WO2021042204A1 - Biomarqueurs pour une thérapie de blocage de cd47 - Google Patents

Biomarqueurs pour une thérapie de blocage de cd47 Download PDF

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WO2021042204A1
WO2021042204A1 PCT/CA2020/051187 CA2020051187W WO2021042204A1 WO 2021042204 A1 WO2021042204 A1 WO 2021042204A1 CA 2020051187 W CA2020051187 W CA 2020051187W WO 2021042204 A1 WO2021042204 A1 WO 2021042204A1
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expression
subgroup
level
cancer
kir
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Lisa Danae Schultz JOHNSON
Sandra Lauren BLITZ
Gloria Hoi Ying LIN
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Trillium Therapeutics Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • P59803PC00_SequenceListing.txt (18,334 bytes), created August 28, 2020, is hereby incorporated by reference.
  • This invention relates to medical therapies that use inhibitors of the interaction between CD47 and SIRPa. More particularly, the invention relates to diagnostic and prognostic methods and means that are useful to identify and stratify subjects more likely to respond to a CD47 blocking agent.
  • CD47 is an immune checkpoint that binds to signal regulatory protein alpha
  • SIRPa a CD47-binding protein drug that inhibits the interaction between CD47 and SIRPa.
  • This CD47 blocking agent is a form of human SIRPa that incorporates a particular region of its extracellular domain linked with a particularly useful form of an IgGl-based Fc region.
  • SIRPaFc shows dramatic effects on the viability of cancer cells that present with a CD47+ phenotype.
  • AML acute myelogenous leukemia
  • lymphomas as well as solid tumors, and in many other types of cancer.
  • a soluble form of SIRPa having significantly altered primary structure and enhanced CD47 binding affinity is described in WO2013/109752.
  • CD47 blocking agents have been described in the literature and these include various CD47 antibodies (see for instance Stanford’s US 8,562,997, and InhibRx’ WO2014/123580), each comprising different antigen binding sites but having, in common, the ability to compete with endogenous SIRPa for binding to CD47, thereby to allow interaction with macrophages and, ultimately, to increase the rate of CD47+ cancer cell depletion.
  • CD47 antibodies have activities in vivo that are quite different from those intrinsic to SIRPa- based drugs. The latter, for instance, display negligible binding to red blood cells whereas the opposite property in CD47 antibodies creates a need for strategies that accommodate the drug “sink” that follows administration.
  • CD47Fc fusion proteins see Viral Logic’s WO2010/083253
  • SIRPa antibodies as described in UHN’s WO2013/056352, Stanford’s WO2016/022971, Eberhard’s US 6913894, and elsewhere.
  • CD47 blockade approach in anti-cancer drug development shows great promise. It would be useful to provide methods and means for improving the effect of these drugs, and in particular for directing the use of the CD47 blocking agents, especially those that incorporate SIRPa.
  • CD47 blocking agents To advance therapeutic applications of CD47 blocking agents, it would be useful to provide a basis for identifying and selecting subjects for treatment. More particularly, it would be helpful to provide a method whereby subjects most likely to respond favorably to treatment with a CD47 blocking agent could be identified, and then selected for subsequent or continued therapy.
  • a marker gene useful in predicting positive, beneficial response to CD47 blockade therapy is one that encodes signal transducer and activator of transcription 2 (STAT2).
  • KIR inhibitory killer cell immunoglobulin-like receptors
  • KIR inhibitory killer cell immunoglobulin-like receptors
  • KIR inhibitory killer cell immunoglobulin-like receptors
  • Subjects selected for treatment on this basis can be expected to respond beneficially to CD47 blockade therapy, for instance by treatment with a CD47 inhibitor in the form of a CD47 binding agent such as a CD47-binding form of SIRPa or an antibody to CD47.
  • a CD47 inhibitor in the form of a CD47 binding agent such as a CD47-binding form of SIRPa or an antibody to CD47.
  • the present method accordingly permits the identification and selection of subjects suitable for commencement of CD47 blockade therapy. Samples taken pre-treatment from these subjects will reveal an altered level of marker gene expression. Other subjects that are not likely to respond to CD47 blockade therapy, as indicated by normal levels of marker gene expression, can be prescribed a different course of therapy.
  • the marker is a gene having an expression level relative to normal that is selected from (1) decreased/reduced STAT2, (2) increased/elevated species within the KIR inhibitory subgroup 1 (KIR-1) and (3) increased/elevated gene species within KIR inhibitory subgroup 2 (KIR-2).
  • the marker is an expression product of a marker gene, such as an RNA transcript or protein that is expressed from that marker gene.
  • a method of predicting responsiveness to treatment with a CD47 blocking agent comprising determining, in a sample of cancer and especially a cancer that is CD47+ obtained from a subject requiring treatment, the level of expression of one or more of the marker genes selected from STAT2, KIR Inhibitory Subgroup 1 and KIR Inhibitory Subgroup 2 and comparing that expression level to a normal level thereof, whereby an alteration in the level of expression of at least one marker gene predicts that the cancer cell in the subject will respond to therapy with a CD47 blocking agent.
  • a method for identifying a subject responsive to treatment with a CD47 blocking agent comprising determining the level of expression of one or more of the marker genes STAT2, KIR inhibitory subgroup 1 and KIR inhibitory subgroup 2, whereby the subject is identified as a CD47 blocking agent responder when expression of STAT2 is reduced and/or when expression of KIR inhibitory subgroup 1 or any species thereof is elevated, and/or when expression of KIR inhibitory subgroup 2 or any species thereof is elevated, relative to a normal level thereof.
  • a treatment method that comprises selecting for treatment a subject identified by the present method, and treating that subject with a CD47/SIRPa blocking agent.
  • a method for treating a subject with a CD47 blocking agent comprising testing a sample obtained from the subject to determine the expression level of one or more of the marker genes STAT2, inhibitory subgroup 1 and KIR inhibitory subgroup 2, and administering the CD47 blocking agent to the subject having an alteration in the expression level of at least one marker gene.
  • the detection methods used to quantify gene expression levels can be any of those in standard use for this purpose.
  • the entity detected by these methods can be DNA that encodes the marker gene or a cDNA counterpart, messenger RNA (mRNA) translations thereof, or the proteins expressed therefrom.
  • mRNA messenger RNA
  • the present invention provides a kit useful in predicting patient response to therapy with a CD47 blocking agent, the kit comprising at least one reagent useful in measuring the expression level of a marker gene, and instructions for the use thereof in the present methods.
  • the reagents can include nucleic acid primers useful to amplify DNA or RNA obtained from a subject having or suspected of being at risk for cancer, e.g.
  • the primers have a nucleic acid sequence adapted to amplify a characterizing part or all of a marker gene encoding KIR inhibitory subgroup 1 and/or KIR inhibitory subgroup 2 and STAT2 and/or an antibody to the marker gene-encoded protein, together with instructions for the use thereof in determining the expression level of at least one of those marker genes.
  • the CD47 blocking agent is a CD47-binding form of SIRPa, including a CD47-binding trap, such as SIRPaFc.
  • the present method is most usefully applied to identify those cancers, and subjects presenting therewith, that will respond to CD47 blocking agent therapy.
  • the present method can equally reveal cancers that are predicted not to respond to such therapy, in that the target tissue does not reveal an altered marker gene expression level before administration of a CD47 blocking agent. This will indicate that a different therapeutic approach should be pursued.
  • FIG. 1 Shows that higher baseline expression of KIR inhibitory subgroup 1 correlates with clinical response.
  • the right panel depicts the percent decrease in CAILS from baseline (Composite Assessment of Index Lesion Severity) for mycosis fungoides patients on the y-axis and the gene expression count (log2) on the x-axis. The relationship held up after study close when there were 11 in CTCL patients in CAILS with a >50% decrease in CAILS and 16 patients with ⁇ 50% decrease in CAILS.
  • FIG. 2 Shows that higher baseline expression of KIR inhibitory subgroup 2 correlates with clinical response.
  • the right panel depicts the percent decrease in CAILS from baseline (Composite Assessment of Index Lesion Severity) for mycosis fungoides patients on the y-axis and the gene expression count (log2) on the x-axis. The relationship held up after study close when there were 11 in CTCL patients in CAILS with a >50% decrease in CAILS and 16 patients with ⁇ 50% decrease in CAILS.
  • FIG. 3 Shows that lower baseline expression of Stat2 correlates with better clinical response.
  • the right panel depicts the percent decrease in CAILS from baseline (Composite Assessment of Index Lesion Severity) for mycosis fungoides patients on the y-axis and the gene expression count (log2) on the x-axis.
  • FIG. 4 shows that KIR inhibitory subgroup 1, KIR inhibitory subgroup 2, and STAT2 yield very similar differentiation between CTCL patients with > 50% decrease and those with ⁇ 50% decrease in CAILS within the range of values observed in this study sample.
  • the 3 panels show the distribution of log2 (gene expression) values at baseline for KIR inhibitory subgroup 1, KIR inhibitory subgroup 2, and STAT2 relative to each respective optimal cut-off value as determined through ROC analysis.
  • Figure 5 The left panel shows pre-treatment log2 (gene expression) values using
  • the right panel shows pre-treatment log2(gene expression) values using Nanostring’s PanCancer Immune Profiling panel for KIR inhibitory subgroup 2 on the x- axis and STAT2 on the y-axis.
  • the tables report the sensitivity and specificity achieved when using 1 optimal cutoff as well as when the optimal cutoff-values for both genes/subgroups are met.
  • FIG. 6 The panel shows gene expression data using Nanostring’s PanCancer
  • Sensitivity was also calculated when the optimal cutoff values for KIR inhibitory subgroup 1 and STAT2 were both met as well as when the optimal cutoff values for KIR inhibitory subgroup 1 and STAT2 were met. Specificity was also calculated when the optimal cutoff values for either KIR inhibitory subgroup 1 or STAT2 were not met as well as when the optimal cutoff values for KIR inhibitory subgroup 1 and STAT2 were not met.
  • the present invention provides an improved method for treating a subject presenting with CD47+ disease cells such as cancer cells and tumors that have a CD47+ phenotype.
  • subjects are first screened to determine the level(s) of expression of at least one of three different marker genes or gene groups, designated STAT2, KIR inhibitory subgroup 1, and KIR inhibitory subgroup 2.
  • STAT2 the level(s) of expression of at least one of three different marker genes or gene groups
  • KIR inhibitory subgroup 1 KIR inhibitory subgroup 2
  • KIR inhibitory subgroup 2 KIR inhibitory subgroup 2 expression.
  • the present method enables drug therapy to commence selectively in a particular group of subjects who would benefit from such therapy, to the exclusion of subjects unlikely to respond favourably to CD47 blockade.
  • the method can be applied for instance by testing a biopsy obtained from a prospective patient and comparing that result with a control, such as a non-cancerous sample counterpart.
  • subjects receive a CD47 blocking agent such as
  • SIRPaFc if their cancer tissue exhibits (1) an decrease in the presence or level of an expression product from STAT2; and/or (2) an increased and or elevated level of an expression product from a gene that encodes a protein within the KIR inhibitory subgroup 1, and/or (3) an increased and or elevated level of an expression product from a gene that encodes a protein within the KIR inhibitory subgroup 2.
  • the KIR subgroups comprise a variety of KIR genes. These genes are grouped as belonging to a particular subgroup depending on their relatedness at the DNA level.
  • KIR Subgroup 1 includes KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL3) and KIR Subgroup 2 includes KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL3, KIR2DL1, KIR2DL2, KIR3DL1, and KIR3DL2). Any one of the genes within these inhibitory subgroups can be the basis for a screen that seeks to reveal whether reductions in the expression products of these genes as occurred or is present. [0031] The identifying reference numbers for the gene and the protein of each marker gene is set out below in Table 1, and full details of these sequences are incorporated herein by reference.
  • KIR receptors are named based on the number of their extracellular Ig-like domains (2D or 3D) and by the length of their cytoplasmic tail (long (L), short (S), or pseudogene (P)). The number following the L, S, or P in the case of a pseudogene, differentiates KIR receptors with the same number of extracellular domains and length of cytoplasmic tail. Finally, the asterisk after this nomenclature indicates allelic variants.
  • KIR receptors changes the site of termination for the gene, causing the cytoplasmic tail to be long or short, depending on the site of the stop codon.
  • KIR2DL4 which has both activating and inhibitory capabilities, KIR receptors with long cytoplasmic tails are inhibitory and those with short tails are activating.
  • KIR species There are numerous subtypes of KIR species. Grouped within Inhibitory KIR subtype 1 are the gene species designated: ⁇ two domains, long cytoplasmic tail: KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4,
  • KIR3DL1 • three domains, long cytoplasmic tail: KIR3DL1, KIR3DL2, KIR3DL3
  • Grouped within the Inhibitory KIR subtype 2 are the gene species designated:
  • an elevated or increased level of a KIR subtype 1 species thus can be an increased or elevated level, relative to the level in a normal or wildtype sample, of a KIR subtype 1 species that is KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, and/or KIR3DS1.
  • an elevated or increased level of a KIR subtype 2 species can be an increased or elevated level, relative to the level in a normal or wildtype sample, of a KIR subtype 2 species that is KIR2DL3, KIR2DL4, KIR2DL5, KIR2DL1, KIR2DL2, KIR3DL3, KIR3DL1, and/or KIR3DL2.
  • the feature of interest is an altered level, i.e., a reduced or increased level, of an expression product from a marker gene, where the expression product is, for instance, protein or RNA produced via that gene.
  • a “reduced level” or a “reduction” or “decrease” means that the level of a marker gene expression product is statistically significantly lower than a normal level or a “wild type” level (baseline).
  • the reduction of interest is also a reduction in the amount of gene expression product in a subject before treatment with a CD47 blocking agent. Reduction can include an absence in the amount of the gene expression product in a CD47+ disease tissue relative to a healthy normal tissue counterpart (baseline) or to an untreated sample counterpart.
  • marker gene expression levels are determined in a sample that is preferably a sample of the cancer tissue presented by the subject before treatment, or a determination of those levels in a sample take from a subject during stages of treatment with a CD47 blocking agent.
  • An increase in the level of expression is, mutatis mutandis, determined using similar approaches and assays.
  • the present method enables drug therapy to be applied to a particular group of subjects who would benefit most from such therapy in terms of reductions in cancer/tumor burden, distribution, overall survival, time to progression and the like.
  • the “level” of a marker gene expression product can be determined using various biological samples, such as blood and other liquids of biological origin, solid tissue samples such as a biopsy specimens and tissue cultures or cells derived or isolated therefrom.
  • the sample can have been manipulated, such as by treatment with reagents; washed; or enriched for certain cell populations, such as cancer cells.
  • the sample can be one that is enriched for particular types of molecules, e.g., nucleic acids such as RNA, polypeptides, etc. Samples include clinical samples.
  • sample types include tissue obtained by surgical resection or by biopsy, cells in culture, cell supernatants, cell lysates, organs, bone marrow, urine, saliva, blood, plasma, serum, cerebrospinal fluid, an aspirate, and the like.
  • a sample can include biological fluids derived from cells (e.g., a CD47+ cancerous cell, an (virally) infected cell, etc.), e.g., a sample comprising polynucleotides and/or polypeptides that is obtained from such cells (e.g., a cell lysate or other cell extract comprising polynucleotides and/or polypeptides).
  • a sample can be obtained by physical extraction or isolation of a sample from a subject. Methods for isolating samples, e.g., blood, serum, plasma, biopsy, aspirate, etc. are all well-known and useful for purposes of the present invention.
  • the sample can also be provided by another source that does the isolation of the sample and provides it in a format ready to test.
  • the level of a given marker can be an expression level of a marker gene or its product, which may be RNA, a protein, etc., in a sample is measured (i.e., “determined”).
  • expression level or “level” is meant the level of gene product (e.g. the absolute and/or normalized value determined for the RNA expression level of a marker gene or for the expression level of the encoded polypeptide, or the concentration of the protein in a biological sample).
  • gene product or “expression product” are used herein to refer to complementary DNA (cDNA) and the RNA transcription products (RNA transcripts, e.g.
  • a gene product can be, for example, an unspliced RNA, an mRNA, a splice variant mRNA, a microRNA, a fragmented RNA, a polypeptide, a pre-polypeptide, a propolypeptide, a prepropolypeptide, a post-translationally modified polypeptide, a splice variant polypeptide, etc.
  • testing are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative, semi-quantitative and/or qualitative determinations. For example, “testing” can be determining whether the expression level is less than or “greater than or equal to” a particular threshold, (the threshold, or baseline, can be pre-determined or can be determined by assaying a control sample).
  • assaying to determine the expression level of a marker gene can involve determining a quantitative value (using any convenient metric) that represents the level of expression (i.e., expression level, e.g., the amount of protein and/or RNA, e.g., mRNA) of a particular marker gene.
  • the level of expression can be expressed in arbitrary units associated with a particular assay (e.g., fluorescence units, e.g., mean fluorescence intensity (MFI)), or can be expressed as an absolute value with defined units (e.g., number of mRNA transcripts, number of protein molecules, concentration of protein, etc.).
  • the level of expression of a marker gene can be compared to the expression level of one or more additional genes (e.g., nucleic acids and/or their encoded proteins) to derive a normalized value that represents a normalized expression level.
  • RNA levels the amount or level of an RNA, such as an RNA transcript, in the sample is determined.
  • the expression level of one or more additional RNAs may also be measured, and the level of marker gene expression compared to the level of the one or more additional RNAs to provide a normalized value for marker gene expression level.
  • Any convenient protocol for evaluating RNA levels may be employed wherein the level of one or more RNAs in the assayed sample is determined.
  • Distinctive marker gene fragments can also be a detection target. These are regions of the marker gene that are unique to that gene, so that amplification, and the conditions selected for amplification, yields an amplicon that is representative of that marker gene only.
  • the distinctive fragment can be a unique portion of the protein-encoding region of the marker gene, such as an extracellular region, or a portion residing in the upstream elements that regulate expression of that gene, or a portion residing in the downstream region that regulates termination of transcription, among other regions.
  • RNA e.g., mRNA, expression levels in a sample many useful approaches are known for measuring RNA e.g., mRNA, expression levels in a sample and any of these methods can be used. These methods include: hybridization- based methods such as Northern blotting, array hybridization (e.g., microarray); in situ hybridization; in situ hybridization followed by FACS; and the like; RNAse protection assays; PCR-based methods including reverse transcription PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), real-time RT-PCR; nucleic acid sequencing methods, e.g., massive parallel high throughput sequencing, such as Illumina's reversible terminator method, Roche's pyrosequencing method, Life Technologies' sequencing by ligation (the SOLID platform), Life Technologies' Ion Torrent platform; and the like.
  • hybridization- based methods such as Northern blotting, array hybridization (e.g., microarray); in situ hybridization; in situ hybridization followed
  • the raw sample can be tested.
  • nucleic acid of the biological sample is amplified (e.g., by PCR) prior to testing.
  • techniques such as PCR (Polymerase Chain Reaction), RT-PCR (reverse transcriptase PCR), qRT-PCR (quantitative RT-PCR, real time RT-PCR), and the like can be used before hybridization methods and/or the sequencing methods.
  • the starting material is typically total RNA or poly
  • RNA isolation can be performed using a purification kit, buffer set and protease from commercial manufacturers, according to the manufacturer's instructions.
  • RNA from cell suspensions can be isolated using Qiagen RNeasy mini-columns, and RNA from cell suspensions or homogenized tissue samples can be isolated using the TRIzol reagent-based kits (Invitrogen), MasterPureTM Complete DNA and RNA Purification Kit (EPICENTRETM, Madison, Wis.), Paraffin Block RNA Isolation Kit (Ambion, Inc.) or RNA Stat-60 kit (Tel-Test).
  • TRIzol reagent-based kits Invitrogen
  • MasterPureTM Complete DNA and RNA Purification Kit EPICENTRETM, Madison, Wis.
  • Paraffin Block RNA Isolation Kit Ambion, Inc.
  • RNA Stat-60 kit Tel-Test
  • Various ways of determining/measuring mRNA levels are known in the art, e.g. as employed in the field of differential gene expression analysis.
  • One protocol for measuring mRNA levels is array-based gene expression profiling.
  • Such protocols are hybridization assays in which a nucleic acid that displays “probe” nucleic acids for each of the genes to be assayed/profiled in the profile to be generated is employed.
  • a sample of target nucleic acids is first prepared from the initial nucleic acid sample being assayed, where preparation may include labeling of the target nucleic acids with a label, e.g., a member of signal producing system.
  • target nucleic acid sample preparation Following target nucleic acid sample preparation, the sample is contacted with the array under hybridization conditions, and complexes are formed between target nucleic acids that are complementary to probe sequences attached to the array surface. The presence of hybridized complexes is then detected, either qualitatively or quantitatively.
  • Specific hybridization technology which may be practiced to generate the expression profiles employed in the subject methods includes the technology described in U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800,992; the disclosures of which are herein incorporated by reference.
  • an array of “probe” nucleic acids that includes a probe for each of the marker gene is contacted with target nucleic acids as described above.
  • Stringent assay conditions use binding pairs of nucleic acids, e.g., surface bound and solution phase nucleic acids, of sufficient complementarity to provide for the desired level of specificity in the assay while being less compatible to the formation of binding pairs between binding members of insufficient complementarity to provide for the desired specificity.
  • the resultant pattern of hybridized nucleic acid provides information regarding expression for each of the marker genes that have been probed, e.g., at least one or more of STAT2, KIR inhibitory subgroup 1 and KIR inhibitory subgroup 2, where the expression information is in terms of whether or not the gene is expressed and, typically, at what level.
  • Non-array-based methods for quantitating the level of one or marker gene products in a sample may be employed. These include those based on amplification protocols, e.g., Polymerase Chain Reaction (PCR)-based assays, including quantitative PCR, reverse- transcription PCR (RT-PCR), real-time PCR, and the like, e.g. TaqMan® RT-PCR, MassARRAY® System, BeadArray® technology, and Luminex technology; and those that rely upon hybridization of probes to filters, e.g. Northern blotting and in situ hybridization.
  • PCR Polymerase Chain Reaction
  • the amount or level of a polypeptide in the biological sample is determined.
  • concentration is a relative value measured by comparing the level of one protein relative to another protein, or the level of the protein in one sample versus the level of the same protein in a different sample.
  • An enhanced or elevated level of gene expression is relevant when it has statistical significance, and especially when its increase over base line or difference between subjects with objective responses has a p value less than 0.05 or better. Elevation is evident when the marker gene expression level is increased, such as by about 50% or greater, e.g. at least one-fold, two-fold, three-fold greater than marker gene expression baseline, or has a p- value less/smaller than 0.05, e.g. 0.001).
  • the cells are removed from the biological sample, e.g., via centrifugation, via adhering cells to a dish or to plastic, etc., before testing.
  • the intracellular protein level is measured by lysing the removed cells of the biological sample to measure the level of protein in the cellular contents.
  • both the extracellular and intracellular levels of protein are measured by separating the cellular and fluid portions of the biological sample (e.g., via centrifugation), measuring the extracellular level of the protein by measuring the level of protein in the fluid portion of the biological sample, and measuring the intracellular level of protein by measuring the level of protein in the cellular portion of the biological sample (e.g., after lysing the cells).
  • the total level of protein i.e., combined extracellular and intracellular protein
  • the presence, concentration or level of one or more additional proteins may also be measured, and marker gene-expressed protein levels are compared to the level of the one or more additional proteins to provide a normalized value for the maker gene product/protein concentration. Any convenient protocol for evaluating protein levels may be employed wherein the level of one or more proteins in the assayed sample is determined.
  • ELISA an antibody-based method.
  • one or more antibodies specific for the proteins of interest may be immobilized onto a selected solid surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate.
  • the assay plate wells are coated with a non-specific “blocking” protein that is known to be antigenically neutral with regard to the test sample such as bovine serum albumin (BSA), casein or solutions of powdered milk.
  • BSA bovine serum albumin
  • the immobilizing surface is contacted with the sample to be tested under conditions conducive to immune complex (antigen/antibody) formation. Following incubation, the antisera-contacted surface is washed so as to remove non- immunocomplexed material. The occurrence and amount of immunocomplex formation may then be determined by subjecting the bound immunocomplexes to a second antibody having specificity for the target that differs from the first antibody and detecting binding of the second antibody.
  • the second antibody will have an associated enzyme, e.g.
  • the amount of label is quantified, for example by incubation with a chromogenic substrate such as urea and bromocresol purple in the case of a urease label or 2,2'-azido-di-(3-ethyl-benzothiazoline)-6-sulfonic acid (ABTS) and H2O2, in the case of a peroxidase label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • the ELISA or EIA format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with specificity for the primary antibody.
  • the solid substrate upon which the antibody or antibodies are immobilized can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate may be chosen to maximize signal to noise ratios, to minimize background binding, as well as for ease of separation and cost.
  • Washes may be effected by removing a bead, emptying or diluting a reservoir such as a microtiter plate well, or rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent.
  • Non-ELISA based-methods for measuring the levels of one or more proteins in a sample may be employed. Representative exemplary methods include Western blotting, proteomic arrays, xMAPTM microsphere technology (e.g., Luminex technology), immunohistochemistry, flow cytometry, and the like as well as non-antibody-based methods (e.g., mass spectrometry).
  • the level of the same marker in a different sample can also be determined.
  • the different sample can be taken from a subject that is healthy and tumor-free, or from a subject that is selected to undergo CD47 blockade therapy but has yet to be so treated.
  • the marker gene product level can be determined at intervals including pre treatment, commencement of treatment, e.g., after first dose, and during treatment and post treatment.
  • the level of marker gene expression product is determined using the NanoString® approach described in the examples herein.
  • RNA from a sample taken from a subject is assayed using multiplex gene expression analysis with 770 genes from 24 different immune cell types including tumor cell infiltrating lymphocytes, common checkpoint inhibitors, CT antigens, and genes covering both the adaptive and innate immune response.
  • this approach identifies sample-borne RNA using hybridizing probes having the sequences noted below.
  • KIR2DL1 (NM 014218.2), also recognizes KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5, KIR3DLland KIR3DL3)
  • KIR2DL3 (NM 0014511.3), also recognizes KIR2DL2, KIR2DL5B, KIR2DL5A, KIR3DL3, and KIR2DL4
  • a method useful to identify a CD47+ cancer subject that will respond to treatment with a CD47 blocking agent comprising obtaining a sample of that cancer, measuring in that sample the level of expression of a marker gene selected from one, or two or more of KIR inhibitory subgroup 1, KIR inhibitory subgroup 2 and STAT2, and identifying for treatment with the CD47 blocking agent the cancer subject that presents with a reduced STAT2 level or an increased KIR inhibitor subgroup 1 or KIR inhibitor subgroup 2 level of marker gene expression.
  • the subject to be treated presents with both reduced STAT2 and increased KIR subgroup 1, or presents with both reduced STAT2 and increased KIR subgroup 2.
  • a method of predicting responsiveness to treatment of a CD47+ cancer with a CD47 blocking agent comprising determining the level of expression of one, two or all of the marker genes KIR inhibitory subgroup 1, KIR inhibitory subgroup 2 and STAT2 in a sample of that cancer obtained from that subject, wherein elevated KIR inhibitory subgroup 1 or elevated KIR inhibitory subgroup 2 or reduced STAT2 expression predicts the cancer will respond to a CD47 blocking agent.
  • the method is applied to a subject that has yet to be dosed with CD47 blocking agent, Subjects that exhibit an increase in marker gene expression are identified for continuing treatment. Treated subjects that fail to exhibit an increase in marker gene expression are withdrawn from such therapy and can prescribed a different therapy.
  • a method for treating a subject with a CD47 blocking agent comprising determining in a sample obtained from the subject the expression level of one, any two, all three of the marker genes KIR inhibitory subgroup 1, KIR inhibitory subgroup 2 and STAT2, and administering the CD47 blocking agent to the subject in which the level of expression of a marker gene is altered as herein described, relative to a normal level.
  • a CD47 blocking agent to treat a subject that has elevated KIR inhibitory subgroup 1 or elevated KIR inhibitory subgroup 2 or reduced STAT2 expression.
  • the level of STAT2 is relevant particularly to CTCL: a reduced STAT2 level indicates that a CD47 blocking agent will work particularly well in treating the CTCL in that patient.
  • the elevated levels of the KIR inhibitor subgroups 1 and 2 are relevant to numerous types and varieties of cancer, both solid and liquid and can be used to indicate anti-CD47 treatment in many subjects whether evaluated alone or in combination, but separately from the STAT2 analysis.
  • anti-CD47 agent or “CD47 blocking agent” or “CD47 blockade drug” refers to any agent that interferes with the binding of CD47 (e.g., on a target cell) to SIRPa (e.g., on a phagocytic cell).
  • suitable anti-CD47 reagents include SIRPa reagents, including without limitation high affinity SIRPa polypeptides, anti-SIRPa, antibodies, soluble CD47 polypeptides, and anti-CD47 antibodies or antibody fragments.
  • a suitable anti-CD47 agent e.g. an anti-CD47 antibody, a SIRPa reagent, etc. specifically binds CD47 to reduce the binding of CD47 to SIRPa.
  • a suitable anti-CD47 agent e.g., an anti-SIRPa, antibody, a soluble CD47 polypeptide, etc. specifically binds SIRPa to reduce the binding between CD47 and SIRPa.
  • a suitable anti-CD47 agent is one that binds SIRPa but does not activate SIRPa.
  • CD47+ (UniProtKB gene ref Q08722) is used herein with reference to the phenotype of cells targeted for treatment with a CD47 blocking agent.
  • Cells that are CD47+ can be identified by flow cytometry using CD47 antibody as the affinity ligand. Labeled CD47 antibodies are available commercially for this use (for example, clone B6H12 is available from Santa Cruz Biotechnology).
  • the cells examined for CD47 phenotype can be standard tumor biopsy samples including particularly liquid and tissue samples taken from the subject suspected of harbouring CD47+ cancer cells.
  • CD47 disease cells of particular interest as targets for therapy with the present combination are those that “over-express” CD47.
  • CD47+ cells typically are disease cells, and present CD47 at a density on their surface that exceeds the normal CD47 density for a cell of a given type.
  • CD47 overexpression will vary across different cell types, but is meant herein to refer to any CD47 level that is determined, for instance by flow cytometry or by immunostaining or by gene expression analysis or the like, to be greater than the level measurable on a counterpart cell having a CD47 phenotype that is normal for that cell type.
  • a subject that would “benefit” from CD47 blockade therapy will display an improvement in cancer burden when treated with a CD47 blocking agent. This can manifest as a reduction in tumor size or number, rate of growth, distribution and the like including improvement/s in survival, time to progression, etc.
  • a “CD47 blocking agent” is any drug or agent that interferes with and dampens or blocks signal transmission that results when CD47 interacts with macrophage-presented SIRPa.
  • the CD47 blocking agent is an agent that inhibits CD47 interaction with SIRPa.
  • the CD47 blocking agent is preferably an agent that binds CD47 and blocks its interaction with SIRPa.
  • the CD47 blocking agent can be an antibody or antibody- based antagonist of the CD47/SIRPa signaling axis, such as an antibody that binds CD47 and blocks interaction of CD47 with SIRPa.
  • the CD47 blocking agent comprises a constant region, i.e., an Fc region, that can be bound by macrophages that are activated to destroy cells to which the CD47 blocking agent is bound, such as cancer cells.
  • the CD47 blocking agent Fc region preferably has effector function, and is derived preferably from either IgGl or IgG4 including IgG4 (S228P).
  • the Fc region can be one that is altered by amino acid substitution to change effector function, e.g., to an inactive state.
  • CD47-binding forms of human SIRPa are the preferred CD47 blocking agents for use in the combination herein disclosed. These drugs are based on the extracellular region of human SIRPa. They comprise at least a part of the extracellular region sufficient to confer effective CD47 binding affinity and specificity. So-called “soluble” forms of SIRPa, lacking the membrane anchoring property in SIRPa, are useful and include those referenced in Novartis’ WO 2010/070047, Stanford’s WO2013/109752, Merck’s W02016/024021 and Trillium’s WO2014/094122.
  • the SIRPaFc drug useful in the present method can be bispecific or it can be monomeric, homodimeric or heterodimeric form of a single chain polypeptide comprising an Fc region of an antibody and a CD47-binding region of human SIRPa.
  • the SIRPaFc polypeptide has the properties discussed below. More particularly, the polypeptide suitably comprises a CD47-binding part of human SIRPa protein in a form fused directly, or indirectly, with an antibody constant region, or Fc (fragment crystallisable).
  • the term “human SIRPa” as used herein refers to a wild type, endogenous, mature form of human SIRPa. In humans, the SIRPa protein is found in two major forms. One form, the variant 1 or VI form, has the amino acid sequence set out as NCBI RefSeq NP_542970.1 (residues 27-504 constitute the mature form).
  • Another form, the variant 2 or V2 form differs by 13 amino acids and has the amino acid sequence set out in GenBank as CAA71403.1 (residues 30-504 constitute the mature form).
  • These two forms of SIRPa constitute about 80% of the forms of SIRPa present in humans, and both are embraced herein by the term “human SIRPa”.
  • Also embraced by the term “human SIRPa” are the minor forms thereof that are endogenous to humans and have the same property of binding with, and triggering signal transduction through CD47.
  • the present invention is directed most particularly to the drug combinations that include the V2 form of SIRPa.
  • useful CD47 blocking agents are SIRPaFc fusion polypeptides that comprise at least one of the three so-called immunoglobulin (Ig) domains within the extracellular region of human SIRPa. More particularly, the present
  • SIRPaFc polypeptides preferably incorporate residues 32-137 of human SIRPa (a 106-mer), which constitute and define the IgV domain of the V2 form according to current nomenclature.
  • the SIRPaFc fusion protein incorporates the IgV domain as defined by SEQ ID NO:4, and additional, flanking residues contiguous within the wild type human SIRPa sequence.
  • This preferred form of the IgV domain represented by residues 31-148 of the V2 form of human SIRPa, is a 118-mer having SEQ ID NO:5 shown below:
  • the Fc region of the SIRPaFc fusion polypeptide preferably does have effector function.
  • Fc refers to “fragment crystallizable” and represents the constant region of an IgG antibody comprised principally of the heavy chain constant region and components within the hinge region. Suitable Fc components thus are those having effector function.
  • An Fc component “having effector function” is an Fc component having at least some effector function, such as at least some contribution to antibody-dependent cellular cytotoxicity or some ability to fix complement. Also, the Fc will at least bind to one or more types of Fc receptor.
  • Functional assays include the standard chromium release assay that detects target cell lysis.
  • Fc region that is wild type IgGl or IgG4 has effector function, whereas the Fc region of a human
  • the Fc is based on human antibodies of the IgGl isotype. In an alternative embodiment, the Fc is based on the IgG4 isotype, and includes the
  • Fc region of these antibodies will be readily identifiable to those skilled in the art.
  • the Fc region includes the lower hinge-CH2-CH3 domains.
  • the Fc region is based on the amino acid sequence of a human IgGl set out as P01857 in UniProtKB/Swiss-Prot, residues 104-330, and has the amino acid sequence shown below and referenced herein as SEQ ID NO:6:
  • the Fc region has either a wild type or consensus sequence of an IgGl constant region.
  • the Fc region incorporated in the fusion protein is derived from any IgGl antibody having a typical effector-active constant region.
  • sequences of such Fc regions can correspond, for example, with the Fc regions of any of the following IgGl sequences (all referenced from GenBank), for example: BAG65283 (residues 242-473), BAC04226.1 (residues 247-478), BAC05014.1 (residues 240-471), CAC20454.1 (residues 99-320), BAC05016.1 (residues 238-469), BAC85350.1 (residues 243- 474), BAC85529.1 (residues 244-475), and BAC85429.1 (residues (238-469).
  • the Fc region has a sequence of a wild type human IgG4 constant region.
  • the Fc region incorporated in the fusion protein is derived from any IgG4 antibody having a constant region with effector activity that is present but, naturally, is less potent than the IgGl Fc region.
  • the sequences of such Fc regions can correspond, for example, with the Fc regions of any of the following IgG4 sequences: P01861 (residues 99-327) from UniProtKB/Swiss-Prot and CAC20457.1 (residues 99-327) from GenBank.
  • the Fc region is based on the amino acid sequence of a human IgG4 set out as P01861 in UniProtKB/Swiss-Prot, residues 99-327, and has the amino acid sequence shown below and referenced herein as SEQ ID NO: 7:
  • the Fc region incorporates one or more alterations, usually not more than about 5 such alterations, including amino acid substitutions that affect certain Fc properties.
  • the Fc region incorporates an alteration at position 228 (EU numbering), in which the serine at this position is substituted by a proline (S228P), thereby to stabilize the disulfide linkage within the Fc dimer.
  • Other alterations within the Fc region can include substitutions that alter glycosylation, such as substitution of Asn297 by glycine or alanine; half-life enhancing alterations such as T252L, T253S, and T256F as taught in US62777375, and many others including the 409 position. Particularly useful are those alterations that enhance Fc properties while remaining silent with respect to conformation, e.g., retaining Fc receptor binding.
  • the Fc incorporates at least the S228P mutation, and has the amino acid sequence set out below and referenced herein as SEQ ID NO: 8:
  • the CD47 blocking agent used in the combination is thus a SIRPaFc fusion protein useful to inhibit binding between human SIRPa and human CD47, thereby to inhibit or reduce transmission of the signal mediated via SIRPa-bound CD47, the fusion protein comprising a human SIRPa component and, fused therewith, an Fc component, wherein the SIRPa component comprises or consists of a single IgV domain of human SIRPa V2 and the Fc component is the constant region of a human IgG, wherein the constant region preferably has effector function.
  • the fusion protein comprises a SIRPa component consisting at least of residues 32-137 of the V2 form of wild type human SIRPa, i.e., SEQ ID NO:4.
  • the SIRPa component consists of residues 31-148 of the V2 form of human SIRPa, i.e., SEQ ID NO:5.
  • the Fc component is the Fc component of the human IgGl designated P01857, and in a specific embodiment has the amino acid sequence that incorporates the lower hinge-CH2-CH3 region thereof i.e., SEQ ID NO:6.
  • the present method utilizes a CD47 blocking agent that is a SIRPaFc fusion polypeptide, as both an expressed single chain polypeptide and as a secreted dimeric fusion thereof (homodimer), wherein the fusion protein incorporates a SIRPa component having SEQ ID NO:4 and preferably SEQ ID NO:5 and, fused therewith, an Fc region having effector function and having SEQ ID NO:6.
  • SIRPa component is SEQ ID NO:4
  • this fusion protein comprises SEQ ID NO:9, shown below:
  • this fusion protein comprises SEQ ID NO:5
  • the Fc component of the fusion protein is based on an IgG4, and preferably an IgG4 that incorporates the S228P mutation.
  • the fusion protein incorporates the preferred SIRPa IgV domain of SEQ ID NO:5
  • the resulting IgG4[S228P]-based SIRPa-Fc protein has SEQ ID NO: 11, shown below: EEELQ VIQPDK S V S V A AGE S AILHC T VT SLIP V GPIQ WFRGAGP ARELIYN QKEGHFPR
  • the fusion protein comprises, as the SIRPa IgV domain of the fusion protein, a sequence that is SEQ ID NO:5.
  • the preferred SIRPaFc is SEQ ID NO: 10.
  • the SIRPa sequence incorporated within the CD47 blocking agent can be varied, as described in the literature. That is, useful substitutions within SIRPa will typically enhance binding affinity for CD47, and can include one or more of the following: L4V/I, V6I/L, A21V, V27I/L, 131T/S/F, E47V/L, K53R, E54Q, H56P/R, S66T/G, K68R, V92I, F94V/L, V63I, and/or FI 03V. Still other substitutions include conservative amino acid substitutions in which an amino acid is replaced by an amino acid from the same group.
  • the SIRPa sequence can also be truncated or extended, so long as CD47 binding affinity is retained.
  • the SIRPaFc fusion polypeptide the SIRPa component and the Fc component are fused, either directly or indirectly, to provide a single chain polypeptide that is ultimately produced as a homodimer in which the single chain polypeptides are coupled through intrachain disulfide bonds formed between the Fc regions of individual single chain SIRPaFc polypeptides.
  • the nature of the fusing region that joins the SIRPa region and the Fc is not critical.
  • the fusion may be direct between the two components, with the SIRP component constituting the N-terminal end of the fusion and the Fc component constituting the C-terminal end.
  • the fusion may be indirect, through a linker comprised of one or more amino acids, desirably genetically encoded amino acids, such as two, three, four, five, six, seven, eight, nine or ten amino acids, or any number of amino acids between 5 and 100 amino acids, such as between 5 and 50, 5 and 30 or 5 and 20 amino acids.
  • a linker may comprise a peptide that is encoded by DNA constituting a restriction site.
  • the linker amino acids typically and desirably will provide some flexibility to allow the Fc and the SIRPa components to adopt their active conformations. Residues that allow for such flexibility typically are Gly, Asn and Ser, so that virtually any combination of these residues (and particularly Gly and Ser) within a linker is likely to provide the desired linking effect.
  • a linker is based on the so-called G4S sequence (Gly-Gly- Gly-Gly-Ser) (SEQ ID NO: 12) which may repeat as (G4S)n where n is 1, 2, 3 or more, or is based on (Gly)n, (Ser)n, (Ser-Gly)n or (Gly-Ser)n and the like.
  • the linker is GTELSVRAKPS (SEQ ID NO: 13).
  • This sequence constitutes a SIRPa sequence that C -terminally flanks the IgV domain (it being understood that this flanking sequence could be considered either a linker or a different form of the IgV domain when coupled with the IgV minimal sequence described above). It is necessary only that the fusing region or linker permits the components to adopt their active conformations, and this can be achieved by any form of linker useful in the art.
  • the CD47 blocking agent can also be an antibody that specifically binds CD47, a suitable anti-CD47 antibody does not activate CD47 upon binding.
  • suitable antibodies include clones B6H12, 5F9, 8B6, and C3 (for example as described in WO 2011/143624, herein specifically incorporated by reference.
  • Suitable anti-CD47 antibodies include fully human, humanized or chimeric versions of such antibodies. Humanized antibodies (e.g., hu5F9-G4, Magrolimab) are especially useful for in vivo applications in humans due to their low antigenicity.
  • These gene markers are useful to identify cancers that will respond favourably to therapy with a CD47 blocking agent.
  • Those subjects or cancers that “respond favourably” are those cancers or subjects that respond to administration of the inhibitor with improvements in the symptoms of the disease being treated.
  • the response could manifest as an improvement in cancer cell or tumor properties or dynamics, such as a reduction in tumor growth rate, in cancer cell or tumor size or number, in cancer cell or tumor distribution and/or in overall cancer cell or tumor burden, for example, and/or as an extension of survival or an improvement in quality of life of the subject presenting with cancer.
  • the subjects to whom the method is most appropriately applied are subjects, such as mammals including pets, horses, livestock, primates and particularly humans, presenting with cancer and particularly a CD47+ cancer including a CD47+ hematological cancer or a CD47+ solid cancer such as a malignant tumor.
  • the subject can be one that presents with any disease that can be treated with a CD47 blocking agent.
  • the disease is a blood cancer selected from a lymphoma, a leukemia or a myeloma, and can be further selected from Hodgkin’s lymphoma, both indolent and aggressive non-Hodgkin’s lymphoma, Burkitt's lymphoma, follicular lymphoma (small cell and large cell), promyelocytic leukemia, chronic and acute myeloid leukemia (AML), acute and chronic lymphoid leukemia, multiple myeloma (MM), giant cell myeloma, heavy-chain myeloma, and light chain or Bence-Jones myeloma as well as diffuse large B-cell lymphoma (DLCBL), T-cell acute lymphoblastic leukemia (T-ALL), and T-cell lymphoma including cutaneous T cell lymphoma (CTCL), mycosis fungoides and Sezary syndrome.
  • Hodgkin’s lymphoma both ind
  • the cancer is also referenced herein as a tumor or as a cancer cell.
  • Other diseases include infection such as viral infection, as well as other diseases involving aberrant CD47 protein, which can include those mediated by altered CD47 protein gene expression or CD47 protein mutation or the like.
  • the prediction based on expression of the present maker genes that a given tumor will respond to CD47 blockade is particularly accurate when the cancer is a solid or at least palpable tumor or blood cancer, and one of those cancers identified herein, e.g., above.
  • Solid cancers including lung, prostate, breast, bladder, colon, ovarian, glioblastoma, medulloblastoma, leiomyosarcoma, and head & neck squamous cell carcinomas, melanomas; etc.
  • these CD47 blocking agents are formulated with a pharmaceutically acceptable carrier using standard practices and ingredients.
  • the formulated drug will be administered parenterally such as by injection or infusion, or orally in the form of tablets, capsules liquids and the like. Dosing and dosing regimens will be standard for drugs in this same category.
  • the cancer for which prediction of response is determined is one that is a hematological cancer and particularly one that is selected from the group consisting of Hodgkin’s lymphoma, indolent and aggressive non-Hodgkin’s lymphoma, Burkitt's lymphoma, follicular lymphoma, promyelocytic leukemia, chronic and acute myeloid leukemia, acute and chronic lymphoid leukemia, multiple myeloma (MM), giant cell myeloma, heavy-chain myeloma, and light chain or Bence-Jones myeloma, diffuse large B-cell lymphoma (DLCBL), cutaneous anaplastic large cell lymphoma (pcALCL), Sezary Syndrome, T-cell acute lymphoblastic leukemia (T-ALL), and T-cell lymphoma including particularly cutaneous T cell lymphoma (CTCL).
  • Hodgkin’s lymphoma indolent and aggressive non-Hod
  • the markers and their assay and their analysis are applied for the benefit of treating subjects presenting specifically with cutaneous T cell lymphoma (CTCL).
  • CTCL cutaneous T cell lymphoma
  • CTCL can cause rash-like skin redness, slightly raised or scaly round patches on the skin, and, sometimes, skin tumors.
  • cutaneous T-cell lymphoma Several types exist. The most common type is mycosis fungoides. Sezary syndrome is a less common type that causes skin redness over the entire body. Some types of cutaneous T-cell lymphoma, such as mycosis fungoides, progress slowly and others are more aggressive. The type of cutaneous T-cell lymphoma determines which treatments are most useful, and can include skin creams, light therapy, radiation therapy and systemic medications, such as chemotherapy. Cutaneous T-cell lymphoma is one of several types of lymphoma collectively that form part of the present invention and are called non- Hodgkin's lymphoma such as Sezary Syndrome and mycosis fungoides.
  • the cancer to be treated can be in relapsed/refractory form.
  • a subject presenting with a cancer having a CD47+ phenotype, such as a form of cutaneous T cell lymphoma is identified for treatment with, for instance, a SIRPaFc (Gl)
  • SIRPaFc Gl
  • Eligible patients recruited for such treatment are those presenting with CTCL that also reveal an altered level of one or more of the marker genes noted herein.
  • the alteration in marker levels found in the tumor/s can be determined by comparing the level with a level determined to be baseline or wildtype, as determined from a pool of samples from the normal population.
  • SIRPaFc Before or following administration of a first dose of SIRPaFc to the selected CTCL patient group, or additional doses if desired, biopsied tissue or a liquid sample of blood, serum, plasma, urine, semen and the like can be tested to determine whether expression of any one or more of the marker genes is at a level that is altered relative to a pre-treatment level. If there is alteration in at least any one marker gene expression, then SIRPaFc therapy can continue since the subject is deemed a responder to CD47 blockade therapy.
  • a “dose” or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy).
  • a therapeutically effective dose can be administered in one or more administrations.
  • a therapeutically effective dose of an anti-CD47 agent is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state (e.g., cancer or chronic infection) by increasing phagocytosis of a target cell (e.g., a target cell).
  • a therapeutically effective dose of an anti-CD47 agent reduces the binding of CD47 on a target cell, to SIRPa on a phagocytic cell, at an effective dose for increasing the phagocytosis of the target cell.
  • An effective dose or a series of therapeutically effective doses would be able to achieve and maintain a serum level of anti-CD47 agent.
  • a therapeutically effective dose of SIRPaFc agent can depend on the specific agent used, but is usually about 2 mg/kg body weight or more (e.g., about 2 mg/kg or more, about 4 mg/kg or more, about 8 mg/kg or more, about 10 mg/kg or more, about 15 mg/kg or more, about 20 mg/kg or more, about 25 mg/kg or more, about 30 mg/kg or more, about 35 mg/kg or more, or about 40 mg/kg or more), or from about 10 mg/kg to about 40 mg/kg (e.g., from about 10 mg/kg to about 35 mg/kg, or from about 10 mg/kg to about 30 mg/kg).
  • the dose required to achieve and/or maintain a particular serum level is proportional to the amount of time between doses and inversely proportional to the number of doses administered. Thus, as the frequency of dosing increases, the required dose decreases.
  • the optimization of dosing strategies will be readily understood and practiced by one of ordinary skill in the art.
  • a sub -therapeutic dose is a dose (i.e., an amount) that is not sufficient to effect the desired clinical results.
  • a sub-therapeutic dose of an anti-CD47 agent is an amount that is not sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state.
  • a sub-therapeutic dose of an anti-CD47 agent can depend on the specific agent used, but is generally less than about 10 mg/kg.
  • continuous treatment i.e., continue therapy
  • planned or current course of treatment e.g., continued administration of an anti-CD47 agent
  • additional therapy means replacing current therapy with either no therapy or a different CD47 therapeutic or a different drug altogether.
  • the anti-CD47 agent can be administered to an individual any time after a pre treatment biological sample is isolated from the individual.
  • the anti-CD47 agent may be administered simultaneous with or as soon as possible (e.g., about 7 days or less, about 3 days or less, e.g., 2 days or less, 36 hours or less, 1 day or less, 20 hours or less, 18 hours or less, 12 hours or less, 9 hours or less, 6 hours or less, 3 hours or less, 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 2 minutes or less, or 1 minute or less) after a pre-treatment biological sample is isolated (or, when multiple pre-treatment biological samples are isolated, after the final pre-treatment biological sample is isolated).
  • Suitable anti-CD47 agents can be provided in pharmaceutical compositions suitable for therapeutic use, e.g. for human treatment.
  • pharmaceutical compositions of the present invention include one or more therapeutic entities of the present invention or pharmaceutically acceptable salts, esters or solvates thereof.
  • the use of an anti-CD47 agent includes use in combination with another therapeutic agent (e.g., another anti-infection agent or another anti-cancer agent).
  • Therapeutic formulations comprising one or more anti-CD47 agents of the invention are prepared for storage by mixing the anti-CD47 agent having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
  • the anti-CD47 agent composition will be formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the anti-CD47 agent can be “administered” by any suitable means, including topical, oral, parenteral, intrapulmonary, intralesional and intranasal.
  • Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration.
  • compositions comprising an active therapeutic agent and another pharmaceutically acceptable excipient.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions can also include, depending on the formulation desired, pharmaceutically- acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles can also be prepared.
  • the preparation also can be encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above.
  • the agents can be administered as a depot injection or implant preparation which can be formulated for sustained or pulsatile release of the active ingredient.
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with regulatory agencies.
  • kits for use in the present methods include a tool e.g., a marker gene-hybridizing and optionally labeled oligonucleotide, or a PCR primer pair specific for a marker gene expression product such as RNA, or an antibody that specifically binds to a marker gene expressed protein, and the like) for determining the expression level of at least one of the present marker genes.
  • a kit can also include an anti-CD47 agent, such as SIRPaFc.
  • An anti-CD47 agent can be provided in a dosage form (e.g., a therapeutically effective dosage form, e.g., stick pack, dose pack, etc.).
  • kits may further include instructions for practicing the present methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like.
  • Yet another form of these instructions is a computer readable medium on which the information has been recorded.
  • Yet another form of these instructions that may be present is a website address.
  • treatment with a CD47 blocking agent is indicated when there is an alteration within the cancer of the level at which at least one of the genes, messages or proteins of STAT2, KIR inhibitory subgroup 1 and KIR inhibitory subgroup 2 is expressed.
  • treatment with the CD47 blocking agent can be recommended.
  • a different therapy should be adopted.
  • Example 1 Gene expression was evaluated in a clinical trial setting.
  • Intratumoral injection of SIRPaFc (with IgGl Fc, TTI-621) in percutaneously accessible tumors was performed in an investigational setting based on a modified 3+3 scheme with escalating doses sequentially through predefined levels of 1, 3, and 10 mg per injection. Injection frequency can be sequentially increased from single injections through 3 or 6 injections administered over 1 or 2 weeks. Dose expansion testing of the maximally assessed SIRPaFc dose and schedule proceeded with six 10 mg doses administered MWF over 2 weeks (induction therapy), in each of 6 cohorts.
  • CAILS Composite Assessment of Index Lesion Severity
  • Biopsies were collected per protocol prior to SIRPaFc treatment with a screening period of 14 days, at maximum induration, and end of induction therapy (7 days following the last injection). Adjacent, uninjected lesions were also biopsied at the same timepoints. For patients who went onto continuation therapy, additional biopsies could be taken at the investigator’s discretion.
  • RNA from blood and formalin fixed paraffin embedded (FFPE) biopsies was extracted. RNA quality and concentration were assessed. For each sample, mRNA transcript abundance was quantified using the NanoString nCounter Human PanCancer Immune Profiling Panel according to the manufacturer’s protocol from 100 ng of total RNA. Normalization to housekeeping genes and subsequent analysis was performed using nSolver software (NanoString, Seattle). Total counts were log2 transformed. In the example below, dose was not taken into account. Differences in pre-treatment log2(Expression) between subjects who had at least 50% decrease in Composite Assessment of Index Lesion Severity (CAILS) scores were compared with subjects who had less than 50% decrease in CAILS score using Wilcoxon rank-sum test.
  • CAILS Composite Assessment of Index Lesion Severity
  • These initial results included 9 subjects diagnosed with CTCL who had at least 50% decrease in CAILS and 12 who did not. The results held when analyses were performed with the full study population at the close of the study (11 subjects diagnosed with CTCL who had at least 50% decrease in CAILS and 16 who did not).
  • KIR inhibitory subgroup 1 and KIR inhibitory subgroup 2 levels were elevated in those who had at least a 50% reduction in CAILS as compared to those who did not ( Figure 1 and 2). In the example below, dose was not taken into account. [00120] As shown in Figure 3, gene expression data using Nanostring’s PanCancer
  • Immune Profiling panel compares baseline expression levels of STAT2 in CTCL patients with > 50% decrease in CAILS (left) or ⁇ 50% decrease in CAILS (right). The right panel depicts the percent decrease in CAILS from baseline (Composite Assessment of Index Lesion Severity) for mycosis fungoides patients on the y-axis and the gene expression count (log2) on the x-axis.

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Abstract

L'invention concerne des procédés utiles dans le traitement d'un sujet présentant un cancer lié àCD47+, par sélection pour le traitement d'un agent bloquant CD47 pour un sujet ayant un niveau d'expression modifié d'un gène marqueur choisi parmi un, deux ou tous les trois parmi : un niveau réduit d'expression STAT2 ; et un niveau élevé d'expression de sous-groupe 1 d'inhibiteur de KIR ; et un niveau élevé d'expression de sous-groupe 2 d'inhibiteur de KIR. En plus de l'utilisation de points de référence uniques, des patients susceptibles d'obtenir un bénéfice thérapeutique par l'administration d'un agent de blocage comprennent ceux qui, avant traitement, présentent un niveau STAT2 étant inférieur à la référence et soit un niveau élevé d'au moins l'une d'une espèce de sous-groupe 1 d'inhibiteur de KIR, soit un niveau élevé d'au moins l'une d'une espèce de sous-groupe 2 d'inhibiteur de KIR.
PCT/CA2020/051187 2019-09-04 2020-08-31 Biomarqueurs pour une thérapie de blocage de cd47 WO2021042204A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018176132A1 (fr) * 2017-03-28 2018-10-04 Trillium Therapeutics Inc. Thérapie par blocage de cd47
WO2020107115A1 (fr) * 2018-11-29 2020-06-04 Trillium Therapeutics Inc. Biomarqueurs pour une thérapie de blocage de cd47

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018176132A1 (fr) * 2017-03-28 2018-10-04 Trillium Therapeutics Inc. Thérapie par blocage de cd47
WO2020107115A1 (fr) * 2018-11-29 2020-06-04 Trillium Therapeutics Inc. Biomarqueurs pour une thérapie de blocage de cd47

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FENG M ET AL.: "Phagocytosis checkpoints as new targets for cancer immunotherapy", NAT REV CANCER, vol. 19, no. 10, 28 August 2019 (2019-08-28), pages 568 - 586, XP036888518, DOI: 10.1038/s41568-019-0183-z *
FOLKES AS ET AL.: "Targeting CD 47 as a cancer therapeutic strategy: the cutaneous T- cell lymphoma experience", CURR OPIN ONCOL, vol. 30, no. 5, September 2018 (2018-09-01), pages 332 - 337 *
JOHNSON LDS ET AL.: "Targeting CD 47 in Sezary syndrome with SIRPaFc", BLOOD ADV, vol. 3, no. 7, April 2019 (2019-04-01), pages 1145 - 1153, XP055800077 *

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