Patent Application
20240280560
The present invention relates to diagnostic markers of acute myeloid leukaemia, to a diagnostic screen based on said markers, and to the use of said screen in diagnostic and prognostic methods.
Human Acute Myeloid Leukaemia (AML) is an aggressive cancer of white blood cells and is the most common adult acute leukaemia. In more detail, AML is a cancer of the myeloid line of blood cells. It is characterized by the rapid growth of an abnormal white blood cell population. Approximately 80% of AML patients are over the age of 60 and the overall survival of this patient group lies at only approximately 5%.
AML can be classified into several subgroups. By way of example, classification according to the World Health Organization (WHO) criteria is based on examination of bone marrow aspirate or a blood sample via light microscopy. Alternatively, bone marrow or blood may be tested for chromosomal translocations by routine cytogenetic methods or fluorescent in situ hybridisation (FISH), and for specific genetic mutations (such as mutations in the FLT3, NPM1 and CEBPA genes) may be detected by polymerase chain reaction (PCR). Immunophenotyping is another method that may be used to identify the AML subtype, which involves detection of cell surface and cytoplasmic markers using flow cytometry.
Flow cytometry is a technique for counting and examining microscopic particles such as cells by suspending them in a stream of fluid and capturing the light that emerges from each cell as it passes through a laser beam. Cell surface molecules often referred to as “cluster of differentiation” (CD) molecules may be exploited in flow cytometry to characterise cell populations. For example, in fluorescence-activated cell sorting, a diagnostic antibody (labelled with a fluorophore) is employed, which binds to a surface molecule (e.g. a CD molecule) present on and characteristic of the cell population in question. Thereafter, the flourophore (attached to the antibody) is activated by a laser beam and the fluorescence signal detected by the flow cytometer. In this manner, fluorescently-labelled antibodies can be used to detect and sort cells displaying a specific CD molecule (or set of CD molecules).
Current AML therapies typically involve induction chemotherapy followed by post-induction therapy. The goal of induction chemotherapy is to reduce the amount of leukaemic cells to less than 5% of all the nucleated cells in a bone marrow sample. Regrettably, this level of reduction of leukaemic cells is not enough to prevent disease recurrence (i.e. relapse) and almost all patients relapse without post-induction therapy. Post-induction therapy typically involves further cycles of chemotherapy, and in some cases, a hematopoietic stem cell transplant that aims to eliminate minimal residual disease (MRD). MRD is the population of leukaemic cells that is recaltricant to therapy. It is thought that this population of cells contains a sub-population of cells termed a leukaemic stem cell (LSC) population that is largely quiescent and serves to sustain disease.
Current methods used to detect MRD include real time quantitative PCR (RQ-PCR) or by multi-parameter flow cytometry (MFC). However, RQ-PCR based MRD assessment is not possible in approximately half of patients with AML. In addition, and despite recent technical developments, there is still a lack of a validated MFC methodology demonstrating clinical utility-current sensitivity levels of MFC are at least 1 log below real time that of RQ-PCR assays.
There is, therefore, a need to provide an alternative and/or improved diagnostic screen for acute myeloid leukaemia. In addition, there is a need to provide an alternative and/or improved method for diagnosis and/or prognosis of acute myeloid leukaemia. In particular, there is a need to provide an alternative and/or improved method to detect and monitor MRD for acute myeloid leukaemia.
The present invention solves one or more of the above mentioned problems.
In one aspect, the invention provides a diagnostic screen for detecting acute myeloid leukaemia, wherein said screen detects the presence (+) or absence (−), as indicated below, of the following cell surface polypeptide markers:
A cell surface polypeptide marker may be displayed (at least in part) on the extracellular surface of a cell. Markers of the present invention may include CD34, CD45RA, CD90, CD123, CD38, CD19, CD47, CCR8, RHAMM and/or CD86. CD34 is a heavily glycosylated, 105-120 kDa transmembrane glycoprotein expressed on hematopoietic progenitor cells, vascular endothelial cells and some fibroblasts. The CD34 cytoplasmic domain is a target for phosphorylation by activated protein kinase C, suggesting a role for CD34 in signal transduction. CD34 may also play a role in adhesion of certain antigens to endothelium. CD45R, also designated CD45 and PTPRC, has been identified as a transmembrane glycoprotein, broadly expressed among hematopoietic cells. Multiple isoforms of CD45R are distributed throughout the immune system according to cell type including CD45RA. CD45R functions as a phosphotyrosine phosphatase, a vital component for efficient tyrosine phosphorylation induction by the TCR/CD3 complex. CD90 is a 25-37 kDa heavily N-glycosylated, glycophosphatidylinositol (GPI) anchored conserved cell surface protein originally discovered as a thymocyte antigen. The CD123 antigen (also known as interleukin-3 receptor) is a molecule found on cells which helps transmit the signal of interleukin-3, a soluble cytokine important in the immune system. CD38, also known as cyclic ADP ribose hydrolase is a glycoprotein found on the surface of many immune cells (white blood cells. CD38 is thought to function in cell adhesion, signal transduction and calcium signaling. CD19 is a 95 kDa type-I transmembrane glycoprotein that belongs to the immunglobulin superfamily. It is expressed on B cells throughout most stages of B cell differentiation and associates with CD21, CD81, and CD225 (Leu-13) forming a signal transduction complex. CD19 functions as a regulator in B cell development, activation, and differentiation. CD47 is an integral membrane protein that plays a role in the regulation of cation fluxes across cell membranes. It is also a receptor for the C-terminal cell binding domain of thrombospondin (SIRP). CD47 is expressed on hemopoietic cells, epithelial cells, endothelial cells, fibroblasts, brain and mesenchymal cells. Chemokine receptor 8, also known as CCR8 is a member of the beta chemokine receptor family CCR8. HMMR hyaluronan-mediated motility receptor (RHAMM) is a cell surface receptor. The CD86 gene encodes a type I membrane protein that is expressed on antigen-presenting cells.
The present inventors have unexpectedly found that a combination of the above-mentioned cell surface markers represents a robust diagnostic screen for acute myeloid leukaemia. Diagnostic capacity in this context may also embrace prognostic capacity and diagnosis/monitoring of MRD.
A screen as defined above has many useful applications including diagnostic and prognostic applications such as in clinical guidance and for determining therapy, for patient management and for assessing treatment efficacy.
In one embodiment, the invention provides a diagnostic screen as defined above, wherein the marker iii) is CD90−.
In another embodiment, the invention provides a diagnostic screen as defined above, wherein the marker iii) is CD123+.
In another embodiment, the invention provides a diagnostic screen as defined above, wherein the marker iii) is CD90− and CD123+.
In a further embodiment, the invention provides a diagnostic screen as defined above, further comprising the cell surface polypeptide marker CD38+. In an alternative embodiment, the invention provides a diagnostic screen as defined above, further comprising the cell surface polypeptide marker CD38− (i.e. CD38− instead of CD38+).
In one embodiment, the invention provides a diagnostic screen as defined above, further comprising one or more (or two or more, or three or more, or four or more) of the cell surface polypeptide markers selected from CD19−, CD47+/−, CCR8+/−, CD86− and/or RHAMM+/−. In one embodiment, the invention provides a diagnostic screen as defined above, comprising the cell surface polypeptide marker CD47+. In an alternative embodiment, the invention provides a diagnostic screen as defined above, comprising the cell surface polypeptide marker CD47− (i.e. CD47− instead of CD47+). In one embodiment, the invention provides a diagnostic screen as defined above, comprising the cell surface polypeptide marker CCR8+. In an alternative embodiment, the invention provides a diagnostic screen as defined above, comprising the cell surface polypeptide marker CCR8− (i.e. CCR8− instead of CCR8+). In one embodiment, the invention provides a diagnostic screen as defined above, comprising the cell surface polypeptide marker RHAMM+. In an alternative embodiment, the invention provides a diagnostic screen as defined above, comprising the cell surface polypeptide marker RHAMM− (i.e. RHAMM− instead of RHAMM+).
In one embodiment, the diagnostic screen comprises one or more antibodies that bind to one or more of the identified markers. Thus, said one or more antibodies may be used to confirm the presence (+) or absence (−) of said cell surface polypeptide markers. In one embodiment, the presence (+) of a marker refers to an elevation in the levels of marker in a sample above a background level. Likewise, the absence (−) of a marker refers to a reduction in the levels of a marker in a sample below a background level. In one embodiment, the elevation in the levels of marker in a sample above a background level is 1 or more (such as 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25) flourescence units. In one embodiment a reduction in the levels of a merker in a sample below a background level is 1 or more (such as 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25) flourescence units. In this regard, it would be routine for a skilled person in the art to determine the background level of marker expression in a sample. Thus, in one embodiment, said cell surface polypeptide markers may be detected by specific binding of said one or more antibodies.
In one embodiment, the screen comprises one or more antibodies that bind to one or more cell surface polypeptide markers selected from CD34, CD45RA, CD90, CD123, CD38, CD19, CD47, CCR8, CD86 and/or RHAMM.
In one embodiment, the screen comprises a first antibody that binds to CD34, a second antibody that binds to CD45RA, and a third antibody that binds to CD90 and/or to CD123. In another embodiment, the screen comprises a first antibody that binds to CD34, a second antibody that binds to CD45RA, a third antibody that binds to CD90− and/or to CD123, and a fourth antibody that binds to CD38.
Any one or more of said antibodies may bind to one of said markers and not (substantially) to any of the other markers. For example, each of the employed antibodies may bind to one of said markers and not (substantially) to any of the other markers. Alternatively, any one or more of said antibodies may bind to two, three, four, five, six, seven, eight, nine or all ten of said markers.
In one embodiment, the screen comprises three antibodies, wherein:
In an alternative embodiment, the screen comprises three antibodies, wherein:
In one embodiment, the antibodies of the present invention recognise and bind to specific epitopes of the above mentioned cell surface polypeptide markers. For example, an antibody of the present invention may bind to an epitope in the N-terminal/C-terminal/mid-region domains/extracellular domains of CD34/CD45RA/CD 90/CD123/CD38/CD19/CD47/CD86/CCR8 and/or RHAMM. The sequence of CD34, CD45RA, CD90, CD123, CD38, CD19, CD47, CD86/CCR8 and RHAMM are available from the NCBI website (http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/human/index.shtml). These protein sequences are provided as SEQ ID NOs: 1-10. The corresponding nucleic acid sequences are provided as SEQ ID NOs: 11-20.
In one embodiment, the antibodies of the present invention may bind to a CD34/CD45RA/CD 90/CD123/CD38/CD19/CD47/CCR8, CD86 and/or RHAMM molecules comprising an amino acid sequence having at least 80% (such at least 85%, 90%, 95%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 1-10, or a fragment thereof.
Conventional methods for determining nucleic acid sequence identity are discussed in more detail later in the specification.
In one embodiment, the antibodies are polyclonal and/or monoclonal antibodies.
In one embodiment, an antibody that binds to one of the above-mentioned cell surface polypeptide markers is one capable of binding that marker with sufficient affinity such that the antibody is useful as a diagnostic/and or prognostic agent. In one embodiment, the term ‘binds’ is equivalent to ‘specifically binds’. An antibody that binds/specifically binds to a cell surface polypeptide marker of interest is one that binds to one of the above mentioned markers with an affinity (Ka) of at least 104 M.
Suitable antibodies of the present invention may include FITC or PE-Cy7 conjugated anti-CD38, PE or FITC-conjugated anti-CD45RA, PE-Cy7-conjugated or APC conjugated anti-CD123, biotin-conjugated anti-CD90, PE-Cy5 or PERCP-conjugated anti-CD34, PE-conjugated CD47, CD19 Horizon V450 and APC-Alexa Fluor 750 or APC-eFluor 780 conjugated streptavidin which are available from a number of different commercial suppliers including BD Biosciences Europe ebioscience, Beckman Coulter and Pharmingen.
In a preferred embodiment, the antibody is a labelled antibody, such as a fluorescently labelled antibody. Suitable labelled compounds include conventionally known labelled compounds, such as fluorescent substances such as cyanine dyes Cy3 (registered trademark of Amersham Life Science), fluorescein isothiacyanate (FITC), allophycocyanin (APC), rhodamine, Phycoerythrin (PE), PE-Cy5 (Phycoerythrin-Cy5), PE-Cy7 (Phycoerythrin-Cy7), APC-Alexa Fluor 750, APC-eFluor 780, Pacific Blue, Horizon V450 and quantum dot, biotin-conjugated; light scattering substances such as gold particles; photo-absorptive substances such as ferrite; radioactive substances such as <125> I; and enzymes such as peroxidase or alkali phosphatase.
In one embodiment of the invention, different antibodies are labelled respectively with mutually distinguishable labels. Labelling may be conducted by binding a labelled compound directly to each antibody. Preferably, the antibodies are labelled with different fluorescent dyes with different fluorescence wavelengths to enable easy discrimination from one another. For example a first antibody may be labelled in red (for example PE-Cy5), a second antibody in orange (for example PI, APC, R-PE) and a third antibody in green (for example Alexa488, FITC). Suitable labelling strategies are routine and known to a person skilled in the art. By way of example, the Lightening Link™ antibody labeling kit may be used (Innova Biosciences, UK).
Methods suitable for detection of the cell surface polypeptide markers of the present invention using labelled antibodies are conventional techniques known to those skilled in the art. For example, when a fluorescent label is used, an antibody that specifically binds to a marker may be detected by observing the emitted fluorescence colour under a microscope. A fluorescent label can also be detected by irradiating a sample with an exciting light-if the label is present, fluorescence is emitted from the sample. Thus, whether a cell is positive or negative for a particular cell surface marker may be judged by using a labelled antibody specific for said marker and observing the emitted fluorescence colour under a microscope. In a preferred embodiment of the invention, fluorescence-activated cell sorting (FACS) is used for detection of the cell surface polypeptide markers/labeled antibodies of the present invention.
In one aspect, the present invention provides a screen (as defined above) for use in a method of diagnosis of acute myeloid leukaemia.
In a related aspect, the invention provides a method for diagnosing acute myeloid leukaemia, said method comprising:
In one embodiment, the method of diagnosis comprises:
All embodiments described above for the diagnostic screen apply equally to the method of diagnosis aspect. By way of example, the latter aspect may further comprise identification of the cell surface polypeptide marker CD38+. Alternatively, the latter aspect may further comprise the cell surface polypeptide marker CD38− (i.e. CD38 instead of CD38+).
In another aspect, the present invention provides a screen (as defined above) for use in a method of prognosis of acute myeloid leukaemia.
In a related aspect, the invention provides a method for prognosis of acute myeloid leukaemia, said method comprising:
In one embodiment, the method of prognosis comprises:
All embodiments described above for the diagnostic screen apply equally to the method of prognosis aspect. By way of example, the latter aspect may further comprise identification of the cell surface polypeptide marker CD38+. Alternatively, the latter aspect may further comprise the cell surface polypeptide marker CD38− (i.e. CD38− instead of CD38+).
In another aspect, the present invention provides a screen (as defined above) for use in a method of identifying a therapeutic candidate for the treatment of acute myeloid leukaemia.
In a related aspect, the invention provides a method of identifying a therapeutic candidate for the treatment of acute myeloid leukaemia, said method comprising:
In one embodiment, the method of identifying a therapeutic candidate for the treatment of acute myeloid leukaemia comprises:
All embodiments described above for the diagnostic screen apply equally to the method of identifying a therapeutic candidate aspect. By way of example, the latter aspect may further comprise identification of the cell surface polypeptide marker CD38+. Alternatively, the latter aspect may further comprise the cell surface polypeptide marker CD38− (i.e. CD38− instead of CD38+).
In another aspect, the present invention provides a screen (as defined above) for use in a method of monitoring efficacy of a therapeutic molecule in treating acute myeloid leukaemia.
In a related aspect, the invention provides a method for monitoring efficacy of a therapeutic molecule in treating acute myeloid leukaemia, said method comprising:
In one embodiment, the invention provides a method for monitoring efficacy of a therapeutic molecule in treating acute myeloid leukaemia, said method comprising:
All embodiments described above for the diagnostic screen apply equally to the method for monitoring efficacy of a therapeutic molecule in treating acute myeloid leukaemia aspect. By way of example, the latter aspect may further comprise identification of the cell surface polypeptide marker CD38+. Alternatively, the latter aspect may further comprise the cell surface polypeptide marker CD38 (i.e. CD38− instead of CD38+).
In one aspect, the invention provides a kit for diagnosis and/or prognosis of acute myeloid leukaemia, said kit comprising at least one antibody that binds to a cell surface polypeptide marker selected from:
In one embodiment, said kit comprises a first antibody that binds to CD34, a second antibody that binds to CD45, and a third antibody that binds to CD90− and/or CD123. In one embodiment, each of said antibodies is different. In another embodiment, each of said antibodies does not substantially bind to any other marker of the present invention—for example: the first antibody does not substantially bind to any of CD45RA, CD90, or CD123; the second antibody does not substantially bind to any of CD34, CD90, or CD123; and the third antibody substantially binds only to one of CD90 or CD123, wherein the third antibody does not substantially bind to either of CD34 or CD45RA. The third antibody may be present that binds to CD90− and not substantially to any of CD34, CD45RA or CD123. A fourth antibody may be present that binds to CD123 and not substantially to any of CD34, CD45RA, or CD90.
In one embodiment, the kit may further comprise instructions explaining how to use the antibodies thereof in a diagnostic/prognostic method of the invention.
All embodiments described above for the diagnostic screen apply equally to the kit aspect. By way of example, the latter aspect may further comprise an antibody that binds to the cell surface polypeptide marker CD38. Thus, in one embodiment, said antibody may constitute a fifth antibody of the kit. In one embodiment, said fifth antibody does not substantially to any other (aforementioned) marker of the invention.
A kit of the present invention may optionally comprise suitable labels as described above (e.g. a fluorophore label) in addition to the one or more antibodies. The kit may optionally contain an instruction manual instructing the user to perform the methods of the present invention.
In one embodiment, acute myeloid leukaemia includes all AML samples that contain the CD34 cell surface marker.
In one embodiment, the term ‘diagnosis’ is used to mean determining the incidence of AML by examining whether one or more of the cell surface polypeptide markers of the diagnostic screen is present. In one embodiment, diagnosis of AML embraces diagnosis of minimal residual disease (MRD). Accordingly, in one embodiment, reference herein to acute myeloid leukaemia (AML) embraces MRD.
In one embodiment, a sample is obtained from a mammal, such as a human. A suitable sample is a bone marrow or blood sample. The white blood cell population of the sample is preferably extracted or enriched prior to detection of the marker-set with antibodies of the present invention. Methods suitable for extraction of enrichment of the white blood cells from a sample are conventional techniques known to those skilled in the art. By way of example, one approach is to deplete a sample of of red cells by red cell lysis. Another approach is to isolate a mononuclear by density centrifugation using a density media like Ficoll. CD34+ cells can be then be purified from mononuclear cells by incubation with magnetic beads coated with CD34 antibody and separating CD34+ cells using a magnet.
In one embodiment, the methods referred to herein are performed in vitro. In one embodiment, the methods referred to herein are performed ex vivo.
The term “antibody” is used in the broadest sense and specifically covers monoclonal and polyclonal antibodies (and fragments thereof) so long as they exhibit the desired biological activity. In particular, an antibody is a protein including at least one or two, heavy (H) chain variable regions (abbreviated herein as VHC), and at least one or two light (L) chain variable regions (abbreviated herein as VLC). The VHC and VLC regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991, and Chothia, C. et al, J. Mol. Biol. 196:901-917, 1987). Preferably, each VHC and VLC is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDRI, FR2, DR2, FR3, CDR3, FR4. The VHC or VLC chain of the antibody can further include all or part of a heavy or light chain constant region. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are interconnected by, e.g., disulfide bonds. The heavy chain constant region includes three domains, CHI, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The term “antibody” includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda. The term antibody, as used herein, also refers to a portion of an antibody that binds to one of the above-mentioned markers, e.g., a molecule in which one or more immunoglobulin chains is not full length, but which binds to a marker. Examples of binding portions encompassed within the term antibody include (i) a Fab fragment, a monovalent fragment consisting of the VLC, VHC, CL and CHI domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fc fragment consisting of the VHC and CHI domains; (iv) a Fv fragment consisting of the VLC and VHC domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, Nature 341:544-546, 1989), which consists of a VHC domain; and (vi) an isolated complementarity determining region (CDR) having sufficient framework to bind, e.g. an antigen binding portion of a variable region. An antigen binding portion of a light chain variable region and an antigen binding portion of a heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science |AI-ATi-A|β; and Huston et al. (1988) Proc. Natl. Acad. ScL USA 85:5879-5883). Such single chain antibodies are also encompassed within the term antibody. These may be obtained using conventional techniques known to those skilled in the art, and the portions are screened for utility in the same manner as are intact antibodies.
The antibodies of the present invention can be obtained using conventional techniques known to persons skilled in the art and their utility confirmed by conventional binding studies. By way of example, a simple binding assay is to incubate the cell expressing an antigen with the antibody. If the antibody is tagged with a fluorophore, the binding of the antibody to the antigen can be detected by FACS analysis.
Antibodies of the present invention can be raised in various animals including mice, rats, rabbits, goats, sheep, monkeys or horses. Blood isolated from these animals contains polyclonal antibodies—multiple antibodies that bind to the same antigen. Antigens may also be injected into chickens for generation of polyclonal antibodies in egg yolk. To obtain a monoclonal antibody that is specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from an animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas, and will continually grow and secrete antibody in culture. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies. Methods for producing monoclonal antibodies are conventional techniques known to those skilled in the art (see e.g. Making and Using Antibodies: A Practical Handbook. GC Howard. CRC Books. 2006. ISBN 0849335280). Polyclonal and monoclonal antibodies are often purified using Protein A/G or antigen-affinity chromatography.
Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M-A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004). Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).
The percent identity is then calculated as:
The present invention will now be described, by way of example only, with reference to the accompanying Examples and Figures, in which:
In A-C, the % values are the average for all samples within the group.
Below, representative FACS analysis of cell populations when CD38− CD45RA+ and GMP-like populations have been cultured for either 4 days (ii-left) or 8 days (iii-right). Top panel, CD34 and CD38 expression; bottom panel, CD90− and CD45RA expression in CD34+ CD38− cells. The percentages shown are the mean for gated populations for all 5 AML samples studied.
Multiplex quantitative PCR data of indicated genes on FACS sorted HSC, CD38− CD45RA+, GMP and MEP cells. All data were normalized to the expression of GAPDH. Results represent the mean value from 5 replicates from 2 control samples. Data from genes affiliated with: (i) HSC and CD38−CD45RA+ cells (green bars); (ii) granulocyte-monocyte lineage cells (blue bars); (iii) lymphoid lineage cells (yellow bars); (iv) erythroid-megakaryocyte lineage cells (red bars).
Bone marrow samples from AML patients and patients undergoing orthopaedic surgery with normal blood counts/films were obtained with informed consent (protocols 06/Q1606/110 and 05/MRE07/74). Bone marrow samples were received in EDTA (Heparin is a suitable alternative) and were lysed with ammonium chloride prior to immunostaining.
Samples received in culture medium were washed and the sample resuspended in PBS before analysis. To do this the sample was transferred to a conical tube (10 ml), which was topped up with PBS and centrifuged for 5 minutes at 3000 rpm. The supernatant was removed using a pipette and the cells resuspended in PBS to ˜2 ml.
Occasionally diagnostic panels were run on peripheral blood samples if the blast count was high enough. The technique used was identical to that for bone marrow.
Samples were prepared as soon as possible after receipt.
Ammonium Chloride was used for lysis:
Made up to 1 Litre with Distilled Water for a working solution which was stable for 1 month.
Adjusted the white cell count of samples for diagnostic studies to ˜5×109/l by dilution with PBS.
Red cell contamination will interfere with scatter plots and reduce overall MRD %
Excessive lysis will result in changes to FSC/SSC scatter properties
We used cell surface markers to compare patterns of stem/progenitor-cell immunophenotypes in CD34+ primary AML and normal control samples. The immunophenotypic gating strategy is illustrated in
For the first time, we showed that there are two major immunophenotypic groups in primary human CD34+ AML with respect to these markers (
Within these two major groups there are variations of CD38 expression between AML samples (
The central novel finding is that 82% of a broad range of primary CD34+ expressing AML samples have one major distinct CD38−CD45RA+ population within the CD34+ CD38− compartment and a corresponding GMP-like population within the CD34+ CD38+ compartment.
Given the CD45RA+ expanded group is the major group, we focused on dissecting which populations within this group had LSC activity in a xenotransplant assay. Previous data had shown that in CD34+ AML LSC activity resides in the CD34 compartment. Therefore, FACS-sorted CD38−CD45RA+ and GMP-like cells from 6 patients (10 populations in total) were injected intravenously into NOD-SCID mice treated with anti-CD122 antibody to remove residual NK-cells (
FACS analysis confirmed that for both CD38−CD45RA+ and GMP-like injected populations, CD34+ and CD34-cells were detected in bone marrow (
To prove that CD38−CD45RA+ and GMP-like AML populations have leukemic stem cell activity defined by secondary engraftment, human cells were harvested from primary recipients and injected into secondary NOD-SCID hosts treated with anti-CD122 antibody (
To confirm the in vivo observations, CD38−CD45RA+ cells and GMP-like populations were FACS-sorted from 5 AML patients (10 populations) and each population was cultured on MS5 stroma with cytokines (
After 4 days of culture of CD38+ CD45RA+ cells nearly all cells remained CD38+ and some cells had already lost CD34 expression (FIG. 6Bii). There was little differentiation of CD34+ CD38+ cells into CD34+ CD38− cells.
Thus, the sum of the in vivo and in vitro data suggest that a CD38−CD45RA+ population lies at the top of hierarchy in most cases of primary human AML and differentiates into GMP-like population but notably both populations have LSC activity.
We then obtained global mRNA expression profiles from 22 FACS-sorted AML CD38−CD45RA+, 21 GMP-like populations from 22 patients. In 18 patients we were able to obtain both CD38−CD45RA− and GMP-like AML populations allowing us to compare expression profiles between the two populations within each patient, thus negating the effect of genetic and epigenetic changes between patients. We also obtained from 5 normal HSC, MPP, CMP, GMP and CD38− CD45RA+ populations from 5 different age-matched human marrow samples. We asked two questions: first, are the two AML LSC populations (CD38− CD45RA+ and GMP-like) molecularly distinct; and second which normal populations are the two AML LSC populations most closely related to at a molecular level.
We used two approaches to determine if the expression profiles for the two AML LSC populations were distinct. First, we used a paired t-test (cut-off 0.01) to obtain a list of differentially expressed genes (917 probes; 748 mapped genes) between CD38−CD45RA+ and GMP-like cells from the subset of 18 AML cases where both populations were available from the same patient. The expression profiles of these differential genes was displayed by 3D Principal Component Analysis (PCA) (
Secondly, to obtain a more quantitative measure of the difference in gene expression between the two AML populations with LSC activity, we compared expression between CD38−CD45RA+ and GMP-like populations from the same patient, by a non-parametric (rank product) method (
To determine which normal populations the two AML LSC populations most closely resemble molecularly, we used ANOVA to curate a 2628 gene set (2789 probes) that maximally distinguished normal stem and progenitor populations. 3-D PCA was then used to display the profiles from the ANOVA curated gene set from normal populations (
Next, we used the same ANOVA gene set as a classifier to ask which normal population did each individual AML LSC populations most closely resemble (
Taken together, the gene expression profiles of both AML populations with LSC activity do not map most closely to normal HSC.
Normal CD38−CD45RA+ Population has Cells with Lymphoid Primed Multipotential (LMPP) Potential.
The gene expression data above showed that global expression profiles of CD38−CD45RA+ AML most closely resemble normal CD38−CD45RA+ cells. The expression data suggested that the normal CD38−CD45RA+ population was distinct fro other stem/progenitor cells. Previous studies had not shed light on the function of normal CD38−CD45RA+ cells. To investigate the lineage potential of CD38−CD45RA+ cells, we performed colony assays (
The B-lymphoid potential of CD38−CD45RA+ cells was tested by co-culture on MS5 stroma with cytokines. As positive control both HSC and MPP populations differentiated into myeloid (CD33-expressing) and B-lymphoid cells (CD19-expressing) cells whereas CMPs only gave rise to CD33+ cells (data not shown). CD38−CD45RA+ cells differentiated into both CD33+ and CD19+ cells (data not shown). The frequency of cells with myeloid, B- and T-lymphoid potential in the CD38−CD45RA+ population was determined by limiting dilution analysis (
Murine multi-potential stem/progenitor cells express low levels of multiple lineage-affiliated gene expression programmes concordant with their lineage potentials (termed multilineage priming). As these cells pass through lineage restriction points, losing lineage potential, there is gradual, concomitant, extinction of lineage-affiliated gene expression programmes. In a refinement of this concept it has been suggested that there is a cascade of lineage-affiliated transcriptional signatures, initiated in HSCs and propagated in a differential manner in lineage-restricted progenitors (
Whether this also occurs in human haemopoietic stem/progenitor cells has not been previously reported. Therefore, we used quantitative RT-PCR to study expression of select lineage-affiliated genes shown in mouse to be representative of lineage-affiliated gene expression programmes, in 10 and 100 FACS sorted normal HSC, CD38−CD45RA+, GMP and MEP cells (
Consistent with previous data we found that in 10 FACS-sorted cells, MPL and HLF were most highly expressed in HSC (
These are reminiscent of stem cell/myelolymphoid genes. The next pattern of myeloid gene expression is seen primarily in CD38−CD45RA+ and GMP cells (CSFR2A and GFI1) (i.e. restricted myelo-lymphoid) and, finally, the last layer of myeloid gene expression are late myeloid-specific genes (MPO and CSFR1), which are mainly expressed in GMPs only (differentiated myeloid). The pattern of early lymphoid gene expression is more focused; CD79A, ETS1, VPREB1, sterile IGHM, FLT3, NOTCH1 and RUNX3 all maximally expressed in CD38−CD45RA+ with little expression in other cell types (FIG. 10Biii). Finally, early stem-erythroid-megakaryocyte gene expression is epitomized by VWF, TAL1 and GATA2, which are all expressed in HSC and MEP. Both VWF and TAL1 are expressed at much lower levels in CD38−CD45RA+ cells, though GATA2 continues to be expressed in these cells, consistent with its broader role in haemopoietic progenitors (including GMP) biology. The erythroid-specific cytokine receptor EPOR, the erythroid-megakaryocyte transcription factor GATA1 and late erythroid transcription factor KLF1 show a more restricted pattern with expression principally in MEPs. Importantly, none of these three genes is expressed in CD38−CD45RA+ cells. Identical patterns of gene expression were obtained when either 10 or 100 FACS sorted cells where used (
The sum of the gene expression data is consistent with multilineage priming in primary human stem/progenitors in a manner analogous to mouse. Moreover, these data are consistent with lymphoid and myeloid (GM), but lack of erythroid-megakaryocyte lineage potential of CD38−CD45RA+ cells described earlier. The sum of the functional and molecular data suggests that CD38−CD45RA+ cells are most similar to mouse LMPP cells.
A 49 year old male suffering from symptoms of pancytopenia presents himself to hospital. 10 ml of blood and/or 2 mls of bone marrow is removed for diagnostic. are for flow cytometery evlauation. The biological samples are treated either as in Example 1 or with red cell lysis buffer to remove red cells. Then the nucleated cells are incubated with antibodies as described in Example 1 that are either directly conjugated or indirectly conjugated. Excess unbound antibody is washed off. The stained cells are then put through a flow cytometer. Data is then collected and prognosis is made.
A 33 year old with known Acute Myeloid Leukaemia present himself in hospital. 10 ml of blood and/or 2 mls of bone marrow is removed to monitor residual leukaemia stem cells for flow cytometery evlauation. The biological samples are treated either as in Example 1 or with red cell lysis buffer to remove red cells. Then the nucleated cells are incubated with antibodies as described in Example 1 that are either directly conjugated or indirectly conjugated. Excess unbound antibody is washed off. The stained cells are then put through a flow cytometer. Data is then collected and the effect of a therapeutic candidate assessed.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1021623.2 | Dec 2010 | GB | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15842240 | Dec 2017 | US |
| Child | 18451671 | US | |
| Parent | 13995347 | Sep 2013 | US |
| Child | 15842240 | US |
