Classifications

• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
  • G16B50/20—Heterogeneous data integration
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics

Definitions

• the present invention provides a phenotype and biological marker identification system and methods for identifying and using novel patterns of biological markers related to disease, disease progression, response to therapy and normal biological functions.
• novel patterns of biological markers will result in more cost-effective drug development, including the improvement of patient selection in clinical trials and the identification of therapeutics with greatly improved safety and efficacy.
• Phenotype information and biological markers can also be used in diagnostic applications.
• the phenotype for a given individual includes, in theory, all measurable characteristics of such individual at all points in time.
• One use of such phenotype information is the identification of biological markers.
• Bio markers are characteristics that when measured or evaluated have, inter alia, a discrete relationship or correlation as an indicator of normal biologic processes, pathogenic processes or pharmacologic responses to a therapeutic intervention.
• Pharmacologic responses to therapeutic intervention include, but are not limited to, response to the intervention generally (e.g., efficacy), dose response to the intervention, side effect profiles of the intervention, and pharmacokinetic properties. Response may be correlated with either efficacious or adverse (e.g., toxic) changes.
• Biological markers include patterns of cells or molecules that change in association with a pathological process and have diagnostic and/or prognostic value. Biological markers may include levels of cell populations and their associated molecules, levels of soluble factors, levels of other molecules, genotypic information, gene expression levels, genetic mutations, and clinical parameters that can be correlated with the presence and/or progression of disease.
• biological markers may provide a more rapid and quantitative measurement of a drug's clinical profile.
• Single biological markers currently used in both clinical practice and drug development include cholesterol, prostate specific antigen ("PSA"), CD4 T cells and viral RNA. Unlike the well known correlation between high cholesterol and heart disease, PSA and prostate cancer, and decreased CD4 positive T cells and viral RNA in AIDS, the biological markers correlated with most other diseases have yet to be identified. As a result, although both government agencies and pharmaceutical companies are increasingly seeking development 3 of biological markers for use in clinical trials, the use of biological markers in drug development has been limited to date.
• Phenotyping requires the instrumentation and assays required to measure hundreds to thousands of parameters, an informatics system to allow this data to be easily accessed, software to correlate the patterns of information with clinical data and the ability to utilize the resulting information in the drug development process.
• the present invention provides such a technology.
• the present invention relates to phenotyping an organism or a class or subclass of organisms.
• the present invention also includes the identification of biological markers that are measured and evaluated as an indicator of normal biologic processes, pathogenic processes or pharmacologic responses to a therapeutic intervention.
• This invention includes technology capable of providing quantitative, sensitive reproducible and rapid measurements of multiple and diverse biological markers that could accurately profile an organism's phenotype or a patient's disease status and response to therapy. Further, because blood is the single most information rich tissue and is easily and readily accessible for testing, the invention focuses on identifying biological parameters from small samples of blood.
• the invention includes a multidisciplinary format comprising three principal elements: instrumentation, assay development and clinical informatics.
• Figure 1 is a schematic representation of the types of information that are assimilated to obtain one embodiment of a biological marker identification system.
• Figure 2 depicts a schematic representation of the improved MLSC instrument of the invention (term “SurroScan” instrumentation).
• Figure 3 depicts the integrated information infrastructure for analyzing the data obtained in the present invention.
• Figures 4 A - C depict the results obtained in Example 1 showing that CD27 + and
• CD27 CD8 T cells vary among samples. Blood samples from three different donors
• Figures 5A and B depict robust cellular measurements with 2-color MLSC.
• Figure 5 A demonstrates the consistency of CD8 T cell counts from 6 different capillaries. Cy5.5 anti-CD8 was combined with a different Cy5 conjugated antibody for each of the capillaries (anti-CD3, CD25, CD7, CD45RA, CD62L, CD69). Fifty different blood samples were analyzed. The box-and-whiskers plots show that the distributions of cell counts are very similar for each of the capillaries. Pair-wise linear regression also shows a high degree of consistency for these assays (data not shown).
• Figure 5 shows the consistency of two measures of B cells, one with Cy5.5 anti-CD20 and one with Cy5.5 anti-CD19. The 95% confidence interval (dotted line) of the linear regression includes a slope of 1 and the fit has a correlation coefficient of 0.97.
• Figure 6 shows a classification matrix comparing CD8 T cells and CD4 T cells in RA patient samples and blood bank samples.
• Figures 7 A and B show results of a three color cellular assay on the SurroScan instrument.
• Figures 8 A - C shows the results of staining intracellular molecule as measured with MLSC technology.
• Figures 9A - C show the results of a 3 detection channel analysis using MLSC technology.
• the present invention is directed to the phenotyping of an organism or a class or subclass of organisms.
• the phenotyping of an organism includes obtaining all measurable characteristics of said individual, past and present. While the complete phenotyping of any organism is not practical or even possible, the phenotyping disclosed and described herein provides an unprecedented quantity of an unprecedented number of types of parameters or characteristics so as to provide a resource of information that will allow for the analysis of normal biological functions, disease, disease progression and changes associated with virtually any perturbation to the organism.
• One utility of the phenotyping system taught by the present invention is to the identification of biological markers for normal biological processes, diseases or medical 5 conditions.
• it is necessary to have i) biological information from a population of individuals, ii) an adequate amount of data from each individual, preferably obtained by multiple sampling over time, and iii) an information storage and retrieval system that a) can integratively incorporate a wide variety of types of information and b) can perform meaningful correlation analysis of the disparate types of data.
• Figure 1 depicts information that is useful to create a biological marker identification system.
• the present invention has the potential to identify and trace changes in patterns of biological markers reflecting both genetic and environmental factors from small samples of blood. Furthermore, the present invention helps decipher genetic components of disease susceptibility, disease progression and response to therapy.
• the present invention is capable of monitoring cells, proteins, organic molecules, genotype, soluble factors, clinical and environmental factors, all of which have been used as biological markers in drug development and as disease markers.
• known biological marker include the monitoring for decreases in CD4 positive T cells and viral RNA levels in AIDS, elevated cholesterol levels as an accepted biological marker for heart disease and changing levels of PSA as a protein marker found in the blood of prostate cancer patients. Since the biological characteristics or parameters that might be discovered to be a biological marker or part of a marker "grouping" are often not predictable, it is essential that the appropriate database contain information regarding as many parameters as possible.
• the present invention extends to a phenotype of a given organism, methods for assembling such phenotype and methods for utilizing such phenotype.
• the phenotype of an organism or class or subclass or organisms comprises a large compilation of data relating to the organism or class or subclass of organisms.
• the novel aspect of the present invention lies in the disparate nature of the data and the quantity of data from each of the various categories of data available on an organism.
• a phenotype can only reach its full usefulness if the data defining the phenotype is extensive. For example, a phenotype for a human patient containing a standard blood profile and clinical factors routinely obtained from a physical examination can not provide enough information to fully exploit such a phenotype.
• a phenotype comprises greater than 40 biological parameters, more preferably greater than 100 parameters, and most preferably greater than 200 different parameters, and in some cases greater than 300 different parameters.
• the phenotype must contain biological parameters that include information from cellular assays, soluble factor assays and clinical information.
• the results of at least 20 cellular assays incorporating measurements of at least 20 cell populations and/or cell associated molecules and the results of at least 20 soluble factor assays are included in the phenotype, along with clinical information.
• the results of at least 40 cellular assays incorporating measurements of at least 40 cell populations and/or cell associated molecules and at least 40 soluble factor assays are included, preferably with an extensive battery of clinical and environmental parameters.
• a rich and readily accessible source of biological information for a patient is the blood.
• blood At the present time, there are over 200 identified discrete leukocyte cell surface antigens with identified antibodies.
• proteins and other soluble factors and small molecules that can be identified in blood. The problem, therefore, is not in finding enough informational content in the blood, but in efficiently extracting all of the available information from limited quantities of blood.
• An additional application of the present invention is in monitoring dose response studies.
• a population of individuals is evaluated before and after the administration of drug and after increasing doses of the drug.
• the selected population may be healthy individuals, and the anticipated biological dose response endpoint is toxicity or side effect profiles.
• markers may be identified for efficacy along with the negative effects of the drug.
• biological markers may provide a more rapid and quantitative measurement of a drug's clinical profile.
• longitudinal studies of individuals receiving a drug or treatment for the prevention or treatment of a disease or medical condition could constitute the population of individuals being evaluated.
• Another application of the present invention is the use of biological markers to identify patients who have very early clinical signs of a disease. This would be extremely valuable for a multitude of disease states where a patient may have "subchnical" signs and symptoms which are not severe enough to bring them to a doctor's office. However, if a patient had a marker which was discovered in their blood and they were advised to seek medical attention, their "subchnical" signs could be identified as their earliest phenotypic 8 presentation of a disease. For many diseases, it is extremely advantageous to diagnose a disease as early as possible so that therapeutic drugs may be started and generally lead to reduced morbidity and mortality of that disease entity for that individual.
• a possible scenario would be if a patient could take a blood test to see if they have a biological marker for Rheumatoid Arthritis. If the marker were present, they could then seek treatment during the "subchnical" stage where they may only have a sensation of warmth in their joints instead of waiting until they have joint pain, swelling and deformity. That individual would likely have a much better long-term outcome for Rheumatoid Arthritis in comparison to someone who waits until they have a much later stage of the disease before seeking treatment.
• the present invention is directed to the phenotyping of an organism or class or subclass of organisms.
• the phenotype is made up of data from a large number of data categories.
• the principle categories of data included within the scope of this invention are i) levels of cell populations including their cell associated molecules in biological fluid, ii) levels of soluble factors in the biological fluid, iii) drug dosing and pharmacokinetics (measurement of a drug and its metabolites in a body) and iv) clinical parameters.
• Additional categories of data may include, but are not limited to, i) levels of small molecule compounds in biological fluid, ii) genotype information regarding the individual, including the individual's genetic makeup and gene expression (mRNA or transcripts) levels, and iii) data obtained from assays of urine components.
• data categories may include images such as x-ray, CAT scans of the brain or body, or MRIs, or information obtained from biopsies, EKGs, stress tests, endoscopies, ultrasound exams, laparascopic procedure, orthroscopic surgeries, PET scans, or any other measurement of an individual's condition.
• the clinical parameters included in the database of the present invention would include, but not be limited to, the individual's age, gender, weight, height, body type, medical history (including comorbidities, medication, etc.), manifestations and categorization of disease or medical condition (if any) and other standard clinical observations made by a physician. Also included among the clinical parameters would be environmental and family history factors.
• Clinical parameters could be further characterized by the source from which the information which is obtained.
• Patient obtained clinical parameters may include information that the patient provides via a questionnaire such as the WOMAC for 9 osteoarthritis, and the Health Assessment Questionnaire for Rheumatoid Arthritis which may be filled out in a doctor's office.
• electronic or web-based questionnaires addressing all of a patient's current clinical symptoms could be completed by the patient prior to a clinic visit.
• Information obtained by a nurse would include vital signs, information from a variety of tests including allergy testing, pulmonary function testing, stress-thallium testing, or ECG tests.
• Clinical parameters collected from a physician includes a detailed history of prior illnesses, surgeries, hospitalizations, medications, reactions to medications, family history, social history, alcohol/drug/smoking history, as well as other behavior which would put a patient at high risk for HIV or Hepatitis.
• a thorough physical exam is also performed by a clinician and is a crucial component of a patient's clinical parameters.
• the levels of cell populations and their associated molecules are identified by microvolume laser scanning cytometry.
• Such data can also be obtained by flow cytometry, but the volume of blood necessary to perform the flow cytometry assays places a serious limit on the number of assays that can be performed on blood taken from a given individual at one time.
• the sample preparation required for performing flow cytometry assays is time consuming, expensive, and may interfere with the measurement result.
• the levels of soluble factors can be measured by any suitable technique.
• the levels of soluble factors is measured by standard immunoassay techniques, such as ELISA techniques.
• microvolume laser scanning cytometry is used to obtain levels of soluble factors.
• Soluble factors can be detected by immunoassays such as MLSC, ELISA, etc., mass spectrometry, 2D gel electrophoresis, combinations of mass spectrometry and immunosorption, and chemical assays.
• cell populations are detected by MLSC assays and soluble factors are detected by immunoassays or mass spectrometry.
• the invention includes improved instrumentation for the rapid, reproducible and quantitative evaluation of biological parameters from a small quantity of blood; miniaturized, high sensitivity assays compatible with improved instrumentation for the detection of hundreds to thousands of biological parameters in blood; a broad clinical strategy to collect extensive medical information content from patients who are followed over time; software, databases and data mining tools to correlate patterns of parameters with normal biological functions, specific diseases, disease progression and response to 10 therapy; databases of clinical data and biological markers in collaboration with academic centers and clinical research institutes for use in drug development; development of diagnostic tests using proprietary patterns of markers and the ability to improve the efficiency of drug development by enabling more informed decisions in choosing lead compounds and identifying patients more likely to benefit from a given therapy.
• the unique ability to phenotype an organism and to conduct reproducible and rapid measurements of large numbers of biological parameters is essential for the present invention to identify novel patterns of biological markers from small samples of blood.
• the invention further includes studies of patient populations related to particular diseases. These studies are based upon statistical analyses of disease patterns and require the collection of large numbers of blood samples from affected individuals.
• the present invention has utility for phenotyping and identifying biological markers in plants and animals and for assisting in preclinical studies.
• phenotype or “phenotyping” refers to a compilation comprising a substantial subset of all measurable characteristics of an organism. Such characteristics or parameters include, but are not limited to, levels of cell populations and their associated molecules, levels of soluble factors, levels of other molecules, genotype information, gene expression levels, genetic mutations, and clinical parameters. Such characteristics or parameters include all historical data and present data. For example, an organism's complete phenotype includes all measurable characteristics at the present time, as well as all such characteristics at all past points of time.
• the phenotype can include an organism's feelings or emotions (in the case where the organism is a human, the phenotype includes the individual's mental state, e.g., depression, pain, agitation, mental illnesses, chemical dependencies); diet and changes in diet, injuries, relational history, sexual practices, socio-economic status.
• an organism refers to all plants, animals, viruses and exoterrestial materials. Included within this definition, but not limited in any way, are humans, mice, rats, rabbits, companion animals, natural and genetically engineered plants, and natural and genetically engineered animals.
• a given phenotype might include a compilation of characteristics of a single organism or a class or subclass of organisms.
• the phenotypic data may be obtained from a single male individual who has been diagnosed with cancer before and after therapeutic intervention, a group of males between the age of 15 and 55, or a group of males between the ages of 15 and 55 diagnosed with cancer.
• the phenotype may be specific to a given individual, or may represent the average or typical condition of a combined group of individuals.
• the phenotype of an individual organism or group of organisms may be used for a variety of purposes.
• the phenotype is looked at longitudinally and evaluated after some perturbation to the organism.
• the comparison of the phenotype of an individual before and after exhibiting symptoms of asthma could be used to identify biological markers associated with asthma.
• the phenotype of an individual who has asthma can be compared with the phenotype of a population of normal adults.
• the phenotype of a naturally occurring plant can be compared with the phenotype of a genetically altered plant to determine what measurable characteristics are altered by the introduction of the genetic alteration.
• a further example of the use of phenotyipng information would be to periodically monitor well-patient status of an individual and to track measures of biological aging processes.
• the potential uses for comprehensive phenotypic data for an organism are almost infinite.
• the present invention includes phenotypes for an organism or class or subclass of organisms, methods for obtaining such phenotypes and methods for utilizing such phenotypes, including for the identification of biological markers.
• biological marker or “marker” or “biomarker” means a characteristic or parameter that is measured and evaluated as an indicator of normal and abnormal biologic processes, pathogenic processes or pharmacologic responses to a therapeutic intervention.
• Pharmacologic responses to therapeutic intervention include, but are not limited to, response to the intervention generally (e.g., efficacy), dose response to the intervention, side effect profiles of the intervention, and pharmacokinetic properties. 12 Response may be correlated with either efficacious or adverse (e.g., toxic) changes.
• Biological markers include patterns or ensembles of cells or molecules that change in association with a pathological process and have diagnostic and/or prognostic value.
• Biological markers include, but are not limited to, levels of cell populations and their associated molecules, levels of soluble factors, levels of other molecules, gene expression levels (mRNA or transcripts), genetic mutations, and clinical parameters that can be correlated with the presence and progression of disease, normal biologic processes and response to therapy.
• Single biological markers currently used in both clinical practice and drug development include cholesterol, PSA, CD4 T cells, and viral RNA. Unlike the well known correlations between high cholesterol and heart disease, PSA and prostate cancer, and CD4 positive T cells and viral RNA and AIDS, the biological markers correlated with most other diseases have yet to be identified.
• the use of biological markers in drug development has been limited to date.
• biological markers are often thought of as having discrete relationships with normal biological status, a disease or medical condition, e.g., high cholesterol correlates with an increased risk of heart disease, elevated PSA levels correlate with increased risk of prostate cancer, reduced CD4 T cells and increased viral RNA correlate with the presence/progression of AIDS.
• useful markers for a variety of diseases or medical conditions may consist of significantly more complex patterns. For example, it could be discovered that lowered levels of one or more specific cell surface antigens on specific cell type(s) when found in conjunction with elevated levels of one or more soluble factors - - cytokines, perhaps - - is indicative of a particular auto-immune disease. Therefore, for the purposes of this invention, a biological marker may refer to a pattern of a number of indicators.
• biological marker identification system means a system for obtaining information from a patient population and assimilating the information in a manner that enables the correlation of the data and the identification of biological markers.
• a biological marker identification system comprises an integrated database comprising a plurality of data categories, data from a plurality of individuals corresponding to each of said data categories, and processing means for correlating data within the data categories, wherein correlation analysis of data categories can be made to identify the data category or 13 categories where individuals having said disease or medical condition may be differentiated from those individuals not having said disease or medical condition, wherein said identified category or categories are markers for said disease or medical condition.
• markers may be identified by comparing data in various data categories for a single individual at different points of time, e.g., before and after the administration of a drug.
• data category means any type of measurement that can be discerned about an organism.
• data categories useful in the present invention include, but are not limited to, numbers and types of cell populations and their associated molecules in the biological fluid of an organism, numbers and types of soluble factors in the biological fluid of an organism, information associated with a clinical parameter of an organism, cell volumetric counts per ml of biological fluid of an organism, numbers and types of small molecules in the biological fluid of an individual, genomic information associated with the DNA of an organism and gene expression levels.
• a single data category would represent the concentration of IL- 1 in the blood of an organism.
• a data category could be the level of a drug or its metabolites in blood or urine.
• biological fluid means any biological substance, including but not limited to, blood (including whole blood, leukocytes prepared by lysis of red blood cells, peripheral blood mononuclear cells, plasma, and serum), sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, sweat, feces, synovial fluid and whole or manipulated tissue.
• biological fluid typically contains cells and their associated molecules, soluble factors, small molecules and other substances. Blood is the preferred biological fluid in this invention for a number of reasons.
• Blood replenishes, in part, from progenitors in the marrow over time. Blood is responsive to antigenic challenges and has a memory of antigenic challenges. Blood is centrally located, recirculates and potentially reports on changes throughout the body. Blood contains numerous cell populations, including surface molecules, internal molecules, and secreted molecules associated with individual cells. Blood also contains soluble factors that are both self, such as cytokines, antibodies, acute phase proteins, etc., and foreign, such as chemicals and products of infectious diseases. 14 As used herein the term "cell population" means a set of cells with common characteristics. The characteristics may include the presence and level of one, two, three or more cell associated molecules, size, etc.
• One, two or more cell associated molecules can define a cell population. In general some additional cell associated molecules can be used to further subset a cell population. A cell population is identified at the population level and not at the protein level. A cell population can be defined by one, two or more molecules. Any cell population is a potential marker.
• cell associated molecule means any molecule associated with a cell. This includes, but is not limited to: 1) intrinsic cell surface molecules such as proteins, glycoproteins, lipids, and glycohpids; 2) extrinsic cell surface molecules such as cytokines bound to their receptors, immunoglobulin bound to Fc receptors, foreign antigen bound to B cell or T cell receptors and auto-antibodies bound to self antigens; 3) intrinsic internal molecules such as cytoplasmic proteins, carbohydrates, lipids and mRNA, and nuclear protein and DNA (including genomic and somatic nucleic acids); and 4) extrinsic internal molecules such as viral proteins and nucleic acid.
• the preferred cell associated molecule is typically a cell surface protein.
• leukocyte cell surface proteins or antigens there are hundreds of leukocyte cell surface proteins or antigens, including leukocyte differentiation antigens (including CD antigens, currently through CD 166, see, Leucocyte Typing VI, Kishimoto, T. et al. ED, 1997), antigen receptors (such as the B cell receptor and the T cell receptor), and major histocompatibility complex.
• leukocyte differentiation antigens including CD antigens, currently through CD 166, see, Leucocyte Typing VI, Kishimoto, T. et al. ED, 1997)
• antigen receptors such as the B cell receptor and the T cell receptor
• major histocompatibility complex Each of these classes encompass a vast number of proteins.
• Table 1 is merely an illustration of the vast number of cell surface proteins and is in no way intended to be a comprehensive list.
• soluble factor means any measurable component of a biological fluid or tissue that is not a cell population or cell associated molecule.
• Soluble factor includes, but is not limited to, soluble proteins, carbohydrates, lipids, lipoproteins, steroids, other small molecules, including metallic, inorganic, ionic and metallorganic species and complexes of any of the preceding components, e.g., cytokines and soluble receptor; antibodies and antigens; and a drug complexed to anything.
• Soluble factors can be both self, such as cytokines, antibodies, acute phase proteins, etc., and foreign, such as chemicals, products of infectious diseases and intestinal flora and fauna.
• Soluble factors may be intrinsic, i.e., produced by the organism, or extrinsic such as a virus, drug or environmental toxin. Soluble factors can be small molecule compounds such as 15 prostaglandins, vitamins, metabolites (such as iron, sugars, amino acids, etc.), drugs and drug metabolites.
• a list of exemplary soluble proteins is provided in Table 6, which is merely an illustration of the vast number of soluble proteins and is in no way intended to be a comprehensive list.
• soluble factors may be either known or unknown entities. A variety of techniques are available where a given species may be identifiable, but the chemical identity of the species is unknown.
• the chemical identity of the soluble factor need not be currently known or known at the time the assay is performed to determine its presence or absence.
• small molecule or "organic molecule” or “small organic molecule” means a soluble factor or cell associated factor having a molecular weight in the range of 18 to 10,000. Small molecules can include, but are not limited to, prostaglandins, vitamins, metabolites (such as iron, sugars, amino acids, etc.), drugs and drug metabolites.
• disease or medical condition means an interruption, cessation, disorder or change of body functions, systems or organs.
• disease or medical conditions include, but are not limited to, immune and inflammatory conditions, cancer, cardiovascular disease, infectious diseases, psychiatric conditions, obesity, and other such diseases.
• immune and inflammatory conditions include autoimmune diseases, which further include rheumatoid arthritis (RA), multiple sclerosis (MS), diabetes, etc.
• perturbation means an exterior or interior measurable event that can occur to an organism.
• a simple example would be the administration of a therapeutic agent to an individual, or an individual that was healthy and then developed asthma.
• a perturbation may also include differences between an individual or groups of organisms that are being compared. For example, a population of animals may be considered to be normal, and their phenotype is being compared to the phenotype of a similar but genetically altered animal. The individual genetically altered animal was perturbed in the sense that its genetic alteration was perturbed from normal. In many cases the perturbation is not a single event that occurs at a discrete point in time. The perturbation may occur over an extended period of time, and/or may be cyclical or intermittent.
• clinical parameter means information that is obtained that may be relevant to a disease or medical condition. Such information may be supplied by 16 the patient or by a medical or scientific observer. Examples of clinical parameters for humans include, but are not limited to, age, gender, weight, height, body type, medical history, ethnicity, family history, genetic factors, environmental factors, manifestation and categorization of disease or medical condition, and any result of a clinical lab test, such as blood pressure, MRI, x-ray, etc.
• Clinical parameters could be further characterized by the source of information which is obtained.
• Patient obtained clinical parameters may include information that the patient provides via a questionnaire such as the WOMAC for osteoarthritis, and the Health
• genetictype information means any data relating to the organisms genetic makeup, gene mutations, gene expression, e.g., mRNA or transcription levels, and any other measure or parameter associated with the genetic material of the organism.
• clinical endpoint means a characteristic or variable that measures how a patient feels, functions, or survives.
• Clinical endpoint There are several mechanism which are commonly used to measure how a patient feels or functions with a specific disease and they often include validated clinical questionnaires. These may be self administered such as the Beck's depression questionnaire or the International Prostate Questionnaire to determine if changes in urination are due to prostatic hypertrophy v. bladder outlet obstruction. These tools may be given by a health care provider who is judging features such as facial expression, inability of patient to sit down for more than 10 minutes, level of agitation etc., while completing the Carrol Questionnaire to determine if a patient is manic.
• MLC Microvolume Laser Scanning Cytometry
• the MLSC system has several key features that distinguish it from other technologies: 1) only small amounts of blood (5-50 ⁇ l) are required for many assays; 2) absolute cell counts (cells/ ⁇ l) are obtained; and, 3) the assay can be executed either directly on whole blood or on purified white blood cells. Implementation of this technology will facilitate measurement of several hundred different cell populations from a single harvesting of blood.
• the MLSC technology is described in United States Patent Numbers 5,547,849 and 5,556,764 and in Dietz et al.
• Laser scanning cytometry with microvolume capillaries provides a powerful method for monitoring fluorescently labeled cells in whole blood, processed blood, and other fluids.
• the present invention further improves MLSC technology by improving the capacity of the MLSC instrument to do simultaneous measurement of multiple biological markers from a small quantity of blood.
• a schematic of the improved SurroScan optical system is shown in Figure 2.
• tag means any entity or species, including but not limited to an atom, a molecule, a fragment of molecule or a functional group; a particle or combination of particles; a single or sequence of electromagnetic pulses; or any other form of matter associated with, attached to (either covalently or non-covalently), or otherwise connected to a component of a biological system (a molecule or collection of molecules such as cell, a cation, an anion, an atom, or any supramolecular assembly, including but not limited to non-covalent complexes between biological molecules) that is used to, identify, 18 quantify, associate, recognize, follow, spot, make out, see, name, track, or otherwise distinguish (henceforth I/Q) said component.
• a biological system a molecule or collection of molecules such as cell, a cation, an anion, an atom, or any supramolecular assembly, including but not limited to non-covalent complexes between biological molecules
• Tags are often extrinsic, i.e. not part of the component under investigation.
• a fluorescent dye molecule is often used as a tag, either for tracking, quantitation, or both.
• biotin or streptavidin as tag, linked to a secondary species such as an enzyme for ELISA, is widespread.
• Other forms of tags include, but are not limited to, isotopic mass tags for protein I/Q by mass spectrometry, Raman-active tags for
• I/Q by Raman scattering particulate tags for I/Q by light scattering, fluorescence, agglutination, energy transfer, and a variety of other detection mechanisms, including surface plasmon resonance.
• particulate tags there are almost an infinite number of particulate tags, only a small number of which have been previously used.
• nanoparticle science is in its infancy (as was organic chemistry two centuries ago)
• particulate tags might rival the organic molecules currently used as bead tags in combinatorial chemistry, in other words thousands to hundreds of thousands or even millions of uniquely identifiable tags.
• We further anticipate that such tags will become small enough to allow all intracellular measurements. For example, there are now roughly one-half dozen different luminescent semiconducting quantum dot nanoparticles, each fluorescing at a different wavelength. In theory, one could anticipate production of thousands or millions of such orthogonal nanoparticulate optical tags, although the detection mechanism may or may not involve fluorescence (or even other optical methods).
• tags could comprise individual molecules either covalently or non-covalently associated with biological components. For example, one could imagine using electrochemically-active redox tags to uniquely identify components. If one had 10 different molecules, each with a different redox potential, and each pre-functionalized to react with a particular biological component, then one could carry out multiplexed tag I/Q, using the detection of the redox potential as the identifying characteristic. This is identical to the strategy currently used with fluorescence, with redox "space” used in lieu of "wavelength” space.
• a tag can be a functional group, as in a carboxylate, an amine, a sugar, etc., or even a spin associated with a molecule.
• a tag can be a functional group, as in a carboxylate, an amine, a sugar, etc., or even a spin associated with a molecule.
• two samples could be mixed together, with each sample having one or more nuclei imparted with a particular sequence of electromagnetic pulses (of the sort typically used in high-field NMR).
• the pulses for two samples would be long-lived enough to compare them using a method of detection.
• the signatures for the two samples would cancel for all species where the concentrations are identical, leaving behind a signal only for those species where concentrations in the two samples are non-identical.
• a “detection molecule” as defined below can itself be a tag (for example when I/Q is based on mass, as in quartz crystal microbalances or piezo inertial biosensors).
• the term “detection molecule” means any molecule or molecular assembly capable of binding to a molecule or other species of interest, including but not limited to a cell-associated molecule, a soluble factor, or a small molecule or organic molecule.
• Preferred detection molecules are antibodies.
• the antibodies can be monoclonal or polyclonal.
• detection molecules are increasingly being used for molecular recognition, and organic chemists have now synthesized a large number of molecular receptors. Ultimately, these could be used as detection molecules, either by themselves or in association with a tag.
• the terms “dye”, “fluorophore”, “fluorescent dye” are used interchangeably to mean a molecule capable of fluorescing under excitation by a laser.
• the dye is typically directly linked to a detection molecule in the present invention, although indirect linkage is also encompassed herein.
• Many dyes are well known in the art and include, but are not limited to those shown in Table 2.
• fluorophores are used which can be excited in the red region (> 600 nm) of the spectrum. Two red dyes, Cy5 and Cy5.5, are typically used. They have emission peaks of 665 and 695 nanometers, respectively, and can be readily coupled to antibodies. Both can be excited at 633 nm with a helium-neon laser.
• animal model refers to any experimental animal system in which diseases or conditions with similar pathology and progression to human diseases or medical conditions can be developed. Suitable animal systems include, but are not limited to, rats, mice, rabbits, and primates. In some cases, the disease arises spontaneously in the animal model. In other cases, the induction of disease in the animal model can result from exposure to the same conditions—for example, infection with a pathogen, exposure to a toxin, or a particular diet—that causes the disease in humans. Alternatively, the disease or condition can be induced in the animal model with agents that mimic the human disease or medical condition even if the actual initiator(s) of the human disease or medical condition is unknown. The disease or medical condition might also be induced through the use of surgical techniques. Genetic manipulation of experimental animal model systems provides a further tool for the development of the animal models, either standing alone or in combination with the other methods of disease induction.
• biological markers of the progression of a particular human disease could be identified in an experimentally-induced animal model of that disease, e.g., the rat adjuvant model of arthritis (reviewed in Philippe, et al, American Journal of Physiology 273:R1550- 56 (1997)).
• an experimentally-induced animal model of that disease e.g., the rat adjuvant model of arthritis (reviewed in Philippe, et al, American Journal of Physiology 273:R1550- 56 (1997)).
• the efficacy of experimental therapeutics could be determined in the animal model.
• Therapeutics that have a highly specific effect on the expression of biological markers in animals, which markers are prognostic or diagnostic of the same disease in humans, can therefore be identified without conducting early— and hence risky— human clinical trials.
• novel biological markers can be identified in experimental animal models of human disease, and then experiments can be 21 performed to determine whether the same markers, or their human homologues, are prognostic or diagnostic of the same disease or medical condition in humans.
• biological markers identified in humans can be used to facilitate preclinical trials where animal models can be evaluated by the corresponding biological markers.
• the present invention provides methods and instrumentation for performing such analyses.
• the expression of biological markers is studied in an animal model of a human disease.
• the biological markers of interest can be initially identified in preferred embodiments using MLSC.
• the identified markers can then be studied using MLSC to determine the response of the animal to a candidate therapeutic.
• MLSC- based assays typically only require small volumes of biological fluid
• MLSC is uniquely suited for use in animal model systems (especially in rat and mouse) where only limited amounts of fluid can be obtained from an animal without sacrificing it.
• the use of MLSC will permit multiple time point analysis of an experimental animal to determine the pharmacokinetics of a candidate therapeutic.
• the animal homologues of known or newly identified human biological markers of a particular disease are studied in an experimentally-induced animal model of that disease.
• the animal homologues of human molecules will already be known and characterized. For example, through extensive study, a great deal is known about proteins that behave similarly in mouse and in humans.
• the identification of previously unknown animal homologues of human biological markers, and the preparation of reagents that can bind to them, can be accomplished through the use of standard molecular biology techniques well known in the art.
• novel biological markers for example, a previously unknown pattern of expression of known blood cell-associated proteins— may be initially observed in an animal model of a human disease.
• the relevancy of the identified markers to the progression or development of the human disease can be determined by identifying human homologues of the biological markers, and then studying their expression in humans suffering from the disease of interest. If the identified animal biological markers appear to be relevant to the human disease, then they can serve: 1) as the basis of new diagnostic and prognostic assays for the disease in humans; and 2) 22 as a means for evaluating the specificity and efficacy of candidate therapeutics in the animal model of the disease.
• new and improved animal models may be developed based on biological markers identified in humans. For example, utilizing the biological marker identification system of the present invention it can be found that for a given disease or medical condition that the level of given soluble factor in serum is greatly increased, while the level of certain cell population is decreased. Based on this information, animal models can be tailored — for example by the use of genetic knockouts of homologous factors — to better simulate the disease in the animal serum.
• the phenotyping system of the present invention may also be useful in the identification of new or improved animal models. For example, by phenotyping a number of genetically altered animals, a fuller picture of the manifestations of the genetic alternations can be recognized. Utilization of this knowledge can be useful in identifying new or improved animal models.
• the present invention can be used in any animal model of a human disease.
• the present invention can be used to identify and analyze biological markers in animal models of many aspects of cardiovascular disease, including hypertension, artherosclerosis, cardiac hypertrophy, atherogenesis, and thrombosis.
• Many animal models of congestive heart failure and hypertrophy are currently being developed, and a number are reviewed in: Carmeliet, Artherosclerosis, 144:163-93 (1999); Young et al, Molecular Basis of Cardiovascular Disease, 37-85 (K.R. Chien, Editor) (1999);
• SHR Spontaneously hypertensive rat
• SHR strains carrying a portion of chromosome 13 (including the renin gene) from normotensive rats can be used to investigate the interaction between high blood pressure and dyslipidemia in cardiovascular disease. St. Lezin et al, Hypertension, 31:373-377 (1998).
• Rat, guinea pig, rabbit, dog, sheep, and baboon models of preeclampsia have been used to study the pathophysiology this hypertensive disorder of human pregnancy. Reviewed in: Hypertension in Pregnancy 12:413-37 (1993).
• the present invention is used to identify and analyze biological markers in animal models of inflammatory diseases such as arthritis and multiple sclerosis.
• the present invention When used to screen candidate therapeutics, the present invention has a number of significant advantages over more traditional screening methodologies. Firstly, clinical testing comes at a relatively late stage in the development of the therapeutic, at which point the therapeutic is known to have a highly specific effect on the expression of analogous animal biological markers; this minimizes the risks to the clinical participants. Secondly, using experimental animal models to analyze patterns of biological marker expression 24 means that only relatively small quantities of the potential therapeutic need be synthesized initially, thus reducing the cost of therapeutic development.
• the methods and systems of the present invention are used to identify markers of disease or medical conditions in animals for veterinary purposes.
• the identified markers can then be used to screen for candidate therapeutics directed against that disease or condition.
• This embodiment can be applied to domesticated animals, livestock and plants.
• MLSC Microvolume Laser Scanning Cytometry
• the MLSC technology is used with a bead based capture system or with various types of enzyme linked immunosorbent assays (such as ELISA) to obtain data for soluble proteins.
• Another preferred means for obtaining data for compounds, particularly small molecules includes the use of mass spectrometry.
• the MLSC technology used in this invention is a powerful method for monitoring fluorescently labeled cells and soluble proteins in blood. This technology is currently used in clinical laboratories for the identification of one or two cellular markers for diagnostic applications.
• the present invention uses MLSC to facilitate the identification of biological parameters.
• the present invention improves MLSC technology by improving the capacity of the MLSC instrument to do simultaneous measurement of multiple biological characteristics or parameters from a small quantity of blood. Specific enhancements achieved with the instrument of the invention (termed
• “SurroScan instrument” include the following: 1) two additional fluorescence color channels allow simultaneous detection and measurement of up to four fluorescent colors; 2) higher laser excitation power improves sensitivity and throughput; 3) disposable capillary arrays allow more assays per patient sample using less blood per assay; 4) improved software and system integration automates sample measurements and data analysis; 5) the capacity of SurroScan instruments is expanded to handle higher volumes of patient samples for database creation and biological marker discovery. 25
• the MLSC technology is described in United States Patent Numbers 5,547,849 and 5,556,764 and in Dietz et al. (Cytometry 23:177-186 (1996)), each of which is incorporated herein in its entirety.
• the Imagn 2000 system commercially available from Biometric Imaging Inc., is an example of a MLSC system.
• Laser scanning cytometry with microvolume capillaries provides a powerful method for monitoring fluorescently labeled cells in whole blood, processed blood, and other fluids.
• the present invention further improves MLSC technology by improving the capacity of the MLSC instrument to do simultaneous measurement of multiple biological markers from a small quantity of blood.
• a schematic of the improved SurroScan optical system is shown in Figure 2.
• a capillary array 10 contains samples for analysis.
• collimated excitation light is provided by one or more lasers.
• excitation light of 633nm is provided by a He-Ne laser 11. This wavelength avoids problems associated with the auto fluorescence of biological materials.
• the power of the laser is increased from 3 to 17 mW. Higher laser power has two potential advantages, increased sensitivity and increased scanning speed.
• the collimated laser light is deflected by an excitation dichroic filter 12. Upon reflection, the light is incident on a galvanometer- driven scan mirror 13. The scan mirror can be rapidly oscillated over a fixed range of angles by the galvanometer e.g. +/- 2.5 degrees.
• the scanning mirror reflects the incident light into two relay lenses 14 and 15 that image the scan mirror onto the entrance pupil of the microscope objective 16.
• This optical configuration converts a specific scanned angle at the mirror to a specific field position at the focus of the microscope objective.
• the +/- degree angular sweep results in a 1 mm scan width at the objective's focus.
• the relationship between the scan angle and the field position is essentially linear in this configuration and over this range of angles.
• the microscope objective focuses 26 the incoming collimated beam to a spot at the objective's focus plane.
• the spot diameter which sets the optical resolution, is determined by the diameter of the collimated beam and the focal length of the objective.
• Fluorescence samples placed in the path of the swept excitation beam emit stokes- shifted light. This light is collected by the objective and collimated. This collimated light emerges from the two relay lenses 14 and 15 still collimated and impinges upon the scan mirror which reflects and descans it.
• the stokes-shifted light then passes through a dichroic excitation filter (which reflects shorter wavelength light and allows longer wavelength light to pass through) and then through first long pass filter 17 that further serves to filter out any reflected excitation light.
• the improved instrument of the instant invention uses a series of further dichroic filters to separate the stokes-shifted light into four different emission bands.
• a first fluorescence dichroic 18 divides the two bluest fluorescence colors from the two reddest. The two bluest colors are then focussed onto first aperture 19 via a first focusing lens 20 in order to significantly reduce any out-of- focus fluorescence signal.
• a second fluorescence dichroic 21 further separates the individual blue colors from one another. The individual blue colors are then parsed to two separate photomultipliers 22 and 23.
• the two reddest colors are focused onto a second aperture 24 via a second long pass filter 25, a mirror 26, and a second focusing lens 27 after being divided from the two bluest colors by first fluorescence dichroic 28.
• the reddest colors are separated from one another by third fluorescence dichroic 28.
• the individual red colors are then parsed to photomultipliers 29 and 30. In this way, four separate fluorescence signals can be simultaneously transmitted from the sample held in the capillary to individual photomultipliers. This improvement, for the first time, allows four separate analytes to be monitored simultaneously.
• Each photomultiplier generates an electronic current in response to the incoming fluorescence photon flux.
• These individual currents are converted to separate voltages by one or more preamplifiers in the detection electronics.
• the voltages are sampled at regular intervals by an analog to digital converter in order to determine pixel intensity values for the scanned image.
• the four channels of the instant invention are named channel 0, 1, 2, and 3.
• the new optical layout has four detection channels to allow simultaneous measurement of up to 4 fluorescently labeled molecules.
• multiple-color assays are used. Typically 3 or more fluorescent colors are used in each 27 assay. Under circumstances where appropriate dye combinations are available, the instrument is capable of supporting 4-color assays.
• An XY translational stage is used to move an array of capillaries relative to the optical system.
• the SurroScan system translation stage holds two arrays, each of which has the footprint of a 96-well plate.
• Capillary arrays have been designed which have 32 fixed capillaries each and spacing that is compatible with multi-channel pipettes. The operator is able to load two plates of 32 capillaries at a time. No operator intervention is needed while the plates are scanned and the images are processed.
• 16 individual capillaries designed for the Imagn 2000 (VCI 20) are loaded into alternative holders.
• Image processing software accommodates images with either 2, 3, or 4 colors of fluorescent dyes.
• the software automatically identifies and parameterizes particles detected in any of the individual colors.
• the measured parameters describing each particle are saved in a list-mode format, which is made compatible with conventional cytometry analysis software, such as FlowJo.
• This capillary cartridge is used in Examples 5, 7 and 8.
• the design currently in use, called Flex-32 contains 32 capillaries. Fill holes in the FLEX32-plates have the same 9 mm spacing as 96-well plates and multichannel pipetting devices. It is constructed from 2 layers of mylar sandwiched together with a double-sticky adhesive layer which is die-cut to define the capillary inner dimensions.
• the resulting cartridge can be manufactured at low cost in high volumes.
• the cartridge is flexible, which allows it to be held onto an optically flat baseplate by vacuum pressure, removing the requirements for flatness in the manufacturing process.
• the capillary spacing was designed to retain compatibility with multi-channel microplate pipetters and robotics. 28
• the invention includes cellular assays, many of which are antibody based, that are compatible with instrumentation, preferably MLSC instrumentation and are capable of measuring hundreds to thousands of cell populations and their cell associated molecules from a single 10 mL tube of blood.
• instrumentation preferably MLSC instrumentation
• any type of detection molecule and assay format compatible with MLSC is encompassed in this invention, including, but not limited to cell surface proteins including markers of activation and adhesion, intracellular molecules, assays to distinguish changes in activation states of cells, assays to concentrate and identify rare white cells, assays for use with whole blood, and assays for detection of soluble factors, such as proteins, in blood.
• fluorophore-labeled antibodies specific for cell surface antigens are used to identify, characterize and enumerate specific populations.
• the reaction can be done in whole blood. In general, there is no need to wash the reagent away; quantitative dilution of the blood-antibody mixture is usually sufficient sample preparation.
• the cell-antibody mixture is loaded into an optical-quality capillary of known volume and analyzed with a laser-based fluorescence imaging instrument. In order to operate with whole blood, fluorophores are used which can be excited in the red region (> 600 nm) of the spectrum. Purified white blood cells can also be analyzed with the instrument. In contrast to flow cytometry, the laser scans over stationary cells rather than cells flowing past the laser.
• a small cylindrical laser spot is scanned across the capillary in one direction while the capillary is translated relative to the optical system in a second direction.
• Photomultiplier tubes are used to detect the fluorescent signal.
• Multi-color capability allows more cell populations to be identified with a given amount of blood than the original 2-color system.
• all populations identified in two 2-color assays can be identified in one 3-color assay.
• unique cell populations can be defined by the simultaneous expression of three or more antigens.
• CD8 T cells can be subsetted into 4 different populations based on the differential expression of
• CD45RA and CD62L are CD45RA and CD62L.
• Immunoassays can be run in a variety of formats and any appropriate format is envisioned in the present invention. Two examples are given below.
• the MLSC system can be used with microsphere-based immunoassays.
• the microsphere In this sandwich assay, the microsphere is used as a solid support for an analyte-specific capture antibody. Analyte from a biological fluid is bound to the antibody-coated microsphere and detected with a second antibody, which is directly labeled with a fluorescent molecule such as Cy5, and which binds to a distinct epitope on the analyte.
• a protocol using amino beads and a heterobifunctional crosslinker to covalently attach antibodies via their hinge region works well in multiple assays.
• Biotinylated antibody specific for a second epitope on the same analyte is added, incubated and washed followed by an avidin- alkaline phosphatase conjugate. The level of analyte is revealed with a chemiluminescent alkaline phosphatase substrate. Plates are read in a Wallac Victor2 luminometer or similar instrument.
• the MLSC system is designed to allow rapid staining of cells using minimal quantities of blood. Reagents directed against scores of different cell surface antigens are 30 developed, which when combined can identify hundreds of different cell populations. The strategy for reagent and combination development is discussed below.
• a set of monoclonal antibody reagents are employed which are suitable for developing more than 100 cellular assays.
• many (about 120) different monoclonal antibodies directed against numerous (about 80) different cell surface antigens have been successfully identified and tested with the 2-color MLSC instrument.
• the small organic dyes like Cy5 and Cy5.5 are readily coupled to the amino groups of antibodies using single-step NHS chemistry and well established procedures.
• Preferred dye-to-antibody ratios have been determined for Cy5, Cy5.5, and Cy7 reagents, and are generally in the range of one to four.
• Protein fluorochromes like APC, are linked to the sulfhydryl groups of moderately reduced antibody in a 3-step procedure using the heterobifuntional crosslinking reagent SMCC. Preparation of reagents containing other fluorophores is also possible.
• the preparation of Cy7-APC and (Cy7-APC)-antibody conjugates for flow cytometry applications has been previously described.
• the antibody- fluorophore coupling chemistry is the same as for APC. All protein-protein conjugates are purified by traditional means, such as, by gel filtration on an Akta FPLC. Fluorescent microspheres can also be investigated. Antibodies are coupled with 2-step carbodiamide chemistry to carboxylated microspheres.
• New monoclonal antibodies reagents are titrated on both whole blood and lysed red blood cells. Reagent specificity, and lack of non-specific binding, is confirmed with appropriate counter stains. Analysis is done with any appropriate software program, including FlowJo cytometry software (Treestar, Inc available as an Internet download at http://www.treestar.com/ flowio/). From the titration the optimal amount of each reagent per assay (typically 0.01 to 2 ⁇ g/ml) and preliminary analysis criteria is determined. In the preferred embodiment, all assays are conducted in homogenous (no wash) mode.
• each antibody reagent has a titer point of ⁇ 1 ⁇ g/ml so that the fluorescence background is not too high.
• a potential difficulty may be that a particular reagent may not be amenable to conjugation or may have too high of a titer point.
• a panel of about 50-100 (or greater) cellular assays is developed for monitoring a disease or medical condition. Such assays enable one to enumerate hundreds of different cell populations.
• the cell surface antigens being evaluated for use may be divided into different subsets based on the types of cellular antigens recognized.
• antigens found on the major leukocyte subtypes including T cells, B cells, antigen- presenting cells, NK cells, and granulocytes, as well as relevant receptors and structures found on these cells are included. These may include activation molecules, co- stimulatory molecules, adhesion molecules, antigen receptors, cytokine receptors, etc.
• a representative, but not exhaustive, list of the antigens that may be evaluated for RA is provided in Table 1.
• the cellular assays described above are designed in either of two formats, whole blood or RBC-lysed blood.
• the assays are done in whole- blood or RBC-lysed blood format.
• the minimal manipulation ensures that the most accurate absolute cell counts (cells/ ⁇ l of blood) are obtained.
• Furthermore only small amounts of blood are required per assay so that many assays can be run from a single tube of blood.
• an alternative assay format, RBC-lysed blood will be preferable.
• These include particular antigen-antibody pairs for which soluble factors (free Ig, soluble cytokine receptors, etc.) contained in the sera interfere with cell labeling and populations of cells that are present in very low frequency.
• This procedure is useful for activated cells expressing CD25 or CD69 which are essentially undetectable in whole blood from normal individuals but are increased ten-fold in the lysed format and have been shown to be increased in various autoimmune states. Improved detection of other minor cell populations such as NK cells has also been demonstrated and should prove particularly useful in analyses. As an example, for a panel of 96 assays, it is estimated that 64 will be done on whole blood and 32 on lysed blood. Alternative sample processing may include, preparation of PBMC by Ficoll gradient, ex vivo stimulation with polyclonal or antigen specific activators. 32 Combining antibody reagents is important for the identification of novel cell populations that may contribute to the pathogenesis, or be a marker for, diseases or medical conditions, such as autoimmune diseases.
• adhesion molecules can be differentially expressed on T cells thought to be involved in the autoimmune process.
• several studies have indicated that there may be an increase in the number of memory CD4 T cells in patients with autoimmune disease.
• the assays of the present invention it is possible to simultaneously look at differential levels of adhesion molecules (e.g., CD1 la + ) specifically on a subset of memory (i.e. CD45RO + ) T cells of the HLA class Il-restricted lineage (i.e. CD4 + ). This should increase the ability to identify relevant disease-related cell populations.
• Multiple-color capability also allows one to look for novel populations of cells by choosing combinations of antigens not typically found together on a given cell type or markers found on the same cell type at different stages of ontogeny.
• any appropriate fluorescent dye is within the scope of the present invention.
• Two commonly used dyes are cyanine dyes Cy5 (em 667) and Cy5.5 (em 703).
• Cy5 em 667)
• Cy5.5 em 703
• a single dichroic filter to split the emission signal at 685 nm is used. More filters will be required when more than two dyes are employed.
• Dyes are evaluated to determine their compatibility in the MLSC system. As an example, a variety of dyes were evaluated to determine an appropriate overall 3-color set (see table 2).
• Parameters to consider when evaluating dyes include 1) spectral separation of the 3 dyes, 2) signal-to- noise ratio as a function of laser power, 3) suitability of the available filters, 4) ease of conjugation, and 5) specificity of the resulting antibody- fluorophore conjugates.
• Cy5 and APC are appropriate for the first color and Cy5.5 is appropriate for the second color.
• Several potential dyes are appropriate for the third color.
• Cy7-APC is expected to be suitable for the MLSC system. Preliminary results with the Imagn 2000 system demonstrate that this dye is detectable in the long wavelength channel (>685 nm) and distinct from both Cy5 and APC. Emission spectra indicate that overlap with Cy5.5 should not be a problem given appropriate filters for the new instrument.
• Fluorescent microspheres offer a wide variety of alternative colors and have been used successfully in some cytometry applications. Conjugation methods will be used which minimize the nonspecific binding that occasionally occurs with microsphere reagents. Typically, each of the 33 fluorophores are evaluated in the context of fluorophore-antibody conjugates using a few select antibodies e.g. anti-CD3, anti-CD4 and anti-CD20.
• MALDI-TOF matrix-assisted laser desorption/ionization
• Chemical derivatization can be selectively employed to activate components in a mixture that are not ionized enough to yield an ESI mass spectra.
• sterols typically devoid of acidic or basic residues that do not ionize under electrospray conditions have been coupled with ferrocene carboxylic acids, the electrochemical nature facilitating ionization.
• Derivatization can also serve as an handle to differentiate between stereoisomers (isobaric species) by using different fragmentation patterns in their daughter and granddaughter ions of the parents.
• the identification and correlation of biological markers with clinical measurements requires the integration of vast amounts of biological and medical data and a search engine that makes such data accessible and usable.
• the instrumentation and assays developed in the present invention have the ability to identify hundreds to thousands of independent markers from a small sample of blood.
• the present invention includes developing a broad clinical strategy to collect extensive medical information from patients that are followed over the time of disease progression and response to therapy.
• the present invention includes software, databases and data mining tools to correlate patterns of markers with specific diseases, disease progression and responses to therapy, including, but not limited to, databases of assays and clinical information, data conversion and statistical analysis tools, and medical questionnaire prototypes.
• the information system of the 35 present invention is designed to use common language and common formats for entry of disparate types of data and is structured for data-mining purposes.
• SnoMed-RT The universal medial language which will likely become widely used in the next several years is SnoMed-RT. This language will be readily adaptable with the current information system of the present invention. Similarly, the present invention is adaptable in that as other languages or technologies become available, they may also become incorporated into the database. An example would be the eventual development of tools to integrate digital x-rays, mammograms, or a virtual colonoscopy which is obtained via a
• the technical challenges in developing an informatics system capable of handling the vast amounts of biological and clinical information necessary to correlate biological markers with disease include modeling and integrating a number of diverse, complex, and often incompatible information sources, adapting to rapid advances in scientific and medical knowledge and methods, and developing a user-friendly interface, proper format and powerful search tools.
• the informatics system provided by the present invention meets these technical challenges.
• the data output from the cellular analyses includes both numbers of cells per ⁇ l of whole blood for each population identified, the mean intensity of staining for each cell associated molecule, which gives an estimate of the antigen density for a given population, the mean size of cells, and the expression levels of a particular molecule. Each number will be analyzed, because, as explained above, both the actual cell numbers as well as expression levels of a particular molecule may vary in a given disease state.
• markers cell counts or staining intensity
• levels of soluble factors associated with categorical clinical variables (such as diagnosis of disease)
• continuous-valued clinical variables such as levels of soluble factors
• regression techniques are used.
• stepwise variable selection and cross-validation are used to identify those markers that are most closely associated with the clinical variable of interest.
• demographic and clinical variables such as age, gender, concomitant drugs, etc.
• genetic parameters are included as covariates in the models. 36
• the architecture of the integrated informatics infrastructure comprises a multi-tiered structure.
• the lowest level consists of a set of data sources.
• the first source comprises the scientific data which includes, but is not limited to, cellular assay data and soluble factor assay data.
• the second source may be semi structured data which is in a combined form of textual and tabular data describing protocols for assay development and protocols for the execution of clinical studies.
• the structure may be encoded as a data type definition (DTD), defining tags that serve both for information indexing and querying as well as selective information display on web browsers.
• the DTD tags also define an information exchange model enabling the high- level electronic sharing of the information with other parties.
• the third data source is the clinical data gathered and restructured to meet the clinical study requirements.
• Clinical questionnaires that are optimized to maximize, under time constraints, the collection of useful and quantifiable information from patients, are used to gather information and to provide the necessary quality control. If necessary, the questionnaires will be multi lingual and adapted to physical challenges (e.g., the inability to use a computer keyboard) that the respondents may have to face.
• the technology of choice for this data source may be XML.
• the clinical information gathering system also comprises of non-textual means of input. A respondent may interact via visual and graphical displays to provide health related information by pointing at images of the human anatomy so as to indicate a problem without having to articulate it. Other means, e.g.
• the fourth source of data is the instrumentation data containing all of the relevant parameter settings required for the execution of the scientific assays on a combination of different instruments, such as Imagn, SurroScan and the ELISA plate reader.
• data can be collected and recorded in lists. In list form, measurement values for each individual cell are recorded. This facilitates identification and analysis of individual cell populations that express a complex set of different molecules. Alternative analysis schemes are readily explored, facilitating optimal data analysis.
• the complete set of patient data (cell populations, soluble factors, medical history, clinical parameters, etc.) can be stored in lists for each patient sample. 37 As indicated in Figure 3, these data sources are integrated and warehoused using a common schema. This schema coordinates the interpretation of the information from the constituent data sources. The interpretation is in a manner that is independent of the logical or physical storage detail of each of the constituent data sources.
• the common schema provides that data sources can be added or modified over time (management of change) without significantly affecting the tool set or user interface that ultimately use the compiled data.
• the common schema provides a buffer between the ever changing data sources and the application programs which use the compiled data and derive knowledge from the data.
• the schema is augmented with an ontology of common concepts and their relationships in immunology and related clinical areas.
• the ontology will be used by the data mining tools and by the user interface to assist in the interpretation of user specified requests for information from the underlying data sources and for the specification of data mining tasks.
• the ontology will also be utilized in the verification of the collected clinical data.
• the toolkit of programs includes programs for statistical analysis, for data mining and for the visualization of the results.
• a result of the analysis by the toolkit programs provides a set of rules relating a set of conditions to a set of consequences. These rules are applied over a statistically significant portion of the underlying data and are of the form: if condl and cond2 and ... and condN then consequencel, consequence2, ...
• the toolkit when applied to the cellular data source and the clinical data source, the toolkit can derive relationships between cellular assay and soluble factor measurements that were previously unknown. The results of the analysis by the toolkit are recycled to the users and to the database reuse in the future.
• the architecture is intended to improve the knowledge discovery process by storing the accumulated discovery experience and by integrating this experience for continued improvement.
• cytometry tools of the present invention may examine list mode data across an assay from multiple patient samples in order to determine the optimal set of 38 circumscribed population (gates).
• the system is coupled with a multi dimensional visualization system that will simultaneously project the computed clusters on selected subsets of two-dimensional and three-dimensional views.
• the final tier is a user interface. This part of the system serves the user interaction and is used to plan and execute tasks related to clinical studies. Tasks supported at the user interface level include, knowledge discovery from study data, clinical study planning, protocol planning and evaluation and assay development.
• the user interface will accept requests for information in a uniform way. It may combine a graphical interface and may allow for "drilling down" of information from the abstract concept level to the stored detail. It may allow for information requests that include both data and text (e.g., documentation pertaining to assay protocol planning) and may allow for interaction over a network.
• the present invention can be used to identify biological markers for rheumatoid arthritis (RA).
• Microvolume laser scanning cytometry (MLSC) is used to help create data for identifying biological markers for RA. Marker discovery efforts are focused on readily accessible biological fluids, most notably blood. A two-color instrument and antibody- based assays have demonstrated the potential of this technique for identifying and enumerating scores of different cell populations with only a small amount of whole blood. Multiparameter cell analysis, in combination with multiple assays for soluble factors, small molecules and an extensive clinical database, is a powerful tool for future biological marker discovery. Such markers have the potential to lead to new and more effective ways to predict and monitor disease activity and responses to therapy.
• Rheumatoid Arthritis is a chronic inflammatory disorder of the small joints, which also has pronounced systemic consequences. Although the etiology of the disease is unknown, its pathology evolves with common characteristics over time. Early events appear to include an inflammatory response initiated by unknown mediators. Activated CD4 + T cells appear to amplify and perpetuate the inflammation. The presence of activated T cells can induce polyclonal B-cell activation and production of Rheumatoid Factor (RF). Tissue damage accrues, releasing autoantigens, and the extent of the T cell 39 response broadens. Eventually, the constant inflammatory environment may lead to transformation of the synovial fibroblasts, yielding destructive potential that is independent of T cells and macrophages. The pro-inflammatory cytokines, produced mainly by macrophages in the joint and the cytokines they induce such as IL-6, are systemically active, present in the serum and augment hepatic synthesis of acute-phase proteins.
• RF Rheuma
• the present invention is useful to identify biological markers of diagnostic and prognostic value for Rheumatoid Arthritis. Such markers are required for classifying different forms of the disease, for example identifying the subset of patients in whom joint erosion occurs more rapidly than in others. Furthermore, the markers are critical for evaluating the efficacy of intervention and developing early, non-toxic and successful therapies. Many investigations have been made of cells and soluble factors in blood, synovium and urine that are candidate markers for the disease.
• each assay combination consists of one or more reagents to identify the major cellular subsets (left column of Table 1). Some of these antigens, e.g. CD4, are targeted in multiple assays. The major markers are combined with different subsetting antibodies (right column of Table 1) in order to maximize information about the sample. Properties of the fluorochomes and the target antigens are considered in developing each assay combination. For example, brighter fluorochromes are used with less abundant antigens. For other assays it is important to use reagents with the best spectral differences for certain targets.
• FlowJo software is used to analyze 1 to 3 different 3-color combinations (e.g., Cy5 CD3, Cy5.5 CD4, Cy7APC-CD45RA vs. Cy5 CD45RA, Cy5.5 CD4, Cy7APC-CD3) to determine the best combination for distinguishing the different cell populations.
• Cy5 CD3, Cy5.5 CD4, Cy7APC-CD45RA vs. Cy5 CD45RA, Cy5.5 CD4, Cy7APC-CD3 to determine the best combination for distinguishing the different cell populations.
• T cells The major antigens being evaluated in a T cell panel include CD2, CD3,
• CD4, CD5, CD7, and CD8 are CD4, CD5, CD7, and CD8.
• CD4 Many kinds of molecules on these T cell subpopulations can be investigated. These include surface antigens which help to distinguish naive (CD45RA) vs. memory cells (CD45RO, CD26), and antigens that play a role in activation (CD25,
• CD69, CD71, HLA class II) or co-stimulation CD27, CD28.
• markers that may play a role in adhesion to inflammatory sites are assayed (CD62L, CD 1 la/CD 18, CD44, CD54, and CD58).
• Subpopulations of T cells based on expression of ⁇ TCR, ⁇ TCR, and a panel of V ⁇ TCR genes are evaluated.
• B cells The major antigens being evaluated in a B cell panel include CD 19, CD20, CD21, CD22, CD23, and CD72.
• various markers on these B cell subsets including markers that may indicate a more activated phenotype (CD40, CD80, CD86, HLA class II, CD5) and those that have been implicated in lymphocyte homing and adhesion (CD62L, CD44, CD 11 a/CD 18) are analyzed.
• IgM, IgG, and IgA receptors for specific antigens are also evaluated.
• Antigen-presenting cells are evaluated using markers to the major antigens CD13, CD14, CD15, and CD33.
• a variety of adhesion molecules CD1 la, CD18, CD29, CD44, CD54, CD58, CD62L
• co-stimulatory molecules CD80, CD86
• Other relevant receptors including CD 16 (Fc ⁇ RIII), CD32 (Fc ⁇ RII), and CD64 (Fc ⁇ RI) are assayed.
• NK subpopulations using the markers CD16, CD56, CD57, and NKBl are analyzed.
• Granulocytes, including neutrophils and eosinophils may be phenotyped using CD13, CD15, and CD16.
• a panel of adhesion molecules and receptors similar to that described above is used to further subset these populations.
• T cells from RA patients show higher levels of the adhesion receptor LFA-1 (CD 1 la/CD 18) but no change in the expression of the IL2 receptor (CD25), which is normally increased on activated cells, or a marker for activation and co-stimulation (CD 80).
• CD 1 la/CD 18 the adhesion receptor LFA-1
• CD25 the IL2 receptor
• CD 80 a marker for activation and co-stimulation
• T cells There are several lines of evidence that implicate T cells in RA (Fox, D.A.
• B cells Phenotypic analysis of B cells has also been performed in RA patients.
• a B cell subpopulation expressing the pan T cell marker CD5 has been shown to be elevated (Sowden, J.A., Roberts-Thomson, P.J. and Zola, H. (1987) Rheumatol Int 7, 255-9, Hardy, R.R., Hayakawa, K., Shimizu, M., Yamasaki, K. and Kishimoto, T. (1987) Science 236, 81-3 and Casali, P., Burastero, S.E., Nakamura, M., Inghirami, G. and Notkins, A.L. (1987) Science 236, 77-81).
• Circulating B cells from RA patients also demonstrate increased expression of HLA DR molecules, again indicative of an activated B cell phenotype (Eliaou, J.F., Andary, M., Favier, F., Carayon, P., Poncelet, P., Sany, J., Brochier, J. and Clot, J. (1988)
• Three-color assay are able to monitor increased HLA class II expression specifically on CD5 T CD19 + B cells.
• Antigen-presenting cells include monocytes, macrophage, dendritic cells, B cells and other cells induced to express class II antigens. In general these cells show an activated phenotype demonstrated by increased expression levels of HLA class II antigens in patients with autoimmune disease (Lipsky, P.E., Davis, L.S., Cush, J.J. and Oppenheimer-Marks, N. (1989) Springer Semin Immunopathol 11, 123-62). Antigen-presenting cells are abundant in the synovial 43 compartment (Viner, N.J. (1995) Br Med Bull 51, 359-67) and blood-derived macrophages have been associated with human cartilage glycoprotein 39 expression in some studies
• Soluble factor assays provide an additional battery of potential biological markers. There are many important soluble factors that have been identified in RA patients. These include levels of circulating cytokines such as TNF ⁇ and
• IL-6 cytokine receptors
• chemokines rheumatoid factors of different isotypes
• immunoglobulin with different forms of glycosylation hormones, acute-phase proteins such as C-reactive protein and serum amyloid A, and soluble adhesion molecules, as well as matrix metalloproteinases and their inhibitors.
• Many of these soluble factors are known to be present at varying levels in RA patients at different stages of disease (Choy, E.H. and Scott, D.L. (1995) Drugs 50, 15-25, Feldmann, M., Brennan, F.M. and Maini, R.N. (1996) Annu Rev Immunol 14, 397-440, and Wollheim, F.A. (1996) Apmis 104, 81-93). Therefore, assays can be conducted to measure these soluble factors and look for statistical correlations with the cell populations identified.
• the clinical parameters will include information on age, gender, stage of disease, outside laboratory tests such as ESR, previous therapy and any concomitant drugs or therapies. This information is relevant to the evaluation.
• immunosuppressive drugs such as those often taken by RA patients, can have a profound effect on the expression of cell surface antigens.
• Patients treated with methotrexate show a decrease in CD 19" and CD5 + 19 + B cells.
• Patients treated with cyclophosphamide show a decrease in activated T cells expressing CD25 or HLA DR.
• Patients treated with prednisone express several changes in cell surface phenotype.
• IgM rheumatoid factor and the percentage of CD5 B cells (Youinou, P., Mackenzie, L., Katsikis, P., Merdrignac, G., Isenberg, D.A., Tuaillon, N., Lamour, A., Le Goff, P.,
• IgA rheumatoid factor is associated with the level of CD5 B cells as well as CD4 + CD45RO + T cells (Arinbjarnarson, S., Jonsson, T., Steinsson, K., Sigfusson, A., Jonsson, H., Geirsson, A., Thorsteinsson, J. and Valdimarsson, H. (1997) J Rheumatol 24, 269-74). Simultaneous measurement of multiple parameters increases the probability of identifying key variables for segregating patient groups.
• This generic example illustrates that this invention is uniquely suited for identifying ensembles of biological markers to characterize diseases.
• the MLSC technology which requires only a very small sample volume, provides that numerous assays can be completed on a single blood sample and ensures that the maximum amount of biological information can be acquired.
• the biological marker identification system can accommodate a mixture of assay types, including whole blood and RBC-lysed blood, among others. The assays conducted are considered relevant for the clinical indication and allow a broad survey. Relevant biological markers can be identified using the technology of the present invention.
• the present invention can be used to identify biological markers for Multiple Sclerosis (MS).
• MS is an autoimmune inflammatory disease of the central nervous system. MS is characterized clinically by relapsing and remitting episodes of neurologic dysfunction. The etiology of the disease remains unknown, however the 45 presence of inflammatory cells in the brain, spinal cord, and cerebrospinal fluid implies that an immune attack against CNS myelin is central to the pathogenesis of MS.
• the hallmark of the MS lesion is an area of demyelination called a plaque that may be found throughout the brain and spinal cord. Inflammatory cells are seen at the edges of the plaque and scattered throughout the white matter.
• the main inflammatory cells include activated lymphocytes and monocyte derived macrophages.
• CD4 T cells accumulate at the edges of the plaque; CD8 T cells are not found as frequently in active disease, but are present in longstanding lesions.
• Autoreactive T cells recognizing myelin basic protein and other non-myelin self-antigens circulate in the blood and upon activation can pass through the blood-brain barrier. Up-regulation of adhesion molecules, histocompatabihty antigens, and other markers of lymphocyte and monocyte activation (IL2R, FcR) are all connected with the activation and homing process.
• proinflammatory cytokines that serves to amplify the immune response.
• the autoimmune response also includes pronounced B cell stimulation.
• the autoantibodies produced can activate the complement system and promote demyelination. Throughout the various stages of disease, there are changes in the molecules and cells in the CNS and the blood that have potential to be markers of disease.
• the present invention can identify disease markers of diagnostic and prognostic value for Multiple Sclerosis. Such markers are valuable for classifying different forms of the disease, for example identifying the subset of patients with relapsing-remitting disease who are most likely to develop those secondary progressive disease. Furthermore, the markers are valuable for evaluating the efficacy of intervention and developing early, non- toxic and successful therapies. Many investigations have been made of cells and soluble factors in blood, cerebrospinal fluid (CSF) and urine that are candidate markers for the disease. In general, one to several markers at a time have been investigated. While some factors, such as oligoclonal immunoglobulin in the CSF, have been associated with MS, there is no consensus panel of MS-specific markers. There is a strong need to simultaneously evaluate multiple candidate markers.
• CSF cerebrospinal fluid
• T cells There are several lines of evidence that implicate T cells in MS. Such evidence includes the association of MS with MHC class II (particularly HLA DR) alleles (Hauser, S.L., Fleischnick, E., Weiner, H.L., Marcus, D., Awdeh, Z., Yunis, E.J. and Alper, CA. (1989) Neurology 39, 275-7). Since CD4 + T cells recognize antigen bound to MHC class II antigens, the association of MS with expression of specific class II molecules 46 implies a role for CD4 T cells in MS. In addition, studies in animal models of MS such as mouse or rat experimental allergic encephalomyelitis have shown that myelin antigen specific CD4 T cells can induce disease when adoptively transferred to na ⁇ ve animals
• MS patients have shown that strategies aimed at eliminating T cells or interfering with T cell function can slow progression of MS.
• T cells in the cerebrospinal fluid and peripheral blood show a memory phenotype with high levels of CD45RO and CD29 on both the CD4 and CD8 T cell populations (Vrethem, M., Dahle, C, Ekerfelt, C, Forsberg, P., Danielsson, O. and Ernerudh, J. (1998) Acta Neurol Scand 97, 215-20).
• CD4 + , CD4 + SLAM + , and CD4 + CD7 + cells are increased in MS patients relative to controls (Ferrante, P., Fusi, M.L., Saresella, M., Caputo, D., Biasin, M., Trabattoni, D., Salvaggio, A., Clerici, E., de Vries, J.E., Aversa, G., Cazzullo, CL. and Clerici, M. (1998) J Immunol 160, 1514-21).
• B cells Phenotypic analysis of B cells has also been performed in MS patients. A B cell subpopulation expressing the pan T cell marker CD5 has been shown to be elevated
• CD5 + B cells do not preferentially produce autobodies (Suzuki, N., Sakane, T. and Engleman, E.G. (1990) J Clin Invest 85, 238-47) and the role of CD5 + B cells in the pathogenesis of autoimmunity in humans is still unclear, perhaps reflecting the presence of activated B cells (Werner-Favre, C, Vischer, T.L., Wohlwend, D. and Zubler, R.H. (1989) Eur J Immunol 19, 1209-13). Consistent with this conclusion, high levels of the memory marker CD45RO were found on circulating CD20 + B cells from patients with MS
• CD80 + B cells The number of circulating CD80 + B cells is also increased significantly in MS patients with active disease, but is normal in stable MS (Gene, K., Dona, D.L. and Reder, NT. (1997) J Clin Invest 99, 2664-71).
• Antigen-presenting cells Several cell types can serve as antigen-presenting cells, including monocytes, macrophage, dendritic cells, B cells and other cells induced to express class II antigens.
• Soluble factor assays provide an additional battery of potential biological markers. There are many important soluble factors that have been identified in MS patients. For example, levels of soluble Apo A-l/Fas (Ferrante, P., Fusi,
• Proinflamatory cytokines like TNF ⁇ and IFN ⁇ are known to be present at varying levels in MS patients at different stages of disease (Navikas, V. and Link, H. (1996) J Neurosci Res 45, 322-33).
• Other relevant proteins such as cytokines and cytokine receptors, chemokines, matrix metalloproteinases and their inhibitors, neopterin, and myelin basic protein, have also been shown to be present at varying levels in MS patients at different stages of disease and healthy controls. Therefore, assays can be conducted to measure these soluble factors and look for statistical correlations with the cell populations identified.
• the clinical history will include information on age, gender, stage of disease, outside laboratory evidence (magnetic resonance imaging, cerebrospinal fluid analysis for oligoclonal immunoglobulin and evoked potential recordings), previous therapy and any concomitant drugs or therapies. This information is relevant for segregating patient populations.
• the cellular assays allowed us to identify approximately 100 different cell populations including sets of T cells, B cells, NK cells, monocytes, and granulocytes. Methods Cellular assays The panel of assays is shown in Table 3. Each reagent is tested and titrated before preparing the reagent combinations in order to optimize assay performance. Sample preparation
• Serum levels of C-reactive protein were measured on the Imagn 2000 with a bead- based immunoassay. Beads coated with anti-CRP antibody were used to capture the analyte. Cy5 conjugated anti-CRP antibody was used reveal the captured analyte.
• FIG 4 The cells were stained with CD27 conjugated to Cy5 in combination with CD8 conjugated to Cy5.5. This combination allowed CD8 + T cells (MHC class I restricted) to be monitored, which are CD27 + (activated) and CD27 " . CD8 " , CD27 + cells (which are actually activated CD4, MHC class II restricted, T cells) are also detected. Although there is variation among the donors, a single gating strategy can be implemented. Three cell populations are identified which differ among the donors. In Figure 4A the majority of CD8 + T cells are CD27 negative. In Figure 4B the majority of CD8 + cells are CD27 positive. Finally, in Figure 4C, the CD8 population is split between those that are CD27 positive and those that are CD27 negative.
• RA rheumatoid arthritis
• the RBC-lysed sample preparation is useful for activated cells expressing CD25 or CD69 which are essentially undetectable in whole blood from normal individuals but are increased ten-fold in the lysed format and are likely to be increased in various autoimmune states. Improved detection of other minor cell populations such as NK cells has also been demonstrated.
• the cellular assays included a panel of 60 2-color combinations comprising 46 whole blood assays and 14 RBC-lysed whole blood. A total of 39 different antibody reagents (30 conjugated to Cy5 and 9 conjugated to Cy5.5), targeting 35 distinct cell surface antigens, were used. All assays are done in homogeneous mode (no wash after 53 staining). This assay panel enables the identification of more than 150 different cell populations.
• the reagent combinations and the cell populations that can be identified are provided in Table 5.
• Soluble Factor Assays Sera are aliquoted and frozen for each blood sample for subsequent measurement of multiple soluble factors. These include levels of circulating cytokines such as TNF ⁇ and
• IL-6 IL-6
• cytokine receptors cytokine receptors
• chemokines rheumatoid factors (RF) of different isotypes
• immunoglobulin immunoglobulin
• acute-phase proteins such as C-reactive protein and serum amyloid A
• soluble adhesion molecules as well as matrix metalloproteinases and their inhibitors.
• Table 6 The initial panel of 22 soluble factors assayed is shown in Table 6. Additional targets are also provided in Table 6. All assays are done in a sandwich ELISA format using matched antibody pairs to ensure the required sensitivity and specificity.
• More assays can be run on the 4- channel SurroScan instrument. Assays are developed using 3 color reagent combinations. Effective dye combinations include Cy5, Cy5.5 and Cy7 and Cy5, Cy5.5 and Cy7-APC allow simultaneous and independent measurment of three target antigens. Three color combinations facilitate the acquisition of more information per capillary than 2 color combinations by 1) eliminating redundancy (e.g. measuring CD3, CD4 and CD8 in one capillary instead of measuring CD3 + CD4 and CD3 + CD8 in two capillaries) and 2) identifmg new populaitons that are defined by the simultaneous expression of 3 antigens (e.g. na ⁇ ve CD4 + T cells that express both CD45RA and CD62L).
• Effective dye combinations include Cy5, Cy5.5 and Cy7 and Cy5, Cy5.5 and Cy7-APC allow simultaneous and independent measurment of three target antigens.
• Three color combinations facilitate the acquisition of more information per capillary than 2 color combinations by 1) eliminating redundancy (e.g. measuring CD3, CD4 and
• Figure 7 provides the results of a 3-color assay on the SurroScan instrument.
• Assays on the SurroScan instrument can be executed with capillary arrays which use about 1/3 less sample than the VCI 20 capillaries.
• For whole blood assays it is possible to process 10 uL or less per 3 color assay, giving the potential for up to 1000 54 assays per 10 mL tube of blood.
• 3-color assays with 50 or more target antigens is under development using both whole blood and lysed formats. It should allow identification of more than 200 cell populations.
• Intracellular molecules can be measured with MLSC technology.
• PBMC peripheral blood mononuclear cells
• ionomycin adenosarcoma
• Cells were stained with Cy5.5 anti-CD8 to identify cytoxic T cells, fixed, permiablized, and stained with Cy5 anti- inerferon-gamma (IFN- ⁇ ) to detect the intracellular cytokine.
• IFN- ⁇ Cy5 anti- inerferon-gamma
• IFN- ⁇ is detected only in stimulated cells.
• a control reagent (MOPC) does not label the cells.
• MOPC control reagent
• the present invention can be used to identify biological markers for allergic asthma.
• Asthma is common chronic lung disease of uncertain etiology. It is characterized by inflammation of the airways leading to symptoms of coughing, wheezing, chest tightness, and shortness of breath. These clinical symptoms are thought to be due to hyper- responsiveness of the airways and a long-term inflammatory process causing obstruction of airflow. The disease causes extreme discomfort and can at times be fatal in the absence of appropriate treatment.
• the clinical manifestations of asthma are thought to result from the superimposition of a variety of environmental factors on genetic predispositions that increase the likelihood of developing asthma. Atopy, the hypersensitivity to environmental allergens, is common in asthma, but not all atopic individuals develop asthma. The relative importance of allergic mechanisms is not completely understood.
• Corticosteroids are efficacious in asthma but have associated with perceived and real side effects that limit their usefulness. A more complete understanding of response to corticosteroids might allow for the development of drugs with only local effects within the lungs or drugs that have beneficial effects without side effects. 55 A study has been designed to identify biological markers of atopy, asthma and the response to corticosteroid therapy. Subjects are screened for four study groups of 20: 1) mild asthmatics who have tested positive to skin test allergens, 2) mild asthmatics who have tested negative to skin test allergens, 3) non-asthmatics who have tested positive to skin test allergens, and 4) non-asthmatics who have tested negative to skin test allergens
• Mild asthmatics have a 1) FEVi > 80% predicted, 2) documented diagnosis of asthma or history of any of the following: cough, worse particularly at night, recurrent wheeze, recurrent difficult breathing, recurrent chest tightness and 3) a positive methacholine challenge test (Cockcroft DW, et al Clin Allergy 1977; 7:235 and Juniper EF, et al Thorax 1984; 39:556).
• Non asthmatics have a 1) FEVi > 80% predicted 2) no history of asthma and 3) a negative methacholine challenge test.
• Allergic subjects have a positive skin test to at least one of a panel of allergens.
• Examples of clinical data include Haematology: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (HCT), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC) platelet count, neutrophil count lymphocyte count, monocyte count, eosinophil count, basophil count and ESR - erythrocyte sedimentation rate; blood biochemistry: alkaline phosphatase, alanine transaminase, aspartate transaminase, gamma-glutamyl transpeptidase, albumin, total protein, total bilirubin, urea, creatinine, sodium, potassium, glucose; urinalysis: protein, glucose, ketones, bilirubin, blood, leucocytes; Hepatitis and HIV testing: HIV I and II, Hepatitis B surface antigen, Hepatitis C antibody. All clinical history and test parameters will be
• Atopic asthma is an immunologic disease mediated by IgE antibodies.
• Exposure to allergen causes B cells to synthesize IgE, which binds to the high affinity receptor mast cells residing in the mucosa of the airways.
• antigen- antibody interactions on the surface of the mast cells triggers release of mediators of anaphylaxis stored in mast cell granules, including: histamine, tryptase, PGD 2 , leukotriene 56 C 4 and D 4 , and platelet activating factor (PAF).
• PAF platelet activating factor
• These soluble factors induce contraction of air smooth muscle and cause an immediate fall in the FEVi.
• Re-exposure to allergens also leads to the synthesis and release of a variety of cytokines: IL-4, IL-5, GM-CSF, TNF- ⁇ ,
• TGF- ⁇ TGF- ⁇ , from T cells and mast cells.
• cytokines attract and activate B cells, which leads to the production of more IgE, and eosinophils and neutrophils, which produce eosinophil cationic protein (ECP), major basic protein (MBP) and PAF.
• ECP eosinophil cationic protein
• MBP major basic protein
• PAF eosinophil cationic protein
• ECP eosinophil cationic protein
• MBP major basic protein
• PAF eosinophil cationic protein
• FEVi about 4-6 hours after exposure.
• a broad panel of cellular and soluble factor measurements are applied to the subject blood samples with the goal of discovery biomarkers.
• the study design supplies information of inter-individual variability within groups, and inter-group differences in marker expression. It is believed that the inter-group differences (e.g. allergic non- asthmatic versus non-allergic non-asthmatic) will be greater than inter-individual variability within groups. It is further believed that prednisone therapy will result in significant intra-individual changes in marker expression.
• Immunoassays in the sandwich-based chemiluminescent ELISA format, are used for the following targets:
• Cytokines, chemokines and their soluble receptors IL-1 alpha, IL-1 beta, IL-1 RA, IL-1 sRI, IL-1 sRII, IL-2, IL-2sR, IL-3IL-4, IL-5, IL-6, IL-6 sR, IL-8, IL-10, IL-12 p40, IL-12 p70, IL-13, IL-16, IL-17, MIF, MIP-1 alpha, MIP-1 beta, RANTES, sTNFalpha RI *, sTNFalpha RII *, TGF beta, TNF alpha, alpha, TGF beta2, TGF beta3, Oncostatin M, M-CSF, GM-CSF, IGF-1, PDGF-BB, FGF-4, FGF-6, FGF-7, Fas, VEGF, MCP-1.
• IgAl Kappa IgAl Lambda, IgAl, 2 Kappa, IgAl, 2 Lambda, IgA2 Kappa, IgA2 Lambda, IgE total, IgGl Kappa, IgGl Lambda, IgGl total, IgG2 Kappa, IgG2 Lambda, IgG2 total, IgG3 Kappa, IgG3 Lambda, IgG3 total, IgG4 Kappa, IgG4 Lambda, IgG4 total, IgG total, IgG total , IgG total Kappa, IgG total Lambda, IgM Kappa, IgM Lambda, IgM total, RFIgA, RFIgG, RFIgM, RF total, Acute phase proteins: 57 CRP, SAA; Matrix metalloproteinases and their inhibitors: M
• TIMP-2 Soluble adhesion molecules: sCD54 (ICAM-1), sCD62E, sCD62P.
• Additional soluble factors which are measured by immunoassays or mass spectroscopy assay include, but are not limited to, Cytokines, chemokines and their soluble receptors: IL-9, IL-11, IL-14, IL-15, IL-18, sCD23, eosinophil proteins: ECP, MBP,
• Immunoglobulin Allergen specific IgE, carbohydrate modified Ig; a variety of prostaglandins; a variety of leukotrienes, histamine.
• Data output from the cellular assays, soluble factor assays, medical histories and screening labels are combined into a single database.
• potential biological markers cell counts, antigen intensity on particular cell types, soluble factor concentrations, etc
• categorical clinical variables disease status, prednisone or placebo, before or after therapy
• demographic and clinical variables can be included as covariates in the models.
• Techniques can be implemented with SAS, Statistica, Statview or similar statistical analysis software packages.
• the present invention can be used to identify biological markers for evaluating the effects of drug administration on cellular and soluble factors to be performed on small samples of peripheral blood. It is expected that these assays will make possible analysis of the effects of different doses of drugs on cellular and soluble markers in human peripheral blood.
• aspirin acetylsahcyhc acid
• aspirin is administered to human volunteers. Different doses of the drug will be orally administered; blood is drawn before and at various time points after administration, and panels of cellular and soluble factor assays are undertaken.
• the aspirin is expected to cause changes in the cellular and soluble components of blood.
• Aspirin is routinely used for two main indications: 1) to reduce the risk of coronary and cerebral thrombosis and 2) as an analgesic/anti-inflammatory agent.
• the mechanism underlying the first indication is believed to be irreversible inhibition of the enzyme PGH- synthatase in platelets.
• a prostaglandin product of this enzyme in platelets is converted to 58 thromboxane A2, which facilitates platelet aggregation and thrombosis.
• a side effect of prostaglandin synthesis is the generation of oxygen free radicals, which in the presence of redox-oxidative metals convert unsaturated fatty acids into aldehydes.
• a relatively stable product of lipid oxidation is malondialdehyde (MDA).
• This compound is routinely assayed colorimeterically or fluorometrically following interaction with thiobarbituric acid (TBA.
• TAA thiobarbituric acid
• Aspirin by inhibiting prostaglandin synthesis, is expected to decrease MDA levels in peripheral blood platelets. This is one parameter that is expected to change following aspirin administration. Changes in other markers of platelet activation such as changes in the expression of CD62P and CD63 may also occur. E-type prostaglandins suppress lymphocyte activation and the production of tumor necrosis factor- ⁇ (TNF- ⁇ ) by the cells of the monocyte-macrophage lineage. If there is some level of lymphocyte activation and TNF- ⁇ production in normal healthy persons, this may be increased after aspirin treatment and detectable in the peripheral blood.
• TNF- ⁇ tumor necrosis factor- ⁇
• the study is designed to identify the effects of aspirin on blood parameters. Eligible subjects are randomly assigned to orally administer aspirin according to one of three dosing schemes. Group I, 1 dose (325 mg tablet) after breakfast, Group II. 2 doses (650 mg) after breakfast and Group III, 2 doses after breakfast and 2 doses after diner (1300 mg total). There are 10-12 subjects per cohort. Blood samples are taken before, during and after aspirin administration. The schedule is given in Table 8. Subjects are healthy individuals age 18-65 who are not taking other aspirin other non-steroidal anti-inflammatory drugs nor currently under care, which requires the use of anti-inflammatory (steroidal or non- steroidal) drugs. Cellular assays
• a panel of 42 three-color cellular assays are used for the initial study see Table 9.
• the panel includes immune and inflammatory parameters and contains some of the assays listed in Example 7. It also includes a series of assays for platelet function (1-17). These assays include direct measurements in diluted whole blood (WB, 1-9) as well as thrombin stimulation assays (TRT, 10-13) and stimulation controls (NTRT, 14-17). 59 Soluble Factors
• Additional measurements include : Von Willebrand factor, b-Thromboglobulin,
• Thromboxane B2 6-keto PGF and malondialdehyde. Soluble factors will be measured from plasma. In addition, some soluble factors (e.g MDA, prostaglandins leukotrienes) will be evaluated for the stimulated samples and controls.
• MDA prostaglandins leukotrienes

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