Patent Application
20090214420
The present invention relates generally to a method of screening for the level of neoplastic cell death in a subject. More particularly, the present invention provides a method of screening for the level of neoplastic cell death by detecting the level of expression of telomerase protein and/or gene by dead cells in said subject or in a biological sample derived from said subject. The method of the present invention is useful in a range of applications including, but not limited to, assessing a neoplastic condition, monitoring the progression of such a condition, assessing the effectiveness of a therapeutic agent or therapeutic regime and predicting the likelihood of a subject either progressing to a more advanced disease state or entering a remissive state. The present invention also provides diagnostic agents useful for detecting telomerase protein and/or nucleic acid molecules.
The present invention still further provides a means for therapeutic targeting, either in vitro or in vivo, of drugs such as inhibitors of cellular proliferation. The ability to target dead or dying neoplastic cells is useful in a range of immunotherapeutic and immunoprophylactic treatments due to the fact that said dead or dying neoplastic cells localise with live neoplastic cells.
Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.
Malignant tumours, or cancers, grow in an uncontrolled manner, invade normal tissues, and often metastasize and grow at sites distant from the tissue of origin. In general, cancers are derived from one or only a few normal cells that have undergone a poorly understood process called malignant transformation. Cancers can arise from almost any tissue in the body. Those derived from epithelial cells, called carcinomas, are the most common kinds of cancers. Sarcomas are malignant tumours of mesenchymal tissues, arising from cells such as fibroblasts, muscle cells, and fat cells. Solid malignant tumours of lymphoid tissues are called lymphomas, and marrow and blood-borne malignant tumours of lymphocytes and other hematopoietic cells are called leukemias.
Cancer is one of the three leading causes of death in industrialised nations. As treatments for infectious diseases and the prevention of cardiovascular disease continues to improve, and the average life expectancy increases, cancer is likely to become the most common fatal disease in these countries. Therefore, successfully treating cancer requires that all the malignant cells be removed or destroyed without killing the patient. An ideal way to achieve this would be to induce an immune response against the tumour that would discriminate between the cells of the tumour and their normal cellular counterparts. However, immunological approaches to the treatment of cancer have been attempted for over a century with unsustainable results.
Accordingly, current methods of treating cancer continue to follow the long used protocol of surgical excision (if possible) followed by radiotherapy and/or chemotherapy, if necessary. The success rate of this rather crude form of treatment is extremely variable but generally decreases significantly as the tumour becomes more advanced and metastasises. Further, these treatments are associated with severe side effects including disfigurement and scarring from surgery (e.g. mastectomy or limb amputation), severe nausea and vomiting from chemotherapy, and most significantly, the damage to normal tissues such as the hair follicles, gut and bone marrow which is induced as a result of the relatively non-specific targeting mechanism of the toxic drugs which form part of most cancer treatments.
Further, most anti-cancer treatments, which include cytotoxic chemotherapeutic agents, signal transduction inhibitors, radiotherapy, monoclonal antibodies and cytotoxic lymphocytes, kill cancer cells by apoptosis. Although tumours may contain a proportion of apoptotic cells and even areas of necrosis before anti-cancer treatment is given, an increased number of apoptotic cells and larger areas of necrosis are anticipated in tumours that respond to the anti-cancer treatment. However, when cytotoxic chemotherapeutic agents are used for the treatment of advanced cancer, the degree of cell kill and thus the response of the tumour to the first treatment is frequently difficult to assess. Conventionally, patients receive a minimum of three cycles of chemotherapy before a clinical and radiological assessment of tumour response is made. Usually, only a minority of patients with advanced cancer responds to cytotoxic drugs and so patients may experience the side effects of treatment without obtaining benefit. Hence, there is an unmet medical need for a diagnostic method that would enable rapid, convenient and reliable detection of tumour cell kill after the first cycle of treatment that would predict treatment response, which in turn often predicts survival. For example, the use of positron emission tomography with fluoro-deoxyglucose (FDG-PET) in patients with oesophageal adenocarcinoma, who received chemoradiotherapy before surgery, differentiated treatment responders from non-responders with >90% sensitivity and specificity and tended to predict those who would subsequently undergo a curative resection of their tumours. Knowing whether the tumour is responding early would spare the majority of patients from ineffective and potentially toxic treatment. Then, non-responding patients can be offered second line treatments or clinical trials of investigational agents.
Accordingly, there is and urgent an ongoing need to develop new methods of diagnosing and treating cancers in a targeted manner. This notion of effective targeted killing of malignant cells has been, to date, unattainable.
In work leading up to the present invention it has been surprisingly determined that telomerase expression is upregulated in dead cancer cells, in particular cancer cells which have died as a result of an anti-cancer treatment. This finding is quite distinct from the prior art in which the use of telomerase as an indicator of the presence of cancer has been directed to its use as a diagnostic tool in the context of screening live cancer cells for telomerase activity. Accordingly, this determination has facilitated the development of a means for identifying dead and dying neoplastic cells based on screening for increased levels, relative to normal levels, of telomerase expression by dead cells. This is particularly important in the context of monitoring the progress of a therapeutic treatment regime which is directed to killing neoplastic cells. Still further, these findings have also enabled the use of telomerase as a means of facilitating the administration of anti-tumour therapy in a highly targeted and specific manner.
Throughout this specification, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.
The subject specification contains nucleotide sequence information prepared using the programme PatentIn Version 3.1, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (eg. <210>1, <210>2, etc). The length, type of sequence (DNA, etc) and source organism for each sequence is indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO:1, SEQ ID NO:2, etc.). The sequence identifier referred to in the specification correlates to the information provided in numeric indicator field <400> in the sequence listing, which is followed by the sequence identifier (eg. <400>1, <400>2, etc). That is SEQ ID NO:1 as detailed in the specification correlates to the sequence indicated as <400>1 in the sequence listing
One aspect of the present invention is directed to a method for detecting non-viable neoplastic cells in a subject, said method comprising screening for the level of telomerase protein and/or gene expression by non-viable cells in said subject or in a biological sample derived from said subject wherein an increase in the level of non-viable cells expressing said telomerase relative to normal levels is indicative of the presence of non-viable neoplastic cells.
Another aspect of the present invention provides a method for detecting dead neoplastic cells in a subject, said method comprising screening for the level of telomerase protein and/or gene expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said telomerase relative to normal levels is indicative of the presence of dead neoplastic cells.
Yet another aspect of the present invention provides a method for detecting dead neoplastic cells in a subject, which cell death was induced by an anti-neoplastic cell treatment regime, said method comprising screening for the level of telomerase protein and/or gene expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said telomerase relative to normal levels is indicative of the presence of dead neoplastic cells.
Still another aspect of the present invention provides a method for detecting dead neoplastic cells in a subject, said method comprising screening for the level of hTERT protein and/or gene expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said hTERT relative to normal levels is indicative of the presence of dead neoplastic cells.
Yet another aspect of the present invention provides a method for detecting dead neoplastic cells in a subject, said method comprising screening for the level of hTR RNA expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said hTR relative to normal levels is indicative of the presence of dead neoplastic cells.
Still yet another aspect of the present invention provides a method for assessing and/or monitoring a neoplastic condition in a subject, said method comprising screening for the level of telomerase protein and/or gene expression by non-viable cells in said subject or in a biological sample derived from said subject wherein an increase in the level of non-viable cells expressing said telomerase relative to normal levels is indicative of the presence of non-viable neoplastic cells.
Yet still another aspect of the present invention provides a method for assessing and/or monitoring a neoplastic condition in a subject, said method comprising screening for the level of telomerase protein and/or gene expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said telomerase relative to normal levels is indicative of the presence of dead neoplastic cells.
A further aspect of the present invention provides a method for assessing and/or monitoring a neoplastic condition in a subject, said method comprising screening for the level of hTERT protein and/or gene expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said hTERT relative to normal levels is indicative of the presence of dead neoplastic cells.
Another further aspect of the present invention provides a method for assessing and/or monitoring a neoplastic condition in a subject, said method comprising screening for the level of hTR RNA expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said hTR RNA relative to normal levels is indicative of the presence of dead neoplastic cells.
Yet another further aspect of the present invention is directed to assessing and/or monitoring the effectiveness of a neoplasm therapeutic treatment regime in a subject said method comprising screening for the level of telomerase protein and/or gene expression by non-viable cells in said subject or in a biological sample derived from said subject wherein an increase in the level of non-viable cells expressing said telomerase relative to normal levels is indicative of the induction of neoplastic cell death.
Still another further aspect of the present invention provides a diagnostic kit for a biological sample comprising in compartmental form a first compartment adapted to contain an agent for detecting telomerase or a nucleic acid molecule encoding telomerase and a second compartment adapted to contain reagents useful for facilitating the detection by the agent in the first compartment. Further compartments may also be included, for example, to receive a biological sample. The agent may be an antibody or other suitable detection molecule.
Yet still another further aspect of the present invention provides the use of an interactive molecule directed to telomerase in the manufacture of a quantitative or semi-quantitative diagnostic kit to detect dead neoplastic cells in a biological sample from a patient. The kit may come with instructions for use and may be automated or semi-automated or in a form which is compatible with an automated machine or software.
Still yet another further aspect of the present invention is directed to a method of treating a neoplastic condition in a subject said method comprising administering to said subject an effective amount of an interactive molecule directed to telomerase or antigenic portion thereof, which interactive molecule is linked, bound or otherwise associated with an effector mechanism, for a time and under conditions sufficient to treat said condition.
Another aspect of the present invention provides a method of treating a neoplastic condition in a subject, said method comprising administering to said subject an effective amount of an immunointeractive molecule directed to hTERT protein or antigenic portion thereof, which immunointeractive molecule is linked, bound or otherwise associated with an effector mechanism, for a time and under conditions sufficient to inhibit, reduce or otherwise down-regulate the growth of the neoplasm.
Yet another aspect of the present invention provides a method of treating a neoplastic condition in a subject, said method comprising administering to said subject an effective amount of an immunointeractive molecule directed to hTERT or hTR RNA or antigenic portion thereof, which immunointeractive molecule is linked, bound or otherwise associated with an effector mechanism, for a time and under conditions sufficient to inhibit, reduce or otherwise down-regulate the growth of the neoplasm.
Still another aspect of the present invention provides a method of therapeutically treating a metastatic cancer in a subject, said method comprising administering to said subject an effective amount of an interactive molecule directed to telomerase or antigenic portion thereof, which interactive molecule is linked, bound or otherwise associated with a therapeutic effector mechanism, for a time and under conditions sufficient to inhibit, reduce or otherwise down-regulate the growth of said metastatic cancer.
Still yet another aspect of the present invention contemplates the use of an anti-telomerase interactive molecule conjugated to an effector mechanism, in the manufacture of medicament for the treatment of a neoplastic condition in a subject wherein said effector mechanism treats said condition.
In yet another further aspect, the present invention contemplates a pharmaceutical composition comprising the modulatory agent as hereinbefore defined together with one or more pharmaceutically acceptable carriers and/or diluents.
Yet another aspect of the present invention relates to the agent as hereinbefore defined, when used in the method of the present invention.
Notes: (i) Bead separation appeared to fail at the 24 h time point. (ii) In the case of both control and test samples at the 48 and 72 h time points, we observed an identical and highly fluorescent subpopulation of events, which was suspected to result from autofluorescence of beads associated non-specifically with dead cells or dead cell fragments. The R2 region for those samples was adjusted to exclude this subpopulation.
Note: As evidenced from the appearance of 7-AAD+ events in the blood of the normal control, the red cell lysis step has contributed to an increase in the number of dead cells recorded at the 0 h time point.
The present invention is predicated, in part, on the surprising determination that telomerase expression can be detected in dying and dead neoplastic cells and, more particularly, that the induction of neoplastic cell death, for example by a cytotoxic agent such as a DNA damaging reagent, results in an increase in the expression of telomerase. Accordingly, the detection of increased levels of telomerase expression by dead cells, relative to normal levels, provides a convenient and precise mechanism for qualitatively and/or quantitatively assessing dead neoplastic cell levels. These findings therefore provide a highly sensitive and accurate means for assessing a neoplastic condition in a mammal, in particular in the context of monitoring the progression of such a condition or assessing the effectiveness of a therapeutic agent or therapeutic regime. Also facilitated is a highly specific means of targeting therapeutic treatments to sites of neoplastic cells and achieving killing of neoplastic cells via a bystander killing mechanism.
Accordingly, one aspect of the present invention is directed to a method for detecting non-viable neoplastic cells in a subject, said method comprising screening for the level of telomerase protein and/or gene expression by non-viable cells in said subject or in a biological sample derived from said subject wherein an increase in the level of non-viable cells expressing said telomerase relative to normal levels is indicative of the presence of non-viable neoplastic cells.
Reference to a cell being “non-viable” should be understood as a reference to the subject cell being dead or dying. In relation to the latter, some killing mechanisms result in a series of stages leading to complete cell death. For example, apoptosis is marked by a series of cellular events which occur subsequently to the onset of the apoptotic signal but prior to final cell death. Reference to “dying” is intended to encompass reference to any cell which has received a signal or other stimulus which has resulted in commitment to the cell death events. Without limiting the present invention to any one theory or mode of action, neoplastic cells are generally characterised by both unlimited replicative potential and evasion of apoptosis that would otherwise be induced by DNA damage and failure of checkpoint controls. Nevertheless, even in the absence of a therapeutic treatment regime, a tumour mass will usually be characterised by a percentage of dead or dying cells which may result, for example, from the known heterogeneity of tumour blood supply. More commonly, however, neoplastic cell death is induced via exogenous means such as therapeutic radiotherapy or chemotherapy. The mechanisms by which these treatment regimes achieve neoplastic cell death are variable and depend on the specific nature of the treatment regime which as been selected for use. Although DNA damage resulting from ionising radiation and/or cytotoxic drugs would normally produce apoptosis of the affected cell, an intrinsic feature of malignant cells is the disabling of apoptosis pathways. Therefore, it would be expected that malignant cells would be resistant to the pro-apoptotic effects of DNA damaging agents. Nonetheless, cell death often still occurs in response to treatment with ionising radiation and/or cytotoxic drugs because alternative mechanisms of cell death are activated such as necrosis, mitotic catastrophe, autophagy and premature sensecence. Hence, the induction of neoplastic cell apoptosis is a less common outcome of these widely used treatment regimes than previously believed. Recent data indicate that if the malignant cell does not die an early death from apoptosis then there will be time for DNA repair. However, if there is lack of repair or misrepair of DNA that is sensed during mitosis then post-mitotic cell death will occur (Brown and Attardi, 2004, Nature Reviews Cancer 5:231-237). However, the induction of neoplastic cell apoptosis is a common outcome in the context of any of the more widely used treatment regimes. Reference to an “apoptotic” cell should be understood as a reference to a cell which is undergoing, or has undergone, apoptosis. Without limiting the present invention to any one theory or mode of action, apoptosis is an active process requiring metabolic activity by the dying cell. Apoptosis is often characterised by shrinkage of the cell, cleavage of the DNA into fragments (which give a “laddering pattern” on gels) and by condensation and margination of chromatin.
Unfortunately, due to the relatively non-specific effects of the treatment regimens (in particular systemically administered chemotherapy) rapidly dividing cellular populations (normal and malignant), the patent may experience severe treatment related toxicities. Accordingly, the design of means for both more accurately and rapidly monitoring the effectiveness of a treatment regime (or even monitoring the effectiveness of the endogenous immune response) is of significant value in that it provides a more effective means of assessing and/or tailoring a treatment regime. In the context of the present invention, it should therefore be understood that a “non-viable” neoplastic cell is one which has been rendered non-viable by any means, including both apoptotic and non-apoptotic means. An example of apoptotic non-viable neoplastic cells are these which have been killed via administration of a drug which induces cellular apoptosis while an example of non-apoptotic non-viable neoplastic cells are those which have been lysed via the classical complement pathway. Preferably, said non-viable neoplastic cell is a dead neoplastic cell.
The present invention therefore more particularly provides a method for detecting dead neoplastic cells in a subject, said method comprising screening for the level of telomerase protein and/or gene expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said telomerase relative to normal levels is indicative of the presence of dead neoplastic cells.
Still more particularly, the present invention therefore still more particularly provides a method for detecting dead neoplastic cells in a subject, which cell death was induced by an anti-neoplastic cell treatment regime, said method comprising screening for the level of telomerase protein and/or gene expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said telomerase relative to normal levels is indicative of the presence of dead neoplastic cells.
Reference to “anti-neoplastic cell treatment regime” should be understood as a reference to any treatment regime which is directed to killing neoplastic cells. The subject treatment regime may be specific or non-specific in this regard. Examples of such treatment regimes include chemotherapy and radiotherapy based regimes.
Reference to a “neoplasm” should be understood as a reference to an encapsulated or unencapsulated growth of neoplastic cells. Reference to a “neoplastic cell” should be understood as a reference to a cell exhibiting abnormal growth. The term “growth” should be understood in its broadest sense and includes reference to proliferation.
The phrase “abnormal growth” in this context is intended as a reference to cell growth which, relative to normal cell growth, exhibits one or more of an increase in the rate of cell division, an increase in the number of cell divisions, a decrease in the length of the period of cell division, an increase in the frequency of periods of cell division or uncontrolled proliferation and evasion of apoptosis. Without limiting the present invention in any way, the common medical meaning of the term “neoplasia” refers to “new cell growth” that results as a loss of responsiveness to normal growth controls, eg. to neoplastic cell growth. Neoplasias include “tumours” which may be either benign, pre-malignant or malignant.
The term “neoplasm” should be understood as a reference to a lesion, tumour or other encapsulated or unencapsulated mass or other form of growth which comprises neoplastic cells.
The term “neoplasm”, in the context of the present invention should be understood to include reference to all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs irrespective of histopathologic type or state of invasiveness.
The term “carcinoma” is recognised by those skilled in the art and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine system carcinomas and melanomas. Exemplary carcinomas include those forming from tissue of the breast. The term also includes carcinosarcomas, e.g. which include malignant tumours composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumour cells form recognisable glandular structures.
The neoplastic cells comprising the neoplasm may be any cell type, derived from any tissue, such as an epithelial or non-epithelial cell. Reference to the terms “malignant neoplasm” and “cancer” and “carcinoma” herein should be understood as interchangeable.
The term “neoplasm” should be understood as a reference to a lesion, tumour or other encapsulated or unencapsulated mass or other form of growth which comprises neoplastic cells. The neoplastic cells comprising the neoplasm may be any cell type, derived from any tissue, such as an epithelial or non-epithelial cell. Examples of neoplasms and neoplastic cells encompassed by the present invention include, but are not limited to central nervous system tumours, retinoblastoma, neuroblastoma and other pediatric tumours, head and neck cancers (e.g. squamous cell cancers), breast and prostate cancers, lung cancer (both small and non-small cell lung cancer), kidney cancers (e.g. renal cell adenocarcinoma), oesophagogastric cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias (e.g. adenocarcinomas and islet cell tumours), colorectal cancer, cervical and anal cancers, uterine and other reproductive tract cancers, urinary tract cancers (e.g. of ureter and bladder), germ cell tumours (e.g. testicular germ cell tumours or ovarian germ cell tumours), ovarian cancer (e.g. ovarian epithelial cancers), carcinomas of unknown primary, human immunodeficiency associated malignancies (e.g. Kaposi's sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas, endocrine tumours (e.g. of thyroid gland), mesothelioma and other pleural or peritoneal tumours, neuroendocrine tumours and carcinoid tumours.
As detailed hereinbefore, the present invention is predicated on the determination that the induction of neoplastic cell death leads to upregulation of telomerase expression. Without limiting the present invention to any one theory or mode of action, telomerase is a ribonucleoprotein complex which comprises a catalytic subunit protein known as human telomerase reverse transcriptase (hTERT) and 451 base-pair RNA template for telomere extension known as human telomerase integral RNA (hTR). hTERT is the critical or rate limiting enzyme for the activity of telomerase. Telomerase is expressed normally by cells in the body that have high replicative potential such as activated lymphocytes, germ cells that lead to the creation of new organisms and stem cells that are responsible for the ongoing renewal of many tissues even in the adult organism.
Reference to “telomerase” should therefore be understood as a reference to all forms of the telomerase complex or its substituent components hTERT and hTR and to fragments, mutants or variants thereof. It should also be understood to include reference to any isoform which may arise from alternative splicing of hTERT mRNA or mutant or polymorphic forms of hTERT. Reference to “telomerase” is not intended to be limiting and should be read as including reference to all forms of the telomerase complex or its substituent components including any protein, RNA or mRNA form, any subunit polypeptides such as precursor forms which may be generated, whether existing as a monomer, multimer, fusion protein or other complex. Accordingly it should be understood that one may screen for telomerase in its form as a hTERT/hTR complex or as an isolated hTERT or hTR subunit. Preferably, said telomerase is hTERT or hTR.
The present invention therefore preferably provides a method for detecting dead neoplastic cells in a subject, said method comprising screening for the level of hTERT protein and/or gene expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said hTERT relative to normal levels is indicative of the presence of dead neoplastic cells.
Preferably, said hTERT is the protein form.
In another preferred embodiment there is provided a method for detecting dead neoplastic cells in a subject, said method comprising screening for the level of hTR RNA expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said hTR relative to normal levels is indicative of the presence of dead neoplastic cells.
Preferably, said cell death was induced by an anti-neoplastic cell treatment regime.
More preferably said neoplasm is central nervous system tumours, retinoblastoma, neuroblastoma and other pediatric tumours, head and neck cancers (e.g. squamous cell cancers), breast and prostate cancers, lung cancer (both small and non-small cell lung cancer), kidney cancers (e.g. renal cell adenocarcinoma), oesophagogastric cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias (e.g. adenocarcinomas and islet cell tumours), colorectal cancer, cervical and anal cancers, uterine and other reproductive tract cancers, urinary tract cancers (e.g. of ureter and bladder), germ cell tumours (e.g. testicular germ cell tumours or ovarian germ cell tumours), ovarian cancer (e.g. ovarian epithelial cancers), carcinomas of unknown primary, human immunodeficiency associated malignancies (e.g. Kaposi's sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas, endocrine tumours (e.g. of thyroid gland), mesothelioma and other pleural or peritoneal tumours, neuroendocrine tumours and carcinoid tumours.
Still without limiting the invention in any way, in most eukaryotic cells, the telomere comprises the specialised structure at the linear end of each chromosome, which contains a 5-20 kilobase stretch of the repetitive DNA sequence, TTAGGG, and which is bound by a large number of specific proteins. Although broken chromosomes have sticky ends that fuse to each other, the telomere functions to prevent chromosomal fusions. In recognition of its protective function, the DNA-protein complex at the telomere is also known as the telomeric ‘cap’. Conversely, telomere dysfunction or ‘uncapping’ may manifest as chromosomal end fusions that generate dicentric chromosomes, which may break unevenly during miosis to produce so-called breakage-fusion-bridge cycles. The resulting unequal distribution of genetic material between daughter cells creates genomic instability and may also induce a lethal event known as mitotic catastrophe. Consequently, the specialised DNA-protein complex at the telomere is required to ‘cap’ or protect the chromosome ends from exposure as double-stranded breaks, which initiates a DNA damage response that may result in DNA repair and, if the damage cannot be repaired, apoptosis of the cell.
The telomere is a complex structure, which incorporates both a novel higher order DNA structure, the T-loop, and a large cast of both telomere associated proteins and telomerase associated proteins. Telomere Repeat binding Factor-2 (TRF2) is a particularly important component of telomere structure and function, which unlike hTERT, is also expressed in all human normal cell types. TRF2 plays an important role in limiting the DNA damage response at telomeres. Other telomere associated proteins, which are expressed in normal cells, include Telomere Repeat binding Factor-1 (TRF1), tankyrase, TRF1 interacting protein (TIN2), hrap1, the Mre11/Rad50/Nbs1 DNA repair complex, Ku70/80 heterodimer. Telomerase associated proteins include the vault protein TEP1, the chaperone proteins p23 and hsp90, the small nucleolar (sno)RNA binding proteins, dyskerin and hGar1, the heterogeneous nuclear ribonucleoproteins (hnRNPs) C1/2, the La ribonucleoprotein and the L22 ribosomal protein and the double stranded RNA binding protein, hStau.
Reference herein to a “subject” should be understood to encompass humans, primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory rest animals (e.g. mice, rabbits, rats, guinea pigs), companion animals (e.g. dogs, cats) and captive wild animals (e.g. foxes, kangaroos, deer). Preferably, the mammal is a human.
Reference to a “biological sample” should be understood as a reference to any sample of biological material derived from an animal such as, but not limited to, cellular material, biofluids (eg. blood), feces, tissue biopsy specimens, surgical specimens or fluid which has been introduced into the body of an animal and subsequently removed (such as, for example, the solution retrieved from lung lavage or an enema wash). The biological sample which is tested according to the method of the present invention may be tested directly or may require some form of treatment prior to testing. For example, a biopsy or surgical sample may require homogenisation prior to testing or it may require sectioning for in situ testing. Alternatively, the dead cell sample may require permeabilisation prior to testing. Further, to the extent that the biological sample is not in liquid form, (if such form is required for testing) it may require the addition of a reagent, such as a buffer, to mobilise the sample.
To the extent that the target molecule is present in a biological sample, the biological sample may be directly tested or else all or some of the nucleic acid material present in the biological sample may be isolated prior to testing. In yet another example, the sample may be partially purified or otherwise enriched prior to analysis. For example, to the extent that a biological sample comprises a very diverse cell population, it may be desirable to select out a sub-population of particular interest, such as enriching for dead cells or enriching for the cell population of which the neoplastic cell forms part. It is within the scope of the present invention for the target cell population or molecules derived therefrom to be pretreated prior to testing, for example, inactivation of live virus or being run on a gel. It should also be understood that the biological sample may be freshly harvested or it may have been stored (for example by freezing) prior to testing or otherwise treated prior to testing (such as by undergoing culturing).
The choice of what type of sample is most suitable for testing in accordance with the method disclosed herein will be dependent on the nature of the situation, such as the nature of the condition being monitored. Preferably, said sample is of blood, urine, cerebrospinal fluid, pleural or peritoneal effusions and ascites, washings and brushings form oropharynx, lung, biliary tree, colon or bladder, biliary, pancreatic and mammary aspirates, and biopsies and surgical resections.
The present invention is predicated on the unexpected finding that dead neoplastic cells exhibit upregulated levels of telomerase expression. Accordingly, this finding now provides a means of monitoring a neoplastic condition in the context of the levels of dead and dying neoplastic cells which exist at a given point in time. This is particularly useful, for example, in the context of assessing the effectiveness of a therapeutic treatment regime, thereby providing a highly sensitive and rapid means for facilitating the optimisation of a treatment regime. In this regard, the person of skill in the art will understand that one may screen for changes to the levels of telomerase at either the protein (e.g. hTERT) or the encoding nucleic acid molecule level (e.g. hTERT or hTR mRNA). To the extent that it is not always specified, reference herein to screening for the level of “telomerase” should be understood to include reference to screening for either the relevant protein or its encoding primary RNA transcript or mRNA.
As hereinbefore described, telomerase is normally expressed by cells in the body which have high replicative potential such as activated lymphocytes, germ cells and stem cells. Telomerase is not active in most somatic cells. Accordingly, in the context of the present invention, the presence of telomerase expression in the dead cell population of many types of biological samples which are comprised of predominantly somatic cells will, in its own right, be indicative of the presence of dead neoplastic cells. For example, a population of normal peripheral blood mononuclear cells exhibit negligible telomerase levels in the dead cell component. However, in some biological samples, such as lymph node biopsy samples, there may be found higher proportions of non-neoplastic dead cells which are expressing telomerase due to the higher replicative potential required of lymphocytes during an immune response.
Accordingly, it should therefore be understood that the present invention is directed to the correlation of the level of telomerase relative to control levels of this molecule. “Control” levels may be either “normal” levels or the levels obtained from the same patient but at a previous point in time. The “normal” level is the level of telomerase protein or encoding nucleic acid molecule in a biological sample corresponding to the sample being analysed of any individual who has not developed a neoplastic condition. This result therefore provides the background levels, if any, of telomerase which are not due to neoplastic cell death but merely correspond to non-neoplastic dead cells which expressed telomerase due to their high replicative activity. It is expected that in the context of most samples these “normal” or “background” levels will be negligible. The method of the present invention should therefore be understood to encompass all suitable forms of analysis such as the analysis of test results relative to a standard result which reflects individual or collective results obtained from healthy individuals. In a preferred embodiment, said normal reference level is the level determined from one or more subjects of a relevant cohort to that of the subject being screened by the method of the invention. By “relevant cohort” is meant a cohort characterised by one or more features which are also characteristic of the subject who is the subject of screening. These features include, but are not limited to, age, gender, ethnicity, smoker/non-smoker status or other health status parameter. As detailed hereinbefore, said test result may also be analysed relative to a control level which corresponds to an earlier telomerase level result determined from the body fluid of said subject. This is a form of relative analysis (which may nevertheless also be assessed relative to “normal” levels) which provides information in relation to the rate and extent of neoplastic cell death over a period of time, such as during the course of a treatment regime.
Said “normal level” may be a discrete level or a range of levels. Individuals exhibiting dead cell telomerase levels higher than the normal range are generally regarded as having undergone neoplastic cell death, this corresponding to an encouraging prognosis since it may indicate treatment responsiveness and/or the move to a remissive state. In this regard, it should be understood that telomerase levels may be assessed or monitored by either quantitative or qualitative readouts. The reference level may also vary between individual forms of telomerase, such as hTERT vs hTR.
Accordingly, the present invention provides means for assessing both the existence and extent of a population of dead neoplastic cells in a subject. As detailed hereinbefore, this has extremely important implications in terms of assessing the effectiveness of a therapeutic treatment regime. To this end, although a one-off analysis of neoplastic dead cell levels in a biological sample provides information in relation to whether neoplastic cell death has occurred, the present invention is also useful, and particularly valuable, as an ongoing monitor. This can be essential in the context of identifying and monitoring a therapeutic treatment regime where an initial event of neoplastic cell responsiveness to a chemotherapy drug which is being utilised ultimately shifts to neoplastic cell resistance to the chemotherapy drug which is being utilised. The results observed utilising the screening regime herein described may correspond to screening for the existence of a higher level of telomerase over a normal level where a one-off test is being utilised. Alternatively, where a patient is subject to ongoing monitoring and where each successive test result is related to previous results, one may observe a series of increases and decreases in dead cell telomerase expression which map out the on going actions and effectiveness of the treatment regime which has been selected for use. Accordingly, increased dead cell telomerase levels relative to normal levels is indicative of neoplastic cell death—this possibly being due to any one of a number of events including the patient's own immune responsiveness or an effective treatment regime. Increased levels of dead cell telomerase expression relative to a previously analysed sample from that patient may indicate an increasing rate of neoplastic ell death while a decreasing level of telomerase expression in this circumstance may indicate either a loss of effectiveness of the selected treatment regime or the shifting of the patient into a remissive state due to the death and clearance of substantially all neoplastic cells.
Accordingly, in another aspect the present invention provides a method for assessing and/or monitoring a neoplastic condition in a subject, said method comprising screening for the level of telomerase protein and/or gene expression by non-viable cells in said subject or in a biological sample derived from said subject wherein an increase in the level of non-viable cells expressing said telomerase relative to normal levels is indicative of the presence of non-viable neoplastic cells.
More particularly, there is provided a method for assessing and/or monitoring a neoplastic condition in a subject, said method comprising screening for the level of telomerase protein and/or gene expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said telomerase relative to normal levels is indicative of the presence of dead neoplastic cells.
Preferably, said telomerase is hTERT or hTR.
According to this preferred embodiment there is provided a method for assessing and/or monitoring a neoplastic condition in a subject, said method comprising screening for the level of hTERT protein and/or gene expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said hTERT relative to normal levels is indicative of the presence of dead neoplastic cells.
More preferably, said hTERT is the protein form.
In another preferred embodiment there is provided a method for assessing and/or monitoring a neoplastic condition in a subject, said method comprising screening for the level of hTR RNA expression by dead cells in said subject or in a biological sample derived from said subject wherein an increase in the level of dead cells expressing said hTR RNA relative to normal levels is indicative of the presence of dead neoplastic cells.
Preferably, said cell death was induced by an anti-neoplastic cell treatment regime.
More preferably, said neoplastic condition is characterised by a neoplasm of the central nervous system tumours, retinoblastoma, neuroblastoma and other pediatric tumours, head and neck cancers (e.g. squamous cell cancers), breast and prostate cancers, lung cancer (both small and non-small cell lung cancer), kidney cancers (e.g. renal cell adenocarcinoma), oesophagogastric cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias (e.g. adenocarcinomas and islet cell tumours), colorectal cancer, cervical and anal cancers, uterine and other reproductive tract cancers, urinary tract cancers (e.g. of ureter and bladder), germ cell tumours (e.g. testicular germ cell tumours or ovarian germ cell tumours), ovarian cancer (e.g. ovarian epithelial cancers), carcinomas of unknown primary, human immunodeficiency associated malignancies (e.g. Kaposi's sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas, endocrine tumours (e.g. of thyroid gland), mesothelioma and other pleural or peritoneal tumours, neuroendocrine tumours and carcinoid tumours.
Yet another aspect of the present invention is directed to assessing and/or monitoring the effectiveness of a neoplastic therapeutic treatment regime in a subject said method comprising screening for the level of telomerase protein and/or gene expression by non-viable cells in said subject or in a biological sample derived from said subject wherein an increase in the level of non-viable cells expressing said telomerase relative to normal levels is indicative of the induction of neoplastic cell death.
Preferably, said telomerase is hTERT protein or mRNA or hTR RNA.
In the context of this aspect of the invention, and as detailed hereinbefore, an increase in the levels of telomerase expressing dead cells relative to control levels is indicative of the effectiveness of a therapeutic treatment regime which is directed to killing the subject neoplastic cells. Conversely, if telomerase levels remain essentially unchanged relative to normal levels, this would indicate that the subject treatment regime is not being effective. Similarly, a decrease in dead neoplastic cell telomerase levels relative to one or more earlier obtained results may indicate a tapering of or even up-regulation of resistance to the effectiveness of the treatment regime.
The present invention should also be understood to extend to a diagnostic method. That is, in addition to using this method to track changes to dead cell populations during and after the course of a therapeutic treatment regime, the existence of a proportion of telomerase expressing dead cells in all tumours even prior to treatment renders this method useful as a diagnostic.
Accordingly, the present invention further provides a method for diagnosing a neoplastic condition in a subject, said method comprising screening for the level of telomerase protein and/or gene expression by non-viable cells in said subject or in a biological sample derived from said subject wherein an increase in the level of non-viable cells expressing said telomerase relative to normal levels is indicative of the presence of a tumour.
As detailed hereinbefore, one may screen for telomerase at either the protein or mRNA level. To the extent that it is not otherwise specified, reference herein to screening for “telomerase” should be understood to include reference to screening for either the telomerase complex or telomerase subunit protein or its encoding primary RNA transcript or mRNA.
Means of screening for changes in telomerase levels in a subject, or biological sample derived therefrom, can be achieved by any suitable method, which would be well known to the person of skill in the art. Briefly, one may seek to detect hTERT protein or the integral human telomerase RNA (hTR) in a biological sample. Anti-telomerase immuno-interactive molecules may be used to identify hTERT protein directly or provide a means for the isolation of the entire telomerase RNP complex, which may be assayed subsequently for hTERT catalytic activity and/or hTR RNA. Anti-La immuno-interactive molecules provide an indirect means for isolating the entire telomerase RNP complex, which may be assayed subsequently for hTERT catalytic activity and/or hTR RNA. In terms of in vivo analyses, anti-telomerase immuno-interactive molecules could be coupled to medical imaging agents in order to visualise specific binding to dead cancer cells, in particular, following the administration of anti-cancer treatments. More specifically, these methods include, but are not limited to:
It should also be understood that the method of the present invention can be performed as an isolated test or it can be combined with any other suitable diagnostic test which may provide additional diagnostic or prognostic information. For example, and without limiting the application of the present invention in any way, the method of the present invention may be performed together with a technology such as CellSearch®, which efficiently and robustly identifies low frequencies of circulating tumour cells in peripheral blood.
Another aspect of the present invention provides a diagnostic kit for a biological sample comprising an agent for detecting telomerase or a nucleic acid molecule encoding telomerase and reagents useful for facilitating the detection by said agent. The agent may be an antibody or other suitable detection molecule.
The present invention further contemplates the use of an interactive molecule directed to telomerase in the manufacture of a quantitative or semi-quantitative diagnostic kit to detect dead neoplastic cells in a biological sample from a patient. The kit may come with instructions for use and may be automated or semi-automated or in a form which is compatible with an automated machine or software.
In addition to the clear diagnostic benefits of the method of the present invention, the ability to accurately target neoplastic cells now provides a means of delivering therapeutic treatments in a localised and highly targeted manner. To date, such treatments (often referred to as “magic bullets”) have not been possible. In particular, in the context of tumour therapy the notion of targeted treatments has not been possible due to the fact that it has not been possible to identify suitable tumour specific antigens against which an antibody could be directed. However, the present invention overcomes these shortcomings by directing the therapeutic treatment to the dead cells which comprise the subject tumour. As detailed hereinbefore, even in the absence of any treatment, a tumour will usually comprise a proportion of dead cells which can be utilised to provide the target for the therapeutic treatment. Even micrometastases, which reduce the chances that primary treatment such as mastectomy for breast cancer or colectomy for bowel cancer will be curative, contain apoptotic cells. Micrometastases are deposits of cancer usually less than 1 mm in diameter, which are believed to lie dormant because the rate of tumour cell proliferation balances the rate of tumour cell apoptosis. To the extent that a therapeutic regime is underway, the method of the present invention provides an opportunity to design and deliver a second phase treatment, for example a more highly toxic treatment, which is able to be more specifically targeted to the regions of dead neoplastic cells which have been induced by the first round of treatment, but which first round treatment may not have been adequate to induce remission. Still further, this method provides a means for administering a less toxic first round treatment in order to upregulate dead neoplastic cell numbers at the sites of tumours, thereby providing a means to accurately target a significantly more toxic second round treatment. This may provide a means of reducing the systemic side effects which are usually associated with chemotherapy. By selecting therapeutic effector mechanisms which can be coupled to an anti-telomerase interactive molecule (such as La or an anti-telomerase antibody), but which function on cells located proximally to the dead cells, that is the viable tumour cells, effective killing of the tumour can be achieved. The subject effector mechanism may take any suitable form but will preferably deliver a toxic molecule or otherwise kill the proximally located viable tumour cells.
Accordingly, another aspect of the present invention is directed to a method of treating a neoplastic condition in a subject said method comprising administering to said subject an effective amount of an interactive molecule directed to telomerase or antigenic portion thereof, which interactive molecule is linked, bound or otherwise associated with an effector mechanism, for a time and under conditions sufficient to treat said condition.
Preferably, said telomerase is hTERT protein or mRNA or hTR RNA.
More preferably, said interactive molecule is an immunointeractive molecule and even more preferably an anti-telomerase antibody.
Preferably, said neoplastic condition is characterised by nervous system tumours, retinoblastoma, neuroblastoma and other pediatric tumours, head and neck cancers (e.g. squamous cell cancers), breast and prostate cancers, lung cancer (both small and non-small cell lung cancer), kidney cancers (e.g. renal cell adenocarcinoma), oesophagogastric cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias (e.g. adenocarcinomas and islet cell tumours), colorectal cancer, cervical and anal cancers, uterine and other reproductive tract cancers, urinary tract cancers (e.g. of ureter and bladder), germ cell tumours (e.g. testicular germ cell tumours or ovarian germ cell tumours), ovarian cancer (e.g. ovarian epithelial cancers), carcinomas of unknown primary, human immunodeficiency associated malignancies (e.g. Kaposi's sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas, endocrine tumours (e.g. of thyroid gland), mesothelioma and other pleural and peritoneal tumours, neuroendocrine tumours and carcinoid tumours.
Reference to “telomerase”, “neoplastic”, “non-viable” and “subject” should be understood to have the same meaning as hereinbefore provided.
The present invention preferably provides a method of treating a neoplastic condition in a subject, said method comprising administering to said subject an effective amount of an immunointeractive molecule directed to hTERT protein or antigenic portion thereof, which immunointeractive molecule is linked, bound or otherwise associated with an effector mechanism, for a time and under conditions sufficient to inhibit, reduce or otherwise down-regulate the growth of the neoplasm.
In another preferred embodiment, there is provided a method of treating a neoplastic condition in a subject, said method comprising administering to said subject an effective amount of an immunointeractive molecule directed to hTERT or hTR RNA or antigenic portion thereof, which immunointeractive molecule is linked, bound or otherwise associated with an effector mechanism, for a time and under conditions sufficient to inhibit, reduce or otherwise down-regulate the growth of the neoplasm.
Preferably said neoplastic condition is a malignant tumour.
Reference to an “effector mechanism” should be understood as a reference to any suitable mechanism which, when localised to the site of dead cells, either directly or indirectly treats the neoplastic condition in issue, for example, down-regulating the growth of adjacent viable tumour cells. In the context of this preferred embodiment, the effector mechanism is most likely a proteinaceous or non-proteinaceous molecule or group of molecules which achieve this outcome. Examples of effector mechanisms suitable for use in the method of the present invention include, but are not limited to:
It should be understood that the method of the present invention may be performed either in vivo or in vitro. Examples of in vitro applications include, but are not limited to, the in vitro purging of biological samples comprising neoplastic cells. For example, bone marrow and/or blood may be removed from a patient, purged to kill tumour cells in accordance with the method of the present invention and then returned to the patient. Such procedures are currently performed, albeit in a significantly less targeted manner, and may reduce the risks of relapse due to transfer of contaminating malignant cells.
Reference to an effector mechanism being “linked, bound or otherwise associated” with an anti-telomerase antibody or other immunointeractive molecule should be understood as a reference to any covalent or non-covalent interactive mechanism which achieves linking of the two molecules. This includes, but is not limited to the use of peptide bonds, ionic bonds, hydrogen bonds, van Der Waals forces or any other interactive bonding mechanism.
Reference to “growth” of a cell or neoplasm should be understood as a reference to the proliferation, differentiation and/or maintenance of viability of the subject cell, while “down-regulating the growth” of a cell or neoplasm is a reference to the process of cellular senescence or to reducing, preventing or inhibiting the proliferation, differentiation and/or maintenance of viability of the subject cell. In a preferred embodiment the subject growth is proliferation and the subject down-regulation is killing. In this regard, killing may be achieved either by delivering a fatal hit to the cell or by delivering to the cell a signal which induces the cell to apoptose.
Reference herein to “therapeutic” or “prophylactic” treatment is to be considered in its broadest context. The term “treatment” does not necessarily imply that a subject is treated until total recovery. Similarly, “prophylaxis” does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term “prophylaxis” may be considered as reducing the severity or onset of a particular condition. “Treatment” may also reduce the severity of an existing condition.
Without limiting the present invention to any one theory or mode of action anti-cancer treatments usually kill by apoptosis but in many cases of advanced cancer, some cancer cells are resistant to the apoptosis that may be induced by a particular anti-cancer treatment. These apoptosis-resistant tumour cells are the source of clinical relapse of disease that ultimately kills most patients with advanced cancers and a significant proportion of those patients with earlier stage cancers. In those patients with advanced cancers who could be shown to be responding to the initial modality of treatment because tumour cell kill could be documented in vivo, additional gains in survival and quality of life may be made if another non-cross resistant treatment modality were also to be employed. Therefore, diagnosis of responding cancer patients using the method of the present invention can identify those patients who could benefit from supplementary treatment with therapeutic conjugates or hybrid fusion proteins, as detailed.
The use of an anti-telomerase antibody may also be favoured in the adjuvant clinical setting. Although early stage breast and colon cancers, for example, can both be cured by surgery, the risk of overt and incurable systemic relapse is heightened because, in those patients whose primary tumour has certain high-risk features and/or whose regional lymph nodes contain metastases, undetectable systemic micrometastases may already exist. So the use of adjuvant chemotherapy and/or adjuvant hormonal therapy (in the case of breast cancer in particular) cures an additional minor proportion of these patients presumably because the systemic micrometastases are cleared successfully.
Further, although dormant tumours remain small because they lack a blood supply, the tumour cells within the lesion turnover at a rapid rate with the rate of cell division balancing the rate of apoptosis. Therefore, even dormant tumours will be suitable targets for the present technology. Both in the case of clinically evident metastases and micrometastases, apoptosis-resistant cancer cells can be admixed with susceptible cancer cells. Bystander killing of these surviving cancer cells can occur if a non-cross resistant and/or synergistic means of tumour killing were delivered to nearby cancer cells that had been rendered apoptotic by the first treatment. Additional technologies that arm this technology with bystander killing potential can improve its therapeutic efficacy.
A most preferred embodiment of the present invention is therefore directed to the treatment of a metastatic cancer.
According to this preferred embodiment, there is provided a method of therapeutically treating a metastatic cancer in a subject, said method comprising administering to said subject an effective amount of an interactive molecule directed to telomerase or antigenic portion thereof, which interactive molecule is linked, bound or otherwise associated with a therapeutic effector mechanism, for a time and under conditions sufficient to inhibit, reduce or otherwise down-regulate the growth of said metastatic cancer.
As detailed hereinbefore, the present invention should also be understood to extend to the down-regulation of growth of neoplastic cells in an in vitro environment. For example, neoplastic cells may be purged from an inoculum of bone marrow or peripheral blood stem cells before an autologous transplant.
An “effective amount” means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. The amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
The present invention further contemplates a combination of therapies, such as the administration of the antibody together with subjection of the mammal to circulating cytotoxic agents or to radiotherapy in the treatment of cancer. In another example, the synergistic interactions between a radionuclide and a radiosensitising drug is of particular importance. For instance, a radioimmunotherapy regime can be designed in which there are do-administered an anti-telomerase antibody conjugated to a radionuclide that emits ionising radiation and a radiosensitising drug such as gemcitabine.
Administration of the interactive molecule (herein referred to as the “modulatory agent”), in the form of a pharmaceutical composition, may be performed by any convenient means. The modulatory agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the modulatory agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of modulatory agent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered continuously, daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
The modulatory agent may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules). The modulatory agent may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.
Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeally, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, infusion, orally, rectally, via IV drip patch and implant.
In accordance with these methods, the agent defined in accordance with the present invention may be coadministered with one or more other compounds or molecules. By “coadministered” is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. For example, the subject agent may be administered together with an agonistic agent in order to enhance its effects. By “sequential” administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.
Another aspect of the present invention contemplates the use of an anti-telomerase interactive molecule conjugated to an effector mechanism in the manufacture of medicament for the treatment of a neoplastic condition in a subject wherein said effector mechanism treats said condition.
Preferably, said interactive molecule is an immunointeractive molecule and even more preferably an anti-telomerase antibody, such as a monoclonal antibody.
Preferably, said neoplastic condition is characterised by central nervous system tumours, retinoblastoma, neuroblastoma and other pediatric tumours, head and neck cancers (e.g. squamous cell cancers), breast and prostate cancers, lung cancer (both small and non-small cell lung cancer), kidney cancers (e.g. renal cell adenocarcinoma), oesophagogastric cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias (e.g. adenocarcinomas and islet cell tumours), colorectal cancer, cervical and anal cancers, uterine and other reproductive tract cancers, urinary tract cancers (e.g. of ureter and bladder), germ cell tumours (e.g. testicular germ cell tumours or ovarian germ cell tumours), ovarian cancer (e.g. ovarian epithelial cancers), carcinomas of unknown primary, human immunodeficiency associated malignancies (e.g. Kaposi's sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas, endocrine tumours (e.g. of thyroid gland), mesothelioma and other pleural or peritoneal tumours, neuroendocrine tumours and carcinoid tumours.
In yet another further aspect, the present invention contemplates a pharmaceutical composition comprising the modulatory agent as hereinbefore defined together with one or more pharmaceutically acceptable carriers and/or diluents. Said agents are referred to as the active ingredients.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.
The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.
Yet another aspect of the present invention relates to the agent as hereinbefore defined, when used in the method of the present invention.
The present invention is further defined by the following non-limiting examples:
Peripheral blood mononuclear cells (PBMC) were prepared from a buffy coat of a discarded unit of whole blood that had been obtained from the South Australian Red Cross Blood Bank. Initially, PBMC had been separated by density gradient centrifugation over Lymphoprep (Axis-Shield, Dundee, UK) and frozen in aliquots using a controlled rate freezer. Subsequently, the aliquoted cells were thawed and again purified over Lymphoprep to obtain viable cells. PBMC were grown in RPMI supplemented with 10% foetal calf serum (FCS), 50μ/mL penicillin and 50 μg/mL streptomycin sulphate (JRH Biosciences, Lenexa, Kans., USA) for 72 h. Half of the culture was treated with the T cell mitogen, conconavalin A (ConA) (Sigma, St Louis, Mo., USA), 10 μg/mL, and half of the culture was left untreated. After culture, cells were again purified over Lymphoprep to remove dead cells and rendered apoptotic by a further 24 h culture with 1 μM staurosporine (STS) (Sigma). Jurkat cells were rendered apoptotic by 24 h culture in RPMI supplemented with 10% FCS, 50 U/mL penicillin and 50 μg/mL streptomycin sulphate and 0.5 μM STS. For studies of primary monocytic and lymphocytic cells derived from PBMC, PBMC were first purified using the Lymphoprep® separation protocol. Then PBMC were cultured in RPMI-1640 with 5% FCS overnight to separate adherent (predominantly monocytic cells) from suspension cells (predominantly T lymphocytes). Suspension cells were collected and incubated in RPMI-1640 with 5% FCS in the presence or absence of 1 μM STS and Jurkat cells were similarly incubated in the presence or absence of 0.5 μM STS. Adherent monocytic cells and U937 cells (a monocytic lymphoma cell line) were incubated in RPMI-1640 with 5% FCS in the presence or absence of 1 μM and 0.5 μM of STS, respectively. After 24 h and 48 h, untreated (control) and STS-treated cells were collected and the control cells were permeabilised using 2% paraformaldehyde solution followed by a 1:10 dilution with methanol at −20° C. for 5 min.
After treatment, all cells were harvested, washed in PBS and stained with 7-amino actinomycin D (7-AAD) (2 μg/mL) and anti-La 3B9 mAb or its isotype control mAb, Sal5, and then with the secondary anti-mouse antibody conjugated to R-phycoerythrin (Zymed, San Francisco, Calif., USA), or with rat anti-hTERT mAb (Alexis Biochemicals, San Diego, Calif., USA) and then with the secondary anti-rat antibody conjugated to FITC (Chemicon). Cells were analysed using a FACScan (Becton Dickinson, Franklin Lakes, N.J., USA) and the results analysed with WinMDI v2.8 software.
Cytotoxic Treatment of Malignant Human Cell Lines and Subsequent Detection of hTERT in Apoptotic Malignant and Primary Cells.
The Jurkat and Raji cell lines were cultured in RPMI supplemented with 10% FCS, 50 U/mL penicillin and 50 μg/mL streptomycin sulphate and the U2OS and HeLa cell lines were cultured in DMEM supplemented with 10% FCS, 50 U/mL penicillin and 50 μg/mL streptomycin sulphate (JRH Laboratories). The cell lines were treated for 24 or 48-hour periods with various cytotoxic agents as follows: Jurkat and Raji cells with etoposide, 20 μg/mL; HeLa and U2OS cells with cisplatin, 1 μg/mL, and vincristine, 0.1 μg/mL; Jurkat, HeLa and U2OS cells with 0.5 μM STS, and Raji cells with 2 μM STS. The treated cells were stained with propidium iodide (PI) (Sigma) 0.5 μg/mL and FITC-conjugated Sal5 or 3B9 mAb, or mouse anti-TRF2 mAb (clone 36, Becton-Dickinson) and then secondary antibody, anti mouse FITC (Chemicon, Temecula, Calif., USA), or rat anti-hTERT mAb (clone 36-10, Alexis Biochemicals) and then secondary antibody, anti rat FITC (Caltag, Burlingame, Calif., USA). Purified mouse IgG or rat IgG (Institute of Medical and Veterinary Science, Adelaide, Australia) were used as negative controls for TRF2 and hTERT staining, respectively. To further improve the method for detection of hTERT for later use in whole blood staining, which appeared to have been hindered by the presence of autofluorescence presumably emanating from red blood cells, anti-hTERT monoclonal antibody clone 36-10 was conjugated directly to AlexaFluor350 (Molecular Probes), which is excited at 355 nm and emits maximally between 420-460 nm. Anti-hTERT-Alexa350 was combined with the EpCAM monoclonal antibody (BD Biosciences), which is conjugated to PE that excites at 488 nm and emits 560-590 nm, and the nuclear impermeant DNA-binding dye TOPRO-3 (Molecular Probes), which excites at 560 nm and emits maximally in the far red region (>650 nm). This combination of fluorochromes did not require compensation of signals as each of these molecules is excited using a different laser and the emissions are collected using separate filters on the high throughput LSR-II flow cytometer (BD Biosciences).
Anti-La (monoclonal antibody hybridoma 3B9) binding was compared to apoptotic PBMC, which are mainly T cells, and apoptotic Jurkat cells, which is a human T cell leukemia line. It is clear that the fluorescence intensity is greater for apoptotic Jurkat cells than PBMC (
The human T cell leukemia cell line, Jurkat, and the human B lymphoma cell line, Raji, were treated for 24 or 48 hour periods with the following cytotoxic agents: staurosporine, which is a pan-kinase inhibitor, etoposide, which is a topoisomerase II inhibitor, vincristine, which is a microtubule depolymerising agent and cisplatin, which is a DNA cross linking agent. Cells were then stained with propidium iodide (PI) and the anti-La monoclonal antibody (mAb), 3B9, or its isotype control, Sal5, or with mAbs specific for hTERT or TRF2. As negative controls for staining with the hTERT- or TRF2-specific mAbs, rat IgG and anti-rat secondary antibody or mouse IgG and anti-mouse secondary antibody, respectively, were used.
Using flow cytometry, it was found that the various cytotoxic agents induced cell death in both Jurkat (
Human lymphocytes, in particular activated lymphocytes, also express telomerase. As illustrated in
H69 cells were cultured in RPMI-1640 containing 5% fetal calf serum (FCS) and 400 μg/mL etoposide. Aliquots were removed at 24 h, 48 h and 72 h, and washed with phosphate-buffered saline (PBS) and then stained for cytofluographic analysis.
Enrichment of Circulating Tumour Cells from Blood of a Small Cell Lung Cancer (SCLC) Patient
Blood samples (2.5 mL) collected in heparinised tubes were enriched for expression of the BerEP4 epithelial cell adhesion molecule (EpCAM) using the CELLection™ Epithelial Enrich kit as per the manufacturer's instructions (Dynal® Biotech, Invitrogen Corp., USA). Briefly, 250 μL BerEP4-beads were mixed with the blood sample for 30 min. at RT. Beads were bound to a Dynal® magnet and washed 5 times with PBS. Bead-bound cells were released from the magnet using PBS containing 0.1% bovine serum albumin (BSA) and deoxyribonuclease I (DNase I) as described by the manufacturer. The released cells were removed to a fresh tube, centrifuged, and washed with PBS. Samples were incubated for 30 min. at RT with anti-mouse IgG (2 μg/mL) to block any mouse BerEP4 antibody remaining on the isolated cells then washed with PBS before being stained and analysed by flow cytometry.
Red cells in the heparinised peripheral blood samples (2.5 mL) were lysed after diluting the blood 1:10 in red blood cell lysis buffer (8 g/L ammonium chloride and 1.2 g/L Tris-HCl, pH 7.2) and constantly rotating the diluted samples for 15 min at RT. Then cells were pelleted at 3,000×g for 10 min. and washed with PBS before being stained and analysed by flow cytometry.
Cells were incubated for 30 min. at room temperature (RT) with monoclonal antibodies (mAb) at a concentration of 50 μg/mL: APOTEL™, which is a rat anti-hTERT mAb (clone 36-10; Alexis Biochemicals), or a matching isotype (IgG2aκ) control mAb of irrelevant specificity. Cells were washed with PBS and incubated for 30 min. at RT with a biotin-conjugated (Fab′)2 fragment of an anti-rat IgG antibody at a concentration of 2 μg/mL. Cells were washed and incubated for 15 min. at RT with streptavidin-PE or with streptavidin-FITC at a concentration of 2 μg/mL. Finally, cells were washed with PBS and analysed by flow cytometry (FACScan, Becton-Dickinson) after incubation for 15 min with 2 μg/mL 7-amino-actinomycin D (7-AAD) at RT.
Statistical analyses were performed using GraphPad Prism v.4.0 software.
The ability of a monoclonal antibody (mAb), which specifically recognises the human telomerase reverse transcriptase (hTERT), was studied in the context of its ability to bind dead cells of small cell lung cancer (SCLC) both in vitro and in vivo after cell death is induced using cytotoxic drugs. First, the SCLC cell line, H69, which expresses the BerEP4 marker of Epithelial Cell Adhesion Molecule (EpCAM) was studied. H69 was cultured in vitro and treated with etoposide to induce cell death. Loss of viability of the cancer cells was evident 24 h after cytotoxic drug treatment because they bound the DNA-binding dye, 7-AAD (
Next, the binding of APOTEL™ in the peripheral blood of a 73-year-old male patient, GP, who had extensive-stage SCLC was studied. GP, who had not previously been treated for his cancer, was administered cytotoxic chemotherapy using carboplatin at AUC5 on day 1 together with etoposide 120 mg/m2 on days 1, 2 and 3. Heparinised peripheral blood samples were drawn from the patient before chemotherapy (0 h) and 24, 48 and 72 hours after initiation of chemotherapy. To demonstrate that GP's blood contained circulating tumour cells (CTC), his blood was enriched before and after cytotoxic drug treatment for BerEP4-expressing cells, which were not present in the peripheral blood of a normal healthy volunteer (
Although the laboratory findings have not yet been correlated with a formal radiological evaluation of overall tumour response to chemotherapy, GP did have a palpable and painful sternal mass before cytotoxic chemotherapy, which became markedly less painful and shrunk significantly in size within two weeks of cytotoxic chemotherapy administration.
These results indicate that APOTEL™ identifies cancer cells that have died as the result of cytotoxic drug administration and, hence, APOTELT™ is useful for predicting early chemotherapy responses in cancer patients.
In addition to its use in isolation, the APOTEL™ test may also be usefully integrated with the only FDA-approved technology for the detection of circulating tumour cells (CTC), which is an immunomagnetic bead-based method of enumerating circulating tumour cells (CTC) called CellSearch® (Veridex, Warren, N.J., USA). The system is designed to count epithelial cells, which are first separated from the blood by BerEP4-specific antibody-coated magnetic beads. CTC are identified among the isolated cells after fluorescence labelling with the double stranded DNA-specific dye, 4,6-diamidino-2-phenylindole (DAPI), and mAb specific for leukocytes (CD45-allophycocyanin) and epithelial cells (cytokeratin 8-, 18- and 19-phycoerythrin) and analysis using the CellSpotter Analyzer® (Veridex). The technology facilitates specific detection of CTC as shown by a large survey of normal individuals and of individuals with malignant and non-malignant diseases. One of 344 (0.3%) individuals, who were healthy or who had non-malignant diseases, had ≧2 CTC per 7.5 mL of blood. In 2,183 blood samples from 964 metastatic carcinoma patients, CTC ranged from 0 to 23,618 CTC per 7.5 mL (mean, 60±693 CTC per 7.5 mL), and 36% (781 of 2,183) of the specimens had ≧2 CTC. Detection of ≧2 CTC occurred at the following rates: 57% (107 of 188) of prostate cancers, 37% (489 of 1,316) of breast cancers, 37% (20 of 53) of ovarian cancers, 30% (99 of 333) of colorectal cancers, 20% (34 of 168) of lung cancers, and 26% (32 of 125) of other cancers (Allard et al. Clin Cancer Res 10:6897-6904, 2004).
After the initial step of BerEP4 enrichment, the CellSearch® technology enables the detection of cells as shown in a study of the blood of prostate cancer patients. Here, few CTC were intact and most appeared as damaged cells or cell fragments by microscopy. By flow cytometry, these CTC events stained variably with DAPI and frequently expressed the apoptosis-induced, caspase-cleaved cytokeratin-18 (Larson et al. Cytometry Part A 62A, 46-53, 2004).
In a study of CTC in patients with metastatic breast cancer, none of 145 normal control subjects had >2 CTC per 7.5 mL of blood whereas in 177 cancer patients >2 CTC per 7.5 mL of blood were found in 61% of patients. Nonetheless, a cut-off of 5 CTC per 7.5 mL of blood was chosen to distinguish patients with an unfavourable prognosis from patients with a favourable prognosis. The results of this study indicated that the number of CTC before treatment was an independent predictor of progression-free survival and overall survival in patients with metastatic breast cancer (Cristofanilli et al New Engl J Med 351:781-791, 2004).
A further report of this study was published that analysed the significance of CTC found in patients with metastatic breast cancer receiving first-line chemotherapy. In these patients, the presence of ≧5 CTC in 7.5 mL of peripheral blood before the initiation of first-line chemotherapy was associated with short progression free survival (PFS) and overall survival (OS). Median OS at this time was >18 months for patients with <5 CTC vs. 14.2 months for patients with >5 CTC. Moreover, the greatest statistically different differences in median OS were observed for the subset of patients (22%) with visceral disease who were receiving chemotherapy and who were negative for hormone receptors and HER2/neu. At the first follow-up blood draw approximately one month post-treatment, the median PFS for patients with <5 CTC was 9.5 months vs. 2.1 months for patients with ≧5 CTC (P=0.0057), and the median OS for patients with ≧5 CTC was more than 18 months vs. 11.1 months for patients with >5 CTC (P=0.0012). At the first follow-up imaging visit at approximately 9 weeks post-treatment, the median PFS for patients with <5 CTC was 8.9 months vs. 1.8 months for patients with >5 CTC (P=0.0001), and the median OS for patients with <5 CTC was 18 months v 11.1 months for patients with ≧5 CTC (P=0.0001). These data indicated that detection of CTC3-4 weeks post-treatment predicted treatment efficacy, which was determined by medical imaging studies 8-10 weeks post-treatment (Cristofanilli et al. J Clin Oncol. 23:1420-1430, 2005).
However, because APOTEL™ detects dead CTC 48 hours after initiation of cytotoxic chemotherapy, it can give a much more prompt indication of an effective and survival-prolonging chemotherapy response than other methods such as the number of CTC3-4 weeks post-treatment or the results of medical imaging studies 8-10 weeks post-treatment.
Cell culture media, RPMI 1640, DMEM and Trypsin EDTA were obtained from JRH Biosciences Inc (Lexona Kans., USA). Staurosporine and ethidium bromide were obtained from Sigma-Aldrich Co. (St Louis, Mo., USA) Trizol and Superscript II were purchased from Invitrogen (Carlsbad, Calif., USA) and Amplitaq Gold from Applied Biosystems (Foster City, Calif., USA) All primers were manufactured by Geneworks (Adelaide, SA)
Jurkat cells (ATCC #TIB-152) were maintained in RPMI 1640 supplemented with 5% fetal calf serum (FCS). U2OS cells (ATCC #HTB-96) were maintained in DMEM supplemented with 5% FCS and passaged every 48-72 hours after detachment with Trypsin EDTA. Jurkat cells express telomerase and thus have the integral RNA component, hTR, whereas U2OS cells lack telomerase and hTR. Apoptosis was induced by the addition of 0.5 μM staurosporine to the culture media for 24, 48 or 72 hours.
Cells were harvested and washed in PBS before RNA extraction of 5×106 cells with Trizol according to manufacturer's instructions. RNA was resuspended in 10 ul of DEPC water and the concentration determined. Three micrograms of RNA was reverse transcribed using Superscript II and oligo dT according to the manufacturer's instructions.
PCR was performed with primers specific for hTR. Two sets of primers were used to generate PCR products of 111 bp (Chen X Q et al. Clin Cancer Res 6:3823-3826, 2000) and 153 bp (Bodnar A G et al. Exp Cell Res 228:58-64, 1996) in size:
A control set of PCR primers was used to amplify mRNA of the housekeeping gene, GAPDH:
One microlitre of cDNA was subjected to the polymerase chain reaction (PCR) in a 25 μL reaction containing 1×PCR buffer, 10 nmol MgCl2 and 5 nmol dNTPs, 50 pmol of each primer and 0.625U of Amplitaq gold. The reaction conditions for PCR amplification were as follows: an initial denaturation of 95° C. for 7 minutes, followed by 30 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 1 minute, with a final extension of 72° C. for 5 minutes. PCR products were electrophoresed on 1% agarose and visualised by ethidium bromide staining. The negative control for the PCR was water together with primers.
RT-PCR amplification of GAPDH shows detectable mRNA in both cells lines up to 72 hours after the induction of apoptosis. Viability analysis using trypan blue staining indicated that of U2OS cells, 36%, 95% and 100% were dead at 24, 48 and 72 hours, respectively, and of Jurkat cells, 40%, 85% and 100% were dead at 24, 48 and 72 hours, respectively. The RNA for hTR was not detected in the hTR-negative U2OS cells but was detected in the hTR-positive Jurkat cells 24, 48 and 72 hours after the induction of apoptosis.
These data indicate that it is possible to PCR-amplify RNA including that of GAPDH and hTR from dead cells in vitro. There is evidence to indicate that RNA including mRNA is sequestered inside new intracellular fibro-granular structures called Heterogenous Ectopic RNP-Derived Structures (HERDS) that develop during apoptosis. HERDS are extruded into the cytoplasm and move toward the cell surface as part of apoptotic blebs, which are eventually released as apoptotic bodies. Although the formation of HERDS was shown to be a reversible marker of transcriptional arrest, which may occur during processes such as heat shock and other physical stress, nutrient deprivation and use of DNA damaging drugs, HERDS formation and the commitment to apoptosis becomes irreversible when nucleolar proteins are identified in HERDS (Biggiogera et al. Biol Cell 96:603-615, 2004). To date there have been no reports of labelling of the La antigen in HERDS. However, since La can bind hTR, immunoaffinity isolation of La from dead cells should facilitate the PCR amplification of hTR particularly when tumour cell numbers are limiting such as in biofluids, which may include tumour cells circulating in the peripheral blood of even early-stage cancer patients. QZyme™ technology (Clontech) is an example of a quantitative PCR-based assay system, which could usefully be applied to La-bound hTR isolated by immunoaffinity methods, because it is unaffected by biosample complexity and requires no optimisation.
Clonetics® conditioned primary cells and normal human bronchial epithelium with retinoic acid (NHBE) were obtained from Cambrex Corporation (NJ, USA) and maintained in culture using the commercially supplied medium. Cancer cell lines were obtained from ATCC and cultured in recommended media. At confluence, cells were detached using Trypsin-EDTA solution, washed with PBS and permeablised by incubating 5×106 cells/mL with 2% paraformaldehyde for 10 min. at RT then diluting 1:10 with −20° C. absolute methanol.
Cell death was induced by adding cisplatin to the culture medium at a concentration of 20 μg/mL for 48 hours.
Cells were collected, washed with PBS and incubated for 30 min. at RT with APOTEL™ or matched isotype control mAb of irrelevant specificity at 50 μg/mL. Cells were washed with PBS and incubated for 30 min. at RT with anti-rat IgG Alexa488 conjugated antibodies at 2 μg/mL. Cells were washed and incubated for 10 min. at RT with 2 μg/mL 7-AAD then analysed by flow cytometry using a B-D FACScan instrument.
Telomerase expression in primary and malignant bronchial epithelial cells was similar (
These in vitro results indicate the potential for targeting a hTERT-specific mAb carrying a payload such as a radionuclide to dead cancer cells in vivo for imaging and therapeutic purposes.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005901613 | Apr 2005 | AU | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU2006/000431 | 3/31/2006 | WO | 00 | 4/27/2009 |
