Patent Application
20100310450
The present invention relates generally to a method of screening for a neoplastic cell in a subject. More particularly, the present invention provides a method of screening for both viable neoplastic cells and, still further, cytotoxin induced neoplastic cell death by detecting the level of expression of La protein and/or gene by a cellular population 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, diagnosing, prognosing or 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 La protein and/or nucleic acid molecules.
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 industrialized 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 soon after administration. 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.
In work leading up to the present invention it has been surprisingly determined that La expression is upregulated in cancer cells and upregulated, still further, in cancer cells which have undergone cell death as a result of treatment which induces DNA damage, as opposed to cell death caused by other means. This finding is quite distinct from the prior art, where La has been used as a general indicator of the presence of apoptotic cells, since the presence of apoptotic cells is not conclusively diagnostic of the neoplastic nature of that cell. Still further, since apoptosis can be induced by a wide variety of factors, of which DNA damage is merely one type of factor, the findings detailed herein have enabled the development of a means of differentiating between neoplastic cell death resulting from non-DNA damage related means versus that induced by a DNA damaging agent. This distinction is significant when one bears in mind that even in an untreated patient, at any given point in time there exist a proportion of dead and dying neoplastic cells, the apoptosis of which has been induced by factors other than treatment which induces DNA damage. Accordingly, this determination has facilitated the development of a means for identifying neoplastic cells based on screening for increased levels, relative to normal levels, of La expression and still further, screening for DNA damage induced neoplastic cell death based on screening for a yet further increase in La expression levels. This is particularly important in the context of monitoring the progress of a therapeutic treatment regime which is directed to killing neoplastic cells.
Throughout this specification and the claims which follow, 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. Further, as used herein the singular forms of “a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
One aspect of the present invention is directed to a method for detecting a neoplastic cell in a subject, said method comprising screening for the level of La protein and/or gene expression by a cellular population in said subject or in a biological sample derived from said subject wherein an increase in the level of cellular La expression relative to normal La expression levels is indicative of a neoplastic cell.
In another aspect of the present invention there is provided a method for detecting a non-viable neoplastic cell in a subject, which non-viability has been induced by a DNA damaging agent, said method comprising screening for the level of La 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 La expression relative to viable neoplastic cell La expression levels is indicative of DNA damage induced neoplastic cell non-viability.
Yet another aspect of the present invention is directed to a method for detecting a malignant neoplastic cell in a subject, said method comprising screening for the level of La protein and/or gene expression by a cellular population in said subject or in a biological sample derived from said subject wherein an increase in the level of cellular La expression relative to normal La expression levels is indicative of a malignant neoplastic cell.
In still another aspect of the present invention there is provided a method for detecting a non-viable malignant neoplastic cell in a subject, which non-viability has been induced by a cytotoxic agent, said method comprising screening for the level of La 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 La expression relative to viable malignant neoplastic cell La expression levels is indicative of cytotoxicity induced neoplastic cell non-viability.
In yet still 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 La protein and/or gene expression by viable and/or non-viable cells in said subject or in a biological sample derived from said subject wherein an increase in the level of La in viable cells relative to normal levels is indicative of a neoplastic cell and an increase in the level of La in non-viable cells relative to viable neoplastic cell levels is indicative of the presence of DNA damage induced neoplastic cell non-viability.
Yet another aspect of the present invention is directed to assessing and/or monitoring the effectiveness of a neoplastic cytotoxic therapeutic treatment regime in a subject said method comprising for the level of La protein and/or gene expression by viable and/or non-viable cells in said subject or in a biological sample derived from said subject wherein an increase in the level of La in viable cells relative to normal levels is indicative of a neoplastic cell and an increase in the level of La in non-viable cells relative to viable neoplastic cell levels is indicative of the presence of cytotoxicity induced neoplastic cell non-viability.
Another further aspect of the present invention provides a diagnostic kit for a biological sample comprising an agent for detecting La or a nucleic acid molecule encoding La and reagents useful for facilitating the detection by said agent.
The present invention still further contemplates the use of an interactive molecule directed to La in the manufacture of a quantitative or semi-quantitative diagnostic kit to detect dead neoplastic cells in a biological sample from a patient.
and 72 h ▪) for sulforhodamine 101 staining. Inset summarizes the dependence of the protein cross-linking ratio on MDC concentration at 48 h (-) and 72 h (▪▪▪) after cisplatin treatment of Jurkat cells. (E) Jurkat cells incubated with cisplatin in the absence or presence of increasing concentrations of MDC were stained with 3B9. 3B9-specific binding is presented as Net MFI±SEM (n=3). Inset summarizes the dependence of 3B9-specific binding on MDC concentration at 24 h (-), 48 h (--) and 72 h (▪▪▪) after cisplatin treatment of Jurkat cells. Data are representative of three identical and independent assays, which produced similar results.
) and the labelling of both heavy and light chains.
The present invention is predicated, in part, on the surprising determination that La expression is increased upon transformation of a normal cell to a neoplastic state and, further, that the induction of neoplastic cell death by a DNA damaging agent, such as a cytotoxic agent, results in a still further increase in the expression of La. Accordingly, the detection of increased levels of La expression by neoplastic cells, relative to normal levels, provides a convenient and precise mechanism for qualitatively and/or quantitatively assessing neoplastic cell levels while levels of La in dead neoplastic cells which are higher than that of the live cells is specifically indicative of a DNA damage induced cell death. 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.
Accordingly, one aspect of the present invention is directed to a method for detecting a neoplastic cell in a subject, said method comprising screening for the level of La protein and/or gene expression by a cellular population in said subject or in a biological sample derived from said subject wherein an increase in the level of cellular La expression relative to normal La expression levels is indicative of a neoplastic cell.
In a related aspect of the present invention there is provided a method for detecting a non-viable neoplastic cell in a subject, which non-viability has been induced by a DNA damaging agent, said method comprising screening for the level of La 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 La expression relative to viable neoplastic cell La expression levels is indicative of cytotoxicity induced neoplastic cell non-viability.
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. In terms of monitoring the progress of a patient who is suffering from a neoplastic condition, distinguishing between neoplastic cell death induced by a cytotoxic treatment regime and other forms of unrelated cell death is crucial to optimising both patient care and the treatment provided.
Without limiting the present invention to any one theory or mode of action, the mechanisms by which neoplastic treatment regimes achieve neoplastic cell death are variable and depend on the specific nature of the treatment regime which has been selected for use. Although DNA damage would normally induce 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 growth inhibition or death are activated such as necrosis, mitotic catastrophe, autophagy and premature senescence. Hence, the induction of neoplastic cell apoptosis is not the only immediate outcome of these widely used treatment regimes but may manifest after a period of days has elapsed. 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). Nevertheless, the induction of neoplastic cell apoptosis is still the most commonly observed outcome in the context of any of the more widely used treatment regimes.
Unfortunately, due to the relatively non-specific effects of neoplastic treatment regimens (in particular systemically administered chemotherapy) all rapidly dividing cellular populations (normal and malignant) are affected. The patient may therefore experience severe treatment related toxicities. Accordingly, the design of means for both more accurately and rapidly monitoring the effectiveness of treatment regimes is of significant value in that it provides a more effective means of assessing and/or tailoring one of the most commonly administered treatment regimes. 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 due to the actions of a DNA damaging agent, as opposed to other forms of cell death induction. Preferably, said non-viable neoplastic cell is a dead neoplastic cell.
In a related aspect of the present invention there is provided a method for detecting a dead neoplastic cell in a subject, which cell death was induced by a DNA damaging agent, said method comprising screening for the level of La 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 cellular La expression relative to viable neoplastic cell La expression levels is indicative of DNA damage induced neoplastic cell death.
Reference to a “DNA damaging agent” should be understood as a reference to any proteinaceous or non-proteinaceous agent which acts to damage cellular DNA. the agent may be a cytotoxic agent or a non-cytotoxic agent. Without limiting the present invention to any one theory or mode of action, many such agents function via the induction of apoptotic processes. However, this is not the only mechanism by which such agents function and it is conceivable that the subject DNA damage may be induced by some other mechanism. Examples of DNA damaging agents include, but are not limited to, the traditionally understood chemotherapy agents such as Actinomycin D, Arsenic Trioxide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Corticosteroids, Cyclophosphamide, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel, Procarbizine, Raltitrexed, Streptozocin, Thioguanine, Thiotepa, Topotecan, Treosulfan, Vinblastine, Vincristine, Vindesine, Vinorelbine. Other means of inducing DNA damage include ionising radiation as well as the use of molecules such as inhibitors of poly-(ADP ribosyl) transferase (PARP) or agents which induce DNA damage as part of a synergistic process with another agent, for example e.g. Gemcitabine or Irinotecan and CHK1/2 inhibitors such as CBP-501 or AZD7762. In addition, new classes of antineoplastic agents such as histone deacetylase inhibitors (HDACi) e.g. vorinostat, BH3 mimetics e.g. ABT737, and Tumor Necrosis Factor-Related Apoptotis-Inducing Ligand (TRAIL), are pro-apoptotic particularly when administered in conjunction with conventional cytotoxic agents. Hence, singly or in combination, these pro-apoptotic compounds will likely increase the amount of La-specific signal detectable in the malignant neoplasm.
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 enlargement of neoplastic cell size as well as 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 individual cell size and nuclear/cytoplasmic ratio, 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 paediatric 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.
Preferably, said neoplastic cell is a malignant neoplastic cell.
Accordingly, a preferred embodiment of the present invention is directed to a method for detecting a malignant neoplastic cell in a subject, said method comprising screening for the level of La protein and/or gene expression by a cellular population in said subject or in a biological sample derived from said subject wherein an increase in the level of cellular La expression relative to normal La expression levels is indicative of a malignant neoplastic cell.
In a related aspect of the present invention there is provided a method for detecting a non-viable malignant neoplastic cell in a subject, which non-viability has been induced by a DNA damaging agent, said method comprising screening for the level of La 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 La expression relative to viable malignant neoplastic cell La expression levels is indicative of DNA damage induced neoplastic cell non-viability.
In one embodiment, said DNA damaging agent is a cytotoxic agent.
Reference herein to “La” includes reference to all forms of La or their homologues, or orthologs or derivatives. Reference to “La” should be understood to include reference to any isoforms which arise from alternative splicing of La mRNA or mutants or polymorphic variants of La. It should also be understood that “La” is a molecule which is alternatively term SS-B.
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, urine, sputum), cerebrospinal fluid, faeces, 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 neoplastic cells exhibit upregulated levels of La expression and, still further, that DNA damage induced neoplastic cell death is identifiable by virtue of a further increase in the expression levels of La. Accordingly, this finding now provides a means of both diagnosing neoplasias and thereafter monitoring the responsiveness of a neoplastic condition to a DNA damage-based treatment regime. This provides 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 La at either the protein or the encoding nucleic acid molecule level. To the extent that it is not always specified, reference herein to screening for the level of “La” should be understood to include reference to screening for either the relevant protein or its encoding primary RNA transcript or mRNA.
As detailed hereinbefore, the present invention is directed to the correlation of the level of La relative to control levels of this molecule. “Control” levels may be either “normal” levels (eg. in the context of neoplastic cell diagnosis) or the levels obtained from the same patient but at a previous point in time (eg. in the context of monitoring the effectiveness of a cytotoxic treatment regime. The “normal” level is the level of La 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 La which are not due to neoplastic cell transformation but merely correspond to normal intracellular levels. Without limiting the present invention to any one theory or mode of action, La is an abundant, ubiquitous nuclear phosphoprotein that has high binding affinity for the 3′ oligo-U motif of all polymerase (pol) III-catalysed transcripts together with certain small RNAs synthesized by other RNA polymerases. La contains two RNA-binding domains and is almost exclusively located in the nucleoplasm because of the nuclear localisation signal (NLS) located at its carboxy-terminus (J. Maraia. J Cell Biol 153, F13-18, 2001; Wolin S L and Tommy Cedervall T. Annu Rev Biochem 71, 375-403, 2002). The most conserved region of the La protein is the La motif, which is a domain found in several other RNA-binding proteins. A high-resolution X-ray crystallographic structure shows that La motif adopts a winged helix-turn-helix architecture associated with a highly conserved patch of mainly aromatic surface residues. Mutagenesis experiments demonstrate that this patch partly determines the binding specificity of the La protein for RNAs ending in 3′ hydroxyl group, which is one of its defining characteristics (Dong G et al. EMBO J 23, 1000-1007, 2004). Major functions of La include stabilizing newly synthesized small RNAs to protect them from exonuclease digestion and acting as a molecular chaperone for small RNA biogenesis. Thus La facilitates pre-tRNA maturation and assembly of small RNAs into functional ribonucleoproteins (RNPs) and may also enable retention of certain nascent small RNAs in the nucleus. Finally, a still controversial hypothesis posits that La binding of specific mRNAs facilitates initiation of translation (Wolin S L and Tommy Cedervall T. Annu Rev Biochem 71, 375-403, 2002).
The method of the present invention should 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 La 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” or “control level” may be a discrete level or a range of levels. Individuals exhibiting La levels higher than the normal range are generally regarded as having undergone neoplastic transformation. Those patients exhibiting non-viable cells expressing a level of La higher than that of their viable neoplastic cells are regarded as having undergoing DNA damaging agent induced neoplastic cell death. This corresponds to an encouraging prognosis since it may indicate treatment responsiveness and, potentially, the move to a remissive state. In this regard, it should be understood that La levels may be assessed or monitored by either quantitative or qualitative readouts.
Accordingly, the present invention provides means for assessing both the existence and extent of a population of viable neoplastic cells and/or neoplastic cells which have responded to a treatment regime. 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 treatment induced 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 La levels as a one-off test, thereby providing information in relation to relative proportions of viable neoplastic cells versus cells which have undergone DNA damaging agent induced cell death. 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 La expression which map out the on going actions and effectiveness of the treatment regime which has been selected for use. Accordingly, increased dead cell La levels relative to viable neoplastic cell levels is indicative the effectiveness of a treatment regime. Increased levels of dead cell La expression relative to a previously analysed sample from that patient may indicate on going effectiveness of the treatment regime while a loss of effectiveness of the treatment regime would be characterised by a loss of non-viable cells exhibiting La expression levels which are higher than that of viable neoplastic cells.
Alternatively, since overexpression of La in malignant neoplastic cells appears to be closely linked to the increased protein synthesis characteristic of malignant cells, successful treatment with antineoplastic agents that inhibit cellular protein synthesis such as the class of mammalian Target Of Rapamycin (mTOR) inhibitors, e.g. temsirolimus or everolimus, may result in the diminution of the La-specific signal in malignant neoplastic cells compared with normal 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 La protein and/or gene expression by viable and/or non-viable cells in said subject or in a biological sample derived from said subject wherein an increase in the level of La in viable cells relative to normal levels is indicative of a neoplastic cell and an increase in the level of La in non-viable cells relative to viable neoplastic cell levels is indicative of the presence of DNA damage-induced neoplastic cell non-viability.
Preferably, said non-viability is cell death.
More preferably, said neoplastic condition is characterised by central nervous system tumours, retinoblastoma, neuroblastoma and other paediatric 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 La protein and/or gene expression by viable and/or non-viable cells in said subject or in a biological sample derived from said subject wherein an increase in the level of La in viable cells relative to normal levels is indicative of a neoplastic cell and an increase in the level of La in non-viable cells relative to viable neoplastic cell levels is indicative of the presence of DNA damage induced neoplastic cell non-viability.
Preferably, said non-viability is cell death.
In accordance with these embodiments, said DNA damaging agent may be a cytotoxic agent.
As detailed hereinbefore, one may screen for La at either the protein or mRNA level. To the extent that it is not otherwise specified, reference herein to screening for “La” should be understood to include reference to screening for either the La protein or its encoding primary RNA transcript or mRNA.
Means of screening for changes in La 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 La protein in a biological sample. La interacting molecules may be used to identify La protein directly. Examples of such molecules include, but are not limited to, antibodies or fragments thereof, affibodies, phylomers, aptamers, single chain antibodies, deimmunized antibodies, humanized antibodies and T cell associated antigen binding molecules. In terms of in vivo analyses, these molecules could be coupled to medical imaging agents in order to visualise specific binding to neoplastic cells, in particular, following the administration of cytotoxic anti-cancer treatments. More specifically, these methods include, but are not limited to:
In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to the skilled artisan. The substrates to be used with the specific enzymes are generally chosen for the production of, upon hydrolysis by the corresponding enzyme, a detectable colour change. Examples of suitable enzymes include those described supra. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody-antigen complex, allowed to bind, and then the excess reagent washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.
Alternately, fluorescent compounds, such as fluorescein, rhodamine and the lanthanide, europium (EU), may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. The fluorescent-labelled antibody is allowed to bind to the first antibody-antigen complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of an appropriate wavelength. The fluorescence observed indicates the presence of the antigen of interest. Immunofluorometric assays (IFMA) are well established in the art and are particularly useful for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules may also be employed.
The use of antibodies, in particular monoclonal antibodies to detect La is a preferred method of the present invention. Antibodies may be prepared by any of a number of means. For the detection of human La, for example, human-human monoclonal antibody hybridomas may be derived from B cells, which have been obtained from patients who make anti-La autoantibodies because they have systemic autoimmune diseases such as systemic lupus erythematosis (SLE) or Sjorgren's syndrome (Ravirajan et al. Lupus 1(3):157-165, 1992). Antibodies are generally but not necessarily derived from non-human animals such as primates, livestock animals (e.g. sheep, cows, pigs, goats, horses), laboratory test animals (e.g. mice, rats, guinea pigs, rabbits) and companion animals (e.g. dogs, cats). Generally, antibody based assays are conducted in vitro on cell or tissue biopsies. However, if an antibody is suitably deimmunized or, in the case of human use, humanized, then the antibody can be labelled with, for example, a nuclear tag, administered to a patient and the site of nuclear label accumulation determined by radiological techniques. The La antibody is regarded, therefore, as a cellular apoptosis targeting agent. Accordingly, the present invention extends to deimmunized forms of the antibodies for use in cellular apoptosis imaging in human and non-human patients. This is described further below.
Currently available antibodies include SW3 and 3B9.
For the generation of antibodies to La, this molecule is required to be extracted from a biological sample whether this be from animal including human tissue or from cell culture if produced by recombinant means. The La can be separated from the biological sample by any suitable means. For example, the separation may take advantage of any one or more of La's surface charge properties, size, density, biological activity and its affinity for another entity (e.g. another protein or chemical compound to which it binds or otherwise associates). Thus, for example, separation of La from the biological fluid may be achieved by any one or more of ultra-centrifugation, ion-exchange chromatography (e.g. anion exchange chromatography, cation exchange chromatography), electrophoresis (e.g. polyacrylamide gel electrophoresis, isoelectric focussing), size separation (e.g., gel filtration, ultra-filtration) and affinity-mediated separation (e.g. immunoaffinity separation including, but not limited to, magnetic bead separation such as Dynabead™ separation, immunochromatography, immuno-precipitation). Choice of the separation technique(s) employed may depend on the biological activity or physical properties of the La sought or from which tissues it is obtained.
Preferably, the separation of La from the biological fluid preserves conformational epitopes present on the protein and, thus, suitably avoids techniques that cause denaturation of the enzyme. Persons of skill in the art will recognize the importance of maintaining or mimicking as close as possible physiological conditions peculiar to La (e.g. the biological fluid from which it is obtained) to ensure that the antigenic determinants or active sites on La, which are exposed to the animal, are structurally identical to that of the native protein. This ensures the raising of appropriate antibodies in the immunised animal that would recognize the native protein. In a preferred embodiment, La is separated from the biological fluid using any one or more of affinity separation, gel filtration and ultra-filtration.
Immunization and subsequent production of monoclonal antibodies can be carried out using standard protocols as for example described by Kohler and Milstein, Nature 256: 495-499, 1975; Kohler and Milstein, Eur. J. Immunol. 6(7): 511-519, 1976; Coligan et al., Current Protocols in Immunology, John Wiley & Sons, Inc., 1991-1997, or Toyama et al, “Monoclonal Antibody, Experiment Manual”, published by Kodansha Scientific, 1987. Essentially, an animal is immunized with a La-containing biological fluid or fraction thereof by standard methods to produce antibody-producing cells, particularly antibody-producing somatic cells (e.g. B lymphocytes). These cells can then be removed from the immunized animal for immortalization.
Where a fragment of La is used to generate antibodies, it may need to first be associated with a carrier. By “carrier” is meant any substance of typically high molecular weight to which a non- or poorly immunogenic substance (e.g. a hapten) is naturally or artificially linked to enhance its immunogenicity.
Immortalization of antibody-producing cells may be carried out using methods which are well-known in the art. For example, the immortalization may be achieved by the transformation method using Epstein-Barr virus (EBV) (Kozbor et al., Methods in Enzymology 121: 140, 1986). In a preferred embodiment, antibody-producing cells are immortalized using the cell fusion method (described in Coligan et al., 1991-1997, supra), which is widely employed for the production of monoclonal antibodies. In this method, somatic antibody-producing cells with the potential to produce antibodies, particularly B cells, are fused with a myeloma cell line. These somatic cells may be derived from the lymph nodes, spleens and peripheral blood of humans with circulating La-reactive antibodies, and primed animals, preferably rodent animals such as mice and rats. Mice spleen cells are particularly useful. It would be possible, however, to use rat, rabbit, sheep or goat cells, or cells from other animal species instead.
Specialized myeloma cell lines have been developed from lymphocytic tumours for use in hybridoma-producing fusion procedures (Kohler and Milstein, 1976, supra; Shulman et al., Nature 276: 269-270, 1978; Volk et al., J. Virol. 42(1): 220-227, 1982). These cell lines have been developed for at least three reasons. The first is to facilitate the selection of fused hybridomas from unfused and similarly indefinitely self-propagating myeloma cells. Usually, this is accomplished by using myelomas with enzyme deficiencies that render them incapable of growing in certain selective media that support the growth of hybridomas. The second reason arises from the inherent ability of lymphocytic tumour cells to produce their own antibodies. To eliminate the production of tumour cell antibodies by the hybridomas, myeloma cell lines incapable of producing endogenous light or heavy immunoglobulin chains are used. A third reason for selection of these cell lines is for their suitability and efficiency for fusion.
Many myeloma cell lines may be used for the production of fused cell hybrids, including, e.g. P3X63-Ag8, P3X63-AG8.653, P3/NS1-Ag4-1 (NS-1), Sp2/0-Ag14 and S194/5.XXO.Bu.1. The P3X63-Ag8 and NS-1 cell lines have been described by Köhler and Milstein (1976, supra). Shulman et al. (1978, supra) developed the Sp2/0-Ag14 myeloma line. The S194/5.XXO.Bu.1 line was reported by Trowbridge, J. Exp. Med. 148(1): 313-323, 1978.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually involve mixing somatic cells with myeloma cells in a 10:1 proportion (although the proportion may vary from about 20:1 to about 1:1), respectively, in the presence of an agent or agents (chemical, viral or electrical) that promotes the fusion of cell membranes. Fusion methods have been described (Kohler and Milstein, 1975, supra; 1976, supra; Gefter et al., Somatic Cell Genet. 3: 231-236, 1977; Volk et al., 1982, supra). The fusion-promoting agents used by those investigators were Sendai virus and polyethylene glycol (PEG).
Because fusion procedures produce viable hybrids at very low frequency (e.g. when spleens are used as a source of somatic cells, only one hybrid is obtained for roughly every 1×105 spleen cells), it is preferable to have a means of selecting the fused cell hybrids from the remaining unfused cells, particularly the unfused myeloma cells. A means of detecting the desired antibody-producing hybridomas among other resulting fused cell hybrids is also necessary. Generally, the selection of fused cell hybrids is accomplished by culturing the cells in media that support the growth of hybridomas but prevent the growth of the unfused myeloma cells, which normally would go on dividing indefinitely. The somatic cells used in the fusion do not maintain long-term viability in in vitro culture and hence do not pose a problem. In the example of the present invention, myeloma cells lacking hypoxanthine phosphoribosyl transferase (HPRT-negative) were used. Selection against these cells is made in hypoxanthine/aminopterin/thymidine (HAT) medium, a medium in which the fused cell hybrids survive due to the HPRT-positive genotype of the spleen cells. The use of myeloma cells with different genetic deficiencies (drug sensitivities, etc.) that can be selected against in media supporting the growth of genotypically competent hybrids is also possible.
Several weeks are required to selectively culture the fused cell hybrids. Early in this time period, it is necessary to identify those hybrids which produce the desired antibody, so that they may subsequently be cloned and propagated. Generally, around 10% of the hybrids obtained produce the desired antibody, although a range of from about 1 to about 30% is not uncommon. The detection of antibody-producing hybrids can be achieved by any one of several standard assay methods, including enzyme-linked immunoassay and radioimmunoassay techniques as, for example, described in Kennet et al. (eds) Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyses, pp. 376-384, Plenum Press, New York, 1980 and by FACS analysis.
Once the desired fused cell hybrids have been selected and cloned into individual antibody-producing cell lines, each cell line may be propagated in either of two standard ways. A suspension of the hybridoma cells can be injected into a histocompatible animal. The injected animal will then develop tumours that secrete the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can be tapped to provide monoclonal antibodies in high concentration. Alternatively, the individual cell lines may be propagated in vitro in laboratory culture vessels. The culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration or centrifugation, and subsequently purified.
The cell lines are tested for their specificity to detect the La by any suitable immunodetection means. For example, cell lines can be aliquoted into a number of wells and incubated and the supernatant from each well is analyzed by enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique, or the like. The cell line(s) producing a monoclonal antibody capable of recognizing the target La but which does not recognize non-target epitopes are identified and then directly cultured in vitro or injected into a histocompatible animal to form tumours and to produce, collect and purify the required antibodies.
These antibodies are La specific. This means that the antibodies are capable of distinguishing La from other molecules. More broad spectrum antibodies may be used provided that they do not cross react with molecules in a normal cell.
In one embodiment, the subject antibody is anti-human La monoclonal antibodies, 8G3 and 9A5. (Bachmann et al. Proc Natl Acad Sci USA 83 (20):7770-7774, 1986), anti-human La monoclonal antibody (mAb), La1B5 (Mamula et al. J Immunol 143(9):2923-2928, 1989), anti-human La monoclonal antibodies (Carmo-Fonseca et al. ExpCell Res 185(1):73-85, 1989), anti-human and anti-bovine La monoclonal antibodies, SW1, SW3 and SW5 (Pruijn et al. Eur J Biochem 232(2):611-619, 1995), anti-human and anti-rodent La mAb, La4B6 (Troster et al. J Autoimmunity 8(6):825-842, 1995) or anti-human and anti-murine La mAb, 3B9 (Tran et al. Arthritis Rheum 46(1):202-208, 2002) or derivative, homologue, analogue, chemical equivalent, mutant or mimetic thereof.
Since the monoclonal antibody may be destined for in vivo use, it may be desirable to deimmunise the antibody. The deimmunization process may take any of a number of forms including the preparation of chimeric antibodies which have the same or similar specificity as the monoclonal antibodies prepared according to the present invention. Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. Thus, in accordance with the present invention, once a hybridoma producing the desired monoclonal antibody is obtained, techniques are used to produce interspecific monoclonal antibodies wherein the binding region of one species is combined with a non-binding region of the antibody of another species (Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987). For example, complementary determining regions (CDRs) from a non-human (e.g. murine) monoclonal antibody can be grafted onto a human antibody, thereby “humanizing” the murine antibody (European Patent Publication No. 0 239 400; Jones et al., Nature 321: 522-525, 1986; Verhoeyen et al., Science 239: 1534-1536, 1988; Richmann et al., Nature 332: 323-327, 1988). In this case, the deimmunizing process is specific for humans. More particularly, the CDRs can be grafted onto a human antibody variable region with or without human constant regions. The non-human antibody providing the CDRs is typically referred to as the “donor” and the human antibody providing the framework is typically referred to as the “acceptor”. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e. at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanized antibody, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. Thus, a “humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A donor antibody is said to be “humanized”, by the process of “humanization”, because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDRs. Reference herein to “humanized” includes reference to an antibody deimmunized to a particular host, in this case, a human host.
It will be understood that the deimmunized antibodies may have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions may be made according to Table 1.
Exemplary methods which may be employed to produce deimmunized antibodies according to the present invention are described, for example, in references Richmann et al., 1988, supra; European Patent Publication No. 0 239 400; Chou et al. (U.S. Pat. No. 6,056,957); Queen et al. (U.S. Pat. No. 6,180,370); Morgan et al. (U.S. Pat. No. 6,180,377).
Thus, in one embodiment, the present invention contemplates the use of a deimmunized antibody molecule having specificity for an epitope recognized by a monoclonal antibody to La wherein at least one of the CDRs of the variable domain of said deimmunized antibody is derived from the said monoclonal antibody to La and the remaining immunoglobulin-derived parts of the deimmunized antibody molecule are derived from an immunoglobulin or an analogue thereof from the host for which the antibody is to be deimmunized.
This aspect of the present invention involves manipulation of the framework region of a non-human antibody.
The present invention extends to the use of mutants, analogues and derivatives of the subject antibodies but which still retain specificity for La.
The terms “mutant” or “derivatives” includes one or more amino acid substitutions, additions and/or deletions.
As used herein, the term “CDR” includes CDR structural loops which covers the three light chain and the three heavy chain regions in the variable portion of an antibody framework region which bridge β strands on the binding portion of the molecule. These loops have characteristic canonical structures (Chothia et al., J. Mol. Biol. 196: 901, 1987; Chothia et al., J. Mol. Biol. 227: 799, 1992).
By “framework region” is meant region of an immunoglobulin light or heavy chain variable region, which is interrupted by three hypervariable regions, also called CDRs. The extent of the framework region and CDRs have been precisely defined (see, for example, Kabat et al., “Sequences of Proteins of Immunological Interest”, U.S. Department of Health and Human Services, 1983). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. As used herein, a “human framework region” is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of La.
As used herein, the term “heavy chain variable region” means a polypeptide which is from about 110 to 125 amino acid residues in length, the amino acid sequence of which corresponds to that of a heavy chain of a monoclonal antibody of the invention, starting from the amino-terminal (N-terminal) amino acid residue of the heavy chain. Likewise, the term “light chain variable region” means a polypeptide which is from about 95 to 130 amino acid residues in length, the amino acid sequence of which corresponds to that of a light chain of a monoclonal antibody of the invention, starting from the N-terminal amino acid residue of the light chain. Full-length immunoglobulin “light chains” (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a κ or λ constant region gene at the COOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g. γ (encoding about 330 amino acids).
The term “immunoglobulin” or “antibody” is used herein to refer to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include the κ, λ, α, γ (IgG1, IgG2, IgG3, IgG4), δ, ε and μ constant region genes, as well as the myriad immunoglobulin variable region genes. One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions. In addition to antibodies, immunoglobulins may exist in a variety of other forms including, for example, Fv, Fab, Fab′ and (Fab′)2.
The invention also contemplates the use and generation of fragments of monoclonal antibodies produced by the method of the present invention including, for example, Fv, Fab, Fab′ and F(ab′)2 fragments. Such fragments may be prepared by standard methods as for example described by Coligan et al. (1991-1997, supra).
The present invention also contemplates synthetic or recombinant antigen-binding molecules with the same or similar specificity as the monoclonal antibodies of the invention. Antigen-binding molecules of this type may comprise a synthetic stabilised Fv fragment. Exemplary fragments of this type include single chain Fv fragments (sFv, frequently termed scFv) in which a peptide linker is used to bridge the N terminus or C terminus of a VH domain with the C terminus or N-terminus, respectively, of a VL domain. ScFv lack all constant parts of whole antibodies and are not able to activate complement. Suitable peptide linkers for joining the VH and VL domains are those which allow the VH and VL domains to fold into a single polypeptide chain having an antigen binding site with a three dimensional structure similar to that of the antigen binding site of a whole antibody from which the Fv fragment is derived. Linkers having the desired properties may be obtained by the method disclosed in U.S. Pat. No. 4,946,778. However, in some cases a linker is absent. ScFvs may be prepared, for example, in accordance with methods outlined in Krebber et al. (Krebber et al., J Immunol. Methods 201(1): 35-55, 1997). Alternatively, they may be prepared by methods described in U.S. Pat. No. 5,091,513, European Patent No 239,400 or the articles by Winter and Milstein (Winter and Milstein, Nature 349: 293, 1991) and Plückthun et al. (Plückthun et al., In Antibody engineering: A practical approach 203-252, 1996).
Alternatively, the synthetic stabilized Fv fragment comprises a disulphide stabilized Fv (dsFv) in which cysteine residues are introduced into the VH and VL domains such that in the fully folded Fv molecule the two residues will form a disulphide bond therebetween. Suitable methods of producing dsFv are described, for example, in (Glockshuber et al., Biochem. 29: 1363-1367, 1990; Reiter et al., Biochem. 33: 5451-5459, 1994; Reiter et al., Cancer Res. 54: 2714-2718, 1994; Reiter et al., J. Biol. Chem. 269: 18327-18331, 1994; Webber et al., Mol. Immunol. 32: 249-258, 1995).
Also contemplated as synthetic or recombinant antigen-binding molecules are single variable region domains (termed dAbs) as, for example, disclosed in (Ward et al., Nature 341: 544-546, 1989; Hamers-Casterman et al., Nature 363: 446-448, 1993; Davies & Riechmann, FEBS Lett. 339: 285-290, 1994).
Alternatively, the synthetic or recombinant antigen-binding molecule may comprise a “minibody”. In this regard, minibodies are small versions of whole antibodies, which encode in a single chain the essential elements of a whole antibody. Suitably, the minibody is comprised of the VH and VL domains of a native antibody fused to the hinge region and CH3 domain of the immunoglobulin molecule as, for example, disclosed in U.S. Pat. No. 5,837,821.
In an alternate embodiment, the synthetic or recombinant antigen binding molecule may comprise non-immunoglobulin derived, protein frameworks. For example, reference may be made to (Ku & Schutz, Proc. Natl. Acad. Sci. USA 92: 6552-6556, 1995) which discloses a four-helix bundle protein cytochrome b562 having two loops randomized to create CDRs, which have been selected for antigen binding.
The synthetic or recombinant antigen-binding molecule may be multivalent (i.e. having more than one antigen binding site). Such multivalent molecules may be specific for one or more antigens. Multivalent molecules of this type may be prepared by dimerization of two antibody fragments through a cysteinyl-containing peptide as, for example disclosed by (Adams et al., Cancer Res. 53: 4026-4034, 1993; Cumber et al., J. Immunol. 149: 120-126, 1992). Alternatively, dimerization may be facilitated by fusion of the antibody fragments to amphiphilic helices that naturally dimerize (Plünckthun, Biochem. 31: 1579-1584, 1992) or by use of domains (such as leucine zippers jun and fos) that preferentially heterodimerize (Kostelny et al., J. Immunol. 148: 1547-1553, 1992).
The present invention further encompasses chemical analogues of amino acids in the subject antibodies. The use of chemical analogues of amino acids is useful inter alia to stabilize the molecules such as if required to be administered to a subject. The analogues of the amino acids contemplated herein include, but are not limited to, modifications of side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 2.
Crosslinkers can be used, for example, to stabilize 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH).
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. The method may also be applied as part of a treatment regime.
Without limiting the present invention to any one theory or mode of action, it has been observed that the screening of a neoplastic cell sample for La results in fixation of the antibody or other interactive molecule which is used. This provides a robust screening system. It should also be understood that one may seek to screen for La which has been localised to the cellular cytoplasm and/or which is associated with apoptotic bodies.
Another aspect of the present invention provides a diagnostic kit for a biological sample comprising an agent for detecting La or a nucleic acid molecule encoding La 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 La in the manufacture of a quantitative or semi-quantitative diagnostic kit to detect non-viable neoplastic cells in a biological sample. 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.
The present invention is further described by reference to the following non-limiting examples.
Cell culture media, RPMI-1640, DMEM and Ham's F12, and fetal calf serum (FCS) were all purchased from JRH Biosciences Inc. (KS, USA). Trypsin-EDTA solution, trypan blue, propidium iodide (PI), bovine serum albumin (BSA), hydrocortisone and staurosporine (STS) were obtained from Sigma-Aldrich Co. (MO, USA). Hybond-P membrane (PVDF), ECF™ substrate, L-[U-14C]Leucine, D[U-14C]Glucose and Protein G purification columns were purchased from Amersham Biosciences, (NJ, USA). The miniPERM bioreactor was obtained from Vivascience (Hannover, Germany) and the BCA Protein Reagent Assay from Pierce Biotechnology Inc. (IL, USA). Solvable™ and UltimaGold™ were purchased from PerkinElmer Inc. (MA, USA). H2O2. The anti-poly(ADP-ribose) polymerase (PARP) monoclonal antibody (IgG1 mAb) clone C-2-10 was obtained from Oncogene™ Research Products (EMD Biosciences Inc., CA, USA). The anti-actin (N-20) affinity purified goat polyclonal antibody was purchased from Santa Cruz Biotechnology Inc. (CA, USA). Goat anti-mouse IgG alkaline phosphatase (AP)-conjugated antibody and rabbit anti-goat IgG AP-conjugated antibody were purchased from Johnson Laboratories (USA). The anti-La/SS-B IgG mAb 3B9 cell line (Tran et al., 2002), prepared by Dr M. Bachmann (Oklahoma Medical Research Foundation, OK, USA), was a generous gift from Dr T. P. Gordon (Department of Immunology, Allergy, and Arthritis, Flinders Medical Centre, SA, Australia). The irrelevant 1D4.5 mAb Sal5 cell line, prepared by Dr L. K. Ashman (Medical Science Building, University of Newcastle, NSW, Australia), was a kind gift from Dr S. McColl (School of Molecular Biosciences, University of Adelaide, SA, Australia). These mAbs were affinity-purified on Protein G purification columns. Purified 3B9 and Sal5 mAbs were conjugated to fluorescein isothiocyanate (FITC) as described by manufacturer's instructions Sigma-Aldrich Co. (MO, USA). Anti-mouse IgG antibody conjugated to Alexa488 and 7-AAD were purchased from Molecular Probes® (Invitrogen, USA). Lymphoprep™ was purchased from Axis-Shield PoC AS (Oslo, Norway). Etoposide and vincristine for injection (Pfizer Inc., NY, USA) and cyclophosphamide, cisplatin were purchased.
The tumour cell lines, Jurkat (ATCC# TIB-152, acute T cell leukemia), EL4 (ATCC# TIB-39, mouse T-lymphocyte lymphoma), U-937 (ATCC# CRL-1593.2, monocytic leukemia) and Raji (ATCC# CCL-86, Burkitt's lymphoma) were routinely grown as suspension cultures in RPMI-1640 containing 5 FCS and passaged every 48-72 h at 1:4 dilution. The U20S osteosarcoma cell line (ATCC# HTB-96), SAOS-2 osteosarcoma cell line (ATCC# HTB-85) and HeLa cervical adenocarcinoma (ATCC# CCL-2) were routinely cultured in DMEM containing 5% FCS and passaged every 48-72 h after detachment using trypsin-EDTA solution. The squamous cell carcinoma cell line, SCC-25 (ATCC# CRL-1628), was cultured in a 1:1 mixture of DMEM and Ham's F12 medium containing supplemented with 400 ng/mL hydrocortisone and 10% FCS and passaged after detachment using trypsin-EDTA solution. Fresh blood from normal volunteers was subjected to Lymphoprep® separation to isolate peripheral blood monocytic cells (PBMC). PBMC were cultured overnight in RPMI-1640 and 5% FCS to separate suspension and adherent cells. The cells in suspension were defined as lymphocytes because more than 70% were CD3+ whereas the adherent cells were defined as monocytes because more than 70% were CD14+. Mouse thymocytes were obtained from mice. Finally, buccal cells were isolated from the gum lining of healthy volunteers as described.
Apoptosis in all cultures was induced by incubation of these cells in culture media described above and in the presence of specified concentrations of cytotoxic chemotherapy drugs (see Figure legends).
Direct immunofluorescence staining was performed using 1-2×105 cells at 106 cells/mL for 30 min at room temperature in PBS containing 0.1% BSA and 5 μg/mL of FITC-conjugated mAb. Cells were thoroughly washed using PBS and centrifugation at 450×g. Cells were resuspended in PBS containing 0.5 μg/mL PI and acquired immediately by a Becton-Dickinson FACScan™ flow cytometry system (BD Biosciences, CA, USA). Positive staining using the mAb-FITC conjugates was determined in comparison to FITC-conjugated isotype control mAb detected using the FL-1 channel (530-nm filter). Indirect immunofluorescent staining was performed using purified mouse antibodies followed by anti-mouse IgG conjugated to Alexa488 detected using the FL-1 channel (530-nm filter). Cell viability was assessed by the exclusion of PI detected using the FL-2 (585-nm filter) or the exclusion of 7-AAD detected using the FL-3 (>650 nm filter). Flow cytometry data was analysed using WinMDI v 2.8 (Scripps Research Institute, CA, USA). Unless otherwise specified, no gating was performed in any of the analysis shown in this paper.
SDS-PAGE was performed as per manufacturer instructions using the Hoefer® Mighty Small II SE 250 electrophoresis system (Amersham Biosciences, NJ, USA) under reducing condition using 12% resolving polyacrylamide gel as per Laemmli (Nature 227: 680-685, 1970). Transfer of polyacrylamide gel onto Hybond-P membrane was carried out as per manufacturer instruction using the TE 22 Mini Tank Transfer Unit (Amersham Biosciences, NJ, USA). Blotting was performed as per standard procedure using 3B9 or anti-PARP mAbs followed by AP-conjugated anti-mouse IgG mAb or anti-actin polyclonal antibody (pAb) followed by AP-conjugated anti-goat IgG mAb. All blots were developed using the ECF™ substrate and scanned using the FluorImager™ 595 (Molecular Dynamics, Amersham Biosciences, NJ, USA) with 488 nm excitation laser and emission collected using 570 nm filter.
Radioligand Binding Study with Anti-La mAb
Sal5 isotype and 3B9 mAb were labelled with 14C by incubating the hybridoma cells (35 million cells) in 35 ml RPMI-1640 containing 10% FCS in the production module of miniPERM bioreactor and 400 ml of RPMI-1640 containing 10% FCS and 250 μCi of D-[U-14C]glucose and 250 μCi of L[U-14C]Leucine in the nutrient module. The bioreactor was incubated in 5% CO2 humidified air at 37° C. on a bottle-rotating device for 5 days. The medium in the production chamber was collected for antibody purification using protein G purification columns as per manufacturer instructions. Radioactivity of purified antibodies (10 μl sample) was counted in UltimaGold™ scintillation liquid (1 ml) for 20 min. using Tri-Carb 3100 β-counter (Packard, regularly calibrated using supplied 14C standards). Protein concentration was determined using BCA Protein Reagent Assay as per manufacturer instructions. The specific radioactivity of 14C-Sal5 and 14C-3B9 was 120.3 and 130.8 dpm/μg, respectively.
Saturation binding study was performed by incubating apoptotic EL4 cells (5×105 cells) at 24 h after treatment with etoposide and cyclophosphamide with increasing concentration of 14C-3B9 in the absence (total) or presence (non specific) of 50-fold molar excess of unlabelled 3B9. After 30 min, cells were washed thoroughly using PBS and radioactivity was measured using the β-counter as described above. Specific binding was calculated as the difference between total and non-specific binding and plotted as a function of concentration of 14C-3B9. Competition binding curve was constructed by incubating apoptotic EL4 cells with 14C-3B9 in the presence of increasing concentrations of unlabelled 3B9. Radioactivity was measured as described earlier and plotted as a function of unlabelled 3B9 concentration. Association time course was performed by incubation of apoptotic EL4 cells with 14C-3B9 in the absence or presence of 50-fold molar excess of unlabelled 3B9 for the specified times. Samples were washed and radioactivity was measure and specific binding was plotted as function of time.
EL4 cells, established from a lymphoma induced in a C57BL/6 mouse (Gorer, British Journal of Cancer 4: 372-379, 1950), were used to establish subcutaneous tumour implants in 6-8 weeks old C57BL/6 mice. Mice were housed and treated as per protocols approved by the Animal Ethics Committee at The University of Adelaide. Briefly, 105 EL4 cells were injected subcutaneously in the right flank of each mouse. Once the tumour reached 1 cm diameter, mice were randomly divided into two groups one of which received intraperitoneal injection of etoposide and cyclophosphamide to achieve a dose of 76 mg/kg and 100 mg/kg, respectively (time 0). These two groups (untreated or treated) were used for the studied described below.
Treated mice received a second injection of etoposide and cyclophosphamide at 24 h after the first injection while untreated mice were left untreated. After 24 h (i.e. time point 48 h), all mice were euthanised, whole blood was obtained by cardiac puncture and EL4 tumours were excised from these sacrificed mice. Excised tumours tissue was disrupted to produce a single cell suspension, washed with PBS and used for immunofluorescent staining with 3B9 and PI and flow cytometry analysis as described earlier.
In other studies, treated mice that received the first chemotherapy injection at time 0, received a second injection of etoposide and cyclophosphamide at 24 h and an intravenous injection of specified amount of 14C-3B9 or 14C-Sal5. Untreated mice only received the intravenous injection of 14C-3B9 or 14C-Sal5. All mice were euthanised, whole blood was obtained by cardiac puncture and EL4 tumours as well as other organs were collected for radioactivity measurement. Serum and organs were solubilised using 1 ml of Solvable™ for 2 h at 50° C., decolourised using 100 μl of H2O2 (30%). UltimaGold™ scintillation liquid (1 ml) was added and samples were counted for 10 min. using the β-counter.
Cytofluographic analysis of cultured EL4 thymic lymphoblastic lymphoma cells, which were stained with the DNA binding dye 7-AAD as a test of membrane integrity, indicated that fewer than 10% of the cultured cells were spontaneously apoptotic and did not bind La-specific 3B9 mAb (
After cell permeabilisation, it was found that significantly higher levels of 3B9 bound to the malignant EL4 lymphoma cell line than to its normal murine counterpart cell type of the thymocyte (
La-specific mAb, which was biosynthetically labelled with the radioisotope carbon-14 (14C-3B9), bound to apoptotic EL4 cells after they were treated with cyclophosphamide and etoposide in a specific and saturable manner. The concentration of agent required to reach half-maximal saturation of one million apoptotic cells was 18 nM and maximal binding was ˜7500 femtomole/million apoptotic cells (
Single cell suspensions were prepared from tumour explants of EL4 tumour-bearing mice, which had been treated or not with cytotoxic chemotherapy. As illustrated in
La-Specific mAb Targets EL4 Tumours In Vivo Especially after Cytotoxic Chemotherapy
EL4 tumour-bearing mice were used to demonstrate that 3B9 La-specific mAb targeted a tumour mass in vivo and that the 3B9 targeting was enhanced 48 hours after the mice were treated with cytotoxic chemotherapy. As illustrated graphically in
The biodistribution studies of 14C-3B9 in EL4 tumour-bearing mice were extended to include lower doses of the radiolabelled agent. Again, as shown in
Using a murine tumour model, La-specific in vitro labelling of dead malignant cells and La-specific in vivo targeting of malignant tumours, which were both significantly augmented by the use of cytotoxic chemotherapy, have been described. Hence, it was sought to determine if similar findings applied to human malignant cells in vitro. First, it was found that in comparison to the counterpart normal cell type, La was overexpressed in the Jurkat T cell leukemia line (cf. CD3+ lymphocytes), the U-937 monocytic leukemia line (cf. CD14+ monocytes) and the oropharyngeal squamous cell carcinoma line SSC-25 (cf. normal buccal mucosal cells) (
Oncomine™ is a cancer-specific database containing microarray data from 962 studies of which 209 were analysed. The database contains 14,177 microarrays from 35 cancer types (information publicly available at the website www.oncomine.org). Several cancer signatures have been deduced from large scale analysis of data held in the database (Hampton, Jama 292(17): 2073, 2004; Rhodes et al., Proc Natl Acad Sci USA 101(25): 9309-14, 2004a; Rhodes et al., Neoplasia 6(1): 1-6, 2004b; Rhodes and Chinnaiyan, Nat Genet. 37 Suppl: S31-7, 2005; Rhodes et al., Nat Genet. 37(6): 579-83, 2005). 209 studies in the database as described below were analysed in order to investigate certain malignancy signatures, which may provide useful targets for the present invention
In the catalogue of the database Oncomine™ on www.oncomine.org, the tissue of interest was selected and only analysed data are shown. Studies were viewed using the Advanced Analysis module only for overexpressed genes and enrichment for these genes was achieved using the following options: (1) InterPro for analysis of motifs, (2) Gene Ontogeny (GO) molecular function for the analysis of function and (3) GO cellular component for cellular compartmentation analysis. Two parameters were used to describe the gene sets deduced from the above analysis: (a) Odds Ratio and (b) P-value. Only the groups of interest and genes with a P-value lower than 1E-4 were considered significant for reporting herein.
As shown in Table 4 and Table 5, the ribonucleoprotein (RNP-1) motif was identified, which comprises the RNA recognition motif (RRM), to be at higher odds of being associated with a cancer signature compared to the nucleus as a cellular component. Similarly, the RNA binding gene set, which was enriched as a cellular function, was also at high odds of being associated with a cancer signature. Finally and more importantly, nucleolar and heteronuclear ribonucleoprotein (hnRNP) components appeared to have very high odds as gene sets in association with a cancer signature.
Consequently, components with the RNP-1 motif generally and hnRNP complex specifically are suitable targets for the method of the present invention. This recommendation is justified by (1) association of hnRNP with cancer at the gene expression level (mRNA data reported herein), (2) the relative abundance of this protein and its correlation with cancer and cancer progression (see review Carpenter et al., Biochimica et Biophysica Acta 1765(2): 85-100, 2006), and (3) the reorganisation of the hnRNP network during apoptosis into the HERDS (Biggiogera et al., Biologie Cellulaire 96(8): 603-15. 2004) and the accessibility of the hnRNP network for detection with antibodies. Nucleolar proteins generally and specifically nucleophosmin represent suitable targets for this strategy. This is justified by the data shown in the analysis of the Oncomine™ data base as well as the literature regarding nucleolar function in cancer (Maggi and Weber, Cancer Investigation 23(7): 599-608, 2005) and, in particular, nucleophosmin as putative-proto-oncogene (Grisendi et al., Nature Reviews. Cancer 6(7): 493-505, 2006).
The suppliers of the materials are identified in brackets after each material. Cell culture media, RPMI-1640, DMEM and Ham's F12, and fetal calf serum (FCS) (JRH Biosciences Inc., Lenexa, Kans.); Trypsin-EDTA solution, trypan blue, propidium iodide (PI), bovine serum albumin (BSA), BCIP/NBT premixed substrate solution for alkaline phophatase (AP), hydrocortisone, monodansylcadaverine (MDC) and staurosporine (STS) and mouse anti-human β-tubulin mAb (TUB 2.1) (Sigma-Aldrich Co., St. Louis, Mo.). Hybond-P membrane (PVDF) and protein G purification columns (Amersham Biosciences, Piscataway, N.J.). BCA Protein Reagent Assay (Pierce Biotechnology Inc., Rockford, Ill.). Anti-poly(ADP-ribose) polymerase (PARP) monoclonal antibody (mAb) clone C-2-10 and anti-proliferating cell nuclear antigen (PCNA) mAb clone PC10 (Oncogene Research Products, Cambridge, Mass.). Trichostatin A (TSA), anti-phospho-histone H2AX (Ser139) clone JBW301 biotin-conjugated mAb and anti-human H2A polyclonal antibody (Millipore Inc., Billerica, Mass.). Anti-β fodrin mAb (Chemicon International, Temecula, Calif.). Anti-β actin affinity-purified rabbit polyclonal antibody, Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG and AP-conjugated goat anti-rabbit IgG antibodies (Rockland, Gilbertsville, Pa.). The anti-La/SS-B 3B9 mAb hybridoma is a murine IgG2a autoantibody, which is crossreactive with human La and which was prepared by Dr M. Bachmann (Oklahoma Medical Research Foundation, OK, USA), was a kind gift from Dr T. P. Gordon (Department of Immunology, Allergy and Arthritis, Flinders Medical Centre, SA, Australia). The isotype control Sal5 (1D4.5) mAb hybridoma, prepared by Dr L. K. Ashman (Medical Science Building, University of Newcastle, NSW, Australia), was kindly supplied by Dr S. McColl (School of Molecular Biosciences, University of Adelaide, SA, Australia). The mAb were affinity-purified using protein G columns and FITC-conjugates were prepared according to the manufacturer's instructions (Sigma-Aldrich Co., St. Louis, Mo.). Anti-human nucleolin mAb, anti-human nucleophosmin (NPM) mAb, anti-mouse IgG antibody conjugated to Alexa488, streptavidin-Alexa488, streptavidin-PE, 7-amino-actinomycin D (7-AAD) and biotinylated cadaverine (Invitrogen, Carlsbad, Calif.). Lymphoprep (Axis-Shield PoC AS, Norton, Mass.). Etoposide (Pfizer Inc., NY, USA), cyclophosphamide and cisplatin (Bristol-Myers Squibb Company, Princeton, N.J.) and gemcitabine (Eli Lilly, Indianapolis, Ind.) were obtained from the Royal Adelaide Hospital Cytotoxics Pharmacy (Adelaide, SA, Australia).
Suspension cultures of Jurkat and U-937 leukemia cell lines were maintained in RPMI-1640 containing 5% FCS and passaged by splitting at 1:10 every 72 h. Cultures of adherent MDA-MB-231, MCF-7, PC-3, LNCaP and A549 cancer cell lines were maintained in RPMI-1640 containing 5% FCS and passaged every 48-72 h at a 1:4 dilution after detachment with trypsin-EDTA solution. Adherent sub-confluent cultures of the pancreatic adenocarcinoma cell line, PANC-1, were routinely cultured in RPMI-1640 containing 10% FCS and passaged as above. The squamous cell carcinoma cell line, SCC-25, was cultured in a 1:1 mixture of DMEM and Ham's F12 medium supplemented with 400 ng/mL hydrocortisone and 5% FCS and passaged after detachment using trypsin-EDTA solution. Peripheral blood mononuclear cells (PBMC) were isolated from fresh heparinized blood obtained from normal healthy volunteer donors using Lymphoprep separation and cultured overnight in RPMI-1640 containing 5% FCS to separate adherent cells from those remaining in suspension. Cytofluographic analysis indicated that >70% of both suspension and adherent cells were CD3+ and CD14+, respectively. Hence, CD3- and CD14-enriched PBMC preparations will be described as peripheral blood lymphocytes and monocytes, respectively. Clonetics conditioned cultures of primary human cells were maintained according to the manufacturer's instructions (Cambrex Corporation, East Rutherford, N.J.) and included cultures of Human Mammary Epithelial Cells (HMEC), Prostate Epithelial Cells (PrEC) and Normal Human Bronchial Epithelium (NHBE). Finally, buccal cells were isolated from the gum lining of normal healthy volunteer donors as described.
Apoptosis or cell death was induced by adding cytotoxic drugs to culture media at the specified concentrations. In some experiments, cells were starved by serum deprivation. In other experiments, TSA used at the specified concentrations was prepared from 1 mg/mL stock solution in absolute ethanol. Control (untreated) cells had ≧90% viability determined by PI or trypan blue staining (data not shown). Viable cells were fixed and permeabilized by incubating cells at 5×106 cells/mL in 2% w/v paraformaldehyde solution in PBS (150 mM sodium phosphate and 150 sodium chloride, pH 7.2) for 10 min. followed by 1:10 dilution in absolute methanol (at −20° C.) for 1-3 min. before a final wash with PBS.
Indirect immunofluorescence staining was performed using purified mouse antibodies at 5 μg/mL for 30 min. at room temperature (RT) in PBS followed by Alexa488-conjugated anti-mouse IgG at 2 μg/mL for 30 min. at RT in PBS. Fluorescence was detected using the FL-1 channel (530 nm filter). Cell viability was assessed by the exclusion of PI (0.5 μg/mL) and detected using the FL-2 channel (585 nm filter) or by the exclusion of 7-AAD (2 μg/mL for 15 min. at RT) and detected using the FL-3 channel (>650 nm filter). Staining for γH2AX was performed using 0.2 μg/mL of anti-γH2AX-biotin for 30 min. at RT followed by 2 μg/mL of streptavidin-PE or streptavidin-Alexa488. Staining of polyclonal antibodies (anti-actin or anti-H2A) was performed using 5 μl g/mL of antibody solution for 30 min. at RT. Control incubations were performed using protein G purified rabbit IgG from normal rabbit serum (IMVS, SA, Australia). Cells were washed then incubated (30 min. at RT) with 2 μg/mL of FITC-conjugated anti-rabbit IgG antibody, which was detected using the FL-1 channel. Samples were acquired immediately by a Becton-Dickinson FACScan™ flow cytometry system (BD Biosciences, San Jose, Calif.). Acquisition was standardized to 10,000 events or in some cases standardized to a set time for acquisition (in seconds) to allow comparison of cell counts in different incubations. Flow cytometry data were analyzed using WinMDI v2.8 (Scripps Research Institute, La Jolla, Calif.). Unless otherwise specified, no gating was performed in any of the reported analyses. Specific binding of antibodies was calculated as the difference in mean fluorescence intensities (MFI) between the test antibody and the control isotype antibody and expressed as the Net MFI±standard error of the mean (SEM), which was calculated from replicate incubations (n>2).
Cell lysates were prepared in SDS lysis solution (2% w/v SDS, 10% v/v glycerol and 62.5 mM Tris-HCl) and protein concentration was determined using BCA protein reagent assay kit according to the manufacturer's instructions. Bromophenol blue (0.05% w/v) and β-mercaptoethanol (5% v/v) were added to lysates after BCA assay measurement. SDS-PAGE was performed according to the manufacturer's instructions with the Hoefer Mighty Small II SE 250 electrophoresis system (Amersham Biosciences, Piscataway, N.J.) under reducing conditions using 12% resolving polyacrylamide gel as per Laemmli's method. The transfer of the polyacrylamide gel to Hybond-P membrane was carried out according to the manufacturer's instructions using the TE 22 Mini Tank Transfer Unit (Amersham Biosciences, Piscataway, N.J.). Immunoblotting was done using standard methods in which staining with 3B9 was followed by staining with AP-conjugated anti-mouse IgG mAb (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.) or anti-actin (N-20) affinity purified goat polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) followed by AP-conjugated anti-goat IgG mAb (Jackson ImmunoResearch). Some blots were developed using the BCIP/NBT premixed solution as specified by the manufacturer and analyzed using GelPro Analyzer v3.1. (Media Cybernetics Inc., Silver Spring, Md.).
Other blots were developed using the ECF™ substrate (Amersham Biosciences, Piscataway, N.J.) and scanned using the FluorImager™ 595 (Molecular Dynamics, Amersham Biosciences, Piscataway, N.J.) with a 488 nm excitation laser and the emissions were collected using a 570 nm filter.
Transglutaminase 2 (TG2)-mediated protein-protein crosslinking in apoptotic cells was investigated using a modification of a previously described method. Briefly, cells were resuspended at 1×106 cells/mL in PBS containing 1% Triton X-100, 0.2 μg/mL sulforhodamine 101 and 0.1 μM SytoxGreen. Samples were vortexed and incubated for 5 min. before analysis by flow cytometry where fluorescence from Sytox Green and sulforhodamine were detected using FL-1 and FL-3, respectively. The degree of crosslinking in apoptotic cells was calculated as the ratio of the MFI of sulforhodamine staining in apoptotic cells to the MFI in control cells (protein crosslinking ratio). In other experiments, cells were incubated with PBS or 1% Triton X-100 in PBS for 10 min. at RT with intermittent vortexing.
To test inhibition of protein crosslinking, cultured cells were incubated with increasing concentrations of the competitive TG2inhibitor, monodansylcadaverine (MDC). MDC was dissolved in DMSO at 25 mM and then diluted in culture media before it was added to cell cultures at the specified concentrations for the specified duration. Incorporation of the TG2 substrate, biotinylated cadaverine, in cellular proteins was used as an index of TG2 activity. Cells were incubated with increasing concentrations of cisplatin with or without 100 μM cadaverine-biotin (from 25 mM stock solution in DMSO). Cells were collected after 48 h, washed extensively with PBS and incubated with streptavidin-Alexa488 for cytofluographic analysis. In similar assays, cadaverine-biotin labelled cells were lysed for SDS-PAGE with or without prior immunoprecipitation using 3B9 mAb. PAGE gels were transferred to PVDF membranes and probed using streptavidin-AP to visualise both the total pool of TG2 substrates and 3B9-reactive TG2 substrates.
Cells were stained using immunofluorescence methods described above and then spotted onto glass slides using the cytospin method. The prepared slides were mounted with coverslips using non-fluorescence mounting medium (Dako, Carpinteria, Calif.). Slides were analyzed using a BioRad Olympus Confocal microscope with appropriate filters and under constant conditions of laser voltage, iris aperture and photomultiplier tube amplification.
Jurkat cells were incubated in the presence or absence of 20 μg/mL cisplatin and pelleted 3 h after treatment to prepare soluble nuclear and chromatin fractions as described. Samples of these fractions were fractionated using 12% SDS-PAGE for immunoblotting as described above. Membranes were probed with 1 μg/mL 3B9, 2 μg/mL anti-H2A antibodies or 0.2 μg/mL anti-γH2AX-biotin followed by the appropriate AP-conjugated secondary antibodies or strepatividin-AP. The presence of H2A in chromatin and not soluble fractions was used to assess the quality of preparation of the chromatin fractions.
Chamber slides were seeded with PANC-1 cells and incubated overnight in RPMI-1640 containing 10% FCS before replacement with medium containing 20 μg/mL cisplatin alone or in combination with TSA. After 3 h, cells were fixed and permeabilized using paraformaldehyde and methanol as described above. Cells were washed and blocked with 5% BSA solution in PBS then stained with 10 μg/mL 3B9 followed by 2 μg/mL Alexa488-conjugated anti-mouse IgG antibody. Cells were washed and incubated with 0.2 μg/mL anti-γH2AX-biotin followed by 2 μg/mL streptavidin-PE. Finally, cells were washed and coverslips were mounted using non-fluorescent mounting media (Dako, Glostrup, Denmark). Alexa488 and PE were excited using a 488 nm laser and fluorescence was detected using filters 1 and 2, respectively, of an Olympus fluorescence microscope. Samples stained separately with 3B9 or γH2AX showed that there was no fluorescence bleeding between the two different filters using the specified fluorophores (data not shown).
Oncomine is a database of microarray data, which holds data from 962 studies of which 209 were analyzed. The database contains 14,177 microarrays from 35 cancer types (information publicly available at the website www.oncomine.org). Several cancer signatures have been deduced from large scale analysis of data in the database (24-28). We analyzed the 209 studies using the Advanced Analysis module limiting results to overexpressed genes only and gene enrichment was selected using the following options: (1) InterPro for analysis of motifs in the overexpresed genes, (2) Gene Ontogeny (GO) molecular function for analysis of functions of overexpressed genes, and (3) GO cellular component for cellular compartmentation of the overexpressed genes. Two paramaters were used to describe the gene sets deduced from the above analysis (a) Odds Ratio and (b) P-value, which were provided by the database.
Statistical comparisons were performed using GraphPad Prism v4 (GraphPad Software, San Diego, Calif.). Generally, two-way analysis of variance (ANOVA) was used to deduce significant differences among the results. The Bonferroni post-test comparison was used to report P values. P values are denoted as: *, P<0.05; **, P<0.01; ***, P<0.001.
As
Synchronisation of Jurkat cells by double-thymidine block demonstrates that maximal binding of Apomab, and by inference maximal expression of La, occurs during S phase of the cell cycle (
Cytofluographic analysis of etoposide-treated Jurkat cells showed a time-dependent loss of cell membrane integrity, which was measured by binding of propidium iodide (PI) to intracellular nucleic acids. Despite the loss of cell membrane integrity, dead and/or dying cells did not accumulate Sal5 mAb, which is an isotype control antibody of irrelevant specificity (upper panel,
Nonetheless, significant differences in binding levels of the mAb specific for the different intracellular antigens were observed after induction of Jurkat cell apoptosis with the DNA-damaging drug, cisplatin. Antigen-specific mean fluorescence intensity (MFI) of cisplatin-treated Jurkat cells was compared with the MFI of control untreated Jurkat cells, which were fixed and permeabilized (
Further evidence for induction of 3B9 binding by DNA-damaging agents was obtained by comparing serum-deprivation with cisplatin-treatment of various malignant cell types. In MCF-7, MDA-MB-231, A549 and PC-3 cancer cell lines, significantly greater 3B9-specific binding to the dead malignant cells was observed after use of the DNA-damaging agent (
As
Further evidence for the involvement of La in the DNA-damage response was obtained using chemotherapy-resistant PANC-1 cells. Neither gemcitabine (
As illustrated in
The same dose of cisplatin is used to treat Jurkat cells that are subsequently permeabilised and fixed and stained with mAb specific for a number of nuclear antigens (
Resistance of apoptotic cells to the non-ionic detergent, Triton X-100, has been reported and correlates with the activity of tissue transglutaminase or transglutaminase 2 (TG2), which mediates protein-protein crosslinking. It was found that during apoptosis, the proportion of 7-AAD+ cells and the level of 3B9 binding to these cells were comparable irrespective of Triton X-100 treatment (
Next, it was hypothesized that inhibition of TG2 activity reduces 3B9 binding to apoptotic cells because of reduced protein-protein crosslinking. First, the effect of increasing doses of cisplatin on the activity of TG2 in Jurkat cells was investigated using the TG2 substrate cadaverine-biotin. Incorporation of cadaverine-biotin into Jurkat cells showed a dose-dependent relationship to cisplatin concentration (
Protein Crosslinking Covalently Attaches 3B9 to the Interior of Leaky Dead Malignant Cells During Apoptosis
Using etoposide or cisplatin, Jurkat cells were induced to undergo apoptosis in the presence of 3B9 or its Sal5 isotype control mAb. 3B9 binding to apoptotic Jurkat cells was detectable even after stripping of the Jurkat cells with Triton X-100, which suggests that the protein cross-linking process in apoptotic cells includes the cross-linking of bound antigen-specific mAb (
The EL4 murine T-lymphoblastic lymphoma cell line was obtained from American Type Cell Culture (TIB-39) and murine thymocytes were freshly prepared from 6-8 week old C57BL/6 mice. Cells were routinely cultured in RPMI-1640 containing 5% FCS (JRH Biosciences Inc., Lenexa, Kans.) and passaged every 48-72 h at 1:4 dilution. 3B9 and the relevant control (Sal5) were prepared as described previously (first paper). Fluorescein isothiocyanate (FITC) conjugated of these agents was prepared as described by manufacturer's instructions (Sigma-Aldrich Co., St. Louis, Mo.).
Apoptosis in cultures was induced by adding 20 μg/mL etoposide (Pfizer Inc., NY, USA) and 20 μg/mL cyclophosphamide (Bristol-Myers Squibb Company, Princeton, N.J.) to the culture media. Data presented here from control (untreated cells) originated from samples with higher than 90% viability as determined by PI or trypan blue (Sigma-Aldrich Co., St. Louis, Mo.) staining (data not shown). Permeabilization of viable cells was performed by 10 min. incubation in 2% w/v paraformaldehyde solution in PBS (150 mM sodium phosphate and 150 mM sodium chloride, pH 7.2) at 5×106 cells/mL, which was diluted 1:10 with −20° C.-cold absolute methanol (1-3 min.) before washing with PBS.
Direct immunofluorescence staining was performed using 1-2×105 cells at 106 cells/mL for 30 min. at room temperature (RT) in PBS containing 0.1% bovine serum albumin (BSA, Sigma-Aldrich Co., St. Louis, Mo.) and 5 μg/mL of FITC-conjugated 3B9 or FITC-conjugated Sal5 isotype control mAb. Indirect immunofluorescence staining was performed using purified mouse antibodies (5 μg/mL for 30 min. at RT in PBS) followed by anti-mouse IgG conjugated to Alexa488 (Invitrogen, Carlsbad, Calif.) (2 μg/mL for 30 min. at RT in PBS) and detected using the FL-1 channel (530-nm filter). Cell viability was assessed by the exclusion of PI (0.5 μg/mL) and detected using the FL-2 channel (585-nm filter) or by the exclusion of 7-AAD (Invitrogen, Carlsbad, Calif.) (2 μg/mL for 15 min. at RT) and detected using the FL-3 channel (>650 nm filter). Samples were acquired immediately by Becton-Dickinson FACScan™ flow cytometry system (BD Biosciences, San Jose, Calif.). Acquisition was standardized to 10,000 events. Flow cytometry data was analyzed using WinMDI v 2.8 (Scripps Research Institute, La Jolla, Calif.). Unless otherwise specified, no gating was performed in any of the reported analyses. Specific binding of antibodies was calculated as the difference in the mean fluorescent intensity (MFI) from the test antibodies and that from control isotype antibody and was expressed as the Net MFI±standard error of the mean (SEM) calculated from replicate incubations (n>2).
Cell lysates were prepared in SDS lysis solution (2% w/v SDS, 10% v/v glycerol and 62.5 mM Tris-HCl) and protein concentration was determined using BCA protein reagent assay kit according to the manufacturer's instructions (Pierce Biotechnology Inc., Rockford, Ill.). Bromophenol blue (0.05% w/v) and β-mercaptoethanol (5% v/v) (Sigma-Aldrich Co., St. Louis, Mo.) were added to lysates after BCA assay measurement. An amount of 12 μg of lysate was added to each lane for SDS-PAGE. SDS-PAGE was performed according to the manufacturer's instructions with the Hoefer® Mighty Small II SE 250 electrophoresis system (Amersham Biosciences, Piscataway, N.J.) under reducing condition using 12% resolving polyacrylamide gel as per Laemmli's method. The transfer of the polyacrylamide gel to Hybond-P membrane was carried out according to the manufacturer's instructions using the TE 22 Mini Tank Transfer Unit (Amersham Biosciences, Piscataway, N.J.). Immunoblotting was done using standard methods in which staining with 3B9 was followed by staining with alkaline phosphatase (AP)-conjugated anti-mouse IgG mAb (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.). Blots were developed using the BCIP/NBT premixed solution as specified by manufacturer (Sigma-Aldrich Co., St. Louis, Mo.) and analyzed using GelPro Analyzer v3.1 (Media Cybernetics Inc., Silver Spring, Md.).
3B9 and matched control were labelled with 14C by incubating the hybridoma cells (35 million cells) in 35 mL RPMI-1640 containing 5% FCS in the production module of miniPERM bioreactor (Vivascience GmbH, Hannover, Germany) and 400 mL of RPMI-1640 containing 5% FCS and 250 μCi of D[U-14C]glucose and 250 μCi of L-[J-14C]leucine (Amersham Biosciences, Piscataway, N.J.) in the nutrient module. The bioreactor was incubated in 5% CO2 humidified air at 37° C. on a bottle-rotating device for 5 days. The medium in the production chamber was collected for purification using Protein G purification columns. Radioactivity of purified antibodies (10 μL sample) was counted in UltimaGold™ scintillation liquid (1 mL) for 20 min using Packard Tri-Carb 3100 n-counter (PerkinElmer Inc., Wellesley, Mass.) regularly calibrated using supplied 14C standards. Protein concentration was determined using BCA protein reagent assay. The specific radioactivity of 14C-control and 14C-3B9 was 120.3 and 130.8 dpm/μg, respectively.
Saturation binding assays were performed by incubating apoptotic EL4 cells (5×105 cells) after 48 h treatment with etoposide and cyclophosphamide with increasing concentrations of 14C-3B9 in the presence (to measure non-specific binding) or absence (to measure total binding) of a 50-fold molar excess of unlabelled 3B9. After 30 min., cells were washed thoroughly using PBS and radioactivity was measured using the □-counter as described above. Specific binding was calculated as the difference between total and non-specific binding and plotted as a function of the concentration of 14C-3B9. Competition binding assays were carried out by incubating apoptotic EL4 cells with 100 nM 14C-3B9 in the presence of increasing concentrations of unlabelled 3B9. Radioactivity was measured as described above and plotted as a function of unlabelled 3B9 concentration. Association assays were performed by incubation of apoptotic EL4 cells with 100 nM 14C-3B9 in the presence or absence of a 50-fold molar excess of unlabelled 3B9 for the specified times. Samples were washed, radioactivity was measured and specific binding was plotted as function of time. Dissociation assays were performed after incubation of apoptotic EL4 cells with 100 nM 14C-3B9 in the absence or presence of 50-fold molar excess of unlabelled 3B9 for 30 min. at RT. Cells were washed, incubated at 37° C. in PBS and samples were removed at the specified times. These samples were washed with PBS, radioactivity was measured and specific binding was plotted as function of time.
The EL4 thymic lymphoblastic lymphoma is a robust model of apoptosis, which is induced by the cytotoxic drugs, cyclophosphamide and etoposide (14). Subcutaneous EL4 tumour implants were established in 6-8 week old syngeneic C57BL/6 mice where 106 EL4 cells were injected subcutaneously in the right flank of each mouse. Mice were housed and treated as per protocols approved by the Animal Ethics Committee at The University of Adelaide and the Animal Ethics Committee at the Institute of Medical and Veterinary Sciences (IMVS). Once the tumour reached 1 cm diameter, mice were randomly divided into control or treatment groups.
Treatment comprised intraperitoneal injections of cytotoxic chemotherapy given in one of the following five regimens of reducing dose intensity. The respective cyclophosphamide and etoposide doses are indicated and are mg/kg. (i) full dose as published: 100, 76 at 0 h and 24 h; (ii) half dose 2 d: 50, 38 at 0 h and 24 h; (iii) half dose 1 d: 50, 38 at 0 h; (iv) quarter dose 2 d: 25, 19 at 0 h and 24 h; (v) quarter dose 1 d: 25, 19 at 0 h.
Control and chemotherapy-treated mice bearing EL4 tumours were euthanized 48 h after treatment and tumours were excised. Excised tumour tissue was disrupted by incubation in RPMI-1640 containing 2 mg/mL collagenase (Sigma-Aldrich Co., St. Louis, Mo.) for 30 min. at 37° C. with constant shaking. Single cell suspensions were washed with PBS and used for immunofluorescent staining with 3B9-FITC or Sal-FITC and PI and flow cytometry analysis as described earlier.
Control and chemotherapy treated mice received an intravenous injection of 14C-3B9 or 14C-Sal5 (control) at time 0. After 48 h, mice were euthanized, whole blood was collected by cardiac puncture, and EL4 tumours and other tissues were collected for radioactivity measurement. Serum and body tissues were solubilized using 1 mL Solvable™ (PerkinElmer Inc., Wellesley, Mass.) for 2 h at 50° C., and then decolorized using 100 μL H2O2 (30%) (Sigma-Aldrich Co., St. Louis, Mo.). UltimaGold™ scintillation liquid (1 mL) was added and samples were counted for 10 min. using the β-counter.
Protein G purified 3B9 or Sal5 mAb were mixed at 2.5 mg/mL in 0.1M sodium bicarbonate and 0.1M sodium phosphate buffer (pH8.5) with 50-fold molar excess of DOTA-NHS-Ester (Macrocyclics, Dallas, Tex.) dissolved in DMSO (Sigma-Aldrich Co., St. Louis, Mo.). DMSO represented <10% of the final reaction, which was incubated for 2 h at 23° C. with constant vigorous shaking. DOTA-NHS-Ester was added at a 50-fold molar excess to the antibody as this ratio was found to minimize inactivation of 3B9 and showed favorable in vivo biodistribution compared to Sal5 conjugated at the same ratio and to 3B9 conjugated at higher ratios (data not shown). Addition of 1.5M Tris-HCl (pH8.8) at a final dilution of 1:10 was used to stop the reaction, which was loaded onto a 100 kDa-cutoff microconcentrator (Millipore, Billerica, Mass.). Reactions were buffer-exchanged using 5×500 μL washes of PBS. The concentration of IgG was measured using a BCA protein assay kit and the concentration of conjugated DOTA was measured using a modification of a published Cu:Arsenazo(III) assay. The ligand/protein (L/P) ratio of 7.5 was calculated as the ratio of the concentrations (μM) of DOTA-immunoconjugates to IgG. The reaction was stored at 4° C. before radiolabeling.
Purified DOTA-immunoconjugate solutions were concentrated and buffer-exchanged into metal free 0.2M ammonium acetate buffer (pH5.5) (Sigma-Aldrich Co., St. Louis, Mo.) containing 6 mg/mL ascorbic acid (Sigma-Aldrich Co., St. Louis, Mo.). Indium-111 chloride (PerkinElmer Inc., Wellesley, Mass.) was diluted in the same buffer (5 mCi/mL; 185 MBq/mL) and mixed with DOTA immunoconjugates (10 mg/mL). Radiolabeling reactions were incubated at 37° C. for 2 h then mixed with an equal volume of 0.2M ammonium acetate buffer (pH8) containing 5 mM EDTA to quench free and loosely bond radionuclide. Quenched reactions were loaded onto 100 kDa-cutoff microconcentrators and purified by three washes of 500 μL endotoxin free PBS. The incorporation of 111In in the purified reactions was determined using instant thin layer chromatography (ITLC) which was performed using ITLC-SG strips (Pall Corporation, East Hills, N.Y.) in 0.2M ammonium acetate solution (pH 8) containing 5 mM EDTA as mobile phase. Briefly, aliquots (2 μL) of purified reactions and free 111In in 0.2M ammonium acetate and 5 mM EDTA were used for ITLC in the mobile phase and the origin and solvent front halves of strips were counted using a Cobra 5010 gamma counter (PerkinElmer Inc., Wellesley, Mass.) normalized for 111In counting using a 100-350 keV counting window. While 99% of radioactivity on the ITLC-SG strip was at the solvent front when ITLC was performed using 111 InCl3 solution, 99% of radioactivity in the DOTA-conjugates remained at the origin indicating the complete incorporation of 111In in the DOTA-3B9 conjugate (data not shown).1 At 0 h, 111In-DOTA-3B9 (72 μg in 100 μL of PBS) was given by intravenous injection to EL4 tumour-bearing mice, which were left untreated or treated with cytotoxic chemotherapy as described above. Mice were euthanized at selected time points, blood was collected by cardiac puncture, and tumours and organs were collected. Blood and organs were weighed and placed in gamma-counter tubes and radioactivity was measured using normalized Cobra5010 gamma counter. Radioactivity in organs was normalized to the weight of the organs and accumulation was calculated as the percentage of radioactivity per gram in the organs to the radioactivity of the injected dose of 111In-DOTA-3B9 at time 0 (% ID/g). Time activity curves for blood and tumours were constructed using GraphPad Prism v.4 (GraphPad Software, San Diego, Calif.) for each treatment group where accumulation % ID/g (±SEM, n=5) plotted as a function of time. Curves were fitted to one-phase exponential decay for blood and one-phase exponential association for tumours and the half-life (t1/2) was provided from the fitted models. In the case of tumour accumulation, the fitted association model also provided a value for maximal accumulation at saturation together with the corresponding standard error of this measurement, which we report as % ID/g±SEM at saturation. Data points did not deviate from the fitted models as judged by runs test performed using the fitting software and the regression value of the fitted model was provided.
Statistical comparisons were preformed using GraphPad Prism. Two-way analysis of variances (2-way ANOVA) was used to deduce significant differences in the results. The Bonferroni post-test in the 2-way ANOVA function in GraphPad prism was used to report P values. P values less than 0.05 were considered significant where one, two and three asterisks denote P values less than 0.05, 0.01 and 0.001, respectively.
La is Overexpressed in Malignant EL4 Cells and La-SPECIFIC mAb Binding to Dead EL4 Cells is Induced by Cytotoxic Drug Treatment
Cytofluographic analysis of cultured EL4 lymphoma cells, which were stained with the DNA binding dye 7-AAD as a test of membrane integrity, indicated that although <10% of the cultured cells demonstrated evidence of spontaneous cell death, these dead cells did not bind La-specific mAb 3B9 (
Next, biosynthetically labelled 3B9 mAb was used to further characterize the binding of La-specific mAb to dead EL4 cells, which were killed with cytotoxic drugs. It was found that 14C-3B9 bound dead EL4 cells in a specific and saturable manner. The concentration of 14C-3B9 required to reach half-maximal saturation was 18 nM and, maximal binding was ˜7500 femtomole/million dead cells (
The in vitro findings depicted in
3B9 Targets EL4 Tumours In Vivo Especially after Cytotoxic Chemotherapy
The biodistribution of La-specific mAb was studied in the EL4 lymphoma model using intrinsically or extrinsically labelled 3B9 mAb. First, biosynthetically labelled 14C-3B9 mAb was used to demonstrate that tumour accumulation of 3B9 mAb was an inherent property of its antigen-binding activity. EL4 tumour-bearing mice were treated with the full dose schedule of cyclophosphamide and etoposide and simultaneously administered 100 μg each of either 14C-3B9 or 14C-Sal5 isotype control mAb before analysis of the mice after 48 hours. The 14C-Sal5 mAb did not accumulate significantly in any organ or tissue including the tumour. In contrast and compared with all other organs, we found that 14C-3B9 accumulated significantly in serum and in the tumour (P<0.001). Moreover, after the mice were treated with cytotoxic chemotherapy, only the tumours accumulated significantly more 14C-3B9 (P<0.001) (data not shown).
In the second series of experiments, 3B9 conjugated to a metal chelator, DOTA, was radiolabelled with 111In and used to investigate the biodistribution and pharmacokinetics in EL4 tumour-bearing mice before and after chemotherapy treatment (
The specificity of 111In-DOTA-3B9 towards the dying tumours was expressed as the ratio of tumour accumulation to that in normal organs (tumour/organ ratio,
A biotinylated form of cadaverine, which is a polyamine TG2 substrate and which consequently becomes covalently bound to other TG2 substrates, is used to identify protein targets of cross-linking during apoptosis. As shown in
DNA-Damaging Chemotherapy Induces Anti-La Antibody Binding in Primary Human Malignant Cells
Two studies were performed on patient material to indicate that Apomab binding was augmented by DNA-damaging cytotoxic chemotherapy. In the first study, primary ALL blasts from a chemonaïve patient were treated in vitro with cytotoxic drugs and then analysed (
To purify primary ALL blasts for further analyses, Ficoll gradient separation was performed using a 10 mL peripheral blood sample from a newly diagnosed and untreated ALL patient. The buffy coat was collected and washed with culture medium before incubation for 10 min in red cell lysis buffer. Cells were washed, aliquots were removed for immediate analysis while other aliquots were incubated or not in different concentrations of cytotoxic drugs singly or in combination. At 24 h, 48 h, and 72 h after treatment was initiated in vitro, ALL blasts were stained with 10 μg/mL Sal5 control or Apomab then with 2 μg/mL Alexa488-conjugated anti-mouse IgG. Cells were washed and incubated with 2 μg/mL 7-AAD then analysed by flow cytometry immediately.
The level of Apomab binding to permeabilised primary ALL blasts (inset,
Analysis In Vitro of Circulating Small Lung Cancer Cells of a Patient Treated with Cytotoxic Drugs
Anti-La antibody binding to circulating tumour cells in peripheral blood of a 73-year-old male patient, GP, who had extensive-stage SCLC was studied. The patient, GP, who had not previously been treated for his cancer, was administered cytotoxic chemotherapy using carboplatin at AUC 5 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 (Oh) 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 present minimally if at all in the peripheral blood of a normal healthy volunteer (control) (
Although the laboratory findings were not 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.
In conclusion, these results using patient tumour material indicate that anti-La antibody identifies malignant cells that have died as the result of cytotoxic drug administration and, hence, this antibody may be useful for predicting early chemotherapy responses in cancer patients.
DNA-Damaging Antineoplastic Induces the Intrinsic Apoptosis Pathway and Induces Sustained Apomab to Apoptotic Cells Unlike Extrinsic Pathway Activation, Culture Stress, Serum Withdrawal or Primary Necrosis
Apoptotic changes after intrinsic pathway activation took longer than after extrinsic pathway activation. During activation of the intrinsic pathway by cisplatin or γ-radiation, evidence of DNA damage was observed at the early 5 h time point in permeabilised cells by γ-phosphorylation of H2AX and, simultaneously, by significantly increased antibody binding, which reflects increased expression of the La target antigen (
In contrast, cells that were overgrown in vitro (
Further analysis indicated differences in the course of apoptosis after γ-radiation vis à vis cisplatin treatment (
After Fas ligation, similar considerations applied to activation of caspase-3. As cells turned into apobodies, caspase-3 was activated simultaneously. Caspase activation indicated that cells were being dismantled into apobodies and the caspase-mediated processes likely included DNA degradation. Hence, the creation of simultaneous DSB seemed to be responsible for the striking induction of γH2AX foci and the concomitant induction of La. Activated caspase-3 activation persisted in cells as they converted to apobodies. On the other hand, the γH2AX and La induction occurred initially and transiently in all cells, and so appeared to be a process that was executed in response to the death stimulus applied to all cells and then was complete (
In confirmation that cisplatin treatment and γ-radiation of Jurkat cells induces expression of La antigen, immunoblots of cell lysates prepared shortly after apoptosis induction but before loss of cell membrane integrity were probed with anti-La antibodies or anti-actin antibodies as a loading control (
To show that anti-La antibody bound a variety of other dead human malignant cell lines after cytotoxic treatment, anti-La antibody or its Sal5 isotype control mAb was used to stain malignant cells after treatment with the pan-tyrosine kinase inhibitor (STS), the topoisomerase II inhibitor, etoposide, or the tubule depolymerising agent, vincristine (
In vivo studies were performed to quantify dead cells in EL4 tumours before and after CE chemotherapy, and to measure anti-La antibody uptake by these tumours. To provide evidence in vivo of induction of the La antigen by DNA-damaging chemotherapy, the relationship between tumour anti-La antibody uptake and the percentage of dead cells within tumours were examined. Anti-La antibody uptake by control tumours was compared with tumour anti-La antibody uptake after CE chemotherapy to determine if it exceeded the level expected if it were only proportionate to the tumour content of dead cells.
C57BL/6 mice bearing EL4 tumours were left untreated (n=3) or treated by intraperitoneal injection (IPI) of 19.5 mg/kg etoposide and 25 mg/kg of cyclophosphamide (n=3). Mice were killed at specified time points, tumours were excised, minced into pieces <10 mm3 using scissors, and 0.1 g of minced tumour weighed and placed in 10 mL 2 mg/mL solution of collagenase Type 1 in HBSS containing 2.5 mM Ca2+. Solutions were incubated at 37° C. with constant rotation for 1 h. Digested tumour cell suspensions were passed sequentially through syringes with 19 G, 23 G then 25 G needles. Suspensions were centrifuged at 2000 rpm for 5 min and pellets were washed with 10 mL HBSS. Washed pellets were resuspended in 1 mL HBSS and 100 μl, aliquots were stained for 10 min at room temperature (RT) in duplicate with 2 μg/mL 7-AAD. Samples were analysed using flow cytometry for the percentage of cells that bound 7-AAD to measure the percentage of dead cells (7-AAD+).
C57BL/6 mice bearing EL4 tumours were left untreated (n=5) or treated with chemotherapy (n=5) as described above. All mice were also injected intravenously with 111In-Apomab. Mice were killed at specified time points, tumours were excised, weighed, and placed in gamma counting tubes to measure radioactivity. Measured radioactivity was normalised to tumour weight (cpm/g) and accumulation was calculated as the percentage of weight-normalised radioactivity counts to total radioactivity counts of injected dose at time 0 h (% cpm/g in tumour/cpm of injected 111In-Apomab at time 0 h).
In comparison with untreated control mice (
To demonstrate this finding more explicitly, Apomab binding to dead tumour cells was related directly to the tumour content of dead cells at each time point (
In conjunction with the data showing therapeutic efficacy of La-directed monoRIT, at least two significant inferences may be drawn from these observations:
Furthermore, cytotoxic radiosensitising drugs such as cisplatin, the antimetabolites gemcitabine and 5-fluorouracil, and the taxanes, will augment the potency of La-directed radioimmunotherapy (RIT) by lowering the threshold for tumour cell death. Therefore, it would be expected that appropriately scheduled administration of a cytotoxic radiosensitising drug and La-directed RIT would lower the effective dose of RIT required for tumour cell kill.
Other than ionising radiation, the radiomimetic drug, cisplatin, topoisomerase inhibitors, DNA-intercalating agents, which are all antineoplastic treatments well known to induce double-stranded DNA breaks (DSB) and some which have been shown to augment Apomab binding to apoptotic tumour cells in vitro, newer agents induce DSB via novel mechanisms of action. For example, PARP inhibitors induce DSB and sensitise to apoptosis those tumour cells that lack DNA repair mechanisms based homologous recombination (HR) such as BRCA nullizygous tumour cells, or tumour cells with features of ‘BRCAness’ (N Turner et al. Nat Rev Cancer 4, 1-6, 2004) because HR is required to repair endogenous DNA damage (H E Bryant et al. Nature 434, 913-917, 2005; H Farmer et al. Nature 434, 917-921, 2005). The antimetabolite and masked DNA chain terminator, gemcitabine, induces few DSB unless the S-phase checkpoint is abrogated by a class of drug known as CHK1/2 inhibitors when DSB become greatly increased and apoptosis ensues (G McArthur et al. J Clin Oncol 24, 3045a, 2006; M A Morgan et al. Cancer Res 65, 6835-6842, 2005). Similar effects were exerted by CHK1/2 inhibitors after ionising radiation induced G2 and S checkpoints (J Falck et al. Nature 410, 842-847, 2001; H Zhao et al. PNAS 99, 14795-14800, 2002).
Further data are presented in support of this hypothesis using combination treatment with cytotoxic drugs and the histone deacetylase inhibitor (HDACi), trichostatin A (TSA), to show that the combination is very effective in inducing cell death among malignant cells and, therefore, provides the opportunity to maximise target creation.
Adding TSA to cisplatin treatment of Jurkat cells in vitro produces a TSA dose-dependent increase in Apomab-specific binding to dead Jurkat cells (
Similar synergistic interactions were found between gemcitabine and TSA in cells of the pancreatic cancer cell line, PANC-1, in vitro. In these experiments, the TSA doses of 200, 100, 50, 25 and 12.5 ng/mL are equivalent to TSA concentrations of 667, 333, 167, 83 and 42 nM, respectively. Together with the chosen concentrations of gemcitabine, TSA significantly inhibited proliferation of PANC-1 cells in vitro (Piacentini P et al. Virchows Arch 448, 797-804, 2006). Moreover, a moderate dose-dependent effect of TSA was observed on the induction of markers of apoptosis among cultures of PANC-1 cells in vitro (Donadelli M et al. Mol Carcinogenesis 38, 59-69, 2003).
Using the MTS proliferation assay, it was observed that TSA alone inhibited proliferation of PANC-1 cells in vitro. Addition of gemcitabine completely inhibited PANC-1 proliferation at a TSA concentration of 50 nM whereas at a TSA concentration of 200 nM, gemcitabine significantly reduced the numbers of PANC-1 cells in the cultures (
While gemcitabine may not be viewed as a conventional DNA-damaging agent, recent evidence indicates that it produces γH2AX DNA damage foci in a PC-3 tumour xenograft model (McArthur G A et al. J Clin Oncol 24(18S) 3045a, 2006). The synergistic effect of TSA on the induction of cell death among gemcitabine-treated PANC-1 cells together with Apomab-specific binding to the dead cells was evident at the lowest concentration of gemcitabine.
Altogether, it is believed that the data illustrated in
Suspension cultures of Jurkat leukemia cell line and EL4 murine T-lymphoblastic lymphoma cell line were maintained in RPMI-1640 containing 5% FCS (JRH Biosciences Inc., Lenexa, Kans.) and passaged by splitting at 1:10 every 72 h. The anti-La/SS-B 3B9 mAb hybridoma (Tran et al. 2002, Arthritis Rheum. 46(1):202-8) is a murine IgG2a autoantibody, which is crossreactive with human La and which was prepared by Dr M. Bachmann (Oklahoma Medical Research Foundation, OK), was a kind gift from Dr T. P. Gordon (Department of Immunology, Allergy and Arthritis, Flinders Medical Centre, SA, Australia). The isotype control Sal5 (1D4.5) mAb hybridoma, prepared by Dr L. K. Ashman (Medical Science Building, University of Newcastle, NSW, Australia), was kindly supplied by Dr S. McColl (School of Molecular Biosciences, University of Adelaide, SA, Australia). Hybridoma were cultured in RPMI-1640 containing 5% FCS and the produced antibodies were affinity-purified from culture media using protein G columns. FITC-conjugates were prepared according to the manufacturer instructions (Sigma-Aldrich Co., St. Louis, Mo.).
Conjugation of mAb with DOTA-NHS-ESTER
DOTA-NHS-ESTER (Macrocyclics, Dallas, Tex.) was dissolved in DMSO (Sigma-Aldrich Co., St. Louis, Mo.) at 25 mg/mL. Purified 3B9 and Sal5 in 0.1 M sodium phosphate/0.1 M sodium bicarbonate buffer (pH 8.6) were mixed at 2 mg/mL with 50-, 100-, 150- and 200-fold molar excess of DOTA-NHS-ESTER. Control reactions were prepared using equivalent volume of DMSO to replace the NHS-ESTER compound. Reactions were incubated at 23° C. for 2 h with constant rotation and were stopped using 1.5M Tris-HCl (pH 8.3) at 10% v/v final concentration. Removal of unconjugated DOTA and buffer exchange of immunoconjugates was achieved by 5×500 μL PBS washes in 100 kDa cut off microconcentrators as described by manufacturer instructions (Millipore, Billerica, Mass.). Conjugates were stored at 4° C. for further use.
Protein concentration was determined using the BCA protein assay kit as described by manufacturer instructions (Pierce Biotechnology Inc., Rockford, Ill.). Protein concentration was converted from units of mg/mL to μM based on the Mr of IgG (150'000 g/mol). The concentration of DOTA was determined using a modified form of the previously described Arsenazo(III) assay (Pippin et al. 1992, Bioconjug Chem 3(4):342-5; Dadachova et al. 1999, Nucl Med Biol, 26(8):977-82; Brady et al. 2004, Nucl Med Biol, 31(6):795-802). Briefly, assays were performed in 96-wells titre plates to reduce the volume used. Stock solutions of Cu:Arsenazo(III) were prepared using 100 μL of 1 mg/mL standard Cu atomic absorption solution (Sigma-Aldrich Co., St. Louis, Mo.), 0.875 mg Arsenazo(III) (Sigma-Aldrich Co., St. Louis, Mo.) and 3 mL of metal free 5M ammonium acetate (Sigma-Aldrich Co., St. Louis, Mo.) in a final 10 mL volume of milliQ water and stored at room temperature (RT) in the dark. Standard concentrations of DOTA were prepared using DOTA-NHS-ESTER dissolved in milliQ water. Aliquots (10 μL) of conjugation reactions and standard DOTA solutions were mixed with 190 μL of working dilution (see figure legends) of Cu:Arsenazo(III) solution in milliQ water, incubated at 37° C. for 30 min and absorbance was measured at 630 nm. The concentration of conjugated DOTA was interpolated from the standard curve constructed for the relationship between DOTA standard solutions and the absorbance of Cu:Arsenazo(III) reagent. DOTA/antibody ratio in prepared conjugates was calculated as the ratio of DOTA concentration (μM) to IgG concentration (μM).
SDS-PAGE was performed as per manufacturer instructions using the Hoefer® Mighty Small II SE 250 electrophoresis system (Amersham Biosciences, Piscataway, N.J.) under reducing condition using 12% resolving polyacrylamide gel as per Laemmli (Laemmli U. K., 2005, J. Biol Regul Homeost Agents 19(3-4):105-112). Transfer of polyacrylamide gel onto Hybond-P membrane was carried out as per manufacturer instruction using the TE 22 Mini Tank Transfer Unit (Amersham Biosciences, Piscataway, N.J.). After blocking with 5% skim milk, probing was performed using 5 μg/mL 111In-labelled 3B9 (prepared as described below). Membranes were washed then exposed to x-ray films (3 h at RT) which were developed and documented.
Native polyacrylamide gel electrophoresis (native PAGE) was used to determine the relative fractionation (Rf) value for unmodified and conjugated antibody. Briefly, 7% native gel was prepared using 100 μL APS, 20 μL TEMED (Sigma-Aldrich Co., St. Louis, Mo.), 1.75 mL of 40% acrylamide (Biorad®) and 1.25 mL of 1.5M Tris-HCl (pH 8.6) in a final 10 mL volume of milliQ water. Native PAGE running buffer was prepared by dissolving 3.4 g of glycine and 1.2 g of Tris in 500 mL of milliQ water (pH adjusted to 8.5). Unmodified and conjugated antibody were mixed with equal volume of native PAGE loading dye (20% v/v glycerol, 1.5M Tris-HCl and 0.05% w/v bromophenol blue in milliQ water) and samples were loaded onto gel. Electrophoresis was performed at 200V for 2.5 h and gel was stained using brilliant blue R250 (BBR250) solution (1 g of BBR250 [Sigma-Aldrich Co., St. Louis, Mo.] in 200 mL methanol and 100 mL H2O). The Rf values were determined using the gel analysis program, GelPro Analyzer™ v3.1 (Media Cybernetics Inc., Silver Spring, Md.).
Jurkat cell were fixed and permeabilized by incubation at 1×107 cells/mL in 2% paraformaldehyde for 10 min, diluted 1:10 in −20° C. methanol (5 min) then washed extensively using PBS. Direct immunofluorescent staining was performed using 5 μg/mL of 3B9-FITC or 5 μg/mL Sal5-FITC. Indirect immunofluorescent assays were performed using permeabilized cells incubated with 5 μg/mL of unmodified 3B9, 3B9-DOTA or matching irrelevant isotype (Sal5 or Sal5-DOTA) for 30 min at RT. Cells were washed then incubated in 2 μg/mL of goat anti-mouse IgG Alexa488-conjugated antibody (Invitrogen, Carlsbad, Calif.) for 30 min at RT. Cells were washed and samples were acquired using Becton-Dickinson FACScan™ flow cytometry system (BD Biosciences, San Jose, Calif.). Acquisition was standardized to 10'000 events Flow cytometry data was analyzed using WinMDI v 2.8 (Scripps Research Institute, La Jolla, Calif.).
Activity of 3B9-DOTA conjugates was tested by their ability to inhibit the binding of 3B9-FITC. Briefly, fixed/permeabilized Jurkat cells were incubated for 30 min at RT with 5 μg/mL of 3B9-FITC in the absence or presence of 5, 25, 50 and 100 μg/mL of 3B9-DOTA conjugates. Cells were washed and analyzed by flow cytometry as described above.
Samples (0.1 μg) of unmodified or DOTA-conjugated IgG in native form (PBS) were applied onto nitrocellulose membranes (Amersham Biosciences, Piscataway, N.J.). Membranes were dried and blocked with 5% skim milk for 30 min at RT. Membranes were incubated (30 min at RT) with 1 μg/mL of goat antibodies conjugated with biotin and directed against the mouse IgG or against the Fc or (Fab)2 regions of mouse IgG (Rockland, Gilbertsville, Pa.). Membranes were washed and incubated with 0.1 μg/mL streptavidin-alkaline phosphatase (AP) (Rockland, Gilbertsville, Pa.) for 15 min at RT. Membranes were washed and developed using the BCIP/NBT premixed solution as specified by manufacturer (Sigma-Aldrich Co., St. Louis, Mo.) and analyzed using GelPro Analyzer™.
Kinetics of Labelling of mAb-DOTA with Terbium
Arsenazo(III) reagent was utilized to study the kinetics of labelling of 3B9-DOTA with Terbium (Tb). TbCl3 (Sigma-Aldrich Co., St. Louis, Mo.) was diluted in milliQ water at 20 mg/ml. Stock of Arsenazo(III):metal complexes was prepared by mixing 18.75 μL of TbCl3 solution with 1.75 mg Arsenazo(III) in 3 mL of 5M ammonium acetate in a final 10 mL of milliQ water. Stock was diluted 1:10 and 90 μL was added to 17.5 μg 3B9-DOTA solutions (10 μL). Absorbance at 630 nm was measured kinetically at 37° C. every 1 min for 1 h.
Radiolabelling of mAb-DOTA with 111In
3B9-DOTA or Sal5-DOTA conjugates were buffer exchanged into 0.2M ammonium acetate (pH 5.5) containing 6 mg/mL ascorbic acid by four washes in 100 kDa cut off microconcentrators. Indium-111(111 InCl3, PerkinElmer Inc., Wellesley, Mass.) and DOTA-conjugates were incubated for 2 h at 37° C. at concentrations of 2 mCi/mL (74 MBq/mL) of 111In and 2 mg/mL of conjugates in (i.e. 1:1 mCi:mg or 37 MBq:mg ratio) 0.2M ammonium acetate buffer containing 6 mg/mL ascorbic acid. In some cases, ascorbic acid which enhances the incorporation of 111In into the conjugates was eliminated from the radiolabelling reaction as interferes with the BCA protein assay kit. Incorporation of 111In was measured as described in next section.
For purification of 111In-labelled conjugates, radiolabelling reactions were washed twice with 400 μL volumes of 0.2M ammonium acetate containing 5 mM EDTA (pH 8.0) then 3 times with 400 μL volumes of PBS in 100 kDa cut off microconcentrators as described earlier. Protein concentration of purified 111In-labelled conjugates was measured using the BCA assay kit and the radioactivity concentration was measured using a specified volume placed in gamma counting tubes and counted using Cobra5010 gamma counter (PerkinElmer Inc., Wellesley, Mass.) which was normalized for 111In counting at 100-350 keV window. Specific radioactivity was measured as cpm from gamma counter per μg of protein present in the counted volume.
Incorporation efficiency was measured using instant thin layer chromatography (ITLC). Briefly, 2 μL aliquots of labelling reactions were mixed with 2 μL of 0.2M ammonium acetate containing 5 mM EDTA (pH 8.0) and applied at 1 cm from the bottom of 1×9 cm ITLC-SG strips (Pall Corporation, East Hills, N.Y.). Chromatography was performed in 0.2M ammonium acetate containing 5 mM EDTA (pH 8.0) as mobile phase and was stopped when solvent front reached 1 cm from the top of the strips. Strips were cut in two halves where the bottom half represented the origin (ori) and the top half represented the solvent front (SF). The activity of the two halves was measured using gamma counter. Incorporation efficiency was calculated as the percentage of activity remaining at the origin to the total activity of the strip [% ori/(ori+SF)].
We have previously described an in vivo model of apoptosis which we have shown specific and preferential accumulation of 3B9 in tumors of chemotherapy treated mice (Al-Ejeh et al., 2007). Briefly, EL4 thymic lymphoma cells (1×106 cells) were injected subcutaneously in the right flanks of 6-8 weeks old C57BL/6 female mice. Mice were housed and treated as per protocols approved by the Animal Ethics Committee at The University of Adelaide and the Animal Ethics Committee at the Institute of Medical and Veterinary Sciences (IMVS). Once the tumor reached 1 cm diameter, mice were treated by intraperitoneal injections of cytotoxic chemotherapy (38 mg/kg etoposide and 50 mg/kg cyclophosphamide) given at time 0 and 24 h. DOTA-conjugates labelled with 111In (100 μg) was injected intravenously at time 0. Mice were euthanized at 48 h, blood was collected by cardiac puncture, and tumors and organs were collected. Blood and organs were weighed and placed in gamma-counter tubes and radioactivity was measured using gamma counter. Radioactivity in organs was normalized to the weight of the organs and accumulation was calculated as the percentage of radioactivity per gram in the organs to the radioactivity of the injected dose at time 0 (% ID/g).
Statistical comparisons were preformed using GraphPad Prism v.4 (GraphPad Software, San Diego, Calif.). Two-way analysis of variances (2-way ANOVA) was used to deduce significant differences in the results. The Bonferroni post-test in the 2-way ANOVA function in GraphPad prism was used to report P values. P values less than 0.05 were considered significant where one, two and three asterisks denote P values less than 0.05, 0.01 and 0.001, respectively.
Previously described assays for the measurement of metal chelators attached to antibodies uses a standard curve for the absorbance (deep blue color) of Cu:Arsenazo(III) solutions of different concentrations. The absorbance of this reagent when mixed with immunoconjugates is then used to interpolate (from standard Cu:Arsenazo(III) curve) the concentration of Cu remaining after chelation which allows calculation of chelated Cu thus representing the concentration of metal chelator (Pippin et al. 1992 supra; Dadachova et al. 1999 supra; Brady et al. 2004 supra). We found that while Cu:Arsenazo(III) standard curve represented dilutions of the deep blue color of the reagent (
Conjugation of 3B9 with DOTA-NHS-ESTER was performed in three separate occasions. The average DOTA/antibody ratios were 2.8±0.3, 8.2±0.3, 11.6±1.1 and 15.0±0.7 for the conjugation conditions of 50-, 100-, 150- and 200-fold molar excess of DOTA-NHS-ESTER to 3139 (
The avidity of DOTA-conjugated 3B9 was investigated using competition binding assays. As shown in
Detection of 3B9-DOTA conjugates using antibodies against whole IgG molecule or antibodies against the Fc and (Fab)2 regions decreased significantly compared to that of unmodified 3B9. Samples of 3B9 and 3B9-DOTA conjugates fractioned in non-reducing SDS-PAGE showed comparable amounts of protein in these samples as judged by the intensity of BBR250 staining (
Further evidence for the modification of 3B9 during conjugation both in structure and avidity was obtained using indirect immunofluorescent staining of permeabilized Jurkat cells. As shown in
The absorbance of Terbium:Arsenazo(III) reagent mixed with 3B9-DOTA conjugates decreased during 1 h incubation at 37° C. (
There has been reported the specific and saturable accumulation of 3B9 in EL4 tumors after cytotoxic chemotherapy treatment compared to the irrelevant control of matched isotype, Sal5 (Al-Ejeh et al., 2007). This model was used to investigate the significance of the above described modification of antibody properties after conjugation in vivo. Biodistribution of 3B9-DOTA in chemotherapy treated mice was affected when prepared at 200-fold molar excess of DOTA-NHS-ESTER compared to that prepared at 50-fold excess (
EL4 murine lymphoma model: cultured EL4 cells were collected and 1×106 cells injected subcutaneously in the right flank of 6-8 week-old female mice of the syngeneic C57BL/6J strain. Tumours were left to grow for 7-8 days to reach 100 mm3 volume before initiation of any of experiments. (This model yields tumours 100% of the time).
Biodistribution of Apomab in EL4-tumour bearing mice under different schedules of chemotherapy and antibody injection
Accumulation of Apomab labelled with 111Indium was traced overtime in treated mice where Apomab was injected either concurrently with CE chemotherapy or 24 h after chemotherapy (
C57BL/6 mice were injected with EL4 cells. Tumours were grown for 1 week and mice were divided into three groups for treatments described below.
1) Control
2) 9.5 mg/kg etoposide and 12.5 mg/kg cyclophosphamide
3) 19 mg/kg etoposide and 25 mg/kg cyclophosphamide
Mice were sacrificed at 24, 48, 72 and 96 h after chemotherapy administration (3 mice per time point) and tumours fixed in formalin, paraffin-embedded and stored for immunohistochemistry (IHC). Staining was performed by Jim Manavis (IMVS) for the cell death marker (activated caspase-3) (
C57BL/6 mice were injected with EL4 cells. Tumours were grown for 1 week and mice then divided into three groups for treatments described below administered.
1) Control
2) 9.5 mg/kg etoposide and 12.5 mg/kg cyclophosphamide
3) 19 mg/kg etoposide and 25 mg/kg cyclophosphamide
Apomab labelled with 111In was administered in all mice and 3 mice from each group were killed at 3, 24, 72 and 96 after Apomab injection and gamma-camera images were obtained at RAH Department of Nuclear Medicine (
Maximum binding of Apomab in control mice and mice treated with ⅛ 1 d or ¼ 1 d CE chemotherapy was calculated from gamma camera image analysis (
Previous experiments using the EL4 tumour model indicated relationship between tumour size/mass and response to chemotherapy as well as accumulation of Apomab in correlation to the mode of cell death. This current study was design to investigate the effect of tumour mass/volume on Apomab binding to EL4 tumours in control mice and mice treated with CE-chemotherapy at two different doses; ⅛ 1 d (half-dose) or ¼ 1 d (full-dose) regimens.
C57BL/6 female (6-8 weeks old) mice were inoculated with identical number of EL4 cells subcutaneously at the right hindquarter on day 1, 2, 3, 4 and 5 (18 mice per day). On day 7, mice from each inoculation day were divided into 3 groups of 6 mice each to produce 3 cohorts which contained 5 groups of 6 mice per group with each group being inoculated with EL4 cell on a different day (3 cohorts×5 groups [inoculated d1, 2, 3, 4, and 5]×6 mice=90 mice). One cohort was left untreated (control), one was injected with ⅛ 1 d CE treatment (⅛ chemo; half-dose) and one with ¼ 1 d CE treatment (¼ chemo; full-dose). On day 8, each of these three cohorts was further divided into two identical cohorts containing 5 groups of 3 mice per group:
One cohort from A, B and C was injected with 111In-Apomab for radio-imaging and radio-counting studies while the other cohort from A, B and C was left for IHC analysis. On day 10, all IHC mice were killed by cardiac bleeding and cervical dislocation. Tumours were dissected and stored in 10% formalin overnight, embedded in paraffin then subjected to immunohistochemistry staining of activated caspase-3. Tumour volumes were measured using calipers before formalin fixation. On the same day, mice injected with 111In-Apomab were killed by inhalation of anaesthetic and imaged using GE millennium clinical gamma camera (10 min acquisition per image using acquisition windows suitable for 111Indium; peaks at 171 and 245 keV). Two mice from control, ⅛ chemo (half-dose) and ¼ chemo (full-dose) cohorts were selected based on matched tumour volume (measured from IHC study) and dissected immediately after image acquisition to collect tumours. All mice were then dissected and organs collected for radioactivity counting using the Cobra 5010 gamma counter. Organs were weight and accumulation was measured as radioactivity counts standardised to the mass of counted sample and calculated as a percentage of the injected dose (% ID/g).
Mice were imaged using GE Millennium clinical gamma camera before dissection. Images of two representative mice from each group at all time points are shown in
C57BL/6 mice were injected with EL4 tumours on day 0, day 2 or day 4. Mice with EL4 cell implanted on day 0 and day 2 were injected with full or half dose chemotherapy on day 7, respectively. All mice received an i.v. injection of Apomab-biotin or Sal5-biotin (3 mice per treatment per antibody) on day 8 and killed on day 10 for tumour dissection. Control tumours and tumours from mice treated with half and full chemo were grown for 6, 8 and 10 days on day of collection, respectively. Tumours were fixed in formalin, embedded in paraffin and sectioned for probing with streptavidin-HRP to detect antibody binding via IHC (
Imaging of EL4 Tumour Response to Chemotherapy Using Apomab in Fab2 Vs. IgG Forms
Mice to be treated with full dose of chemotherapy were injected with EL4 cells (18 mice). Control mice were injected with EL4 cells (18 mice). Chemotherapy was administered on the 4th of Jun. 2007. Groups of 3 mice were injected with 111In-labelled antibody as follows:
1) Control mice injected with 111In-3B9 (2 groups of 3 mice)
2) Chemo-treated mice injected with 111In-3B9 (2 groups of 3 mice)
3) Control mice injected with Fab2 of 111In-Sal5 (2 groups of 3 mice)
4) Control mice injected with Fab2 of 111In-3B9 (2 groups of 3 mice)
5) Chemo-treated mice injected with Fab2 of 111In-Sal5 (2 groups of 3 mice)
6) Chemo-treated mice injected with Fab2 of 111In-3B9 (2 groups of 3 mice)
One group of cohorts 1 and 2 was imaged (24 h after RIC injection) while the other group was imaged (48 h after RIC injection). One group of cohorts 5-8 were injected with D-lysine solution 4 times at 2 h intervals starting 30 min before RIC injection (40 mg/injection in 200 μL). Groups from cohorts 5-8 (without or with D-Lysine injections) were images at 24 h after RIC injection. All mice were dissected after imaging and biodistribution (% ID/g) was calculated.
As shown in
Biodistribution of IgG Apomab in control and chemotherapy treated mice is shown in
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.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU07/01549 | 10/11/2007 | WO | 00 | 8/31/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60851277 | Oct 2006 | US |
