HK1152563B - Tle3 as a marker for chemotherapy - Google Patents

Abstract

Methods of using TLE3 as a marker for predicting the likelihood that a patient's cancer will respond to chemotherapy. Methods of using TLE3 as a marker for selecting a chemotherapy for a cancer

Description

TLE3 as a marker for chemotherapy

Technical Field

Is free of

Background

The main difficulty in cancer treatment is selecting the chemotherapy with the greatest efficacy and least toxicity for a given patient. Analysis of cell surface markers using, for example, Immunohistochemistry (IHC) has provided a means to classify certain cancers into multiple subclasses. For example, one factor considered in the prognosis and treatment decision for breast cancer is the presence or absence of Estrogen Receptors (ERs). ER-positive breast cancers are generally significantly more responsive to hormonal therapy (e.g., tamoxifen, which acts as an antiestrogen in breast tissue) than ER-negative cancers. Although useful, these assays only partially predict clinical characteristics of breast cancer. Phenotypic differences exist among different cancers, but current diagnostic tools are not able to detect them. Therefore, there is still much debate on how to classify patients according to the possible treatments to maximize their efficacy (see, for example, "NIH consensus Development meeting Statement: Adjuvant Therapy for Breast cancer" (NIHConsenssens Development Statement: Adjuvant Therapy for Breast cancer), 11.1-3.2000, J. Nat. cancer Inst. monograms, 30: 5-15, 2001 and Dilyo (Di Leo), et al, J. International J. Clin. Oncol. 7: 245-253, 2002). In particular, there is currently no tool to predict a patient's likely response to treatment with paclitaxel, a chemotherapeutic drug with particularly adverse side effects. There is a great need for improved methods and reagents to classify cancer and thereby select a treatment regimen with maximum efficacy and minimal toxicity for a given patient.

Disclosure of Invention

The correlation between expression of TLE3 (transducin-like mitogen enhancer 3, intets (Entrez) gene bank number 7090) and cancer response to chemotherapy has been determined in the art. This association has been confirmed using the TLE3 antibody and samples from the breast cancer cohort (including both treated and untreated patients, with known efficacy). The inventors have also observed that binding of TLE3 antibody in a treated ovarian cancer patient sample is associated with an improved prognosis. Thus, in one aspect, the present invention provides a method for predicting the likelihood that a patient's cancer will respond to chemotherapy using TLE3 as a marker. In another aspect, the present invention provides a method of using TLE3 as a marker to determine whether to administer chemotherapy to a cancer patient. In yet another aspect, the present invention provides a method of selecting chemotherapy for a cancer patient using TLE3 as a marker.

Any known method can be used to detect expression of TLE 3. Thus, while the methods of the invention have been exemplified as being useful for detecting TLE3 polypeptides through the use of antibodies, in certain embodiments one or more primers well known in the art may be used to detect TLE3 polynucleotides.

Generally, TLE3 can be used in combination with other markers or clinical factors (e.g., cancer stage, tumor size, ganglion characteristics, age, etc.) to further improve the predictive power of the methods of the invention.

Brief description of the appendix

The present patent application relates to materials including the tables and data presented in appendix a. In particular, appendix A is a table listing a number of markers that may be used in combination with the TLE3 marker in the methods of the invention. The table includes antibody numbers, maternal gene names, intets library numbers, known alias names for the maternal genes, peptides that can be used to make the antibodies, and exemplary antibody titers for staining. The nucleotide (and corresponding amino acid) sequence of each of the maternal genes listed in appendix a from the public databases (e.g., the gene bank (GenBank), the international protein database (Swiss-Prot), or any derivative database of the future of these databases) can be readily obtained by one of skill in the art using the maternal gene name, inteins gene bank numbering, and/or known alias of the maternal gene. The nucleotide sequences and corresponding amino acid sequences of each of the parent genes listed in appendix a are incorporated herein by reference from these public databases. Antibodies numbered initially as S5 or S6 are available from designated commercial sources.

Drawings

FIG. 1 compares IHC images of TLE 3-negative (S0643-) and TLE 3-positive (S0643+) samples from breast cancer patients.

Figure 2 shows a Kaplan-Meier (Kaplan-Meier) recurrence curve obtained using all patients in the henzville Hospital (Huntsville Hospital) (HH) breast cancer cohort after classification based on staining with antibodies raised against the TLE3 marker. The recurrence data for TLE 3-positive and TLE 3-negative patients were used to obtain top and bottom curves, respectively. As shown in the figure, antibodies that bind the TLE3 marker correlated with improved prognosis in this breast cancer cohort (HR 0.573, p < 0.004).

Figure 3 shows a kaplan-meier recurrence curve obtained using all patients in the rossville Park Cancer Institute (RP) breast Cancer cohort after classification based on staining with antibodies raised against the TLE3 marker. The selected patients in the RP cohort were all triple negative patients with ER (estrogen receptor, Instron Genbank accession number 2099), PR (progestin receptor, Instron Genbank accession number 5241) and the HER-2 marker (v-erb-b2 erythroblastic leukemia virus oncogene homolog 2, Instron Genbank accession number 2064). The recurrence data for TLE 3-positive and TLE 3-negative patients were used to obtain top and bottom curves, respectively. As shown in the figure, antibodies that bind the TLE3 marker correlated with an improved prognosis in this breast cancer cohort (HR 0.24, p < 0.011).

Fig. 4 shows a kaplan-meier recurrence curve obtained using patients in the fig. 1HH breast cancer cohort who did not receive chemotherapy. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, the antibody binding TLE3 marker was not associated with prognosis in breast cancer patients who did not receive chemotherapy (HR 0.788, p 0.49).

Fig. 5 shows a kaplan-meier recurrence curve obtained using the patients receiving chemotherapy in the fig. 1HH breast cancer cohort. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, antibodies that bind the TLE3 marker correlated with prognosis recovery in patients receiving chemotherapy (HR ═ 0.539, p < 0.013).

Figure 6 shows a kaplan-meier recurrence curve obtained using patients receiving chemotherapy in the fig. 2RP breast cancer cohort. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, the antibody binding TLE3 marker was associated with an improved prognosis in this subclass of breast cancer patients (HR 0.194, p 0.010). These results are similar to those obtained with the HH queue in fig. 5.

Figure 7 shows a kaplan-meier recurrence curve obtained using patients receiving CMF (cyclophosphamide, methotrexate, and 5-fluorouracil) chemotherapy in the HH breast cancer cohort of figure 5. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, antibodies that bind the TLE3 marker were associated with improved prognosis in patients treated with this subclass (HR ═ 0.398, p < 0.019).

FIG. 8 shows a Kaplan-Meier recurrence curve obtained using patients in the FIG. 5HH breast cancer cohort receiving CA (cyclophosphamide and doxorubicin) or CAF (cyclophosphamide, doxorubicin, and 5-fluorouracil) chemotherapy with or without a taxane. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, the association between the antibody binding the TLE3 marker and prognosis lost significance in patients treated with this subclass (HR ═ 0.666, p ═ 0.22).

Fig. 9 shows kaplan-meier recurrence curves obtained using patients receiving only CA or CAF chemotherapy (i.e., without taxane) in the fig. 8HH breast cancer cohort. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, the antibody binding TLE3 marker was not relevant for the prognosis of patients with this subclass treated (HR 1.03, p 0.95).

FIG. 10 shows a Kaplan-Meier recurrence curve obtained using patients in the FIG. 8HH breast cancer cohort that received CA or CAF and a taxane. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, antibodies that bind the TLE3 marker correlated with prognosis for recovery in patients with this subclass treated (HR 0.114, p 0.038).

Figure 11 shows kaplan-meier recurrence curves obtained using patients in the fig. 6RP breast cancer cohort that received CA chemotherapy alone (i.e., without the use of a taxane). The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, the antibody binding TLE3 marker was not relevant for the prognosis of patients with this subclass treated (HR 0.759, p 0.81).

Figure 12 shows kaplan-meier recurrence curves obtained using patients in the fig. 6RP breast cancer cohort that received CA along with a taxane. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, antibodies that bind the TLE3 marker were associated with an improved prognosis in patients with this subclass treated (HR 0.142, p 0.011).

Figure 13 shows kaplan-meier recurrence curves obtained using patients receiving taxane or CMF in the fig. 6RP breast cancer cohort. Some patients receiving taxanes also received CA. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, antibodies that bind the TLE3 marker were associated with an improved prognosis in patients with this subclass treated (HR 0.137, p 0.011).

Figure 14 shows kaplan-meier recurrence curves obtained using patients receiving neoadjuvant chemotherapy in the fig. 6RP breast cancer cohort. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. The sample size was small (N ═ 12); however, as shown in the figure, antibodies that bind the TLE3 marker showed significant correlation with improved prognosis for patients with this subclass treated when measured using the Fisher Exact Test (p ═ 0.005).

Figures 15-17 show kaplan-meier recurrence curves obtained using patients receiving chemotherapy in the fig. 6RP breast cancer cohort. Recurrence data for TLE 3-positive and TLE 3-negative patients with stage II + (fig. 15), IIb + stage (fig. 16), and III + stage (fig. 17) cancer, respectively, were used to obtain top and bottom curves. In each case, antibodies that bind the TLE3 marker were associated with improved prognosis for patients treated with these subclasses. The sample size was small in the subclass of fig. 17 (N ═ 19); however this association is significant when measured using the fisher's exact test (p ═ 0.020).

FIG. 18 shows a Kaplan-Meier recurrence curve obtained using patients in the ovarian cancer cohort at the University of Alabama atman Birmingham (UAB). All patients received paclitaxel. Most patients also receive platinum chemotherapy (carboplatin or cisplatin). The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, antibodies that bind the TLE3 marker were associated with prognosis in these treated patients (HR ═ 0.64, p < 0.049).

Detailed Description

Definition of

Binding-when the interaction partner "binds" to the label, it is linked by non-covalent direct interaction.

Cancer markers- "cancer markers" or "markers" are molecular entities that can be detected in cancer samples. In general, a marker can be a polypeptide (e.g., TLE3 protein) or a polynucleotide (e.g., TLE3mRNA) that can indicate that a gene (e.g., TLE3 gene) is expressed and is present in a cancer sample, e.g., is present in the cytoplasm or membrane of a cancer cell and/or is secreted from the cell.

Cancer sample-the term "cancer sample" or "sample" as used herein broadly includes a cell or tissue sample taken from a cancer patient (e.g., taken from a tumor, taken from the bloodstream, etc.), cells of tumor origin that may be located elsewhere in the body (e.g., cells in the bloodstream or metastatic sites), or any material derived from the sample. Derivative material may include, for example, nucleic acids or proteins extracted from a sample, progeny cells, and the like. In one embodiment, the cancer sample can be a tumor sample.

Association- "Association" refers to the degree to which one variable can be predicted from another variable, e.g., the degree to which a cancer responds to therapy can be predicted from the expression of a marker in a cancer sample. The correlation between two variables can be measured using a variety of statistical methods such as, but not limited to, the Sinterden t-test, the exact Field test, the Pearson correlation coefficient, the Sperman correlation coefficient, the Chi squared test, and the like. The result is expressed in a conventional manner as a measured correlation coefficient, where the p-value provides a measure of the likelihood of correlation occurring by chance. Associations with p values less than 0.05 are generally considered statistically significant. Preferably, the associated p-value is less than 0.01, particularly preferably less than 0.001.

Hybridization-when a primer and a label "hybridize" to each other physically as described herein, they are linked by non-covalent base pair interactions.

Interaction partner-an "interaction partner" is an entity that binds to a polypeptide label. For example, but not limited to, the interaction partner may be an antibody or fragment thereof that binds to the label. In general, an interaction partner is considered to "specifically bind" to a label if it binds to the label to a detectable degree and does not bind to an unrelated molecular entity (e.g., other labels) to a detectable degree under similar conditions. Whether there is specific binding between the label and the interaction partner generally depends on whether the target label presents a particular structural feature, such as an antigenic determinant or epitope, that the interaction partner recognizes. In general, it is to be understood that specificity is not necessarily absolute. For example, it is well known in the art that in addition to a target epitope, a variety of antibodies can often cross-react with other epitopes. Whether the cross-reactivity is acceptable depends on which application the interaction partner is used for. Thus, the degree of specificity of an interaction partner depends on its context of use. In general, an interaction partner will appear to be specific for a particular label if it has a greater propensity to bind to that label than to other potential partners (e.g., other labels). One skilled in the art is able to select interaction partners with a sufficient degree of specificity to function appropriately in any given application (e.g., for detection of a target marker, for therapeutic purposes, etc.). It will also be appreciated that specificity may be assessed in the context of other factors, such as the affinity of the interaction partner for the target marker versus the affinity of the interaction partner for other potential partners (e.g., other markers). If the interaction partner exhibits a high affinity for the target marker and a low affinity for the non-target molecule, the interaction partner may be an acceptable diagnostic agent even in the absence of specificity.

Primer- "primer" is an oligonucleotide entity that physically hybridizes to a polynucleotide marker. In general, a primer is said to "specifically hybridize" to a label if the primer hybridizes to the label to a detectable degree and does not hybridize to an unrelated molecular entity (e.g., other label) to a detectable degree under similar conditions. Whether there is specific hybridization between the label and the primer depends on the presence of a specific nucleotide sequence in the target label that is complementary to the primer nucleotide sequence. In general, it is to be understood that specificity is not necessarily absolute. The degree of specificity of a primer depends on the context in which it is used. In general, a primer exhibits specificity for a particular label if it has a greater propensity to hybridize to that label than to other potential partners (e.g., other labels). One skilled in the art will be able to select primers with a sufficient degree of specificity to function properly in any given application. It will also be appreciated that specificity may be assessed in the context of other factors, such as the affinity of the primer for the target marker versus the affinity of the primer for other potential partners (e.g., other markers). If the primer exhibits high affinity for the target marker and low affinity for the non-target molecule, the primer may be an acceptable diagnostic agent even in the absence of specificity.

Response-the "response" of a cancer to a therapy may mean any detectable change, such as a change at the molecular, cellular, organ, or organism level. For example, tumor size, patient life expectancy, recurrence, or length of patient survival, etc., are all responses. Responses can be measured in any of a variety of ways, including, for example, non-invasive measurement of tumor size (e.g., CT scan, image enhanced visualization, etc.), invasive measurement of tumor size (e.g., residual tumor resection, etc.), surrogate marker measurement (e.g., serum PSA, etc.), clinical course differences (e.g., measuring patient quality of life, time to relapse, time to survival, etc.).

Small molecule- "Small molecule" is a non-polymeric molecule. Small molecules can be synthesized in the laboratory (e.g., by combinatorial synthetic methods) or obtained from nature (e.g., natural products). Small molecules are generally characterized by containing several carbon-carbon bonds and by a molecular weight of less than 1500Da, but this feature is not intended to be limiting for the purposes of the present invention.

Detailed description of certain preferred embodiments of the invention

As described above, it has been determined that expression of TLE3 (transducin-like mitogen enhancer 3, intel gene bank number 7090) in cancer samples correlates with cancer response to chemotherapy. As described in the examples, this association has been confirmed using the TLE3 antibody and samples from two breast cancer cohorts (including both treated and untreated patients, with known efficacy). This predictive model has also been shown to be consistent across multiple samples for a cohort of treated ovarian cancer patients. TLE3 has also been shown to be useful in predicting response to specific types of chemotherapy, including treatments involving administration of cell cycle specific chemotherapeutic drugs (e.g., methotrexate and taxanes). Since these chemotherapeutic agents are known to be useful in different cancer types, these results indicate that the methods of the invention can also be used to predict the efficacy of the chemotherapeutic agents in different cancer types.

Predicting response to chemotherapy and selecting chemotherapy

In one aspect, the present invention provides a method for predicting the likelihood that a patient's cancer will respond to chemotherapy using TLE3 as a marker. In general, these methods involve providing a cancer sample from a cancer patient, determining whether TLE3 is expressed in the cancer sample, and predicting the likelihood that the patient's cancer will respond to chemotherapy based on the results of the determining step. In one embodiment, the predicting step comprises predicting that the patient's cancer is likely to respond to chemotherapy based on the presence of TLE3 in the cancer sample. In one embodiment, the predicting step comprises predicting that the patient's cancer is unlikely to respond to chemotherapy based on the absence of TLE3 expression in the cancer sample.

In certain embodiments, a negative control sample is provided and the determining step comprises detecting the expression level of TLE3 in the cancer sample and the negative control sample and comparing the expression level of TLE3 in the cancer sample and the negative control sample. In general, a negative control sample can be any sample that does not express TLE3 in a reproducible manner. In one example, a negative control sample can be a sample that does not bind TLE3 antibody in a reproducible manner. In one example, a negative control sample can be a sample that does not reproducibly produce detectable levels of TLE3 mRNA. In one example, a negative control sample can be obtained from a patient with TLE 3-negative cancer. In one embodiment, the negative control sample can be obtained from a patient not suffering from cancer. In certain embodiments, the negative control sample may be derived from a tissue of the same type as the cancer (e.g., breast tissue when referring to breast cancer). In other embodiments, the negative control sample may be derived from a different type of tissue or even a different organism or cell line.

Alternatively or additionally, in certain embodiments, a positive control sample is provided and the determining step comprises detecting the expression level of TLE3 in the cancer sample and the positive control sample and comparing the expression level of TLE3 in the cancer sample to the positive control sample. In general, a positive control sample can be any sample that expresses TLE3 in a reproducible manner. In one example, a negative control sample can be a sample that binds TLE3 antibody in a reproducible manner. In one example, a negative control sample can be a sample that reproducibly produces detectable levels of TLE3 mRNA. In one embodiment, a positive control sample can be obtained from a patient having TLE 3-positive cancer. In certain embodiments, the positive control sample may be derived from a tissue of the same type as the cancer (e.g., breast tissue when referring to breast cancer). In other embodiments, the positive control sample may be derived from a different type of tissue or even a different organism or cell line.

Any known method can be used to determine expression of TLE 3.

In one embodiment, a TLE3 polypeptide can be detected using an interaction partner that binds to a TLE3 polypeptide (e.g., a TLE3 protein or antigenic fragment thereof). For example, TLE3 expression can be detected using TLE3 antibody as an interaction partner and by contacting a cancer sample with the TLE3 antibody, as described below. In such embodiments, the methods of the invention can involve providing a cancer sample from a cancer patient, contacting the cancer sample with an antibody directed against TLE3, and predicting the likelihood that the patient's cancer will respond to chemotherapy based on the binding of the antibody to the cancer sample. In one embodiment, the predicting step can comprise predicting that the patient's cancer will respond to chemotherapy based on the ability of the antibody to bind to the cancer sample. In another embodiment, the predicting step can comprise predicting that the patient's cancer will not respond to chemotherapy based on the inability of the antibody to bind to the cancer sample.

In another example, the TLE3 polynucleotide can be detected using one or more primers that hybridize to the TLE3 polynucleotide (e.g., TLE3mRNA, cDNA, or RNA). In such embodiments, the methods of the invention can involve providing a cancer sample from a cancer patient, contacting the cancer sample with one or more primers that hybridize to TLE3, and predicting the likelihood that the patient's cancer will respond to chemotherapy based on the hybridization of the one or more primers to the cancer sample. In one embodiment, the predicting step can comprise predicting that the patient's cancer may respond to chemotherapy based on the availability of one or more primers to hybridize to the cancer sample. In another embodiment, the predicting step can comprise predicting that the patient's cancer will not respond to chemotherapy based on the inability of the one or more primers to hybridize to the cancer sample.

In another aspect, the present invention provides a method for determining whether to administer chemotherapy to a cancer patient based on the likelihood that the patient's cancer will respond to chemotherapy. In one embodiment, the determining step comprises determining to administer chemotherapy to the cancer patient based on the presence of TLE3 expression in the cancer sample. In one embodiment, the deciding step comprises deciding not to administer chemotherapy to the cancer patient based on the absence of expression of TLE3 in the cancer sample.

In yet another aspect, the invention provides a method of selecting chemotherapy for a cancer patient. In general, these methods comprise providing a cancer sample from a cancer patient, determining whether TLE3 is expressed in the cancer sample, and selecting chemotherapy for the cancer patient based on the results of the determining step. In one embodiment, the selecting step comprises selecting chemotherapy based on the presence of TLE3 expression in the cancer sample.

As described in the examples, TLE3 expression was demonstrated to be associated with response to chemotherapy with methotrexate (see fig. 7) and taxanes (see fig. 10, 12 and 13). Methotrexate and taxanes are considered to be cell cycle specific chemotherapeutic drugs (see, e.g., Goodman and Gilman, pharmacological basis of Therapeutics (The pharmacological basis of Therapeutics), IX. Chemotherapy of neoplastic disease (Chemotherapy of neoplastics), chapter 51, Antineoplastic Agents (Antineoplastic Agents), 11 th edition, main coded lorentn l. brayton (law l. Brunton), side edited John s. lazo (John s. lazo) and kiss l. paque (Keith l. Parker)). In contrast to cell cycle-specific chemotherapeutic drugs which exhibit their mechanism of action during specific phases of the cell cycle, non-cell cycle-specific chemotherapeutic drugs exert an equivalent effect during all phases including the resting phase (G0). Plant alkaloids other than taxanes have also been classified in the literature as cell cycle specific chemotherapeutic agents, as have many other antimetabolites in addition to methotrexate. In contrast, a variety of alkylating agents, such as cisplatin and cyclophosphamide, have been classified as non-cell cycle specific chemotherapeutic drugs. The results of the present invention show that the predictive ability of TLE3 can be extended to other cell cycle specific chemotherapeutic drugs besides methotrexate and taxanes.

In certain embodiments, the methods of the invention are therefore useful for selecting a cell cycle specific chemotherapeutic agent or determining whether to administer a cell cycle specific chemotherapeutic agent. In one embodiment, the methods of the invention can be used to select an antimetabolite or to determine whether to administer an antimetabolite, and in one embodiment, the methods can be used to select a plant alkaloid or to determine whether to administer a plant alkaloid. In one embodiment, the methods of the invention can be used to select or decide whether to administer methotrexate. In another embodiment, the methods of the invention can be used to select a taxane or to determine whether to administer a taxane. In one embodiment, the taxane is paclitaxel. In one embodiment, the taxane is docetaxel.

In each case it will be appreciated that these chemotherapeutic agents may be administered alone or in combination with other chemotherapeutic agents known in the art and described below. It is also to be understood that the invention encompasses methods in which the chemotherapeutic agent of choice is methotrexate or a taxane derivative (i.e., a compound structurally derived from methotrexate or a taxane). The various derivatives may typically share most of the structure of the parent compound, but may include different substituents, heteroatoms, ring fusions, levels of saturation, isomers, stereoisomers, and the like, at one or more positions within the parent compound. The following U.S. patents illustrate, in a non-limiting manner, the preparation of exemplary methotrexate derivatives that can be used in the methods of the invention: U.S. patent nos. 6,559,149 and 4,374,987. The following U.S. patents illustrate, in a non-limiting manner, the preparation of exemplary taxane derivatives useful in the methods of the present invention: U.S. patent No. 7,074,945; 7,063,977 No; 6,906,101 No; 6,649,778 No; 6,596,880 No; 6,552,205 No; 6,531,611 No; 6,482,963 No; 6,482,850 No; 6,462,208 No; 6,455,575 No; 6,441,026 No; 6,433,180 No; 6,392,063 No; 6,369,244 No; 6,339,164 No; 6,291,690 No; 6,268,381 No; 6,239,167 No; 6,218,553 No; 6,214,863 No; 6,201,140 No; 6,191,290 No; 6,187,916 No; 6,162,920 No; 6,147,234 No; 6,136,808 No; 6,114,550 No; 6,107,332 No; 6,051,600 No; 6,025,385 No; 6,011,056 No; 5,955,489 No; 5,939,567 No; 5,912,263 No; 5,908,835 No; 5,869,680 No; 5,861,515 No; 5,821,263 No; 5,763,477 No; 5,750,561 No; 5,728,687 No; 5,726,346 No; 5,726,318 No; 5,721,268 th: 5,719,177 No. C; 5,714,513 No; 5,714,512 No; 5,703,117 No; 5,698,582 No; 5,686,623 No; 5,677,462 No; 5,646,176 No; 5,637,723 No; 5,621,121 No; 5,616,739 No; 5,606,083 No; 5,580,899 No; 5,476,954 No; 5,403,858 No; U.S. Pat. No. 5,380,916; 5,254,703 No; and No. 5,250,722. The entire contents of each of the above-mentioned patents and any other references cited herein are incorporated by reference herein.

Methotrexate functions by inhibiting folate metabolism and has been approved for the treatment of bladder Cancer, breast Cancer, gastric Cancer, choriocarcinoma, head and neck Cancer, meningeal Cancer, leukemia (acute meningeal leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia), lymphoma (Burkitt's lymphoma, childhood lymphoma, non-Hodgkin's lymphoma), mycosis fungoides, unknown primary Cancer and lymphosarcoma (Methotrexate in the BC Cancer center Drug handbook (Methotrexate in B C Cancer Agency Cancer Drug), 2007). Methotrexate has also been shown to be useful in the treatment of esophageal, lung, and testicular cancer (recent Methotrexate in UpToDate, 2007). In certain embodiments, the methods of the invention comprise the step of selecting or determining whether to administer methotrexate and one or more other chemotherapeutic agents. For example, methotrexate is commonly administered to cancer patients in a combination known as CMF, which also includes cyclophosphamide and 5-fluorouracil.

Taxanes are diterpenes produced by plants of the genus Taxus (Taxus). Taxanes may be derived from natural sources or produced synthetically. The taxane includes paclitaxel (TAXOL)^TM) And docetaxel (TAXOTERE)^TM). Taxanes act by interfering with normal microtubule growth during cell division. In certain embodiments, the methods of the invention comprise the step of selecting or deciding whether to administer a taxane (e.g., paclitaxel or docetaxel) and one or more other chemotherapeutic drugs, e.g., a taxane is typically administered to a cancer patient (i.e., administered with CA or CAF) in combination with cyclophosphamide and doxorubicin (doxorubicin) and optionally 5-fluorouracil.

Paclitaxel has been approved for the treatment of breast Cancer, Kaposi's sarcoma, lung Cancer, and ovarian Cancer (Paclitaxel in BC Cancer Drug Manual, 2007 in BC Cancer center, and pakhail (Mekhail) and makman (Markman), the journal of pharmacological therapists oping (Expert opin. pharmacother.) 3: 755-66, 2002). Paclitaxel has also been shown to be useful for treating cervical cancer (pages 1124-34, AHFS 2005 Drug Information (Drug Information), Besserda (Bethesda), Maryland: American Society of Health-systems Pharmacists (American Society of cancer), 2005), endometrial cancer (Paclitaxel in the BC cancer center cancer drugs Manual, 2007), bladder cancer (Paclitaxel in UpToDate, 2007), head and neck cancer (recent Paclitaxel, 2007), leukemia (recent Paclitaxel, 2007), and malignant melanoma (recent Paclitaxel, 2007). Side effects of paclitaxel include hypersensitivity reactions such as facial flushing, skin rash, or shortness of breath. Patients typically receive medications to prevent hypersensitivity prior to paclitaxel administration. Paclitaxel can also cause temporary damage to bone marrow. Bone marrow damage can make a person more susceptible to infection, anemia, and bruising, or bleeding. Other side effects may include joint or muscle pain in the arms and legs; diarrhea; nausea and vomiting; numbness, burning or tingling in the hands or feet; and alopecia.

Docetaxel has been approved for the treatment of breast Cancer (Apollo (Aapro), Oncology Seminars in Oncology 25(5 suppl. 12): 7-11, 1998; Nabholtz et al, J. Clinical Oncology 17 (5): 1413-24, 1999; Shorstrom et al, European Journal of Cancer (Europan Journal of Cancer)35 (8): 1194J. 201, 1999; and Bostan et al, J. Clostracom. 18 (6): 1212-9, 2000), non-small cell lung Cancer (Fossella et al, J. Clin. Oncology 18 (12): 2354-62, 2000; and Hans Walsh (Hainsworth) et al, Cancer (Cancer)89 (2): 328-33, 2000) and ovarian Cancer (Kaye et al, European J. Cancer 33 (13): 2167-70, 1997). Docetaxel has also been shown to be useful in the treatment of mesothelioma (Wolobiov et al, Proc Am Soc Clin Oncol) 19: 578a, 2000), prostate Cancer (Picus et al, Oncology seminar 26(5 J.17): 14-8, 1999; and Parrelike (Petrylak) et al, J.Clin.Oncology 17 (3): 958-67, 1999), urothelial transitional cell carcinoma (Dimopoulos) et al, Annals of Oncology 10 (11): 1385-8, 1999 and Packmoss silk (Pectasids) et al, European Journal of Cancer 36(I) 74-9, 2000), head and neck Cancer (Docetaxel in USP DI), Couxel and British < (2000) J.81, British et al, (J.81: 81), 1999) and small cell lung cancer (smith (Smyth) et al, european journal of cancer 30A (8): 1058-60, 1994).

Improved response to chemotherapy was observed in TLE 3-positive breast and ovarian cancer patients, and this observation indicates that the methods of the invention can be used in different cancer types. We observed that TLE3 expression was associated with an improved response to treatment with methotrexate and taxanes, which further indicates that the methods of the invention can be applied to a variety of cancers that respond to these chemotherapeutic drugs. As noted above, such cancers include, but are not limited to, breast cancer, ovarian cancer, lung cancer, bladder cancer, gastric cancer, head and neck cancer, and leukemia.

In one embodiment, the methods of the invention are useful for cancer patients with breast cancer. In one embodiment, the methods of the invention are useful for cancer patients with ovarian cancer. In one embodiment, the methods of the invention are useful for cancer patients with lung cancer. In one embodiment, the methods of the invention can be used for cancer patients with bladder cancer. In one embodiment, the methods of the invention are useful for cancer patients with gastric cancer. In one embodiment, the methods of the invention can be used for cancer patients with head and neck cancer. In one embodiment, the methods of the invention can be used for cancer patients with leukemia.

As described in the examples, in one embodiment, TLE3 expression was observed in triple negative breast cancer patients with ER (estrogen receptor, Intel Gene Bank number 2099), PR (progestin receptor, Intel Gene Bank number 5241), and the HER-2 marker (v-erb-b2 erythroblastic leukemia virus oncogene homolog 2, Intel Gene Bank number 2064) in association with response to chemotherapy. Thus, in certain embodiments, the methods of the invention are useful for breast cancer patients that fall within this category.

As described in the examples, when treatment was administered in the context of neoadjuvant therapy, a correlation was also found between TLE3 expression and response to chemotherapy. Thus, in certain embodiments, the methods of the invention may be used in patients receiving chemotherapy in the context of neoadjuvant therapy. In other embodiments, chemotherapy may be administered in the context of adjuvant therapy.

As described in the examples, the association between TLE3 expression and response to chemotherapy was also found to be independent of cancer stage. Thus, in certain embodiments, the methods of the invention can be used in patients with stage II + cancer (i.e., stage II or later). In certain embodiments, the methods of the invention are useful for patients with stage IIb + or stage III + cancer.

Detection of TLE3 expression

As described above, any known method can be used to determine expression of TLE 3. In one embodiment, TLE3 expression can be determined by detecting TLE3 polypeptide markers using an interaction partner (e.g., an antibody); in another example, TLE3 expression can be determined by detecting TLE3 polynucleotide marker using primers.

Detection of TLE3 polypeptide marker

The TLE3 polypeptide marker can be detected using any interaction partner that binds to the TLE3 polypeptide marker (which can be the TLE3 protein or an antigenic fragment thereof). Thus, any entity that binds to the TLE3 label to a detectable degree can be employed as an interaction partner of the invention, provided that it binds to the label with a suitable combination of affinity and specificity.

Particularly preferred interaction partners are antibodies or fragments (e.g., F (ab) fragments, F (ab')₂Fragments, Fv fragments, or sFv fragments, etc.; see, for example, einbao (Inbar) et al, proceedings of the national academy of sciences of the united states (proc.nat. acad.sci.usa) 69: 2659, 1972; hockman (Hochman) et al, biochemistry (Biochem.) 15: 2706, 1976; and eurichi (Ehrlich) et al, biochemistry 19: 4091, 1980; houston (Huston) et al, journal of the national academy of sciences of the united states 85: 5879,1998; U.S. Pat. nos. 5,091,513 and 5,132,405 to houston et al; and U.S. patent No. 4,946,778 to Ladner et al, each of which is incorporated herein by reference. In certain embodiments, the interaction partner may be selected from a library of mutated antibodies (or fragments thereof). For example, a collection of antibodies each comprising a different point mutation can be screened for their binding to a marker of interest. In addition, chimeric antibodies may be used as interaction partners, such as "humanized" or "veneered" antibodies, as set forth in more detail below.

Where Antibodies are used as interaction partners, these Antibodies can be prepared by any of a variety of techniques known to those of skill in the art (see, e.g., Harlow and ryan (Lane), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (Cold Spring Harbor Laboratory), 1988, and also see the examples). For example, antibodies can be produced by cell culture techniques, including the production of monoclonal antibodies, or can be made recombinant by transfecting antibody genes into a suitable bacterial or mammalian cell host. In one technique, an "immunogen" comprising the antigenic portion of the marker of interest (or the marker itself) is first injected into any of a number of mammals (e.g., mice, rats, rabbits, sheep, or goats). In this step, the label (or antigenic portion thereof) may be used as an immunogen without modification. Alternatively, particularly for relatively short labels, an excellent immune response may be elicited if the label is linked to a carrier protein, such as bovine serum albumin or Keyhole Limpet Hemocyanin (KLH). The immunogen is injected into the animal host, preferably by inclusion of one or more boosters according to a predetermined protocol and periodic blood draws from the animal. Polyclonal antibodies specific for the label can then be purified from the antiserum, e.g., by affinity chromatography, using the label (or antigenic portion thereof) coupled to a suitable solid support. Exemplary methods are described in the examples.

If desired for diagnostic or therapeutic purposes, for example, Kohler (Kohler) and Milstein (Milstein), european journal of immunity (eur.j. immunol.) 6: 511, 1976 and modifications thereof to prepare monoclonal antibodies specific for TLE 3. Briefly, these methods involve the preparation of immortalized cell lines that produce antibodies with the desired specificity (i.e., reactivity with the target marker). Such cell lines can be generated, for example, using spleen cells obtained from the immunized animals described above. The spleen cells are then immortalized, for example, by fusion with a myeloma cell fusion partner, preferably homologous to an immunized animal. A variety of fusion techniques may be employed. For example, spleen cells and myeloma cells can be combined with a non-ionic detergent for several minutes and then plated at low density on a selective medium that supports hybrid cell growth but not myeloma cell growth. Preferred selection techniques use HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time (typically about 1 to 2 weeks), the hybrid cell colonies are observed. Single colonies were selected and their culture supernatants tested for binding activity to the marker. Preferred are hybridomas having high reactivity and specificity.

Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques can be employed to improve productivity, such as injecting hybridoma cell lines into the abdominal cavity of a suitable vertebrate (e.g., a mouse). Monoclonal antibodies can then be harvested from ascites fluid or blood. Contaminants can be removed from the antibody by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. TLE3 can be used, for example, in an affinity chromatography step in a purification process.

It is to be understood that the present invention is not limited to the use of antibodies or antibody fragments as interaction partners. In particular, the invention also encompasses the use of synthetic interaction partners that mimic antibody function. Several methods of designing and/or identifying antibody mimetics have been proposed and demonstrated in the art (see, for example, review by Schweierson (Hsieh-Wilson) et al, review by chemical research statement (Acc. Chem. Res.) 29: 164, 2000; and Packzuh (Peczuh) and Hamilton (Hamilton), chemical research (Chem, Rev.) 100: 2479, 2000). For example, small molecules that bind to the surface of proteins in a similar manner to natural proteins have been identified in the industry by screening synthetic libraries of small molecules or natural product isolates (see, e.g., Gallop et al, J.Med.chem.). 37: 1233, 1994; Gordon et al, J.Med.Chem.37: 1385, 1994; Dewitt (DeWitt et al, J.Natl.Acad.Sci.90: 6909, 1993; Buning (Bunin) et al, U.S. national academy of sciences 91: 4708, 1994; Bill. and Elman (Elman), J.Chem.Chem.Soc.: 116: 11580, 1994; Wang (Wang.Wake et al, J.Med.Chem.38: 2995, 1995; and Kirk (King) and Elman. Chem.1427: 1995). Similarly, combinatorial approaches have been successfully applied in the industry to screen peptide and protein libraries for the ability to bind a variety of proteins (see, e.g., Call (Cull) et al, Proc. Natl. Acad. Sci. USA 89: 1865, 1992; Mattheakis et al, Proc. Natl. Acad. Scott. and Smith, Science 249: 386, 1990; Daford (Devrin) et al, Science 249: 404, 1990; Cridey (Corey) et al, Gene (Gene) 128: 129, 1993; Braley et al, Tetrahedron letters 31: 5811, 1990; Fodor (Fodor) et al, Science 251: 767, 1991; Houghten et al, Nature 354: 84, 1991; Lam et al, Blakem 354, Blakem et al, Nature Vicker. Biovicon et al, Biovicon 510; conjugated Vickers. Vis. 510; conjugated Livicon. Daviz. et al, 1992; nissan (seeds) et al, Proc. Natl. Acad. Sci. USA 90: 10700, 1993; and orlmeyer (Ohlmeyer), et al, proceedings of the american national academy of sciences 90: 10922, 1993). Similar methods have also been used in the art to study carbohydrate-protein interactions (see, e.g., Oldenburg et al, Proc. Natl. Acad. Sci. USA 89: 5393, 1992) and polynucleotide-protein interactions (see, e.g., Ellington (Ellington) and Shorstak (Szostak), Nature 346: 818, 1990; and thelck (Tuerk) and Gold (Gold), science 249: 505, 1990). These methods have also been extended to the study of the interaction between proteins and non-natural biopolymers (e.g., oligourethanes, oligoureas, oligosulfones, etc.) (see, e.g., Zuckermann et al, J. am. chem. J. 114: 10646, 1992; Simon et al, J. am. academy. 89: 9367, 1992; Zuckerman et al, J. chem. 37: 2678, 1994; Burgess et al, J. German applied Chemicals (Angew. chem., int. Ed. Engl.) 34: 907, 1995; and Chauling (Cho) et al, science 261: 1303, 1993). In addition, alternative protein backbones that are substantially based on the basic folding of antibody molecules have been proposed and can be used to prepare the interaction partners of the invention (see, e.g., ancient (Ku) and Schultz, Proc. Natl. Acad. Sci. USA 92: 6552, 1995). Antibody mimetics have also been constructed that comprise a small molecule backbone such as 3-aminomethylbenzoic acid and substituents consisting of a single peptide loop. Peptide loops play a binding function in these mimetics (see, e.g., smith et al, journal of the american chemical association 116: 2725, 1994). Antibody mimetics comprising a plurality of synthetic peptide loops constructed based on calixarene units have also been described in the art (see, e.g., U.S. patent No. 5,770,380 to hamilton et al).

Any available strategy or system can be used to detect binding between an interaction partner and a TLE3 label. In certain embodiments, binding can be detected by adding a detectable label to the interaction partner. In other embodiments, binding can be detected by using a labeled second interaction partner that specifically binds to the first interaction partner, e.g., as is well known in the art of antigen/antibody detection. The detectable label may be directly detectable or may be indirectly detectable, for example, by the combined action of one or more other members of the signal producing system. Examples of directly detectable labels include radioactive labels, paramagnetic labels, fluorescent labels, light scattering labels, absorptive labels, and colorimetric labels. Examples of indirectly detectable labels include chemiluminescent labels, such as enzymes that convert a substrate to a chromogenic product, such as alkaline phosphatase, horseradish peroxidase, and the like.

Once the labeled interaction partner is bound to the TLE3 label, the complex can be visualized or detected in a variety of ways, with the particular detection mode being selected based on the particular detectable label, with representative detection methods including, for example, scintillation counting, autoradiography, paramagnetic measurements, fluorescence measurements, light absorption measurements, light scattering measurements, and the like.

In general, the binding between the interaction partner and the TLE3 marker can be analyzed by contacting the interaction partner with a cancer sample comprising the marker. Depending on the nature of the sample, suitable methods include, but are not limited to, Immunohistochemistry (IHC), radioimmunoassay, ELISA, immunoblotting and Fluorescence Activated Cell Sorting (FACS). IHC is a particularly suitable detection method in case proteins are to be detected in a tissue sample, e.g. a biopsy sample. Techniques for obtaining tissue and cell samples and performing IHC and FACS are well known in the art.

When processing large numbers of samples (e.g., when processing several samples from the same patient or multiple samples from different patients simultaneously), it is desirable to employ an array and/or automation mode. In certain embodiments, the tissue arrays described in the examples may be used. The tissue array may be constructed according to a variety of techniques. According to one procedure, commercially available mechanical devices (e.g., manual tissue array instrument MTA1, available from beccher Instruments, Sun Prairie, WI) were used to remove 0.6 micron diameter full thickness "cores" from paraffin blocks (donor blocks) prepared from each patient and insert the cores into individual paraffin blocks (recipient blocks) at designated locations on the grid. In a preferred embodiment, cores from up to about 400 patients (or multiple cores from the same patient) can be inserted into a single recipient block; preferably, the core spacing is about 1 mm. The resulting tissue array can be processed into thin sections for staining with interaction partners according to standard methods applicable to paraffin-embedded materials.

Regardless of the mode employed, and regardless of the detection strategy employed, identification of discrimination titers (discriminating ters) can simplify binding studies to assess desirability of using interaction partners. In such studies, the interaction partners are contacted with a plurality of different samples, which preferably have at least one common trait (e.g., tissue origin) and typically have a plurality of common traits (e.g., tissue origin, stage, microscopic characteristic, etc.). In some cases, it is desirable to select a set of samples having at least one common trait and at least one different trait so that titers can be determined that distinguish between the different traits. In other cases, it may be desirable to select a set of samples that do not have a detectably different trait, such that titers can be determined that distinguish between previously indistinguishable samples. However, it will be appreciated by those skilled in the art that the present invention will generally allow these two goals to be achieved even in the study of sample sets having different degrees of similarity and difference.

As discussed above and in the examples, the present inventors have applied these techniques to samples from breast and ovarian cancer patients. The invention also contemplates confirming that the marker secreted from the marker-producing cells may be present in serum, thereby enabling detection of the marker by a blood test without the need for a biopsy sample. The interaction partners that bind to the label represent a particularly preferred embodiment of the invention.

Generally, the results of such an analysis can be represented in any of a variety of modes. The results may be presented in a qualitative manner. For example, a test report may only indicate whether the TLE3 marker was detected, and may also indicate a limit of detection. In addition, the test report may indicate the subcellular location of binding (e.g., cellular nucleus to cytoplasm) and/or the relative level of binding at these different subcellular locations. The results can be expressed in a semi-quantitative manner. For example, different ranges may be defined and scores (e.g., 0 to 5) may be assigned to the ranges, providing a degree of quantitative information. Such a score may reflect various factors such as the number of cells in which the marker is detected, the signal strength (which may indicate the level of expression of the marker), and the like. The results may be expressed in a quantitative manner, e.g., as a percentage of cells in which the marker is detected, as a concentration, etc. It will be appreciated by those skilled in the art that the type of results provided by the test may vary depending on the technical limitations of the test and the biological significance associated with the detection of the marker. For example, in some cases, a purely qualitative result (e.g., whether a marker is detected at a certain level of detection) provides a large amount of information. In other cases, a more quantitative result (e.g., the ratio of the expression levels of the marker in the two samples) is desired.

Detection of TLE3 Polynucleotide markers

Although the use of an interaction partner, such as an antibody, to detect a polypeptide marker may represent the most convenient means of determining whether TLE3 is expressed in a particular sample in a variety of circumstances, the methods of the invention also encompass the use of primers to detect a polynucleotide marker. Various methods of detecting the presence of a particular polynucleotide marker are known in the art and can be used in the methods of the invention. Generally, these methods rely on hybridization of one or more primers to a polynucleotide marker.

Any available strategy or system can be used to detect hybridization between a primer and a TLE3 polynucleotide (which can be TLE3mRNA, cDNA generated from mRNA by RT-PCR, RNA generated from the cDNA, etc.). In certain embodiments, hybridization can be detected by simply adding a detectable label to the primer. In other embodiments, hybridization can be detected by using a labeled second primer that can specifically hybridize to the first primer (e.g., a region of the first primer that does not hybridize to the TLE3 label). In other embodiments, the TLE3 marker within a cancer sample can be amplified by PCR using a set of primers designed to amplify a region in the TLE3 gene. The resulting product can then be detected using, for example, a labeled second primer that can hybridize to the amplification product. Variations of these embodiments will be apparent to those skilled in the art.

Considerations regarding primer design are well known in the art and are described, for example, in the following documents: newton (Newton) et al (editors), PCR: common data sequences (PCR: Essential data Series), John Wiley International publication (John Wiley & Sons); PCR primers: a Laboratory Manual (PCRPrimer: A Laboratory Manual), Cold spring harbor Laboratory Press, Cold spring harbor, NY, 1995; white et al (editors), PCR protocol: current Methods and Applications (PCR Protocols: Current Methods and Applications), Methods in molecular biology (Methods in molecular biology), The Humana Press (The Humana Press), Totorwa, NJ, 1993. In addition, a variety of computer programs known in the art can be used to select suitable primers.

In general, a detectable label may be detected directly or may be detected indirectly, such as by the combined action of one or more other members of a signal producing system. Examples of directly detectable labels include radioactive labels, paramagnetic labels, fluorescent labels, light scattering labels, absorptive labels, and colorimetric labels. Examples of indirectly detectable labels include chemiluminescent labels, such as enzymes that convert a substrate to a chromogenic product, such as alkaline phosphatase, horseradish peroxidase, and the like.

Once the labeled primer is hybridized to the TLE3 label, the complex can be visualized or detected in a variety of ways, with the particular detection mode being selected based on the particular detectable label, with representative detection methods including, for example, scintillation counting, autoradiography, paramagnetic measurements, fluorescence measurements, light absorbance measurements, light scattering measurements, and the like.

In general, hybridization between a primer and a TLE3 marker can be analyzed by contacting the primer with a cancer sample that includes the marker. Depending on the nature of the cancer sample, suitable methods include, but are not limited to, microarray analysis, in situ hybridization, Northern blot, and various nucleic acid amplification techniques, such as PCR, RT-PCR, quantitative PCR, ligase chain reaction, and the like.

Identification of novel therapies

According to the present invention, the predictive ability of TLE3 can be used not only to classify cancers based on their likely responsiveness to known therapies, but also to identify potential new therapies or therapeutic agents that can be used to treat cancer.

Indeed, TLE3 represents an attractive candidate for identifying new therapeutic agents (e.g., by screening to detect compounds or entities that preferably bind or hybridize to a marker with at least a specified affinity and/or specificity, and/or by screening to detect compounds or entities that modulate (i.e., increase or decrease) the expression, location, modification, or activity of a marker). Accordingly, in one embodiment, the present invention provides a method comprising the steps of: contacting a test compound with cells expressing the TLE3 marker (e.g., individually engineered cells or in a tissue background, etc.); and determining whether the test compound modulates the expression, location, modification or activity of the TLE3 marker. In many cases, it can be demonstrated that the interaction partner or primer (e.g., antisense or RNAi primer) is itself a useful therapeutic agent.

Thus, the invention provides interaction partners and primers that are themselves useful therapeutic agents. For example, binding of an antibody raised against TLE3 to a cancer cell can inhibit the growth of the cell. Alternatively or additionally, the interaction partners defined or prepared according to the invention can be used to deliver therapeutic agents to cancer cells. Specifically, an interaction partner (e.g., an antibody raised against TLE3) can be coupled to one or more therapeutic agents. Suitable agents in this regard include radionuclides or drugs. Preferred radionuclides include⁹⁰Y、¹²³I、¹²⁵I、^13II、¹⁸⁶Re、¹⁸⁸Re、²¹¹At and²¹²and (4) Bi. Preferred drugs include chlorambucil (chlorembucil), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), cyclophosphamide, carboplatin, cisplatin, procarbazine (procarbazine), dacarbazine (decarbazine), carmustine (carmustine), cytarabine (cytarabine), hydroxyurea, mercaptopurine, methotrexate, paclitaxel, docetaxel, thioguanine, 5-fluorouracil, actinomycin D (actinomycin D), bleomycin (bleomycin), daunorubicin (daunorubicin), doxorubicin, etoposide (etoposide), vinblastine (vinblastine), vincristine (vincristine), L-asparaginase, adrenal gland enzyme (ADP)Corticosteroids, ganciclovir triphosphate (canciclovir triphosphate), adenine arabinoside triphosphate, 5-aziridinyl-4-hydroxyamino-2-nitrobenzamide, acrolein (acrolein), phosphoramide mustard gas, 6-methylpurine, etoposide, benzoic acid mustard gas, cyanide, and nitrogen mustard.

According to such embodiments, the therapeutic agent may be coupled to the interaction partner by direct or indirect covalent or non-covalent interaction. Where the therapeutic agent and the interaction partner each have substituents that are reactive with each other, there may be a direct interaction between the therapeutic agent and the interaction partner. For example, a nucleophilic group (e.g., amino or mercapto) on one may be capable of reacting with a carbonyl-containing group (e.g., anhydride or acid halide) or an alkyl group containing a good leaving group (e.g., halide) on the other. The indirect interaction may involve a linker group that itself non-covalently binds both the therapeutic agent and the interaction partner. The linker group may serve as a spacer to space the interaction partner from the agent to avoid interference with the binding capacity. Linker groups may also be used to increase the chemical reactivity of substituents on the agent or interaction partner and thereby increase the coupling efficiency. The increase in chemical reactivity may also facilitate the use of agents or functional groups on agents that would not otherwise be possible.

It will be appreciated by those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homofunctional and heterofunctional, such as those described in the catalog of Pierce chemistry (Pierce Chemical), rockford, i11, can be employed as linker groups. For example, coupling can be achieved through amino, carboxyl, sulfhydryl, or oxidized carbohydrate residues. There are a number of references describing such methods, such as U.S. patent No. 4,671,958 to Rodwell et al. It will also be appreciated that the therapeutic agent and the interaction partner may be coupled by non-covalent interactions (e.g., ligand/receptor type interactions). Any ligand/receptor pair that is stable and specific enough to function in the context of the present invention may be used to couple the therapeutic agent and the interaction partner. For example, can be usedThe therapeutic agent is covalently linked to biotin and the interaction partner is covalently linked to avidin. The strong non-covalent binding of biotin to avidin then allows the coupling of the therapeutic agent to the interaction partner. Typical ligand/receptor pairs include protein/cofactor pairs and enzyme/substrate pairs. In addition to the commonly used biotin/avidin pairs, these ligand/receptor pairs include, but are not limited to, biotin/streptavidin pairs, digoxigenin/anti-digoxigenin pairs, FK506/FK506 binding protein (FKBP) pairs, rapamycin/FKBP pairs, cyclosporin receptor (cyclophilin)/cyclosporin pairs, and glutathione/glutathione transferase pairs. Other suitable ligand/receptor pairs can be recognized by those skilled in the art, such as with epitope tags (such as, but not limited to, glutathione transferase (GST), c-myc, gamma-gamma,And Maltose Binding Protein (MBP)) and other epitope tags described in: kessler (Kessler), page 105-152, Advances in Mutagenesis technology (Advances in Mutagenesis), Kessler edition, Schpringer publishing company (Springer-Verlag), 1990; "affinity chromatography: methods and Protocols (Methods of molecular Biology) (Affinity Chromatography: Methods and Protocols (Methods in molecular Biology)) ", edited by Pascal Bailey (Pascal Bailon), Homalner Press, 2000; and "Immobilized Affinity Ligand technology (Immobilized Affinity Ligand technologies)", Hermolus (Hermanson) et al, Academic Press (Academic Press), 1992.

It is desirable to use a linker group that is cleavable during or after internalization into the cell, provided that the therapeutic agent is more effective when isolated from the interaction partner. A variety of different cleavable linker groups have been described in the literature. The mechanism of intracellular release of the agent from these linker groups involves cleavage by: reduced disulfide bonds (e.g., U.S. patent No. 4,489,710 to spatzler (spatler)), irradiated light labile bonds (e.g., U.S. patent No. 4,625,014 to senet (Senter) et al), hydrolysis-derived amino acid side chains (e.g., U.S. patent No. 4,638,045 to cohn (Kohn) et al), serum complement-mediated hydrolysis (e.g., U.S. patent No. 4,671,958 to rodville et al), and acid-catalyzed hydrolysis (e.g., U.S. patent No. 4,569,789 to bradler (Blattler) et al).

In certain embodiments, it may be desirable to couple more than one therapeutic agent to an interaction partner. In one embodiment, a plurality of molecules of the agent are coupled to one molecule of the interaction partner. In another embodiment, more than one type of therapeutic agent may be coupled to one interaction partner molecule. Regardless of the specific example, a formulation having more than one agent can be prepared in a variety of ways. For example, more than one agent may be directly coupled to the interaction partner molecule, or a linker providing multiple attachment sites may be used.

Alternatively, a carrier may be used. The carrier can carry the agent in a variety of ways, including covalently bonded directly or through a linker group. Suitable carriers include proteins (e.g., albumin) (e.g., U.S. Pat. No. 4,507,234 to Kato et al), peptides, and polysaccharides (e.g., aminodextran (e.g., U.S. Pat. No. 4,699,784 to Shih et al.) the carrier may also carry agents by non-covalent binding or by encapsulation in, for example, liposome vesicles (e.g., U.S. Pat. No. 4,429,008 to Martin et al and U.S. Pat. No. 4,873,088 to Mayhew et al.) radionuclides specific for the radionuclide agent include radiohalogenated small molecules and chelating compounds A compound of a radionuclide. For example, U.S. patent No. 4,673,562 to Davison et al discloses representative chelating compounds and their synthesis.

Where the interaction partner itself is a therapeutic drug, it will be appreciated that in many cases any interaction partner that binds the same label may be so used.

In a preferred embodiment of the invention, the therapeutic agent (whether or not an interaction partner) is an antibody, for example, an antibody directed against the TLE3 marker. As is well known in the art, where antibodies or fragments thereof are used for therapeutic purposes, it may be demonstrated that the use of "humanized" or "veneered" forms of the antibodies of interest is beneficial in reducing any potential immunogenic response. In general, "humanized" or "veneered" antibody molecules and fragments thereof can minimize undesired immune responses against anti-human antibody molecules, which can limit the duration and efficiency of therapeutic applications of these moieties in human recipients.

A variety of "humanized" antibody molecules comprising antigen-binding portions derived from non-human immunoglobulins have been described in the art, including chimeric antibodies having rodent variable regions and their associated Complementarity Determining Regions (CDRs) fused to human constant domains (see, e.g., Winterer et al, Nature 349: 293, 1991; Lobuglio et al, Proc. Natl. Acad. Sci. USA 86: 4220, 1989; Shaw et al, J. Immunol. 138: 4534, 1987; and Brown et al, Cancer research (Cancer Res) 47: 3577, 1987), animal CDRs which are grafted into human support Framework Regions (FRs) prior to fusion with suitable human antibody constant domains (see, e.g., Richmann et al, Nature 332: 323, 1988; Verhoeyen et al, Johne et al, Jones et al, naturally 321: 522, 1986), and rodent CDRs supported by recombinant veneered rodent FRs (see, e.g., european patent publication No. 519,596, published 12/23 1992). It is to be understood that the present invention also encompasses the use of XenoMouse according to the techniques described in U.S. Pat. No. 6,075,181^TM"fully human" antibodies generated by the technique (Annenix (AbGenix) Inc., Frimemont, Calif.).

In addition, so-called "veneered" antibodies can be used, including "veneered FRs". The veneering method involves the selective replacement of FR residues in, for example, the murine heavy or light chain variable region with human FR residues to provide a heterologous molecule comprising an antigen-binding portion that retains substantially all of the native FR protein folding structure. Veneering techniques are based on the following understanding: the antigen-binding characteristics of antigen-binding portions depend primarily on the structure and relative arrangement of the sets of heavy and light chain CDRs within the antigen-binding surface (see, e.g., davis et al, annum of biochemistry (ann. rev, Biochem), 59: 439, 1990). Thus, antigen binding specificity can be retained in humanized antibodies only when the CDR structures, their interaction with each other, and their interaction with the rest of the variable region domain are carefully maintained. By using veneering techniques, external (e.g., solvent accessible) FR residues that are readily encountered by the immune system are replaced with human residues to provide hybrid molecules comprising a poorly immunogenic or substantially non-immunogenic veneered surface.

Preferably, the interaction partners suitable for use as therapeutic drugs (or therapeutic agent carriers) exhibit high specificity for the target marker (e.g., TLE3) and low background binding for other markers. In certain embodiments, monoclonal antibodies are preferably used for therapeutic purposes.

Pharmaceutical composition

As described above, the present invention provides novel therapies and methods for identifying these novel therapies. In certain embodiments, the interaction partner or primer may be a useful therapeutic agent. Alternatively or additionally, the interaction partner defined or prepared according to the invention binds to a label (e.g., TLE3) that is used as a target for a therapeutic agent. Likewise, the interaction partners of the invention can be used to deliver therapeutic agents to cancer cells. For example, interaction partners provided according to the invention may be coupled to one or more therapeutic agents.

The invention includes pharmaceutical compositions comprising these inventive therapeutic agents. In general, a pharmaceutical composition may include a therapeutic agent and one or more inactive agents, such as a biocompatible sterile carrier, including, but not limited to, sterile water, saline, buffered saline, or dextrose solution. The pharmaceutical compositions may be administered alone or in combination with other therapeutic agents, including other chemotherapeutic agents, hormones, vaccines and/or radiation therapy. At this point and elsewhere in the specification, "in combination with … …" is not intended to indicate that multiple agents must be administered or formulated together for delivery, but such methods of delivery are within the scope of the present invention. Generally, each agent can be administered in a dose according to a predetermined schedule of the agent. Alternatively, the invention encompasses the delivery of the pharmaceutical compositions of the invention as well as agents that can improve their bioavailability, reduce or alter their metabolism, inhibit their excretion, or alter their distribution in the body. Although the pharmaceutical composition of the present invention can be used to treat any individual in need thereof (e.g., any animal), it is most preferably used to treat a human.

The pharmaceutical compositions of the present invention may be administered to humans and other animals by a variety of routes, including orally, intravenously, intramuscularly, intraarterially, subcutaneously, intraventricularly, transdermally, rectally, intravaginally, intraperitoneally, topically (using powders, ointments, or drops), buccally, or as an oral or nasal spray or aerosol. In general, the optimum route of administration may depend on a variety of factors, including the nature of the agent (e.g., its stability in the gastrointestinal environment), the condition of the patient (e.g., whether the patient is able to tolerate oral administration), and the like. The intravenous route is currently most commonly used for the delivery of therapeutic antibodies. However, the present invention contemplates delivery of the pharmaceutical compositions of the present invention by any suitable route that takes into account the possible advances in drug delivery science.

General considerations for formulating and manufacturing Pharmaceutical agents can be found, for example, in Remington's Pharmaceutical Sciences, 19 th edition, Mack publishing (Mack publishing) Inc., Oriental, PA, 1995.

Example

Example 1: production of antibodies

This example illustrates the method used to generate the TLE3 antibody used in these examples. Similar methods can be used to generate antibodies that bind to any marker of interest (e.g., proteins that bind to or are derived from other markers listed in appendix a). In some cases, antibodies can be obtained from commercial sources (e.g., warecon (Chemicon), daceae (Dako), Oncogene Research Products (Oncogene Research Products), nicomaks (NeoMarkers), etc.) or other publicly available sources (e.g., Imperial Cancer Research technology, etc.).

Materials and solutions

● anisole (Cat. No. A4405, Sigma, St. Louis, Mo)

● 2, 2' -azino-bis- (3-ethyl-benzothiazoline-sulfonic Acid) (ABTS) (Cat. No. A6499, Molecular Probes, Eugeni, OR)

● activated Maleimide keyhole limpet hemocyanin (Cat. No. 77106, pierce, Rockford, IL)

● keyhole limpet hemocyanin (Cat. No. 77600, pierce, Rockford, EL)

● phosphoric acid (H)₃PO₄) (Cat No. P6560, Sigma)

● glacial acetic acid (Cat. No. BP1185-500, Fisher)

● EDC (EDAC) (Cat No. 341006, Calbiochem)

● 25% glutaraldehyde (catalog number G-5882, Sigma)

● Glycine (catalog number G-8898, Sigma)

● Biotin (catalog number B2643, Sigma)

● boric acid (catalog number B0252, sigma)

● Sepharose 4B (catalog number 17-0120-01, LKB/Pharmacia (Pharmacia), Uppsala, Sweden)

● bovine serum albumin (LP) (Cat No. 100350, Boehringer Mannheim, Indianapolis, IN)

● cyanogen bromide (Cat. No. C6388, Sigma)

● dialysis bag Spectra/Por membrane MWCO: 6-8000 (Cat No. 132665, Spectrum Industries, Raguna Hills, CA)

● Dimethylformamide (DMF) (Cat No. 22705-6, Aldrich (Aldrich), Milwaukee, Wis.)

● DIC (catalog number BP 592-500, Fisher)

● ethanedithiol (catalog number 39, 802-0, Aldrich)

● Ether (catalog number TX 1275-3, EM science (EM Sciences))

● ethylenediaminetetraacetic acid (EDTA) (Cat. No. BP 120-1, Fisher, Springfield, NJ)

● 1-Ethyl-3- (3' dimethylaminopropyl) -carbodiimide, HCL (EDC) (Cat. No. 341-006, Kalbel, san Diego, Calif.)

● Freund's complete Adjuvant (Freund's Adjuvant, complete) (Cat. No. M-0638-50B, Li's laboratory (Lee Laboratories), Grayson (Grayson), GA)

● Freund's incomplete Adjuvant (Freund's Adjuvant, incomplete) (Cat. No. M-0639-50B, Li's laboratory)

● fused silica chromatography column (column number 12131011; fused silica number 12131029, Walian Sample Products, harbor City, CA)

● gelatin from cow hide (Cat. No. G9382, Sigma)

● biotinylated goat anti-rabbit IgG (catalog number A0418, Sigma)

● HOBt (catalog number 01-62-0008, Kaire)

● Horseradish peroxidase (HRP) (Cat No. 814393, Boehringer Mannheim)

● HRP-streptavidin (Cat. No. S5512, Sigma)

● hydrochloric acid (Cat. No. 71445-500, Fisher)

● Hydrogen peroxide 30% w/w (Cat. No. H1009, Sigma)

● methanol (Cat. No. A412-20, Fisher)

● 96 well microtiter plate (Cat. No. 2595, Corning-Costar, Princeton, Calif.)

● N-. alpha. -Fmoc protected amino acids, available from Kalbel, see the '97-' 98 catalog, pages 1-45.

● N-. alpha. -Fmoc protected amino acids attached to Wang (Wang) resin were purchased from Kelbert, see '97-' 98 catalog, pages 161-164.

● NMP (catalog number CAS 872-50-4, Bordick and Jackson, Maskigen, MI)

● peptide (synthesized by genetic Research, Inc.; details set forth below)

● piperidine (catalog number 80640, Flulca, available from Sigma)

● sodium bicarbonate (catalog number BP328-1, Fisher)

● sodium borate (Cat. No. B9876, Sigma)

● sodium carbonate (catalog number BP357-1, Fisher)

● sodium chloride (Cat number BP 358-10, Fisher)

● sodium hydroxide (Cat. number SS 255-1, Fisher)

● streptavidin (catalog number 1520, Boehringer Mannheim)

● thioanisole (Cat. No. T-2765, Sigma)

● trifluoroacetic acid (catalog number TX 1275-3, EM science)

● Tween-20 (catalog number BP 337-500, Fisher)

● sink (Rectangular service' Saver)^TMGrade No. 3862, Lebermaid (Rubbermaid), Worcester, OH)

● BBS-Borate buffered saline containing EDTA (adjusted to pH 8.2 to 8.4 with HCl or NaOH), 25mM sodium borate (Borax), 100mM boric acid, 75mM NaCl and 5mM EDTA in distilled water.

● 0.1N HCl in brine, as follows: concentrated HCl (8.3ml/0.917 liter distilled water) and 0.154M NaCl

● Glycine (pH 2.0 and pH 3.0) was dissolved in distilled water and adjusted to the desired pH, i.e., 0.1M Glycine and 0.154M NaCl.

● 5 Xborate and 1 Xsodium chloride in distilled water, i.e., 0.11M NaCl, 60mM sodium borate and 250mM boric acid.

● substrate buffer in distilled water adjusted to pH 4.0 with sodium hydroxide, 50 to 100mM citric acid.

● AA solution: HOBt was dissolved in NMP (8.8 g HOBt in 1L NMP). Fmoc-N-a-amino group at a concentration of 0.53M.

● DIC solution: 1 part of DIC was dissolved in 3 parts of NMP.

● deprotection solution: 1 part of piperidine was dissolved in 3 parts of DMF.

● reagent R: 2 parts of anisole, 3 parts of ethanedithiol, 5 parts of thioanisole and 90 parts of trifluoroacetic acid.

Device

● MRX plate reader (Dynatech, Shantii, VA)

● Hamilton masker (Hamilton Eclipse) (Hamilton instruments, Rino, NV)

● Beckman TJ-6 centrifuge (model TJ-6, Beckman Instruments, Fullerton, CA)

● Chart recorder (recorder 1, item number 18-1001-40, pharmacia LKB Biotech Co., Ltd.)

● UV monitor (ultraviolet absorptometer (Uvicord) SII number 18-1004-50, LKB Biotech Co., Framcia)

● Amikang (Amicon) stirring cell concentrator (model 8400, Amikang, Beverli, MA)

● 30kD MW cut-off filter (Cat. No. YM-30 film, Cat. No. 13742, Amikang)

● multichannel automatic pipette (catalog No. 4880, kang Ning, Cambridge, MA)

● PH meter kangning 240 (Corning Science Products), kangning glass Products (Corning glasses), kangning, NY)

● ACT396 peptide synthesizer (Advanced ChemTech, Louisville, KY)

● vacuum dryer (tank from Bokon (Labconco), Kansasicheng, MO and pump from Alcatel (Alcatel), Larrel, MD).

● Freeze dryer (Unitop)600sl, in series with Freezemobile 12, both from Vitis, Caldina, NY)

Peptide selection

Peptides that produce the corresponding antibodies are selected from within the target protein sequence using a program utilizing the Hopp (Hopp)/wood (Woods) method (described in Hopp and wood, molecular immunology (mol.) 20: 483, 1983; and Hopp and wood, proceedings of the national academy of sciences USA 78: 3824, 1981). The procedure uses a scanning window that identifies a 15-20 amino acid peptide sequence containing several putative epitopes predicted by low solvent accessibility. This is in contrast to the practice of most hopp/wurtz methods, which identify a single short (about 6 amino acids) putative epitope. The predicted solvent accessibility can sometimes be further evaluated by PHD prediction of the loop structure (described in ross (rot) and Sander (Sander), Proteins (Proteins) 20: 216, 1994). Preferably the peptide sequence shows minimal similarity to other known human proteins. Similarity is determined by performing a BLASTP alignment using a word length equal to 2 (set forth in Alzheimer's et al, J. mol. biol.) 215: 403, 1990). All alignments assigned an expectation value (EXPECTvalue) of less than 1000 were examined and those peptides with alignments similarity greater than 60% or greater than four residues in exact consecutive unnotched alignments were forcibly excluded. When it is desired to target a protein to an area outside the cell membrane, the extracellular region of the protein of interest is determined according to the literature or as defined by the transmembrane domain predicted using the hidden markov model (hiddenMarkov model) (described in crohn's et al, journal of molecular biology 305: 567, 2001). When the peptide sequence is located in the extracellular domain, the peptide is excluded if it contains an N-linked glycosylation site. For the preparation of the TLE3 antibody, a single peptide having the amino acid sequence KNHHELDHRERESSAN (SEQ ID No.383) was used, as shown in appendix a. Appendix a provides one to three peptide sequences that can be used to prepare antibodies against other markers.

Peptide synthesis

The sequence of the desired peptide is provided to a peptide synthesizer. The C-terminal residue was determined and an appropriate Wang resin was attached to the reaction vessel. The peptide was synthesized from C-terminus to N-terminus using a synthesis cycle by adding one amino acid at a time. The addition of the amino acids is controlled by the peptide synthesizer, which is controlled according to the peptide sequence entered into its database. The synthesis procedure was carried out as follows:

step 1-resin swelling: 2ml of DMF was added, incubated for 30 minutes and the DMF was drained.

Step 2-Synthesis cycle (repeated according to peptide length)

2 a-deprotection: 1ml of the deprotection solution was added to the reaction vessel and incubated for 20 minutes.

2 b-washing cycle

2 c-coupling: 750ml of an amino acid solution (varying with the sequence listed in the peptide synthesizer receiving instructions) and 250ml of DIC solution were added to the reaction vessel. The reaction vessel was incubated for 30 minutes and washed once. The coupling step was repeated once.

2 d-washing cycle

Step 3-final deprotection: steps 2a and 2b are finally carried out once.

The resin was deswelled in methanol (washed twice in 5ml methanol, incubated for 5 minutes in 5ml methanol, washed in 5ml methanol) and then vacuum dried.

The peptide was removed from the resin by incubation in reagent R for 2 hours and subsequent precipitation in ether. The peptide was washed in ether and then dried under vacuum. Redissolving the peptide in diH₂In O, frozen and lyophilized overnight.

Coupling of peptides to keyhole limpet bonnet hemocyanin

Peptide (6mg) was coupled to keyhole limpet bonnet hemocyanin (KLH). If the peptide of choice includes at least one cysteine, three aliquots (2mg) can be dissolved in PBS (2ml) and coupled to KLH via glutaraldehyde, EDC or maleimide activated KLH (2mg) in 2ml PBS, resulting in a total volume of 4 ml. If the peptide is cysteine-free (e.g., the TLE3 peptide), two aliquots (3mg) can be coupled by glutaraldehyde and EDC methods.

Maleimide coupling can be achieved by mixing 2mg of peptide with 2mg of maleimide activated KLH in PBS (4ml) and incubating for 4 hr.

EDC coupling can be achieved by mixing 2mg of peptide, 2mg of unmodified KLH and 20mg of EDC in 4ml of PBS (pH is lowered to 5 by addition of phosphoric acid) and incubating for 4 hours. The reaction was then stopped by slow addition of 1.33ml acetic acid (pH 4.2). This amount was increased 1.5 fold when 3mg of peptide was coupled using EDC.

Glutaraldehyde coupling occurs when 2mg of peptide is mixed with 2mg of KLH in 0.9ml PBS. 0.9ml of 0.2% glutaraldehyde in PBS was added and mixed for 1 hour. 0.46ml of 1M glycine in PBS was added and mixed for 1 hour. This amount was increased 1.5 fold when glutaraldehyde was used to couple 3mg of peptide.

The coupled aliquots were then pooled again, mixed for 2 hours, dialyzed against 1 liter PBS and lyophilized.

Immunization of rabbits

Two New Zealand white rabbits were injected with 250. mu.g (total amount) of KLH-coupled peptide in a total volume of 1ml of equal volume of Freund's complete Adjuvant and saline, after which 100. mu.g of KLH-coupled peptide in equal volume of Freund's incomplete Adjuvant and saline was injected into three to four dorsal subcutaneous sites in a total volume of 1ml at weeks 2, 6,8 and 12 after the first immunization. The immunization protocol was as follows:

blood was collected before day 0 immunization, and primary immunization was carried out

Day 15, 1 st boost

Day 27, 1 st blood draw

Day 44, 2 boost

Day 57, 2 nd blood draw and 3 rd boost

3 rd blood draw on day 69

4 th boost on day 84

Day 98, 4 th blood draw

Collecting rabbit serum

Rabbits were bled from the ear artery (30 to 50 ml). The blood was coagulated into a clot at room temperature over 15 minutes and serum was separated from the clot by centrifugation at 5000g using IEC DPR-6000. Cell-free serum was carefully decanted into clean tubes and stored at-20 ℃ for affinity purification.

Determination of antibody titer

All solutions except the washing solution were added by means of a Hamilton mask (liquid handling dispenser). Antibody titers were determined in rabbits using ELISA assays with solid phase peptides. Flexible high binding ELISA plates were coated with peptide diluted in BBS (100 μ Ι,1 μ g/well) in a passive manner and plates were incubated overnight in a water bath (air tight container with wet cotton balls) at 4 ℃. The plate was emptied, followed by three washes with BBS containing 0.1% Tween-20(BBS-TW) by repeated filling and emptying using a semi-automated plate washer. The plates were blocked by filling each well with BBS-TW (BBS-TW-BG) containing 1% BSA and 0.1% gelatin and incubating at room temperature for 2 hours. The plate was emptied and both pre-and post-immune sera were added to the wells. The first well contained serum in the BBS at 1: 50. The serum was then titrated on the plate for 11 additional consecutive titrations at a 1: 1 ratio to give a final (12) dilution of 1: 204,800. Plates were incubated overnight at 4 ℃. The plates were emptied and washed three times as described above.

Biotinylated goat anti-rabbit IgG (100 μ l) was added to each test well of the microtiter plate and incubated at room temperature for 4 hours. The plate was emptied and washed three times. Horseradish peroxidase-conjugated streptavidin (100 μ l, diluted 1: 10,000 in BBS-TW-BG) was added to each well and incubated at room temperature for 2 hours. The plate was emptied and washed three times. Fresh ABTS was prepared from the stock by combining 10ml of citrate buffer (0.1M, pH 4.0), 0.2ml of stock (15mg/ml aqueous solution) and 10. mu.l of 30% hydrogen peroxide. ABTS solution (100 μ Ι) was added to each well and incubated at room temperature. After 20 minutes of substrate addition, the plate was read at 414 nm.

Preparation of peptide affinity purification column:

the affinity column was prepared by coupling 5mg of peptide to 10ml of cyanogen bromide activated Sepharose 4B and 5mg of peptide to hydrazine-Sepharose 4B. Briefly, 100 μ l DMF was added to the peptide (5mg) and the mixture was vortexed until the contents were completely wetted. Water (900 μ Ι) was then added and the contents were mixed by rotation until the peptide dissolved. Half of the dissolved peptide (500 μ l) was added to a separate tube containing 10ml of cyanogen bromide activated Sepharose 4B in 0.1ml of borate buffered saline (pH 8.4) (BBS) and 10ml of hydrazine-Sepharose 4B in 0.1M carbonate buffer (adjusted to pH 4.5 using excess EDC in citrate buffer (pH 6.0)). The coupling reaction was allowed to proceed overnight at room temperature. The coupled agaroses were pooled and loaded onto a fused silica column, washed with 10ml BBS, blocked with 10ml 1M glycine and washed with 10ml 0.1M glycine (adjusted to pH 2.5 with HCl) and neutralized again in BBS. The entire volume of the column was washed to bring the optical density to baseline at 280 nm.

Affinity purification of antibodies

Peptide affinity columns were attached to a UV monitor and chart recorder. The titrated rabbit antisera were thawed and pooled. Serum was diluted with one volume of BBS and allowed to flow through the column at 10 ml/min. Non-peptide immunoglobulins and other proteins were washed out of the column with excess BBS until the optical density at 280nm reached baseline. The columns were separated and the affinity purification column was eluted using a step pH gradient from pH 7.0 to 1.0. Elution was monitored at 280nm and antibody-containing fractions (pH 3.0 to 1.0) were collected directly into the excess 0.5M BBS. Excess buffer (0.5M BBS) in the collection tube was used to neutralize the collected antibodies in the acidic fractions of the pH gradient.

The whole procedure was repeated using "specifically treated" serum to ensure maximum recovery of antibodies. Concentration of washed cells using a stirred cell device and a 30kD molecular weight cut-off membraneAnd removing the material. The concentration of the final formulation was determined by reading the optical density at 280 nm. The concentration was determined using the following formula: mg/ml ═ OD₂₈₀/1.4。

It will be appreciated that in certain embodiments, additional steps may be used to purify the antibodies of the invention. In particular, it may prove advantageous to repurify the antibody, for example, against one of the polypeptides used to generate the antibody. It is to be understood that the invention encompasses antibodies that have been prepared by such additional purification or re-purification steps. It will also be appreciated that the purification process may affect the binding between the sample and the antibody of the invention.

Example 2: preparation and staining of tissue arrays

This example illustrates a method for preparing a tissue array for use in each example. This example also illustrates how antibodies can be stained.

Tissue arrays were prepared by: full-thickness cores from a large number of paraffin blocks (donor blocks) containing stop segments from multiple different patients and/or different tissues or tissue segments from a single patient were inserted into blank paraffin blocks (recipient blocks) at designated locations in a grid of grid pattern. Standard slides of paraffin (donor blocks) embedded tissue containing thin sections of the specimen suitable for H & E staining were then made. A trained pathologist or a comparable practitioner skilled in assessing tumor and normal tissue designates a target sampling region (e.g., a tumor region as opposed to stroma) on the tissue array. The core was then removed from the donor block using a commercially available tissue array instrument from Beecher Instruments before it was inserted into the designated location of the recipient block. The process was repeated until all donor blocks had been inserted into the recipient block. The recipient block was then sliced to obtain 50-300 slides containing cores from all cases inserted into the recipient block.

Immunohistochemical staining was then performed with the selected antibodies using dachiakison (Envision) +, peroxidase IHC kit (dacco, kaptet, CA) with DAB substrate according to manufacturer's instructions. FIG. 1 shows exemplary IHC staining images of TLE 3-negative (S0643-) and TLE 3-positive (S0643+) samples.

Example 3: TLE3 expression was associated with cancer patient response to chemotherapy.

Tumor samples from two different breast cancer cohorts, Henzville Hospital (HH) and rossville park cancer institute (RP), were stained with TLE3 antibody from example 1. Treatment and recurrence data were available for all patients in both cohorts. Figure 2 shows kaplan-meier relapse curves obtained using all patients in the HH cohort after classification by staining for TLE3 antibody. The recurrence data for TLE 3-positive and TLE 3-negative patients were used to obtain top and bottom curves, respectively. As shown, the antibody that binds the TLE3 marker correlates with an improved prognosis for this breast cancer cohort (HR 0.573, p 0.004). Figure 3 shows a kaplan-meier relapse curve obtained in a similar manner using all patients in the RP cohort. For the HH cohort, antibodies that bind the TLE3 marker were found to be associated with improved prognosis (HR 0.239, p 0.011).

To determine whether TLE3 expression was correlated with response to chemotherapy, kaplan-meier relapse curves were obtained using HH cohort patients with or without chemotherapy (see fig. 4 and 5, respectively). As shown in figure 4, the antibody that binds the TLE3 marker was not associated with prognosis in patients who did not receive chemotherapy (HR 0.788, p 0.490). However, as shown in fig. 5, the association recovered in patients receiving chemotherapy (HR ═ 0.539, p ═ 0.013). These results demonstrate that expression of TLE3 is associated with an improved response to chemotherapy (i.e., TLE 3-positive cancers are more likely to respond to chemotherapy than TLE-3 negative cancers). Kaplan-meier recurrence curves obtained using patients receiving chemotherapy in the RP breast cancer cohort were consistent with this predictive model (see figure 6, HR 0.194, p 0.010). Kaplan-meier recurrence curves obtained using patients receiving chemotherapy in the UAB ovarian cancer cohort were also consistent with this predictive model (see fig. 18, HR 0.64, p 0.049).

Example 4: specific chemotherapy associations

Since different patients in the HH and RP cohorts received different types of chemotherapy, it was also possible to determine whether TLE3 expression was associated with a response to a particular type of chemotherapy.

Figure 7 shows a kaplan-meier recurrence curve obtained using patients receiving CMF (cyclophosphamide, methotrexate, and 5-fluorouracil) chemotherapy in the HH breast cancer cohort of figure 5. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in figure 7, the antibody that binds the TLE3 marker was associated with an improved prognosis in patients treated with this subclass (HR ═ 0.398, p ═ 0.019). Since the following results demonstrate that the antibody is not associated with patients in the HH cohort treated with CA (cyclophosphamide and adriamycin, HR ═ 1.000) or CAF (cyclophosphamide, adriamycin and 5-fluorouracil, HR ═ 1.000), it can be confirmed that the predictive association in fig. 7 is between TLE3 binding and treatment with methotrexate (see also fig. 9, which combines subclasses of CA and CAF treatment, HR ═ 1.030).

Fig. 8 shows kaplan-meier recurrence curves obtained using patients receiving CA or CAF chemotherapy (with or without taxane) in the fig. 5HH breast cancer cohort. As shown in the figure, the association between the antibody binding the TLE3 marker and prognosis lost significance in patients treated with this subclass (HR ═ 0.666, p ═ 0.22). The significance was further reduced when the profile was obtained with patients receiving only CA or CAF chemotherapy (i.e. without taxane) (see fig. 9, HR 1.030, p 0.95). However, the association was restored in patients receiving CA or CAF and a taxane (see fig. 10, HR 0.114, p 0.038). These results demonstrate that TLE3 binding is associated with treatment with a taxane.

Figure 11 shows kaplan-meier recurrence curves obtained using patients in the fig. 6RP breast cancer cohort that received CA chemotherapy alone (i.e., without the use of a taxane). The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, the antibody binding TLE3 marker was not relevant for the prognosis of patients with this subclass treated (HR 0.759, p 0.81). The association recovered when the curves were obtained using patients receiving CA chemotherapy with taxanes (see fig. 12, HR 0.153, p 0.018). These results confirm the results obtained in figures 8 and 9 using the samples from the HH cohort.

Figure 13 shows kaplan-meier recurrence curves obtained using patients receiving taxane or CMF in the fig. 6RP breast cancer cohort. Some patients receiving taxanes also received CA. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. As shown in the figure, antibodies that bind the TLE3 marker were associated with an improved prognosis in patients with this subclass treated (HR 0.137, p 0.011).

Figure 14 shows kaplan-meier recurrence curves obtained using patients receiving neoadjuvant chemotherapy in the fig. 6RP breast cancer cohort. The recurrence data for TLE 3-positive and TLE 3-negative patients in this subclass were used to obtain top and bottom curves, respectively. The sample size was small (N ═ 12); however, as shown in the figure, antibodies that bind the TLE3 marker showed significant correlation with improved prognosis for this subclass of treated patients when measured using the exact fischer test (p ═ 0.005). In addition, of the 12 patients receiving neoadjuvant chemotherapy, two patients received CA (both patients showed relapse) and ten patients received CA and taxane (seven patients showed relapse, three patients did not). Notably, the three patients who did not show any recurrence were only patients with TLE 3-positive samples. These results are significant because they show that the association between TLE3 binding and response to chemotherapy is independent of whether treatment is administered in the context of adjuvant therapy or neoadjuvant therapy.

Figures 15-17 show kaplan-meier recurrence curves obtained using patients receiving chemotherapy in the fig. 6RP breast cancer cohort. Recurrence data for TLE 3-positive and TLE 3-negative patients with stage II + (fig. 15), IIb + stage (fig. 16), and III + stage (fig. 17) cancer, respectively, were used to obtain top and bottom curves. In each case, antibodies that bind the TLE3 marker were associated with improved prognosis for patients treated with these subclasses. The sample size was small in the subclass of fig. 17 (N ═ 19); however this association is significant when measured using the fisher's exact test (p ═ 0.020). These results are clinically significant because they demonstrate that the predictive power of the TLE3 marker is independent of cancer stage and remains significant even in patients with the worst prognosis (e.g., stage III + patients).

Example 5: two variable analysis

To confirm that the predictive power of TLE3 was independent of other clinical factors (e.g., age, tumor size, ganglion status, necrosis, etc.), the results from the RP breast cancer cohort were used to perform a bivariate statistical analysis. The results are summarized in table 1 below. As shown in the table, prediction using TLE3 maintained significance in all bivariate analyses, confirming that it was independent of other clinical factors.

TABLE 1 two variable analysis

¹Ganglia with metastatic cancer were found.

²Vascular lymphatic invasion.

³A taxane-containing regimen.

OTHER EMBODIMENTS

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope of the disclosure being indicated by the following claims.

Claims (20)

1. Use of an interaction partner that binds to a TLE3 polypeptide or one or more primers that hybridize to a TLE3 polynucleotide in the preparation of a detection composition for predicting the likelihood that a patient's cancer will respond to chemotherapy with a taxane or taxane derivative;

wherein detection of the presence of TLE3 predicts that the cancer will respond and/or detection of the absence of TLE3 predicts that the cancer will not respond;

wherein the cancer patient has breast cancer, lung cancer or ovarian cancer.

2. The use of claim 1, wherein the interaction partner is an antibody.

3. The use of claim 1, wherein the taxane is paclitaxel.

4. The use of claim 1, wherein the taxane is docetaxel (docetaxel).

5. The use of claim 1, wherein the cancer patient has stage II + cancer.

6. The use of claim 1, wherein the cancer patient has stage IIb + cancer.

7. The use of claim 1, wherein the cancer patient has stage III + cancer.

8. Use of an interaction partner that binds to a TLE3 polypeptide or one or more primers that hybridize to a TLE3 polynucleotide in the preparation of a test composition for determining whether to select chemotherapy for a cancer patient;

wherein a taxane or taxane derivative is selected for the cancer patient if expression of TLE3 is present in the sample of the cancer or the taxane or taxane derivative is not administered to the cancer patient if expression of TLE3 is not present in the sample of the cancer;

wherein the cancer patient has breast cancer, lung cancer or ovarian cancer.

9. The use of claim 8, wherein the interaction partner is an antibody.

10. The use of claim 8, wherein the taxane is paclitaxel.

11. The use of claim 8, wherein the taxane is docetaxel.

12. The use of claim 8, wherein the cancer patient has stage II + cancer.

13. The use of claim 8, wherein the cancer patient has stage IIb + cancer.

14. The use of claim 8, wherein the cancer patient has stage III + cancer.

15. Use of a taxane in the manufacture of a medicament for treating breast cancer in a breast cancer patient whose cancer is determined to express TLE 3.

16. The use of claim 15, wherein the expression of TLE3 is detected by an antibody that binds the TLE3 polypeptide.

17. Use of a taxane in the manufacture of a medicament for treating ovarian cancer in an ovarian cancer patient whose cancer is determined to express TLE 3.

18. The use of claim 17, wherein the expression of TLE3 is detected by an antibody that binds the TLE3 polypeptide.

19. Use of a taxane in the manufacture of a medicament for treating lung cancer in a lung cancer patient whose cancer is determined to express TLE 3.

20. The use of claim 19, wherein expression of TLE3 is detected by an antibody that binds to the TLE3 polypeptide.