mice results in complete inhibition of endogenous antibody production. The transfer of the immunoglobulin gene array of the human germline in such mice of mutant germline will result in the production of human antibodies, through challenge with antigen. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immun., 7:33 (1993); and U.S. patents 5,591,669, 5,589,369 and
5,545,807. a rar N—— “"
! Alternatively, phage display technology (see, for example, McCafferty et al., Nature, 348: 552-553 (1990)) can even be used to produce human antibodies and antibody fragments in vitro, using 'repertoires of immunoglobulin variable domain (V) gene from non - humanized donors.
According to this technique, the V domain genes of the antibody are cloned in alignment into both a larger and a smaller gene for the coating protein of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the particle surface. of the phage.
Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody that exhibits these properties.
Thus, 1 the phage mimics some of the properties of the B cell.
Phage display 'can be performed in a variety of formats in the manner described, for example, in Johnson et al., Curr.
Opin.
Struct.
Biol, 3: 564-571 (1993). Various sources of V gene segments can be used for phage display.
See, for example, Clackson et al., Nature, 352: 624-628 (1991). A repertoire of V genes from non-humanized human donors can be constructed and antibodies in a diverse array of antigens (including —auto-antigens) can be isolated following essentially the techniques described in Marks et al., J.
Mol.
Biol, 222: 581-597 (1991); Griffith et al.
EMBO /., 12: 725-734 (1993) and U.S. patents 5,565,332 and 5,573,905. In certain examples, human antibodies can be generated by B cells activated in vitro in the manner described in, for example, - U.S.5.567.610 and 5.229.275 patents. AND.
Antibody fragments Several techniques have been developed for the production of antibody fragments.
Traditionally, these fragments were derived "EEE
: 'Morimoto et al., J. Biochem. Biophys. Meth., 24: 107-117 (1992); and Brennan: et al., Science, 229: 81 (1985)). However, these fragments can now be produced directly using recombinant host cells. Per . For example, antibody fragments can be isolated from the antibody phage display libraries discussed above. Alternatively, Fab'-SH fragments can be recovered directly from E. coli cells and chemically coupled to form F (ab ') fragments, (see, for example, Carter et al., BioTechnology, 10: 163-167 (1992 )). According to another approach, F (ab ') fragments can be isolated directly from human host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is an Fv chain fragment. simple (Fvsc). See, for example, PCT publication WO 93/16185 and U.S. patents 5,571,894 and 5,587,458. The antibody fragment can also be a linear antibody in the manner described, for example, in the U.S. patent.
5,641,870. Such linear antibody fragments can be monospecific or bispecific.
F. Bispecific antibodies Bispecific antibodies are antibodies that exhibit binding specificities with at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes on the same polypeptide of interest. Other bispecific antibodies can combine a binding site with the polypeptide of interest with binding site (s) for one or more additional antigens. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (for example, F (ab '), from bispecific antibodies). Methods for preparing bispecific antibodies are NU ————
: complete bispecific is based on the coexpression of two chain pairs: heavy and immunoglobulin light chain, where the two chains have different MS EsNRS specificities (see, for example, Millstein et al., Nature, 305: 537-. 539 (1983)). Due to the random variety of heavy and light chains — of immunoglobulin, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually carried out by affinity chromatography. Similar procedures are disclosed in PCT publication WO 93/08829 and Traunecker et al., EMBOJ. 10: 3655-3659 (1991).
According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combination sites) are fused into the domain sequences. immunoglobulin constant. The fusion is preferably with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2 and CH3 regions. It is preferable to present the first heavy chain constant region (CHI), which contains the site necessary for light chain binding, present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, is inserted into separate expression vectors, and is co-transfected into a suitable host organism. This provides better flexibility in adjusting the mutual proportions of the three polypeptide fragments, in modalities when unequal ratios of the three polypeptide chains used in the construction provide the ideal yields.
- However, it is possible to insert the coding sequences for two or all three polypeptide chains in an expression vector, when the expression of at least two polypeptide chains in equal ratios results in high yields, or when the ratios have no meaning FI opartislat— HUH
: In a preferred embodiment of this approach, bispecific Ns antibodies are composed of a mr hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a chain pair. heavy-chain hybrid immunoglobulin light (which provides a second - binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only half of the bispecific molecule provides an easy way of separation. See, for example, PCT publication WO 94/04690 and Suresh et al., Meth. Enzymol, 121: 210 (1986).
According to another approach described in the U.S. patent
5,731,168, the interface between a pair of antibody molecules can be - genetically modified to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (for example, tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large — side chain (s) are created at the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (for example, alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer, with respect to unwanted end products, such as homodimers.
Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, and the other to biotin. Heteroconjugate antibodies can be prepared using any convenient “> - - cross-linking method. Suitable crosslinking agents and techniques are NME ————— K
. 'well known in the art and are disclosed, for example, in U.S. patent: 4,676,980.
Suitable techniques for generating bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical bonding. In certain examples, bispecific antibodies can be generated by a procedure in which intact antibodies are cleaved proteolytically to generate F (ab ') fragments, (see, for example, Brennan et al., Science, 229: 81 (1985)) . These fragments are reduced in the presence of the complexing agent dithiol, sodium arsenite, to stabilize vicinal dithiols and prevent the formation of intermolecular disulfide. The Fab 'fragments generated are then converted to thionitrobenzoate (TNB) derivatives. . One of the Fab'-TNB derivatives is then converted to Fab'-thiol by reduction: with mercaptoethylamine, and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
In some embodiments, Fab'-SH fragments can be recovered directly from E. coli and chemically coupled to form bispecific antibodies. For example, a fully humanized bispecific antibody F (ab ') molecule can be produced by the methods described in Shalaby et al., J. Exp. Med., 175: 217-225 (1992). Each Fab 'fragment was secreted separately from E. coli and subjected to targeted chemical coupling in vitro to form the bispecific antibody.
Various techniques for preparing and isolating antibody fragments — bispecific directly from recombinant cell culture have also been described. For example, bispecific antibodies were produced using leucine zippers. See, for example, Kostelny et al., J. Inmunol., 148: 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were “- - Tigados-à Fab portions! of two different antibodies by gene fusion. You EEE
: 'antibody homodimers were reduced in the hinge region to - form monomers, and then reoxidized to form the MEN antibody heterodimers. This method can also be used for the production of "antibody homodimers. The" diabody "technology, described by —MHollingeretal, Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993), has provided an alternative mechanism to prepare bispecific antibody fragments The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker, which is too small to allow pairing between the two domains on the same chain. In this way, the VH and VL domains of a fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. light chain Fv dimers (Fvs) is described in Gruberetal, J. Immunol, 152: 5368 (1994).
Antibodies with more than two valences are also contemplated. Per. example, specific antibodies can be prepared. See, for example, Tutt et al., J. Immunol, 147: 60 (1991).
G. Antibody purification During the use of recombinant techniques, antibodies can be produced within an isolated host cell, in the periplasmic space of a host cell, or secreted directly from a host cell in the medium. If the antibody is produced intracellularly, the particle residues are removed first, for example, by centrifugation or ultrafiltration. Carter et al., BioTech., 10: 163-167 (1992) describe a procedure to isolate the antibodies that are secreted in the E. coli periplasmic space. Briefly, the cell paste is thawed in the presence of sodium acetate (PH 3.5), EDTA and phenylmethylsulfonylfluoride “—————— (PMSE) for about 30 minutes. Cell debris can be removed FOOT
. Ô by centrifugation. Where the antibody is secreted in the medium, the supernatants of: each expression system are concentrated using generally a commercially available Na concentration protein filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor, such as PMSF, can be included in any of the previous steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of unforeseen contaminants. The antibody composition prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography. The suitability of protein A as an affinity linker depends on the species and isotype of any immunoglobulin Fc domain, which is present in the antibody. Protein A can be used to purify antibodies based on human y1, y2 or y4 heavy chains (see, for example, Lindmark et al., J. Immunol. Meth., 62: 1-13 (1983)) . Protein G is recommended for all mouse and human y3 isotypes (see, for example, Guss et al., EMBO 1, 5: 1567-1575 (1986)). The matrix in which the affinity binder is | posted is more often agarose, but other matrices are available. Mechanically stable matrices, such as those with controlled pore life or poly (styrenodivinyl) benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, Bakerbond ABXTM resin (J. T. Baker; Phillipsburg, N. J.) is used for purification. Other techniques for protein purification, such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, silica chromatography, SEPHAROSE'Y heparin chromatography, chromatography on an anionic or cationic resin (such as as a polyaspartic acid column), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation also TT - are available depending on the antibody to be recovered. FOOT
. B After any preliminary purification step (s), the mixture: comprising the antibody and contaminants of interest can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5- 4.5, preferably carried out at low salt concentrations (for example, about 0-0.25 M salt).
Those skilled in the art will understand that any binding molecule with an antibody-like function, for example, a binding molecule or binding partner that is specific to one or more analytes of interest in a sample, can also be used in methods and compositions of the present invention. Examples of suitable antibody-type molecules include, but are not limited to, domain antibodies, single-structure antibodies, nanobodies, shark antigen-reactive proteins, avimers, adnectins, anti-tranquilizers, affinity binders, Phylomers-like structures, aptamers, Affibodies, trinectinss and the like.
VII. Administration methods In accordance with the methods of the present invention, the anti-cancer drugs described herein are administered to a subject by any convenient means known in the art. The methods of the present invention can be used to select a suitable anti-cancer drug, or combination of anti-cancer drugs for the treatment of cancer (e.g., a hematological malignancy) in a subject. The methods of the invention can also be used to identify the response of a cancer (for example, a hematological malignancy) in a subject for treatment with an anti-cancer drug or combination of "anti-cancer drugs. In addition, the methods of the invention can be used to predict the response of a subject with cancer (e.g., a hematological malignancy) to treatment with an anti-cancer drug, or combination of anti-cancer drugs. Furthermore, the methods of the Tr ———-— present invention can be used to identify a subject with cancer FOOT
. To (for example, a hematological malignancy) that is resistant to treatment: with an anti-cancer drug, or combination of anti-cancer drugs. Those skilled in the art will understand that the anti-cancer drugs - described herein can be administered alone or as part of a therapeutic approach combined with conventional chemotherapy, radiation therapy, hormonal therapy, immunotherapy and / or surgery.
In certain embodiments, the anti-cancer drug comprises an anti-signaling agent (ie, a cytostatic drug), such as a monoclonal antibody or tyrosine kinase inhibitor, an anti-proliferative agent, a chemotherapeutic agent (ie, a cytotoxic medicine), a hormonal therapeutic agent, a radiotherapeutic agent, a vaccine and / or any other compound with the ability to reduce or cancel the uncontrolled growth of abnormal cells, such as cancer cells. In some embodiments, the subject is treated with one or more anti-signaling agents, anti-proliferative agent and / or hormonal therapeutic agents in combination with at least one chemotherapeutic agent. Exemplary monoclonal antibodies, tyrosine kinase inhibitors, anti-proliferative agents, chemotherapeutic agents, hormonal therapeutic agents, radiotherapeutic agents and vaccines are described above.
In some embodiments, the anti-cancer drugs described herein may be co-administered with conventional immunotherapeutic agents including, but not limited to, immunostimulants (eg, Calmette-Guerin bacillus (BCG), levamisole, interleukin-2, alpha- —interferon , etc.), immunotoxins (e.g., anti-CD33-calicheamicin monoclonal antibody conjugate, anti-CD22 monoclonal antibody conjugate, Pseudomonas exotoxin, etc.) and radioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugate with " mn, ”y, or 131 FAN FOOT
. Anti-cancer drugs can be administered with: a suitable pharmaceutical excipient, if necessary, and can be performed —— through any of the accepted modes of administration.
Thus, - administration can be, for example, oral, buccal, sublingual, gingival, palatal, intravenous, - topical, subcutaneous, transcutaneous, transdermal, intramuscular, - intra-articular, parenteral, intra-arterial, intradermal, intraventricular, - intracranial, intraperitoneal, intravesical, intrathecal, intralesional, intranasal, rectal, vaginal or by inhalation. "Co-administer" means that an anti-cancer medicine is administered at the same time, just before or exactly after the administration of a second medicine (for example, another anti-cancer medicine, a medicine used to reduce the side effects associated with therapy with anti-cancer medication, a radiotherapeutic agent, a hormonal therapeutic agent, an immunotherapeutic agent, etc.). A therapeutically effective amount of an anti-cancer drug can be administered repeatedly, for example, at least 2, 3, 4, 5, 6, 7, 8 or more times, or the dose can be administered by continuous infusion.
The dose may take solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams , plasters, lotions, gels, aerosols, foams or the like, preferably in single dosage forms suitable for simple administration of precise dosages.
As used herein, the term "single dosage form" - refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined amount of an anti-cancer drug calculated to produce the desired start, tolerability and / or therapeutic effect, in Tr -association-with a suitable pharmaceutical excipient (for example, a | UP—— ==
: ampoule). In addition, the most concentrated dosage forms can be: prepared, from which the most diluted single dosage forms mr can then be produced. The most concentrated dosage forms - will thus contain substantially, for example, at least more than 1, 2,3, 4, S 5,6,7,8,9,100 or more times the amount of the anti-cancer drug.
Methods for preparing such dosage forms are known to those skilled in the art (see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ED., Mack Publishing Co., Easton, PA (1990)). Dosage forms typically include a carrier or conventional pharmaceutical carrier and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers and the like. The appropriate excipients can be adapted to the particular dosage form and route of administration by methods well known in the art (see, for example, REMINGTON'S 15º PHARMACEUTICAL SCIENCES, supra).
Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, —methylcellulose, ethylcellulose, hydroxypropylmethylcellulose and polyacrylic acids such as carbopols, for example, Carbopol 941, Carbopol 980, Carbopol 981, etc. Dosage forms may additionally include lubricating agents such as talc, magnesium stearate and mineral oil, wetting agents, emulsifying agents, suspending agents, preserving agents such as methyl, ethyl and propylhydroxy-benzoates (ie , parabens), pH adjusting agents such as inorganic and organic acids and bases, sweetening agents and flavoring agents. The dosage forms may also comprise biodegradable polymer beads, dextran and TT> -eplexes-including cyelodextrin. FOOT
: Á For oral administration, the therapeutically effective dose can: be in the form of tablets, capsules, emulsions, suspensions, solutions, —— syrups, sprays, tablets, powders and continuous-release formulations. Excipients suitable for oral administration include pharmaceutical grades of - mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate and the like.
In some embodiments, the therapeutically effective dose takes the form of a pill, tablet or capsule, and thus the dosage form may contain, together with an anti-cancer medication, any of the following: a diluent such as lactose, sucrose, phosphate dicalcium and the like; a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such as starch, acacia gum, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof. An anti-cancer drug can also be formulated in a "suppository disposed, for example, in a polyethylene glycol (PEG) vehicle.
Liquid dosage forms can be prepared by dissolving or dispersing an anti-cancer drug and, optionally, one or more pharmaceutically acceptable adjuvants in a vehicle such as, for example, aqueous saline (for example, 0.9% w / v chloride sodium), aqueous dextrose, - glycerol, ethanol and the like, to form a solution or suspension, for example, for oral, topical or intravenous administration. An anti-cancer medication can also be formulated into a retention enema.
For topical administration, the therapeutically effective dose can be in the form of emulsions, lotions, gels, foams, creams, jellies, - solutions, suspensions, plasters and transdermal patches. For administration by inhalation, an anti-cancer medication can be delivered as a dry powder or in liquid form through a nebulizer. For parenteral administration, the therapeutically effective dose may be in the form of rea NNE
:: sterile injectable solutions and sterile packaged powders.
Preferably, the: injectable solutions are formulated at a pH of about 4.5 to about 7.5. aa Therapeutically effective dose can also be provided - in a lyophilized form.
Such dosage forms can include a buffer, for example, bicarbonate, for reconstitution prior to administration, or the buffer can be included in the lyophilized dosage form for reconstitution, for example, with water.
The lyophilized dosage form may additionally comprise a suitable vasoconstrictor, for example, epinephrine.
The lyophilized dosage form can be supplied in a syringe, packaged - optionally in combination with the reconstitution buffer, in such a way that the reconstituted dosage form can be immediately administered to a subject.
A subject can also be monitored at periodic time intervals to assess the efficiency of a certain therapeutic regimen.
For example, the activation states of certain oncogenic fusion proteins, and / or signal transduction molecules, may change based on the therapeutic effect of treatment with one or more of the anti-cancer drugs described here.
The subject can be monitored to assess the response and understand the effects of certain medications or treatments in an individualized approach.
Additionally, subjects who initially respond to a specific anti-cancer drug, or combination of anti-cancer drugs, may become refractory to the drug or drug combination, indicating that these subjects have developed resistance to the drug.
These subjects may have their current therapy discontinued and an alternative treatment may be prescribed, according to the methods of the present invention.
In certain respects, the methods described here can be used in conjunction with panels of gene expression markers that predict “——— - -—- prebability — of the prognosis and / or recurrence of cancer in various N ————— -
: populations. These gene panels can be used to identify subjects who are unlikely to experience recurrence and are therefore unlikely to benefit from adjuvant chemotherapy. Expression panels can - be used to identify subjects who can safely avoid adjuvant chemotherapy, without negatively affecting disease-free results and total survival.
In addition, in certain other respects, the methods described herein can be used in conjunction with panels of gene expression markers that identify the original tumors for cancers of unknown origin (CUP). These gene panels can be used to identify subjects with metastatic cancer, who would benefit from therapy consistent with that provided to subjects initially diagnosed with cancer. Suitable systems include, but are not limited to, the Aviara CancerTYPE ID assay, an RT-PCR abse expression assay, which measures 92 genes to identify the primary site of origin for 39 tumor types, and the tissue tissue test Pathwork ”, which measures the expression of more than 1,600 genes in a microarray, and compares a tumor gene expression“ signature ”against those of 15 known tissue types.
VIII. Examples The following examples are provided to illustrate, but not limit, the claimed invention.
Example 1. Single cell detection using a microarray ELISA with dual proximity detector with tyramide signal amplification.
This example illustrates a microarray sandwich ELISA with a high-throughput, multiplex, dual-proximity detector with a higher dynamic range that is suitable for analyzing the activation states of fusion proteins, and signal transduction molecules, NN—— “ “. .
: i particular, the proximity test is in the form of a microarray: addressable. mr 1) Capture antibody was printed on a 16-pad slide. FAST (Whatman Inc.) with a serial dilution of 1 mg / ml to 0.004 mg / ml. Alternatively, a 2-fold serial dilution of each capture antibody (0.25 mg / mL, 0.125 mg / mL and 0.0625 mg / mL) can be used, and double or quadruple points can be made for each dilution of antibody. To detect the BCR-ABL fusion protein, an exemplary capture antibody comprises a monoclonal or —polyclonal anti-BCR antibody.
2) After drying overnight, the slide was blocked with Whatman blocking buffer.
3) 80 µl of cell lysate was added to each piece with a 10-fold serial dilution. The slide was incubated for two hours at room temperature.
4) After six washes with TBS-Tween, 80 µl of detection antibodies were added to the slides for the proximity assay, diluted in TBS-Tween / 2% BSA / 1% FBS. To detect the BCR-ABL fusion protein, exemplary detection antibodies include: (1) a monoclonal or polyclonal anti-ABL antibody conjugated directly to glucose oxidase (GO); and (2) a monoclonal or polyclonal antibody that recognizes phosphorylated GLA, conjugated directly to horseradish peroxidase (HRP). The incubation took place for 2 hours at room temperature.
5) Alternatively, the detection step can use a biotin-conjugated antibody that recognizes phosphorylated ABL. In these examples, after six washes, an additional sequential incubation step with streptavidin-HRP was included for 1 hour.
6) Alternatively, the detection step can use a HUH
] Both the directly bound conjugate and the biotin-: streptavidin (SA) conjugate of HRP in the phosphorylated ABL antibody can be used. nr 6) For signal amplification, 80 µL of biotin-tiramide at 5 ° C. ug / mL were added and reacted for 15 minutes. The slide was washed six times with W TBS-Tween, twice with 20% DMSO / TBS-Tween, and once with TBS.
7) 80 uL of SA-Alexa 555 were added and incubated for 30 minutes. The slide was then washed twice, dried for 5 minutes, and examined in a microarray viewer (Perkin-Elmer, Inc.).
The microarray format of the proximity assay described here advantageously exhibits very little foundation (for example, compared to two-antibody assays), due to the greater specificity obtained by detecting the proximity between two detection antibodies.
Example 2. Generation of activation profiles for drug selection.
The compositions and methods of the present invention can be applied for drug selection in the treatment of cancer. As a non-limiting example, the present invention can be used to identify the presence and / or activity of one or a plurality of oncogenic fusion proteins associated with hematological malignancies, in order to provide the appropriate treatment for patients with these types of cancer. A typical protocol involves the generation of two profiles, a reference activation profile and a test activation profile, which are then compared to determine the efficiency of a particular drug treatment regimen (see figure 2).
Reference activation profile To produce a reference activation profile, a blood sample is obtained from a patient with a specific type of cancer Tr - for example, leukemia such as CML) before treatment with SEN — .ÕÀ " THE--
. anti-cancer medicine. Tumor cells are isolated from the sample: from blood using, for example, any of the techniques described here with NE. more details. Isolated cells can be stimulated in vitro with one or "more growth factors. The stimulated cells are then lysed to produce a cell extract. The cell extract is applied to a targetable array that contains a serial dilution of a panel of antibodies to capture specifics for one or a plurality of oncogenic fusion proteins (alone or in combination with one or a plurality of signal transduction molecules), whose activation states can be altered in the patient's types of cancer. proximity tests are performed using the appropriate detection antibodies (for example, antibodies independent of the activation state and / or antibodies dependent on the activation state) to determine the activation status of each analyte of interest. , providing the activation states of specific analytes, such as oncogenic fusion proteins in the patient's cancer, in the absence of any anti-cancer drugs.
Test activation profile To obtain a test activation profile, a second blood sample is obtained from the patient with a specific type of cancer (for example, leukemia such as CML) both before treatment with anti-cancer medication and after administration of an anti-cancer drug (for example, at any time throughout the course of cancer treatment). The tumor cells are isolated from the blood sample. If the isolated cells are obtained from a patient who is not receiving treatment with an anti-cancer drug, the isolated cells are incubated with anti-cancer drugs that target one or more of the activated oncogenic fusion proteins, and / or transduction molecules signals, determined from> the reference activation profile described above. For example, NIE
] if determined from the reference activation profile in which the: BCR-ABL fusion protein is activated, before cells can be incubated mr with imatinib (Gleevec ”). Isolated cells can then be stimulated in vitro with one or more growth factors. The isolated cells are then lyzed to produce a cell extract. The cell extract is applied in the targetable array and simple detection or proximity tests are performed to determine the activation status of each analyte of interest. A test activation profile for the patient is thus generated, providing the activation states for specific analytes, such as fusion proteins - homogeneous in the patient's cancer in the presence of specific anti-cancer drugs.
Drug selection Anti-cancer drugs are determined to be suitable or unsuitable for the treatment of the patient's cancer by comparing the test activation profile with the reference activation profile. For example, if drug treatment causes most or all of the oncogenic fusion proteins, and / or signal transduction molecules, to be substantially less activated than in the absence of the drugs, for example, a strong activation change without medications for weak or very weak activation with the medications, so the treatment is determined to be suitable for the patient's cancer. In such examples, treatment is both initiated with the appropriate anti-cancer drug in a patient who has not received drug therapy, and subsequent treatment is continued with the appropriate anti-cancer drug in a patient who is already receiving the drug. However, if drug treatment is considered unsuitable for the treatment of the cancer patient, different drugs are selected and used to generate a new Tr - —-— -activation test profile, which is then compared to the reference activation profile. In E DE a on an e a er aeao
In such examples, treatment is both initiated with an anti-cancer drug: suitable in a patient who has not received drug therapy, and] subsequent treatment is changed to an anti-cancer drug - suitable in a patient currently receiving the drug inappropriate. The protocol described in this example is also used to identify patients who are resistant to therapy with a tyrosine kinase inhibitor, such as imatinib, due to mutations in the target protein kinase (eg, BCR-ABL), non-compliance with the therapeutic regimen. and / or administering a dose of subideal medication. Example 3. Exemplary oncogenic fusion proteins associated with cancer.
This example provides a non-extensive list of translocations in human tumors that cause the formation of oncogenic fusion proteins and their associated neoplasms.
Burkitt's lymphoma: translocation of the c-myc t (8; 14) gene (g24; 932). The common chimeric oncoprotein is c-myc / IGH.
AML: translocation of a portion of chromosome 8 to chromosome 21. The resulting chimeric oncoprotein is RUNXI1 / ETO. Another t (12; 15) translocation (p 13; 925) results in the chimeric oncoprotein —TEL / TrkC (kinase).
CML: Philadelphia chromosome is a translocation that results in BCR / ABL (kinase).
Ewing's sarcoma ;: translocation between chromosomes 11 and
22. The resulting chimeric oncoprotein is EWS / FLI (transcription factor).
ALL: Chimeric oncogenic proteins: TETE er HUH
: mis hand +
A A DA A VARA. cryptic t (12; 21) “TEL / AMLI1 (Kinase) 125.4% | t: 19) 023: p13) E2A / PBX (PBX1) E in 1 (9; 22) (934: 911) BCT / ABL PI85 fusion) - 1.6% | MTB Rato MILA 16% t (8; 14) (924, 932); IGHMYC fusion for IGIQISAD 0 TCR / RBIN2 spindle | DFSP: More than 95% of DFSP tumors have the t (17; 22) chromosomal translocation that results in the chimeric oncoprotein COL1 A1 / PDGF (which binds and activates PDGFR). Leukemia - pro-myelocytic = acute: a translocation determined as t (15; 17) (922, 4 12). The resulting chimeric oncoprotein is RARo / PML (complex transcription protein). Acute B-cell lymphoblastic leukemia: t translocation (17; 19), which results in the chimeric oncoprotein E2A / HLF (apoptosis inhibitor).
Acute B-cell leukemia: t translocation (1; 19). The chimeric oncoprotein is E2A / Pbx1 (kinase substrate).
Rhabdomyosarcoma: t translocation (2:13) (935; 914), which results in the chimeric oncoprotein PAX3 / FKHR (transcription factor).
A soft tissue malignancy in a very young child: t (12;: 15) (p13; 925) rearrangement that results in the following chimeric oncoprotein: protein tyrosine kinase ETV6 / NTRK3 (kinase).
Papillary thyroid carcinoma: the chimeric oncoprotein is RET / PTC (kinase).
Prostate cancer: the chimeric oncoprotein is TMRSS / ERG (kinase).
DA rr rerea es EE EEE
] Additional examples of translocations in human tumors: that cause the formation of oncogenic fusion proteins and their neoplasms - associated: rt ber / abl Chronic myelogenous leukemia, acute lymphocytic leukemia E2A / pbx1 acute myeloid leukemia B PML / RAR Acute pro-myelocytic leukemia B-cell lymphoma CBFB / MYH11 Acute myeloid leukemia aml1 / mtg8 B-cell lymphoma NPM / ALK Large-cell lymphomas Adapted from GM Cooper, Oncogenes, 2nd ed. Boston and London: Jones and Bartlett, 1995. Example 4. Proximity-mediated immunoassay for the detection of total and activated levels of oncogenic fusion proteins. Introduction The BCR-ABL oncogene is formed by a translocation of the normal ABL gene located on the long arm of chromosome 9, in the N-terminal part of the BCR gene located on the long arm of chromosome 22, to form the Philadelphia chromosome. Depending on where the translocated ABL gene is joined at the N-terminal of the BCR gene, different sizes of the BCR-ABL gene products are produced, with the most prevalent being the p210 BCR-ABL gene product, as the oncogene that causes CML and a subset of ALL. Additional gene products, such as p185 and p230 BCR-ABL, are also produced. In particular, the p210 BCR-ABL gene product is responsible for causing AML. This protein contains 1,790 amino acids and is composed of a BCR oligomerization domain (OLTI) at the N-terminus, followed by the BCR S / T kinase domain, a BCR amino acid sequence insert that is not present in normal BCR, Fr AE the ro the was the wing NEEM
'followed by the SH3, SH2 and Y kinase domains of GLA, as well as the domain: rich in C-terminal GLA proline. : Clinical background M Although most patients with chronic myelogenous leukemia (CML) in the chronic phase respond well to imatinib, some patients do not reach the desired end point, and others may eventually succumb to the response or are intolerant. Regarding treatment with 400 mg / day of imatinib (Gleevec): there is a complete cytogenetic response (CCyR) in 70-80% of patients; there is a loss of CCyR at a rate of 4 to 7% / year in the first 3 years, and then 1 to 2% / year thereafter; the complete survival rate after 7 years is 90%, the same rate without survival after 7 years is 81% and the relapse rate is around 30% in 5 years. There is a better response to treatment with 800 mg / day of imatinib (Gleevec) (CCyR = 95%; Relapse rate = 5%), but a dose like this of imatinib is also more toxic. Nilotinib has a similar efficiency with 800 mg / day of imatinib, but it is more toxic than imatinib. Complete inhibition of BCR-ABL yields better responses to Gleevec, but incomplete inhibition of BCR-ABL results in relapse with respect to Gleevec. Therefore, real-time detection of the level of expression and degree of activation of BCR-ABL would benefit patients with CML on targeted therapy, adjusting the dose of the drug to efficiently inhibit the target, while minimizing toxicity. For example, those patients with incomplete inactivation with 400 mg / day of Gleevec can be - placed at a higher dose (for example, 800 mg / day) faster to ensure the response. Diagnostic challenge High baseline levels of BCR and GLA of HUH
'BCR-ABL fusion in abundance. In addition, current assays that: detect BCR-ABL phosphorylation levels lack the sensitivity to detect phosphorylation in clinical blood samples. Furthermore - antibodies against fusion proteins, such as BCR-ABL, are not - very specific for multiple variants of the fusion protein.
New BCR-ABL detection assays Figure 3A illustrates an exemplary proximity test (300) to detect the presence (total level) and / or activation state (phosphorylation level) of an oncogenic fusion protein, such as BCR- GLA (310). Figure 3B illustrates an alternative embodiment (400) of the proximity assays of the present invention to detect the presence (total level) and / or activation state (phosphorylation level) of BCR-ABL (410), using the binding antibodies.
Non-limiting examples of antibodies suitable for use in methods for measuring levels of total and / or activated BCR-ABL protein, as illustrated in Figures 3A-3B, include those shown in Table 1 above.
Results Figure 4 illustrates the BCR-ABL signal in K562 cells (ie, cells from a human chronic myelogenous leukemia cell line) after the removal of full-size free BCR, using an antibody specific for the BCR terminal carboxyl region. of total size combined with the beads, according to the exemplary proximity test shown in figure 3A. In particular, Figure 4 demonstrates that the BCR-ABL signal is not affected by removal of free BCR from a patient sample - using the unprecedented proximity assay of the present invention.
Figure 5 illustrates the BCR signal in K562 cells after removal of free BCR, using an antibody specific for the full-length BCR terminal carboxyl region conjugated to the beads, according to the exemplary proximity-probe shown in figure 3A. The Ú way | - | - NANRRR ————
shown in figure 5, a decrease in the BCR signal in 10,000 cells: demonstrates that only BCR is removed using the unprecedented E proximity assay of the present invention. - Figure 6 illustrates the detection of total and phosphorylated levels of —BCR-ABL in K562 cells, using the exemplary proximity assay shown in figure 3A. Conclusion This example demonstrates that the exemplary proximity assays, shown in Figures 3A and 3B, to detect the presence and / or activation status of BCR-ABL are advantageous for at least the following reasons: (1) full-size BCR proteins they can be removed from a patient sample, such as blood or bone marrow aspirate, using a specific decrease label for the C-terminal region of BCR; (2) once the full-size BCR is removed from the patient sample, the capture antibodies specific to the N-terminal region of BCR can capture the BCR-ABL fusion proteins; and (3) once the BCR-ABL fusion proteins are captured, their levels of expression and activation can be detected with high sensitivity through proximity channeling, in which a simple detectable signal, which is correlated to —protein levels Total or activated BCR-ABL, is generated only by binding all three antibodies, resulting in better assay specificity, less foundation and simplified detection.
Example 5. Detection of total and activated levels of BCR-ABL without interference from BCR and / or full-size ABL.
This example provides additional experimental data demonstrating the advantages of the exemplary proximity assays, shown in figures 3A and 3B, to detect the presence (total level) and / or activation state (phosphorylation level) of BCR-ABL. In a TT rr Rartieules modality, the full-size BCR proteins are removed from a UU NNR extract——
] cells (for example, malignant white blood cells, such as: leukemia cells) obtained from a patient sample or from a cell extract 'obtained from a cell line (for example, a' leukemia cell line), using a specific decrease label for the C-terminal region of BCR.
Once the full-length BCR is removed from the cell extract, the capture antibodies specific to the N-terminal region of BCR can capture the BCR-ABL fusion proteins.
Once the BCR-ABL fusion proteins are captured, their levels of expression and activation can be detected with high sensitivity by means of proximity channeling, in which a simple detectable signal, which is correlated to the total or activated levels of BCR protein -ABL, is generated only by binding the three antibodies, resulting in better assay specificity, less foundation and simplified detection.
Figure 7 illustrates the level of phosphorylation ("BCR-ABL phospho") and the total amount ("BCR-ABL total") of BCR-ABL detected in K562 cells (ie, cells from a human chronic myelogenous leukemia cell line ) after removal of full size free BCR, using an antibody specific for the full size BCR terminal carboxyl region conjugated to the beads according to the exemplary proximity assay shown in figure 3A.
In particular, figures 7A and 7B show that the phosphorylated and total BCR-ABL signals were not altered after putting a K562 cell extract in contact with the antibodies specific for the full-length BCR C termination, coupled with the beads for remove the free BCR protein using the unprecedented proximity assay of - the present invention.
Figure 7C illustrates that the full size free BCR was actually removed from the cell extract after treatment with beads coupled to the C-terminal BCR antibody.
Figure 8 provides another illustration of the phosphorylated BCR-ABL Tr signal in K562 cells after removal of free BCR, using a | EEEDE —...
One antibody specific for the full-length BCR terminal carboxyl region: conjugated to the beads according to the exemplary proximity assay shown in Figure 3A.
In particular, figure 8A provides one. microarray comparison of the phosphorylated BCR-ABL signal detected in - K562 cell lysates, with or without the removal of full-size BCR ("Untreated accounts" = BCR not removed versus "Treated accounts" = BCR removed with accounts that contain specific antibody to the full-length BCR C-terminus conjugated to these). Figure 8B provides a graphical representation of the microarray data with relative fluorescence units (RFU) as a function of the cell number.
These figures demonstrate that the phosphorous BCR-ABL signal was not altered by removing full-size BCR from an extract of K562 cells, using the unprecedented proximity assay of the present invention.
Figure 9 provides another illustration of the total BCR-ABL signal in K562 cells after removal of free BCR, using an antibody specific for the full-size BCR terminal carboxyl region conjugated to the beads according to the exemplary proximity assay represented in figure 3A.
In particular, Figure 9A provides a microarray comparison of the total BCR-ABL signal detected in K562 cell lysates, with or without the removal of full-size BCR ("Untreated accounts" = Untreated BCR versus "Treated accounts" = BCR removed with beads that contains an antibody specific for the C terminus of full size BCR conjugated to them). Figure 9B provides a graphical representation of the microarray data with relative fluorescence units - (RFU) as a function of the cell number.
These figures demonstrate that the total BCR-ABL signal was not altered by removing full-size BCR from an extract of K562 cells, using the unprecedented proximity assay of the present invention.
Wounds P ————
Fig. 10 provides another illustration of BCR removal: free from full size from an extract of K562 cells, after placing the Ô cell extract in contact with beads coupled to the C-terminal antibody. BCR.
In particular, Figure 10A provides a microarray comparison - of the total BCR signal detected in K562 cell lysates, with or without the removal of full size BCR ("Untreated accounts" = Untreated BCR versus "Treated accounts" = BCR removed with beads that contains an antibody specific for the C terminus of full size BCR conjugated to them). Figure 10B provides a graphical representation of the data — microarray arrangement with relative fluorescence units (RFU) as a function of the cell number.
These figures demonstrate that full-size BCR was reduced from virtually the cell extract when treated with beads containing a specific antibody to the full-length BCR C terminus.
In fact, cell extracts that contain, for example, less than 1,000 cells did not produce a BCR signal that was significantly above baseline (0 cells) when incubated with beads coupled to the C-terminal antibody of BCR.
Figure 11 illustrates that the full-size free BCR and ABL proteins, but not the BCR-ABL fusion protein, are present in white blood cells (WBCs). Figure 12 shows that the full size free BCR present in WBCs inhibited the phosphorus BCR-ABL signal in K562 cell extracts, when such K562 cell extracts were increased with WBC extracts.
Figure 13 illustrates the total BCR-ABL signal in K562 cells augmented with WBC extracts after removal of free BCR, using an antibody specific for the full-size BCR terminal carboxyl region conjugated to the beads.
In particular, Figure 13A shows that the free BCR signal was saturated when the K562 cell extracts were increased with the WBC extracts.
After treatment with
- - when coupled to the BCR antibody, the free BCR was removed.
Figure IB
'shows that the BCR-ABL signal was not changed with or without the treatment: with beads in the same experiment. | 'In some embodiments, the full-length GLA protein - can be additionally or alternatively removed from the cell extract (for example, malignant white blood cells, such as leukemia cells) obtained from a patient sample, or from the cell extract obtained from a cell line (for example, a leukemia cell line), before capturing and detecting the expression and / or activation of BCR-GLA, using a specific decrease label for the full-length GLA amino.terminal region. A non-limiting example of a diminishing label like this is an account in which an antibody specific to the full-length NL-terminal region of ABL is affixed.
Example 6. Detection of total and activated levels of BCR-ABL in cells treated with inhibitors of BCR-ABL kinase activity.
This example demonstrates that BCR-ABL inhibitors, such as imatinib (Gleevec ”), nilotinib (Tasigna”), and dasatinib (Sprycel ), Inhibited dose-dependent activation (i.e., phosphorylation), but not expression ( that is, total levels), of BCR-ABL protein in K562 cells (ie, cells of a human chronic myelogenous leukemia cell line).
In particular, Figures 14A and 14B illustrate that the BCR-ABL inhibitor, imatinib (Gleevec ), Inhibited dose-dependent activation (i.e., phosphorylation), but not the expression (i.e., total levels) of protein BCR-ABL in K562 cells. Figure 14C shows the phosphorus / total ratio of BCR-ABL through - inhibition with imatinib, which is correlated with the percentage of inhibition of the phosphorus BCR-ABL signal with treatment with imatinib.
Figures 15A and 15B illustrate that the BCR-ABL inhibitor, nilotinib (Tasigna ”), inhibited dose-dependent activation (ie, NNE
Í GLA in K562 cells. Figure 15C shows the phosphorus / total ratio of BCR-ABL: through inhibition with nilotinib, which is correlated with the percentage of It is inhibition of the phosphorus BCR-ABL signal with treatment with nilotinib.
- Figures 16A and 16B illustrate that the BCR-ABL inhibitor, “dasatinib (Sprycel”), inhibited dose-dependent activation (ie, phosphorylation), but not the expression (ie, total levels) of BCR protein - GLA in K562 cells. Figure 16C shows the phosphorus / total ratio of BCR-ABL upon inhibition with dasatinib, which is correlated with the percentage of inhibition of the phosphorus BCR-ABL signal with treatment with dasatinib. Example 7. Detection of the total and activated levels of the BCR-ABL CRKL substrate in various cancer cell lines.
This example demonstrates the detection of the total and activated (phosphorylated) levels of the BCR-ABL CRKL substrate, in the manner determined by a sandwich ELISA. In other embodiments, the presence and / or activation state of a BCR-ABL substrate, such as CRKL, can be measured using a proximity assay such as a collaborative proximity immunoassay (COPIA) described in PCT application PCT / US2010 / 042182, filed on July 15, 2010, and in US patent publications 20080261829, 20090035792 and 20100167945, the disclosures of which are incorporated by reference in their entirety, for all purposes.
Figure 17 illustrates that CRKL is both present and activated (i.e., phosphorylated) in K562 cells (ie, cells from a human chronic myelogenous leukemia cell line). Figure 18 shows that CRKL is present in A431 cells (ie, cells of a cell line - from human squamous cell carcinoma), and is activated (ie, phosphorylated) by treatment with EGF. Figure 19 shows that CRKL is present in T47D cells (ie, cells from a human breast ductal epithelial tumor cell line), but is not activated (ie, phosphorylated) by >> -— treating-with EGE . Figure 20 shows that CRKL is present in cells HUH
| T47D and is activated (ie, phosphorylated) at low levels by treatment with heregulin (HRG). Figure 21 shows that CRKL is present in MCF-7 cells (ie, cells from a human breast adenocarcinoma cell line) and is activated (ie, phosphorylated) at low levels through - treatment with heregulin (HRG). Figure 22 illustrates the presence of activated CRKL (ie, phosphorylated) in white blood cells (WBC's) from patient samples, with the level of activation being different between donors.
Example 8. Detection of the total and activated levels of the substrate BCR-ABL JAK2 in various cancer cell lines.
This example demonstrates the detection of the activated (phosphorylated) levels of the BCR-ABL JAK2 substrate, in the manner determined by a sandwich ELISA. In other embodiments, the presence and / or status of: activation of a BCR-ABL substrate, such as JAK2, can be measured using a proximity assay, such as a collaborative proximity immunoassay (COPIA) described in the PCT application PCT / US2010 / 042182, filed on July 15, 2010, and patent publication US 20080261829, 20090035792 and 20100167945, the disclosures of which are incorporated herein by reference in its entirety, for all purposes.
Figure 23 illustrates that JAK2 is activated (i.e., phosphorylated) in K562 cells (i.e., cells in a human chronic myelogenous leukemia cell line) and A431 cells (i.e., cells in a human squamous cell carcinoma cell line).
Example 9. Isolation of cells from the blood without dilution of anti-cancer medication.
This example demonstrates the recovery of K562 cells (that is, cells from a human chronic myelogenous leukemia cell line), from the blood, augmented with K562 cells using a magnetic capture bead with anti-CD45 antibodies, followed by the preparation of a lysate TO ———- K562 cehtar -and- determination of the state of expression and / or activation of a —UúÚúú5 or more oncogenic fusion proteins (for example, BCR-ABL), substrates thereof, pathways or combinations thereof. This example also demonstrates the recovery of white blood cells from blood samples from a patient, using magnetic capture counts with anti-CD45 and / or anti-CDI5 antibodies, followed by the preparation of a cell lysate and determination of the state of expression and / or activation of one or more oncogenic fusion proteins (for example, BCR-ABL), substrates thereof, pathways thereof or combinations thereof. Eliminating the need for any of the washing steps after cell isolation, the methods described here are advantageous because cells of interest can be recovered from the blood without changing the intracellular concentration of an anti-cancer drug, such as an inhibitor of tyrosine kinase. As such, the methods described in this example are contrary to accepted practice in the technique of washing cells after isolation (for example, cells attached to the wash bead), and providing cell lysates from recovered cells without substantial dilution of an anti-drug. -cancer, such as a tyrosine kinase inhibitor (eg, Gleevecº, Tasigna ”, Sprycelº, etc.), inside the cells. Recovery of K562 cells from blood ImL using Dynacontas CD45
1. Prepare buffer 1.
1.1 Add 500mg of BSA in 500mL of PBS.
1.2 Add 2 mL of 0.5M EDTA.
2. Prepare blood and boost with K562 cells.
2.1 Obtain 10 mL of donor whole blood.
2.2 Dilute blood dilution 1: 1 with buffer 1.
2.3 Perform a cell count of K562 cells using the automated cell counter.
2.4 Add 5e6, le6, 0.1e6 and O K562 cells separately in ImL of diluted blood for each concentration.
3. Wash with dynacounts (Invitrogen Cat. No. 111.53D).
3.1 Transfer 100 μL of beads to each 1 and 7 cells in a 1.5 mL eppendorf tube.
3.2 Add ImL of buffer 1 and mix gently.
3.3 Place the tube on the magnet for 1 minute.
3.4 Remove the supernatant.
3.5 Remove the tube from the magnet and resuspend in equal amounts of buffer 1, as the initial volume of transferred beads.
4. Cell isolation.
4.1 Add 100 μL of looped beads to each increased K562 blood sample.
4.2 Incubate the samples on a rotor at a cold temperature (or room temperature) for 20 minutes, 2 hours or 1 hour.
4.3 Place samples on the magnet and remove the supernatant.
5. Preparation of cell lysate.
5.1 Add | mL of cold lysis buffer to beads linked to 5 and 6 K562 cells
5.2 Add 500 μl of lysis buffer to the beads connected to the 1 and 6 K562 cells.
5.3 Add 100 μL of lysis buffer to the beads connected to the 0.186 K562 cells.
5.4 Vortex and place on ice for 20; vortex intermittently.
5.5 Centrifuge for 15 'at maximum speed at 4 degrees.
5.6 Transfer the supernatant to an eppendorf tube to run the microarray, for example, by performing the proximity-mediated immunoassay described here Isolation of CML cell from the patient's blood sample using Dynabeads CDA45 and / or CD15
1. Prepare buffer 1.: 1.1 Add 500mg of BSA in 500mL of PBS. 1.2 Add 2 mL of 0.5M EDTA. . 2. Prepare the patient's blood.
2.1 Obtain 2-3mL of the patient's whole blood using EDTA (or heparin) as an anticoagulant.
2.2 Add or not protease inhibitors.
3. Wash DynaBeads (Invitrogen Cat. No. 1 11.53D).
3.1 Transfer 100 µL (200 µL, 300 µL) of beads for each 1 mL of blood sample to a 1.5 mL eppendorf tube.
3.2 Add 1 mL of buffer 1 and mix gently. ã 3.3 Place the tube on the magnet for 1 minute. . 3.4 Remove the supernatant.
3.5 Remove the tube from the magnet and resuspend in 100 μl of 1l buffer as the initial volume of transferred beads.
4. Isolation of cells.
4.1 Add the 100 ul of washed beads in 1 ml of blood sample in a 1.5 ml eppendorf tube.
4.2 Incubate the samples on a rotor in a cold temperature (room temperature) for 20 minutes, 2 hours or 1 hour.
4.3 Place the samples on the magnet and remove the supernatant
5. Preparation of cell lysate.
5.1 Add 100 μL of lysis buffer to the cells bound cells.
5.2 Vortex and place on ice for 20; vortex intermittently.
5.3 Centrifuge for 15 'at maximum speed at 4 degrees. Ea and EE a a E E A paro Sem] and o
5.4 Transfer the supernatant to an eppendorf tube to: run the microarray, for example, by performing the immunoassay mediated by the proximity described here. Figure 24 illustrates that phosphorylated BCR-ABL can be - detected and measured in cell lysates prepared from K562 cells, isolated from blood using magnetic anti-CD45 beads. In particular, the 4G10 antibody (Millipore), which binds to the phospho-tyrosine residue in the ADbI portion of the fusion protein, was used to detect the levels of phosphorylated BCR-ABL. A similar assay can be performed on cell lysates prepared from white blood cells (eg, chronic myelogenous leukemia (CML) cells), isolated from blood samples from. using anti-CD45 and / or anti-CD15 magnetic beads to detect e. measure the presence and / or level of phosphorylated BCR-ABL. Example 10. Detection of both total levels of oncogenic fusion protein and total levels of full-size natural protein in patient samples.
This example demonstrates a method for the simultaneous detection of the total amount and / or activation state of an oncogenic fusion protein, in combination with one or both natural full size proteins that contain sequences or domains found in the oncogenic fusion protein. In a particular modality, the present method makes it possible to detect and / or measure both the total levels of BCR-GLA and the total levels of BCR and / or GLA of natural size in a biological sample, such as a blood or aspirate sample. of “bone marrow.
In certain embodiments, the levels of natural protein (for example, BCR and / or full-length GLA levels) are determined along with the levels of oncogenic fusion protein (for example, BCR-
'modalities, the full-size protein can be isolated: advantageously together with oncogenic fusion protein, in such a way that' the levels of these molecules are determined in the same piece. . Figure 25 illustrates that the total BCR-ABL levels were not altered when an antibody directed to the full-length BCR C terminations (whose C-terminal domain is not present in BCR-ABL) was indicated on the same slide as same piece, as an antibody directed to the N-terminal region of BCR-ABL. Figure 26 illustrates that the natural free BCR signal, detected with a specific BCR antibody for the N-termination, was reduced when an antibody directed to the C-terminus of natural BCR was identified on the same "slide of the same piece.
NR In particular embodiments, the method described in this example can be used to detect and / or measure total BCR-GLA levels, as well as total BCR-GLA levels of total natural size, and a ratio of total BCR-GLA levels to the BCR or GLA levels of full natural size can be calculated. In some instances, the ratio of BCR-ABL levels to BCR or full-size ABL levels is calculated to provide a more accurate determination of response indicators such as, for example, a major molecular response, a complete molecular response , a complete cytogenetic response and combinations of these. In other examples, the method described in this example can be used to monitor changes in the expression of BCR-ABL, with respect to a control, such as full-size BCR or GLA (for example, calculating —a ratio of BCR- Total GLA for BCR or full-size GLA levels) as a therapy function (for example, tyrosine kinase inhibitor therapy). All publications and patent applications cited in this or individual patent application were specifically and individually: indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example with. For the purpose of clarifying the understanding, it will be easily evident to those skilled in the art, in the light of the precepts of this invention, that certain changes and modifications can be made here without departing from the spirit or scope of the attached claims.
.

权利要求:
Claims (27)
[1]
1. Method for determining the level or state of activation of an oncogenic fusion protein, characterized by the fact that it comprises: (a) putting a cell extract in contact with a first fraction - specific connection for a first domain of a first protein total size, under suitable conditions to transform the first full-size protein present in the cell extract into a complex comprising the first full-size protein and the first binding fraction, in which the first domain of the first size protein total does not have a corresponding oncogenic fusion protein, which comprises a second domain different from the first full size protein fused to a first domain of a second protein of different total size; (b) removing the complex from step (a) from the cell extract to form a cell extract devoid of the first full-size protein; (c) placing the cell extract of step (b) in contact with a second, a third and a fourth binding fraction that, under suitable conditions, can transform the oncogenic fusion protein present in the cell extract into a complex comprising the oncogenic fusion protein and the second, third and fourth binding fraction; where the second binding fraction is specific to the second domain of the first protein of different total length, where the second binding fraction is contained in a solid support in an arrangement, where the third binding fraction is labeled with a facilitation fraction and is specific to one of the following: (i) the first domain of the second - protein of different total size; (ii) the second domain is different from the first full-size protein; or (iii) the fusion site between the second domain different from the first protein of full size, and the first domain of the second protein of different size, where the fourth fraction of binding is marked with a first element of a signal amplification pair, and is specific to the first domain of the second protein of different total size, and in which the facilitation fraction generates an oxidizing agent that channels and reacts with the first element of the signal amplification pair; (d) incubating the complex of step (c) with a second element of the signal amplification pair to generate an amplified signal; and (e) detecting the amplified signal generated from the first and second elements of the signal amplification pair, by determining the levels or activation status of the oncogenic fusion protein.
[2]
2. Method according to claim 1, characterized by the fact that the cell extract comprises an extract of cells isolated from a sample optionally selected from whole blood, serum, plasma, urine, sputum, bronchial lavage fluid , tears, breast aspiration, lymph, saliva, fine needle aspiration (FNA) and combinations thereof.
[3]
3. Method according to claim 2, characterized by the fact that the sample is obtained from a cancer patient, optionally caused by the formation of a concogenic fusion protein due to the translocation of the chromosome in the cancer, said cancer optionally being a hematological malignancy, osteogenic sarcoma, or soft tissue sarcoma, hematological malignancy being a leukemia or lymphoma, chronic myelogenic leukemia (ML-ML).
[4]
4, Method according to claim 2, characterized by the fact that the isolated cells are selected from the group consisting of circulating tumor cells, leukocytes and combinations thereof, or in which the isolated cells are stimulated in in vitro with growth factors, or in which isolated cells are incubated with an anti-cancer drug before stimulation with growth factor, or in which isolated cells are lysed after stimulation with growth factor to produce the cell extract, ouem that the isolated cells are not washed before lysis to produce the cell extract.
[5]
5. Method according to any one of claims 1 to 4, characterized by the fact that the oncogenic fusion protein is selected from the group consisting of BCR-ABL, DEK-CAN, E2A-PBX1, RARa-PML, IREL- URG, CBFR-MYH11, AMLI-MTG8, EWS-FLI, LYT-10-Cal, HRX-ENL, HRX-AFA4, NPM-ALK, IGH-MYC, RUNX1I-ETO, TEL-TRKC, TEL-AML1, MLL- AFA4, TCR-RBTN2, COLIA1I-PDGF, E2A-HLF, PAX3-FKHR, ETVG6-NTRK3, RET-PTC, TMRSS-ERG, TPR-MET and combinations thereof.
[6]
6. Method according to any one of claims 1 to 5, characterized by the fact that the oncogenic fusion protein is BCR-ABL.
[7]
Method according to claim 6, characterized by the fact that the first full-size protein is BCR, wherein the first domain of the first full-size protein comprises the carboxyl-terminal region of BCR (BCR-C)., where the second domain different from the first full-size protein comprises the amino-terminal region of BCR (BCR-N), where the second different total size protein is GLA, where the first domain of the second protein is in the case of different total size it comprises the carboxyl-terminal region of ABL (ABL-C), in which the first protein of total size is ABL, in which the second protein of different total size is BCR.
[8]
8. Method according to any one of claims 1 to 7, characterized by the fact that the activation state is selected from the group consisting of a phosphorylation state, state of ubiquitination, state of complexation and combinations thereof, or further comprises further determining the level or state of activation of one or more signal transduction molecules, wherein said one or more signal transduction molecules is a BCR-ABL substrate, wherein said BCR-ABL substrate is selected from the group consisting of CRKL, JAK2, STAT5, VAV, BAP-1 and combinations thereof.
[9]
Method according to any one of claims 1 to 8, characterized in that the first binding fraction comprises a first antibody, said first antibody is affixed to a solid support, wherein the solid support is selected from the group consisting of glass, plastic, chips, pins, filters, beads, paper, membrane, bundles of fibers and combinations thereof.
[10]
Method according to any one of claims 1 to 9, characterized in that the second binding fraction comprises a second antibody, said second antibody is affixed to a solid support, wherein the solid support is selected from the group consisting of glass, plastic, chips, pins, filters, beads, paper, membrane, bundles of fibers and combinations thereof.
[11]
11. Method according to any one of claims 1 to 10, characterized by the fact that steps (c) and (e) comprise an enzyme-linked immunosorbent assay (ELISA), a flow cytometry assay, or a label selection assay, said ELISA optionally comprises a sandwich ELISA, said flow cytometry optionally comprises a fluorescence activated cell separation assay (FACS).
[12]
12. Method according to claim 1, characterized in that the cell extract of step (b) is brought into contact with a serial dilution of the second binding fraction to form a plurality of complexes comprising the oncogenic fusion protein and the second link fraction, wherein said third and fourth link fraction comprise the third and fourth antibodies, respectively, where the third and fourth antibodies are both antibodies independent of the activation state in which the amplified signal generated from the first and second elements of the signal amplification pair, it is correlated with the total amount of the oncogenic fusion protein.
[13]
13. Method according to claim 12, characterized by the fact that the third antibody is an antibody independent of the activation state and the fourth antibody is an antibody dependent on the activation state, or in which the amplified signal, generated from of the first and second elements of the signal amplification pair, it is correlated with the amount of activated oncogenic fusion protein.
[14]
14. Method according to any one of claims 1 to
13, characterized by the fact that the third link fraction is marked directly with the facilitation fraction, or that the fourth link fraction is marked directly with the first element of the signal amplification pair, or that the fourth The binding fraction is marked with the first element of the signal amplification pair through the connection between a first element of a binding pair conjugated to the second detection antibody, and a second element of the binding pair conjugated to the first element of the signal amplification pair.
[15]
15. Method according to any one of claims 1 to 14, characterized by the fact that it further comprises: (f) bringing the cell extract into contact with a fifth specific binding fraction for a second domain of the second protein of different total size , under suitable conditions to transform the second protein of different total size present in the cell extract into a complex comprising the second protein of different total size and the fifth binding fraction, in which the second domain of the second protein of different total size it does not have the oncogenic fusion protein; and (g) removing the complex from step (f) from the cell extract to form a cell extract devoid of the second protein of different total size, in which step (f) is carried out before, during, or after step (a).
[16]
16. Method, according to claim 15, characterized by the fact that the solid support is selected from the group consisting of glass, plastic, chips, pins, filters, beads, paper, membrane, bundles of fibers and combinations thereof.
[17]
17. Method for optimizing therapy and / or reducing toxicity in a subject having cancer and receiving a course of therapy for the treatment of cancer, characterized by the fact that the method comprises: (a) isolating cancer cells after administration of a anti-cancer medication; (b) lyse the isolated cells to produce a cell extract;
(c) measuring a level of expression and / or activation of an oncogenic fusion protein in the cell extract, as defined in any one of claims 1 to 17; (d) comparing the measured level of expression and / or activation of the protein — oncogenic fusion with a level of expression and / or activation of the oncogenic fusion protein, measured at an earlier time, during the period of therapy; and (e) determining a subsequent dose of the therapy period for the subject, or whether a different therapy period can be administered to the subject based on the comparison of step (d).
[18]
18. Method, according to claim 17, characterized by the fact that the oncogenic fusion protein is BCR-ABL, in which the subject is determined to have cancer cells that express the oncogenic fusion protein before receiving the period of therapy, in which the subject is positive for BCR-GLA.
[19]
19. Method, according to claim 17 or 18, characterized by the fact that both the level of expression and the level of activation of the oncogenic fusion protein are measured in the cell extract, in which step (c) additionally comprises calculating a ratio of the levels of activated protein to total oncogenic fusion protein, where step (d) comprises comparing the calculated ratio of the levels of activated protein to total oncogenic fusion protein with a ratio of the levels of activated protein to total oncogenic fusion protein , calculated for the subject at an early stage.
[20]
20. Method, according to claim 19, characterized by the fact that the calculated ratio of the levels of activated protein to total oncogenic fusion protein is correlated with the percentage inhibition of the fusion protein - activated cogen, through treatment with anti-inflammatory drug. cancer, in which the calculated ratio of activated protein levels to total oncogenic fusion protein is related to the level of a control protein in which the calculated ratio of activated protein levels to total oncogenic fusion protein comprises a ratio of the levels of phosphorus / total BCR-ABL protein.
[21]
21. Method according to claim 20, characterized in that the control protein comprises a natural protein of full size that contains sequences or domains found in the oncogenic fusion protein in which the control protein is selected from the group consisting of BCR full size GLA full size and combinations thereof.
[22]
22. Method according to any one of claims 17 to 21, characterized in that less than about 50% inhibition of the level of activation of the oncogenic fusion protein indicates a need to increase the subsequent dose of the therapy period, or to administer a different period of therapy, in which less than about 50% inhibition of the level of activation of the oncogenic fusion protein indicates a subject's lack of agreement with the period of therapy, the existence of possible side effects or toxicity associated with the period of therapy, or combinations thereof, in which more than about 80% inhibition of the level of activation of the oncogenic fusion protein indicates that the subject is in the correct therapy period and in the correct dose.
[23]
23. Method according to any one of claims 17 to 22, characterized by the fact that cancer is chronic myelogenous leukemia (CML), in which the anti-cancer drug is selected from the group consisting of a monoclonal antibody, tyrosine kinase inhibitor, chemotherapeutic agent, hormonal therapeutic agent, radiotherapeutic agent, vaccine and combinations thereof, in which the tyrosine kinase inhibitor is selected from the group consisting of imatinib mesylate (Gleevecº), nilotinib (Tasignaº), dasatinib (Sprycelº), bosutinib (SKI-606) and combinations thereof.
[24]
24. Method according to any one of claims 17 to 23, characterized in that steps (c) to (e) alternatively comprise: (c ') measuring a level of expression and / or activation of a fusion protein oncogenic and one or more signal transduction molecules in its pathway in the cell extract; (d ') to compare the measured level of expression and / or activation of
in the case of oncogenic fusion, and signal transduction molecules, with a level of expression and / or activation of the oncogenic fusion protein, and signal transduction molecules, measured at an initial moment during the period of therapy; and (e ') determining a subsequent dose of the therapy period for the subject, or whether a different therapy period can be administered to the subject based on the comparison of step (d'.
[25]
25. Method for selecting an appropriate anti-cancer drug for the treatment of cancer, or for identifying the response of a cancer to treatment with an anti-cancer drug, or for predicting the response of a subject having cancer to treatment with cancer. an anti-cancer drug, or to determine whether a subject having cancer is resistant to treatment with an anti-cancer drug, characterized by the fact that the method comprises: (a) isolating cells from a cancer after administering a drug anti-cancer, or before incubation with an anti-cancer drug; (b) lyse the isolated cells to produce a cell extract; (c) measuring a level of expression and / or activation of an oncogenic fusion protein in the cell extract, as defined in any one of claims 1 to 24; and (d) determine whether the anti-cancer drug is suitable or unsuitable for the treatment of cancer, by comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference level and / or activation profile generated in the absence of the anti-cancer drug, identify the cancer as responsive or unresponsive to treatment with the anti-cancer drug, by comparing the level of expression and / or activation detected for the fusion protein — homogeneous with a reference level and / or activation profile generated in the absence of the anti-cancer drug, and / or predicting the likelihood that the subject will respond to treatment with the anti-cancer drug, by comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference level and / or activation profile generated in the absence of the anti-cancer drug, and / or determining whether the subject is resistant or sensitive to treatment with the anti-cancer drug, by comparing the level of expression and / or activation detected for the oncogenic fusion protein with a reference level and / or activation profile generated in the absence of the anti-cancer drug or in the presence of the anti-cancer drug in an initial period, respectively.
[26]
26. Method, according to claim 25, characterized by the fact that cancer is chronic myelogenous leukemia (CML) in which the anti-cancer drug is selected from the group consisting of a monoclonal antibody, tyrosine kinase inhibitor, chemotherapeutic agent, hormonal therapeutic agent, radiotherapy agent, vaccine and combinations thereof, in which the monoclonal antibody is selected from the group consisting of trastuzumab (Herceptin ”), alemtu- zumab (Campathº *), bevacizumab (Avastinº) , cetuximab (Erbituxº), gemtuzumab (Mylotargº), panitumumab (V ectibix ”"), rituximab (Rituxanº), tositumomab (BEX-XARº) and combinations thereof, said tyrosine kinase inhibitor is selected from the group consisting of mesylate of imatinib (Gleevecº), nilotinib (Tasignaº), da-satinib (Sprycelº), bosutinib (SKI-606), gefitinib (Iressaº), sunitinib (Sutentº), erlo-tinib (Tarceva ”), lapatinib (Tykerbº), canertinib (CI 1033), semaxinib (SU5416), valtananib (PTK787 / ZK222584), sora fenib (BAY 43-9006), leflunomide (SU101), van-detanib (ZACTIMA ”"; ZD6474) and combinations thereof, in which the chemotherapeutic agent is selected from the group consisting of pemetrexed (ALIMTAº), gemcitabine (Gemzarº), sirolimus (rapamycin), rapamycin analogs, platinum, carboplatin, cisplatin, satraplatin compounds , paclitaxel (Taxolº), docetaxel (Taxotere ), tensirolimus (CCI-779), everolimus (RADO001) and combinations thereof in which the hormonal therapeutic agent is selected from the group consisting of aromatase inhibitors, modulators of the selective estrogen receptor, steroids, phinasteride, agonists of the hormone that releases gonadotropin, pharmaceutically acceptable salts of these, stereoisomers of these, derivatives of these, analogs of these and combinations thereof, in which the radiotherapeutic agent is selected from the group consisting of in “Sc, Cu, Cu, Sr, sy, y, * y 1 Rh," Ag, "im, msn, Pm,
Sm, "Ho," Lu, Re, Re, 2 At, Bi and combinations thereof.
[27]
27. Invention, characterized by any of its embodiments - categories of claim encompassed by the material initially revealed in the patent application or in its examples presented here.

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法律状态:
2021-02-09| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|Free format text: REFERENTE A 10A ANUIDADE. |

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2021-05-25| B08K| Patent lapsed as no evidence of payment of the annual fee has been furnished to inpi [chapter 8.11 patent gazette]|Free format text: EM VIRTUDE DO ARQUIVAMENTO PUBLICADO NA RPI 2614 DE 09-02-2021 E CONSIDERANDO AUSENCIA DE MANIFESTACAO DENTRO DOS PRAZOS LEGAIS, INFORMO QUE CABE SER MANTIDO O ARQUIVAMENTO DO PEDIDO DE PATENTE, CONFORME O DISPOSTO NO ARTIGO 12, DA RESOLUCAO 113/2013. |

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2021-12-14| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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US61/253393|2009-10-20|

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PCT/US2010/053386|WO2011050069A1|2009-10-20|2010-10-20|Proximity-mediated assays for detecting oncogenic fusion proteins|

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