Methods of treating autoimmune and inflammatory diseases
阅读说明：本技术 治疗自身免疫性疾病和炎性疾病的方法 (Methods of treating autoimmune and inflammatory diseases ) 是由 M·汤森德 J·哈克尼 N·拉马穆尔蒂 于 2018-03-23 设计创作，主要内容包括：本文提供用于治疗自身免疫性和/或炎性疾病如狼疮的生物标记物和疗法,及使用BTK抑制剂的方法。特别地,提供用于狼疮中患者选择和预后的生物标记物,以及与之相关的治疗性治疗的方法、制造品和用于产生它们的方法、诊断试剂盒、检测方法和广告方法。(Provided herein are biomarkers and therapies for treating autoimmune and/or inflammatory diseases, such as lupus, and methods of using BTK inhibitors. In particular, biomarkers for patient selection and prognosis in lupus are provided, as well as methods of therapeutic treatment related thereto, articles of manufacture and methods for producing the same, diagnostic kits, detection methods and advertising methods.)
1. A method of treating an individual having an autoimmune or inflammatory disease comprising administering to the individual a therapeutically effective amount of a BTK inhibitor, wherein a sample from the individual has been found to have elevated levels of one or more biomarkers selected from IgJ, Mzb1, and Txndc 5.
2. A method of treating an autoimmune or inflammatory disease in an individual, the method comprising:
(a) determining that a sample from the individual comprises an elevated level of one or more biomarkers selected from IgJ, Mzb1, and Txndc 5; and
(b) administering to the individual an effective amount of a BTK inhibitor, thereby treating the immune disease or disorder.
3. A method of selecting a therapy for an individual having an autoimmune or inflammatory disease comprising determining the level of one or more biomarkers selected from IgJ, Mzb1, and Txndc 5; and selecting the drug based on the level of the biomarker.
4. A method for identifying an individual suffering from an autoimmune or inflammatory disease, who is more or less likely to show benefit from a treatment comprising a BTK inhibitor, by determining the level of one or more biomarkers selected from IgJ, Mzb1 and Txndc5 in a sample from the individual, wherein an elevated level of a biomarker in the sample indicates that the individual is more likely to show benefit from a treatment comprising a BTK inhibitor or a reduced level of a biomarker indicates that the individual is less likely to show benefit from a treatment comprising a BTK inhibitor.
5. An assay for identifying an individual having an autoimmune or inflammatory disease to receive a BTK inhibitor, the method comprising:
(a) determining the level of one or more biomarkers selected from IgJ, Mzb1, and Txndc5 in a sample from the individual; and is
(b) Administration of a BTK inhibitor is recommended based on the level of the biomarker.
6. A diagnostic kit comprising one or more reagents for determining the level of one or more biomarkers selected from IgJ, Mzb1 and Txndc5 in a sample from an individual having an autoimmune or inflammatory disease, wherein detection of an elevated level of the biomarker means increased efficacy in treating the individual with a BTK inhibitor, and wherein detection of a low or substantially undetectable level of the biomarker means decreased efficacy in treating the individual having an autoimmune or inflammatory disease with a BTK inhibitor.
7. The method of any one of claims 3 and 4, wherein the method further comprises administering to the individual an effective amount of a BTK inhibitor.
8. The method, assay and/or kit of any one of claims 1-7, which determines the level of a biomarker by measuring the RNA level of the biomarker relative to a reference level.
9. The method, assay and/or kit of claim 8, wherein measuring RNA levels comprises amplification.
10. The method, assay and/or kit of claim 9, wherein measuring RNA levels comprises quantitative PCR.
11. The method, assay and/or kit of claim 10, wherein measuring the level of RNA comprises amplifying the RNA and detecting the amplified product, thereby measuring the level of RNA relative to a reference level.
12. The method, assay and/or kit according to any one of claims 1-11, wherein the sample is a blood sample.
13. The method, assay and/or kit according to any one of claims 1-12, wherein the BTK inhibitor is an antibody, binding polypeptide, small molecule and/or polynucleotide.
14. The method, assay and/or kit according to claim 13, wherein the BTK inhibitor is a small molecule.
15. The method, assay and/or kit according to claim 14, wherein small molecule is in some embodiments, a small molecule BTK inhibitor is compound (a):
or a pharmaceutically acceptable salt thereof.
16. The method, assay and/or kit of any one of claims 1-15, wherein the autoimmune or inflammatory disease is systemic lupus erythematosus.
17. The method, assay and/or kit of claim 16, wherein the autoimmune or inflammatory disease is lupus nephritis.
18. A method, assay and/or kit according to claim 16, wherein the autoimmune or inflammatory disease is extrarenal lupus.
19. The method, assay and/or kit of any one of claims 1-18, wherein two of the biomarkers are selected.
20. The method, assay and/or kit of any one of claims 1-18, wherein three of the biomarkers are selected.
Technical Field
Provided herein are biomarkers and therapies for treating autoimmune and inflammatory diseases, and methods of using BTK inhibitors. In particular, biomarkers for patient selection and prognosis in autoimmune and inflammatory diseases are provided, as well as methods of therapeutic treatment, articles of manufacture and methods for producing the same, diagnostic kits, detection methods and advertising methods related thereto.
Background
Autoimmune and inflammatory diseases and disorders remain significant threats to human health. Despite the apparent progress in treating autoimmune and inflammatory diseases and conditions, improved therapies are still sought. Many autoimmune and inflammatory diseases show signs of heterogeneity. For example, Systemic Lupus Erythematosus (SLE) is a disease with heterogeneous evidence in a population of patients with SLE. See Kennedy et al, Lupus Sci. & med., 2015; 2: e000080. In view of this heterogeneity, in addition to new methods of treating autoimmune and inflammatory diseases (e.g., SLE), there is a need for methods of identifying certain patients using diagnostic biomarkers that can improve treatment outcome.
Plasmablasts are rapidly dividing, short-lived antibody secreting cells. Increased plasmablasts have been identified in the blood of juvenile lupus patients, and in general, increased abundance of antibody transcripts in lupus patients. Arece et al, J.Immunol.167, 2361-2369 (2001); l.bennett et al, j.exp.med.197, 711-723 (2003). Although plasmablasts account for a small proportion of B cells in the blood, they are responsible for the majority of antibody transcripts present in whole blood mRNA.
Protein kinases, the largest family of human enzymes, encompass well over 500 proteins. Bruton's Tyrosine Kinase (BTK) is a member of the Tec family of tyrosine kinases and is a regulator of B cell early development and mature B cell activation, signaling, and survival. Evidence for the role of BTK in allergic disorders and/or autoimmune and/or inflammatory diseases has been established in a BTK deficient mouse model. For example, in the mouse preclinical model of SLE, BTK deficiency has been shown to lead to significant improvement in disease progression. In addition, BTK deficient mice may also resist the development of collagen-induced arthritis and may be less susceptible to staphylococcal arthritis. A large body of evidence supports the role of the B cell and humoral immune system in the pathogenesis of autoimmune and/or inflammatory diseases. See, for example, WO 2012/118750. Protein-based therapeutics developed to deplete B cells (e.g., Rituxan) represent one approach to the treatment of numerous autoimmune and/or inflammatory diseases. Because of the role of BTK in B cell activation, preparations of BTK can be used as inhibitors of B cell mediated pathogenic activities (e.g., autoantibody production). BTK is also expressed in osteoclasts, mast cells and monocytes and has been shown to be important for the function of these cells. For example, BTK deficiency in mice is associated with impaired IgE-mediated mast cell activation (markedly reduced TNF- α and other inflammatory cytokine release), and BTK deficiency in humans is associated with a dramatic reduction in TNF- α production by activated monocytes.
Inhibition of BTK activity can be used to treat allergic disorders and/or autoimmune and/or inflammatory diseases, such as: SLE, rheumatoid arthritis, various vasculitis, Idiopathic Thrombocytopenic Purpura (ITP), myasthenia gravis, allergic rhinitis, and asthma (Di Paolo et al (2011) Nature chem. biol.7(1): 41-50; Liu et al (2011) journal. of Pharm. and expert. Ther.338(1): 154-. Specific BTK inhibitors (Liu (2011) drug couple and displacement 39(10): 1840-1849; U.S. patent No. 7,884,108, WO 2010/056875; U.S. patent No. 7,405,295; U.S. patent No. 7,393,848; WO 2006/053121; U.S. patent No. 7,947,835; US 2008/0139557; U.S. patent No. 7,838,523; US 2008/0125417; US 2011/0118233; PCT/US2011/050034 "pyridone/pyrazinone, process for producing and method of using the same", PCT/US2011/050013 pyrazinone, process for producing and method of using the same ", filed 2011 8/31, and U.S. series No. 13/102,720" pyridone and aza-pyridone compounds and method of using the same ", filed 2011 5/6).
U.S. patent No. 8,716,274, which is incorporated herein by reference in its entirety, discloses a class of heteroaryl pyridine and aza-pyridone compounds that are useful for inhibiting BTK. Compound (a) shown below is a specific BTK inhibitor compound:
compound (a) is: (S) -2- (3' - (hydroxymethyl) -1-methyl-5- ((5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-yl) amino) -6-oxo-1, 6-dihydro- [3,4' -bipyridine ] -2' -yl) -7, 7-dimethyl-2, 3,4,6,7, 8-hexahydro-1H-cyclopenta [4,5] pyrrolo [1,2-a ] pyrazin-1-one. The chemical structure is dominant in any case of inconsistency between chemical structure and chemical name.
All references, including patent applications and publications, referred to herein are incorporated by reference in their entirety.
SUMMARY
Provided herein is a method of treating an individual having an autoimmune or inflammatory disease comprising administering to the individual a therapeutically effective amount of a BTK inhibitor, wherein a sample from the individual has been found to have elevated levels of one or more biomarkers selected from IgJ, Mzb1, and
Also provided herein is a method of treating an autoimmune or inflammatory disease in an individual, the method comprising:
(a) determining that a sample from the individual comprises an elevated level of one or more biomarkers selected from IgJ, Mzb1, and
(b) Administering to the individual an effective amount of a BTK inhibitor, thereby treating the immune disease or disorder.
Also provided herein is a method of selecting a therapy for an individual having an autoimmune or inflammatory disease, the method comprising determining the level of one or more biomarkers selected from IgJ, Mzb1, and
Also provided herein is a method of identifying an individual having an autoimmune or inflammatory disease who is more or less likely to show benefit from a treatment comprising a BTK inhibitor by determining the level of one or more biomarkers selected from IgJ, Mzb1 and Txndc5 in a sample from the individual, wherein an elevated level of a biomarker in the sample indicates that the individual is more likely to show benefit from a treatment comprising a BTK inhibitor or a reduced level of a biomarker indicates that the individual is less likely to show benefit from a treatment comprising a BTK inhibitor.
Also provided herein is an assay for identifying an individual having an autoimmune or inflammatory disease to receive a BTK inhibitor, the method comprising:
(a) determining the level of one or more biomarkers selected from IgJ, Mzb1, and Txndc5 in a sample from the individual; and is
(b) Administration of a BTK inhibitor is recommended based on the level of the biomarker.
Also provided herein is a diagnostic kit comprising one or more reagents for determining the level of one or more biomarkers selected from IgJ, Mzb1 and Txndc5 in a sample from an individual having an autoimmune or inflammatory disease, wherein detection of an elevated biomarker level means increased therapeutic efficacy in treating the individual with a BTK inhibitor, and wherein detection of a low or substantially undetectable level of the biomarker means decreased therapeutic efficacy in treating the individual having an autoimmune or inflammatory disease with a BTK inhibitor.
In some embodiments of the methods provided herein, the method further comprises administering to the individual an effective amount of a BTK inhibitor.
In some embodiments of the methods, assays, and/or kits provided herein, the sample is a blood sample.
In some embodiments of the methods, assays, and/or kits provided herein, the BTK inhibitor is an antibody, binding polypeptide, small molecule, and/or polynucleotide.
In some embodiments of the methods, assays, and/or kits provided herein, the BTK inhibitor is a small molecule. In some embodiments, the small molecule BTK inhibitor is compound (a) or a pharmaceutically acceptable salt thereof.
In some embodiments of the methods, assays, and/or kits provided herein, the autoimmune disease or inflammatory disease is systemic lupus erythematosus. In some embodiments, the autoimmune or inflammatory disease is lupus nephritis. In some embodiments, the autoimmune or inflammatory disease is extrarenal lupus (extrarenal-renal lupus).
Biomarkers and methods of using them to predict response to treatment with B cell antagonists (e.g., anti-CD 20 antibodies) in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and lupus have been previously disclosed, but plasmablast gene signature (signature) has not been disclosed and has not been disclosed in terms of BTK inhibition. See WO 2012/118750, the entire content of which is hereby incorporated by reference.
As provided herein, the transcript profile of a subpopulation of B cells identifies a gene expression signature specific for plasmablasts. This signature is highly correlated with plasmablast abundance of in vitro spiked (spike) in the experiment. Using FACS analysis of B cell subsets in SLE patients, paired with RNA-sequencing, the existing gene expression signature showed a strong correlation with the frequency of plasmablasts in whole blood. Although plasmablasts account for a small proportion of B cells in the blood, they are responsible for most of the antibody transcripts found in whole blood mRNA. Expanding to two additional phase II clinical trial cohorts, using the SLEDAI disease activity index, plasmablast signatures were found to be associated with disease activity. This association is driven by the association of plasmablasts with the presence of anti-DNA antibodies, low levels of complement, and leukopenia. Elevated plasmablast tags are also associated with high levels of interferon activity. Patient race/ethnicity also predicts plasmablast tag levels, regardless of disease severity. Standard of care medications, particularly mycophenolate, reduce expression of plasmablast marker genes. Treatment of a patient with rituximab, for example, results in a significant, but ultimately transient, reduction in plasmablast tag expression.
Brief Description of Drawings
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FIGS. 1A-1 and 1A-2. plasmablast in vitro differentiation and sorting strategy. Plasmablasts were differentiated from CD20+ CD27+ memory B cells for 7 days in culture conditions containing CpG along with cytokines IL-2, IL-6, IL-10, IL-15, IFN α. Naive B cells (CD20+ CD27-), and FACS-sorted CD20+ CD27+ activated B cells and differentiated CD20loCD38+ plasmablasts were used for gene expression profiling.
Figure 1b heatmap of plasmablast-specific expressed genes. Genes were identified that were expressed at 0.001 FDR by plasmablasts at least 10-fold higher than primary and activated B cells and had expression levels >5RPKM in plasmablasts. Values represent variance stabilization data that has been normalized within each gene for
Figure 2 a.heat map of candidate plasmablast tag genes added to pbmc samples increasing the number of plasmablasts. Plasmablasts were injected into PBMCs from two independent donors, as shown in black and gray in the above heatmap. Values represent the Δ Ct for each gene relative to HPRT1 and were normalized to mean 0 and
FIG. 2B-1, FIG. 2B-2, FIG. 2B-3 and FIG. 2B-4. the expression level of the plasmablast signature gene relative to HPRT1 or the average of all three genes compared to the percentage of plasmablasts present in each sample. The dotted and dashed lines indicate different PBMC donors, while the different symbols represent different donors of plasmablasts. Linear regression analysis was used to predict expression of plasmablast tags, or component genes, with PBMC donors and plasmablast donors incorporated into the model. All four models were highly statistically significant, with p < 1X 10-10. The predictive power of the model is reported as r2 from the linear model.
FIG. 2C-1, FIG. 2C-2, FIG. 2C-3 and FIG. 2C-4. the relative expression of plasmablast gene relative to HPRT1 or the mean of all three signature genes measured in B cell populations isolated from healthy donors one week after receiving influenza vaccine. N ═ naive B cells, M ═ memory B cells, and PB ═ plasmablasts. Plasmablasts have the highest expression of the marker gene compared to the other population. Asterisks indicate statistical significance of differences between B cell populations using linear regression, including donors as covariates; p <0.05, p <0.01, p < 0.001.
FIG. 2D-1, FIG. 2D-2, FIG. 2D-3 and FIG. 2D-4 plasmablast signature and component genes correlate with plasmablast frequency measured by FACS in the blood of lupus patients. In 43 patients, the percentage of IgD-CD19+ CD27+ + CD38+ + plasmablasts as whole blood cells was measured over up to 3 time points, where we accompanied the RNA-sequencing data for a total of 96 samples. Gene expression values are presented as RPKM for each gene or as the geometric mean of the three gene signatures RPKM. The correlation coefficient was calculated using the Spearman rank order (rank-order) method.
Figure 3a plasmablast signature correlates with disease activity measured with SLEDAI. Values represent the average expression of the plasmablast signature gene relative to
3B-1, 3B-2, and 3B-3. the independent components of the SLEDAI composite index correlate with increased plasmablast signature gene expression. Linear regression was used to assess the statistical significance between patients showing each symptom and those not showing each symptom; asterisks indicate the level of significance in this test: p <0.05, p <0.01, p < 0.001.
FIG. 3C-1, FIG. 3C-2, and FIG. 3C-3 serum C3 and C4 complement levels and serum anti-dsDNA antibody titers correlate with plasmablast tag expression. Correlation coefficients were calculated using Spearman rank order.
FIG. 3D-1 and FIG. 3D-2 Whole blood interferon signature expression (ISM) correlates with plasmablast signature values. Correlation coefficients were calculated using Spearman rank order.
Figure 4a. treatment of patients with rituximab reduced plasmablast tag expression levels. The line represents the mean expression level inside the rituximab treatment cohort (dashed line) or placebo cohort (solid line), with the error bars representing the mean standard error. Black arrows indicate when the patient received drug or placebo infusion. Expression of plasmablast signature genes was modeled using a linear mixed effects model incorporating age, race/ethnicity, concomitant drug use, interferon activity, SLEDAI and treatment group (treatment arm) and time points and their interactions as fixed effects and patients as random effects. Red asterisks indicate time points of specifically significant differences in rituximab treatment groups: p <0.05, p <0.01, p < 0.001.
Figure 4b treatment with mycophenolate mofetil and rituximab independently reduced plasmablast tag expression levels. The line represents the mean of the placebo cohort (solid line) or the rituximab treatment cohort (dashed line), and the mean standard error is represented by the error bar. The arrows indicate when the patient received placebo or rituximab infusions. Expression values were modeled using a linear mixed effects model incorporating age, interferon activity and treatment groups and visits and their interactions with patients as random effects. The asterisks at the top of the curve indicate time points where rituximab-treated patients showed a significant decrease from baseline over the placebo group, while the asterisks near the bottom of the curve indicate time points that were different from baseline regardless of treatment: p <0.05, p <0.01, p < 0.001.
Figure 4c. patients with detectable anti-chimeric antibody (HACA) had higher plasmablast marker gene expression. The line indicates the mean expression of plasmablast marker genes in rituximab-treated patients with detectable HACA (solid line) or those patients without detectable HACA (dashed line), and the error bars indicate the mean standard error.
Figure 5a. patients treated with Mycophenolate Mofetil (MMF) or Methotrexate (MTX) showed lower plasmablast expression than patients treated with azathioprine (AZA). Plasmablast label screening values were compared between patients receiving different immunosuppressive dosing regimens. Statistical significance was tested by comparing AZA to the other two treatments separately using linear regression. Asterisks indicate significant differences between treatments: p <0.05, p <0.01, p < 0.001.
Figure 5b patients receiving MMF treatment at screening tended to have lower plasmablast label than those patients not receiving MMF treatment. P-values were calculated using linear regression between patients taking MMF and those not taking MMF at the screening visit.
Figure 6a. european ancestral patients had lower levels of plasmablast expression than other ethnic patients in the explor clinical trial cohort. Values represent the mean expression level of the plasmablast gene relative to
Lunar patients did not show significant differences based on race/ethnicity. Using linear regression, no significant difference across ethnicity was observed.
Figure 6c patients of european descent in the rose clinical trial cohort showed lower plasmablast marker expression. Values represent the geometric mean RPKM of plasmablast genes. Visit values were screened across self-reported race/ethnic comparisons using linear regression against the mean RPKM of the log2 transformation. Asterisks indicate significance of difference compared to patients self-reported as caucasian/caucasian: p <0.05, p <0.01, p < 0.001.
Figure 7 dose-response curves of the BTK inhibitor GDC-0852 inhibiting CD 40L-mediated plasmablast differentiation show that plasmablast differentiation is inhibited in a dose-dependent manner. The percentage of plasmablasts in four healthy donors was determined using FACS analysis to calculate the IC50 value for each donor.
Btk inhibition reduces plasmablast gene signature in fig. 8A, 8B, 8C, and 8d. Memory B cells from 4 healthy donors differentiated into plasmablasts in the presence of DMSO vehicle or 370nM GDC-0852 using the conditions described above. Expression of plasmablast signature gene was measured by Fluidigm and normalized to housekeeping gene (HPRT 1). Expression values were plotted as relative transcript abundance relative to housekeeping genes. The plasmablast signature was calculated as the geometric mean of the relative abundance of three independent genes.
FIG. 9 plasmablast gene expression correlates with plasmablast number. The percentage of plasmablasts as determined by FACS analysis correlated with plasmablast signature as determined in fig. 8D (Spearman rho ═ 0.81). White dots represent samples differentiated in the presence of DMSO, while black dots represent GDC-0852 treated samples.
Figure 10 BTK inhibitor GDC-0852 inhibited CpG-mediated plasmablast differentiation in a dose-dependent manner. The percentage of plasmablasts in four healthy donors was determined using FACS analysis to calculate the IC50 value for each donor.
Detailed Description
I. Definition of
"polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into the polymer by a DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. Modifications to the nucleotide structure, if present, may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "capping", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, e.g., for example, those having uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and having charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties (e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.)), those having intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.), those containing alkylating agents, those having modified bonds (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of the polynucleotide. In addition, any hydroxyl groups typically present in sugars can be substituted (e.g., with phosphonate groups, phosphate groups), protected with standard protecting groups, or activated to prepare additional bonds to additional nucleotides, or can be conjugated to solid or semi-solid supports. The 5 'and 3' terminal OH groups may be phosphorylated or partially substituted with an amine or organic capping group of 1 to 20 carbon atoms. Other hydroxyl groups may also be derivatized to the targetA quasi-protective base. Polynucleotides may also contain similar forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2 '-O-methyl-, 2' -O-allyl, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methylriboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate ester is comprised of P (O) S ("thioester"), P (S) S ("dithioester"), (O) NR2("amidates"), P (O) R, P (O) OR', CO OR CH2("methylal") substitution, wherein each R or R' is independently H or a substituted or unsubstituted alkyl group (1-20C) optionally containing an ether (-O-) linkage, an aryl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, or an arylaldehyde group (araldyl). Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.
"oligonucleotide" as used herein generally refers to a short single-stranded polynucleotide that is less than, but not necessarily less than, about 250 nucleotides in length. The oligonucleotide may be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above polynucleotide descriptions apply equally and fully to oligonucleotides.
The term "primer" refers to a single-stranded polynucleotide capable of hybridizing to a nucleic acid, typically by providing a free 3' -OH group, and then polymerizing the complementary nucleic acid.
The term "small molecule" refers to any molecule having a molecular weight of about 2000 daltons or less, preferably about 500 daltons or less.
The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as the progeny screened or selected for in the originally transformed cell.
As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".
An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromanogr.b 848:79-87 (2007).
An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody constructs, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.
A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
"affinity" refers to the strength of the aggregate non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can be generally represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
An "affinity matured" antibody refers to an antibody having one or more alterations in one or more hypervariable regions (HVRs), wherein such alterations result in an improvement in the affinity of the antibody for the antigen as compared to a parent antibody not possessing such alterations.
The term "detecting" includes any means of detection, including direct and indirect detection.
The term "biomarker", as used herein, refers to an indicator that can be detected in a sample, e.g., a prognostic, diagnostic and/or prognostic indicator. Biomarkers can serve as indicators of particular subtypes of a disease or disorder (e.g., cancer) characterized by certain molecular, pathological, histological, and/or clinical features. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), polypeptides and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and/or glycolipid-based molecular markers.
The terms "biomarker signature," "biomarker expression signature," or "expression signature" are used interchangeably herein and refer to one or a combination of biomarkers whose expression is indicative (e.g., predictive, diagnostic, and/or prognostic). Biomarker signatures can serve as indicators of particular subtypes of a disease or disorder (e.g., cancer) characterized by certain molecular, pathological, histological, and/or clinical features. In some embodiments, the biomarker signature is a "gene signature". The term "gene signature" is used interchangeably with "gene expression signature" and refers to one or a combination of polynucleotides whose expression is an indicator (e.g., prognostic, diagnostic, and/or prognostic). In some embodiments, the biomarker signature is a "protein signature". The term "protein tag" is used interchangeably with "protein expression tag" and refers to one or a combination of polypeptides whose expression is indicative (e.g., predictive, diagnostic, and/or prognostic).
The "amount" or "level" of a biomarker that is associated with an increased clinical benefit to an individual is a detectable level in a biological sample. These can be measured by methods known to those skilled in the art and also disclosed herein. The level or amount of expression of the biomarker assessed can be used to determine a response to treatment.
The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a biomarker in a biological sample. "expression" generally refers to the process by which information (e.g., gene-encoded and/or epigenetic) is converted into structures that are present and operational in a cell. Thus, "expression" as used herein may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide). Transcribed polynucleotides, fragments of translated polypeptides, or fragments of polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered as expressed, whether they are derived from transcripts produced from transcripts that are alternatively spliced or degraded, or from post-translational processing of polypeptides, e.g., by proteolysis. "expressed genes" include those that are transcribed into a polynucleotide as mRNA and subsequently translated into a polypeptide, and also include those that are transcribed into RNA but not translated into a polypeptide (e.g., transport and ribosomal RNA).
"elevated expression," "elevated expression level," or "elevated level" refers to an increased expression or increased level of a biomarker in an individual relative to a control (e.g., one or more individuals not suffering from a disease or disorder (e.g., cancer)) or an internal control (e.g., a housekeeping biomarker).
By "reduced expression," "reduced expression level," or "reduced level" is meant a decrease in expression or a reduced level of a biomarker in an individual relative to a control (e.g., one or more individuals not suffering from a disease or disorder (e.g., cancer)) or an internal control (e.g., a housekeeping biomarker). In some embodiments, the decreased expression is rare or absent expression.
In certain embodiments, the term "at a reference level" refers to a level of a biomarker in a sample from an individual or patient that is substantially the same as the reference level or to a level that differs from the reference level by up to 1%, up to 2%, up to 3%, up to 4%, up to 5%. In some embodiments, the reference level is the median level of the biomarker in the reference population. In some embodiments, the reference level of the marker is the average level of the marker in the reference population. In some embodiments, the reference level of the marker is the mean level of the marker in the reference population.
In certain embodiments, the term "above a reference level" refers to a level of a biomarker in a sample from an individual or patient that is at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or greater than the reference level, as determined by the methods described herein, as compared to the reference level. In some embodiments, the reference level is the median level in the reference population. In some embodiments, the reference level of the marker is the average level of the marker in the reference population.
In certain embodiments, the term "below a reference level" refers to a level of a biomarker that is at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or greater below the reference level in a sample from an individual or patient as determined by the methods described herein, as compared to the reference level. In some embodiments, the reference level is the median level in the reference population. In some embodiments, the reference level of the marker is the average level of the marker in the reference population. In some embodiments, the reference level of the marker is the mean level of the marker in the reference population.
The term "housekeeping biomarker" refers to a biomarker or group of biomarkers (e.g., polynucleotides and/or polypeptides) that are similarly present, typically in all cell types. In some embodiments, the housekeeping biomarker is a "housekeeping gene. "housekeeping gene" refers herein to a gene or group of genes that encode a protein whose activity is essential for maintaining cell function and is typically similarly present in all cell types.
"amplification" as used herein generally refers to the process of producing multiple copies of a desired sequence. "multiple copies" means at least two copies. "copy" does not necessarily mean a perfect sequence that is complementary to or identical to the template sequence. For example, the copies may comprise nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced by primers comprising sequences that are hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.
The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acids obtained from a single source (e.g., an individual) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction.
The "stringency" of the hybridization reaction can be readily determined by one of ordinary skill in the art, and is generally an empirical calculation depending on probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally relies on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. Thus, it follows that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures will tend to make the reaction conditions less stringent. For additional details and explanation of the stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience publishers, (1995).
As defined herein, "stringent conditions" or "high stringency conditions" can be determined by: (1) low ionic strength and high temperature are used for washing, e.g. at 50 ℃, 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate; (2) denaturing agents such as formamide, e.g., 50% (v/v) formamide, together with pH 6.5, 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer, together with 750mM sodium chloride, 75mM sodium citrate, are used during hybridization at 42 ℃; or (3) overnight hybridization at 42 ℃ in a solution using 50% formamide, 5 XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS, and 10% dextran sulfate, while washing in 0.2 XSSC (sodium chloride/sodium citrate) at 42 ℃ for 10 minutes, followed by washing at 55 ℃ for 10 minutes at high stringency consisting of 0.1 XSSC with EDTA.
"moderately stringent conditions" can be determined as described by Sambrook et al, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Press,1989, and include the use of washing solutions and hybridization conditions (e.g., temperature, ionic strength, and% SDS) that are less stringent than those described above. An example of moderately stringent conditions is incubation overnight at 37 ℃ in a solution comprising 20% formamide, 5 XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5 XDenhardt's solution, 10% dextran sulfate, and 20mg/ml denatured sheared salmon sperm DNA, followed by washing of the filters in 1 XSSC at about 37-50 ℃. The skilled person will recognize how to adjust the temperature, ionic strength, etc. as required to accommodate factors such as probe length.
The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., cancer). For example, "diagnosing" may refer to identifying a particular type of cancer. "diagnosis" may also refer to the classification of a particular subtype of cancer, for example, according to histopathological criteria or according to molecular characteristics (e.g., a subtype characterized by expression of a biomarker (e.g., a particular gene or protein encoded by the gene) or a combination thereof).
The term "aiding diagnosis" is used herein to refer to a method that aids in making a clinical determination regarding the presence or nature of a particular type of symptom or condition of a disease or disorder (e.g., cancer). For example, a method of aiding in the diagnosis of a disease or condition (e.g., cancer) may include measuring certain biomarkers in a biological sample from an individual.
As used herein, the term "sample" refers to a composition obtained or derived from a subject and/or individual of interest that contains a cellular entity and/or other molecular entity to be characterized and/or identified, e.g., based on a physical, biochemical, chemical, and/or physiological characteristic. For example, the phrase "disease sample" and variations thereof refers to any sample obtained from a subject of interest that is otherwise expected or known to contain the cellular and/or molecular entities to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.
By "tissue sample" or "cell sample" is meant a group of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample may be solid tissue from a fresh, frozen and/or preserved organ, tissue sample, biopsy sample and/or aspirate; blood or any blood component (e.g., plasma); body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from any time of pregnancy or development in a subject. The tissue sample may also be primary or cultured cells or cell lines. Optionally, a tissue or cell sample is obtained from the diseased tissue/organ. Tissue samples may contain compounds that are not naturally mixed with tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
As used herein, "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refers to a sample, cell, tissue, standard, or level used for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased portion (e.g., tissue or cell) of the body of the same subject or individual. For example, healthy and/or non-diseased cells or tissues adjacent to a diseased cell or tissue (e.g., cells or tissues adjacent to a tumor). In another embodiment, the reference sample is obtained from untreated tissue and/or cells of the body of the same subject or individual. In another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased portion (e.g., tissue or cell) of an individual that is not the subject or individual. In even another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from untreated tissue and/or cells of the body of an individual that is not the subject or the individual.
For purposes herein, a "section" of a tissue sample means a single portion or piece of the tissue sample, e.g., a thin section of tissue or cells cut from the tissue sample. It is understood that multiple sections of a tissue sample may be taken and analyzed, provided that it is understood that the same section of tissue sample may be analyzed at both the morphological and molecular levels, or in the case of both polypeptides and polynucleotides.
"correlation" or "associating" means comparing the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol in any manner. For example, the results of the first analysis or protocol may be used in the implementation of the second protocol and/or the results of the first analysis or protocol may be used to decide whether the second analysis or protocol should be performed. For embodiments of polynucleotide analysis or protocols, the results of the polynucleotide expression analysis or protocol can be used to determine whether a particular treatment protocol should be performed.
Any endpoint can be used to assess an "individual response" or "response," which endpoint indicates that the individual is benefitting, including without limitation (1) inhibiting disease progression (e.g., cancer progression) to some extent, including slowing and complete arrest; (2) reduction in tumor size; (3) inhibit (i.e., reduce, slow, or completely stop) cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibit (i.e., reduce, slow, or completely stop) metastasis; (5) alleviating one or more symptoms associated with a disease or disorder (e.g., cancer) to a certain extent; (6) increasing the length of progression-free survival; and/or (9) reduce mortality at a given point in time after treatment.
As used herein, the term "substantially the same" refers to a sufficiently high degree of similarity between two numerical values such that one of skill in the art would consider the difference between the two values to be of little or no biological significance and/or statistical significance within the context of the biological feature (e.g., Kd value or expression) measured with the values. The difference between the two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10%, as a function of the reference/comparative value.
As used herein, the term "substantially different" refers to a sufficiently high degree of difference between two numerical values such that one of skill in the art would consider the difference between the two values to be statistically significant over the background range of the biological characteristic measured with the values (e.g., Kd values). The difference between the two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50%, as a function of the value of the reference/comparator molecule.
As used herein, the word "label" relates to a detectable compound or composition. The label is typically conjugated or fused, directly or indirectly, to an agent, such as a polynucleotide probe or antibody, and facilitates detection of the agent conjugated or fused thereto. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels), or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that produces a detectable product.
An "effective amount" of an active agent is an amount effective, at dosages and for periods of time as required, to achieve the desired therapeutic or prophylactic result.
The "therapeutically effective amount" of a substance/molecule, agonist or antagonist may vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount wherein any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time as required, to achieve the desired prophylactic result. Generally, but not necessarily, since a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
The term "pharmaceutical formulation" refers to a preparation in such a form as to allow the biological activity of the active ingredient contained therein to be effective, and not containing additional components that are unacceptably toxic to a subject to whom the formulation will be administered.
"pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical formulation other than an active ingredient that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
As used herein, "treatment" (and grammatical variations thereof such as the verb "treat" or the word "treat") refers to a clinical intervention intended to alter the natural process of the individual being treated, and may be intended to be prophylactic or to be administered during the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating a disease state, and ameliorating or improving prognosis. In some embodiments, the antibody is used to delay disease progression or to slow the progression of disease.
The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance, wherein the precursor or derivative is less cytotoxic to tumor cells and is capable of being enzymatically activated or converted to a more active parent form as compared to the parent drug. See, for example, Wilman, "primers in Cancer chemistry" biological Society Transactions,14, pp.375-382, 615th Meeting Belfast (1986) and Stella et al, "primers: A Chemical Approach to Targeted Drug Delivery," direct Drug Delivery, Borchardt et al (eds.), pp.247-267, Human Press (1985). Prodrugs of the present invention include, but are not limited to, phosphate-containing prodrugs, phosphorothioate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs, which may be converted to the more cytotoxic free drug. Examples of cytotoxic drugs that may be derivatized into prodrug forms for use in the present invention include, but are not limited to, those chemotherapeutic agents described above.
An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.
The term "simultaneous" is used herein to refer to the administration of two or more therapeutic agents, wherein at least a portion of the administrations overlap in time. Thus, simultaneous administration includes dosing regimens when one or more active agents continues to be administered after one or more other active agents has ceased.
By "reduce or inhibit" is meant the ability to cause an overall reduction of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more. Decreasing or inhibiting can refer to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.
The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
An "article of manufacture" is any article of manufacture (e.g., a package or container) or kit that comprises at least one agent, e.g., a drug for treating a disease or disorder (e.g., cancer) or a probe that specifically detects a biomarker described herein. In certain embodiments, the articles of manufacture and kits are marketed, distributed, or sold as a unit for performing the methods described herein.
As used herein, the phrase "based on" means that information about one or more biomarkers is used to inform treatment decisions, information provided on package inserts, or marketing/sales guides, etc.
As understood by those of skill in the art, reference herein to "about" a certain value or parameter includes (and describes) embodiments that refer to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".
It is understood that aspects and embodiments of the invention described herein include aspects and embodiments "consisting of … …" and/or "consisting essentially of … …". As used herein, the singular forms "a", "an" and "the" include plural referents unless otherwise specified.
Methods and uses
Provided herein are methods of using plasmablast biomarkers. In particular, methods of using BTK inhibitors and plasmablasts biomarkers are provided. For example, provided is a method of treating a subject having a disease or disorder, the method comprising: administering to the individual a therapeutically effective amount of a BTK inhibitor if the individual has been found to be present with and/or has an elevated level of a plasmablast biomarker. Further provided herein is a method of treating a disease or disorder in an individual, the method comprising: determining that a sample from the individual comprises elevated plasmablast biomarker levels, and administering to the individual an effective amount of a BTK inhibitor, thereby treating the disease or disorder. In some embodiments, the plasmablast biomarker is selected from the group of gene tags consisting of IgJ, Mzb1, and
Provided herein are methods of treating a disease or disorder in an individual, comprising administering to the individual an effective amount of a BTK inhibitor, wherein treatment is based on the presence and/or elevated levels of plasmablast biomarkers in a sample from the individual. In some embodiments, the plasmablast biomarker is expression of one or more of IgJ, Mzb1, and
Further, provided herein are methods of selecting a therapy for an individual having a disease or disorder, the method comprising determining the presence and/or level of a plasmablast biomarker, and selecting a drug based on the presence and/or level of the biomarker. In some embodiments, the drug is selected based on elevated plasmablast biomarker levels. In some embodiments, the plasmablast biomarker is selected from the group of gene tags consisting of IgJ, Mzb1, and
Provided herein is a method of identifying a subject suffering from a disease or disorder who is more or less likely to show benefit from a treatment comprising a BTK inhibitor, the method comprising: determining the presence and/or level of a plasmablast biomarker in a sample from a subject, wherein the presence and/or elevated level of the plasmablast biomarker in the sample indicates that the subject is more likely to show benefit from treatment comprising a BTK inhibitor, or the absence and/or reduced level of the plasmablast biomarker indicates that the subject is less likely to show benefit from treatment comprising a BTK inhibitor. In some embodiments, the plasmablast biomarker is selected from the group of gene tags consisting of IgJ, Mzb1, and
Also provided herein are assays for identifying individuals having a disease or disorder to receive a BTK inhibitor, the methods comprising: (a) determining the presence and/or level of a plasmablast biomarker in a sample from an individual; (b) the BTK inhibitor is recommended based on the presence and/or level of plasmablast biomarkers. In some embodiments, a BTK inhibitor is recommended based on elevated plasmablast biomarker levels. In some embodiments, the plasmablast biomarker is selected from the group of gene tags consisting of IgJ, Mzb1, and
Provided herein are diagnostic kits comprising one or more reagents for determining the level of a plasmablast biomarker in a sample from an individual having a disease or disorder, wherein detection of the presence and/or elevated level of the plasmablast biomarker means increased efficacy when the individual is treated with a BTK inhibitor, and wherein detection of a low or substantially undetectable level of the biomarker means decreased efficacy when the individual having the disease is treated with a BTK inhibitor. Also provided herein are articles of manufacture comprising, packaged together, a pharmaceutical composition comprising a BTK inhibitor and a package insert indicating: BTK inhibitors are useful for treating patients with a disease or disorder based on the expression of plasmablast biomarkers. In some embodiments, the plasmablast biomarker is selected from the group of gene tags consisting of IgJ, Mzb1, and
Further, provided herein are methods of treating a disease or disorder in an individual, comprising administering to the individual an effective amount of a BTK inhibitor, and assessing the level of one or more plasmablast biomarkers in a sample from the individual during BTK inhibitor treatment (e.g., as compared to a reference). Also provided are methods of treating a disease or disorder in an individual, the method comprising administering to the individual an effective amount of a BTK inhibitor, wherein treatment is based on the level of one or more plasmablast biomarkers in a sample from the individual (e.g., as compared to a reference). Provided is a method of monitoring responsiveness to a treatment comprising a BTK inhibitor in an individual, the method comprising: determining the level of one or more plasmablast biomarkers in a sample from the individual, wherein a decreased level of one or more plasmablast biomarkers in the sample (e.g., as compared to a reference) indicates that the individual is more likely to respond to a treatment comprising a BTK inhibitor, or an increased level of one or more plasmablast biomarkers and/or a level that is substantially the same as its pre-treatment level (e.g., as compared to a reference) indicates that the individual is less likely to respond to a treatment comprising a BTK inhibitor. In some embodiments, the plasmablast biomarker is selected from the group of gene tags consisting of IgJ, Mzb1, and
Additionally, a method of determining whether an individual having a disease or disorder should continue or discontinue treatment with a BTK inhibitor is provided, the method comprising measuring the level of one or more plasmablast biomarkers in a sample from the individual, wherein an elevated level and/or a level substantially the same as its pre-treatment level (e.g., as compared to a reference) of the one or more plasmablast biomarkers determines that the individual should discontinue treatment with a BTK inhibitor and a reduced level (e.g., as compared to a reference) of the one or more plasmablast biomarkers determines that the individual should continue treatment with a BTK inhibitor. In some embodiments, the plasmablast biomarker is selected from the group of gene tags consisting of IgJ, Mzb1, and
In some embodiments, the method comprises: (a) measuring the RNA level of one, two or three biomarkers selected from IgJ, TXNDC5 and MZB1 in a biological sample from the patient; (b) comparing the RNA level measured in (a) to a reference level; and (c) identifying the patient as more likely to benefit from the BTK inhibitor therapy when the RNA level measured in (a) is above a reference level. In some embodiments, the RNA is mRNA. In some embodiments, measuring mRNA levels comprises amplification. In some embodiments, measuring mRNA levels comprises quantitative PCR. In some embodiments, measuring mRNA levels comprises amplifying the mRNA and detecting the amplified product, thereby measuring the levels of the mRNA. In some embodiments, the reference level is the median level of each marker in the reference population.
In some embodiments, the reference level of the marker is the median level of the marker in the reference population. In any of the embodiments described herein, the reference level can be the average level (mean level) of each marker in a reference population. In some embodiments, the reference level of the marker is the average level (average level) of the marker in the reference population. Non-limiting exemplary reference populations include immune or inflammatory disease patients, healthy individuals, and groups comprising healthy individuals and immune or inflammatory disease patients. In some embodiments, the reference population comprises SLE patients.
In some embodiments, the method of analyzing or detecting a biomarker has a p-value of less than 0.05. In some embodiments, the method has a specificity greater than 80%. In some embodiments, the method has a sensitivity of greater than 80%. In some embodiments, the method has a ROC of greater than 70%. In some embodiments, the method has an AUC greater than 70%. In some embodiments, the method has a positive predictive value of greater than 70%. In some embodiments, the method has a negative predictive value of greater than 70%. In some embodiments, the reference gene expression profile is from a subject in a reference population of patients and/or healthy volunteers. In some embodiments, the comparing step comprises at least one of: comparing the digital image of the expression profile to a database of comparison expression data.
In some embodiments of any of the foregoing methods, the plasmablast biomarker is IgJ. In some embodiments of any of the foregoing methods, the plasmablast biomarker is
In certain of the above embodiments, the sample is a urine sample. In some embodiments, the sample is a blood sample. In some embodiments, the biological sample is selected from the group consisting of blood, serum, plasma, and Peripheral Blood Mononuclear Cells (PBMCs). In some embodiments, the biological sample is RNA obtained from blood, e.g., whole blood or a cellular fraction of blood (such as PBMCs). In some embodiments, the biological sample is serum or plasma. The sample may be taken before, during or after treatment. Samples may be taken from patients suspected of having or diagnosed with SLE or other immune or inflammatory disease and therefore likely in need of treatment. Alternatively, the sample may be taken from a normal individual who is not suspected of having any disease. In some embodiments, the RNA is extracted from a biological sample described herein prior to detecting or measuring the mRNA level of the marker.
The presence and/or expression level/amount of a biomarker can be determined qualitatively and/or quantitatively based on any suitable criteria known in the art, including but not limited to DNA, mRNA, cDNA, protein fragment, and/or gene copy number. In certain embodiments, the presence and/or expression level/amount of the biomarker in the first sample is increased as compared to the presence/absence and/or expression level/amount in the second sample. In certain embodiments, the presence/absence and/or expression level/amount of the biomarker in the first sample is reduced compared to the presence and/or expression level/amount of the biomarker in the second sample. In certain embodiments, the second sample is a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue. Additional disclosure for determining the presence/absence and/or expression level/amount of a gene is described herein.
In some embodiments of any of the methods, elevated expression refers to an overall increase in the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by prior known standard methods such as those described herein, of about any of: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more. In certain embodiments, elevated expression refers to an increase in the expression level/amount of a biomarker in a sample, wherein the increase is at least about any one of the following for the expression level/amount of each biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue: 1.5X, 1.75X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 25X, 50X, 75X, or 100X. In some embodiments, elevated expression refers to an overall increase of greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold as compared to a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the methods, reduced expression refers to an overall reduction in the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) as measured by existing known standard methods, such as those described herein, as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue by about any of the following: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more. In certain embodiments, reduced expression refers to a reduction in the expression level/amount of a biomarker in a sample, wherein the reduction is at least about any one of the following in the expression level/amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue: 0.9X, 0.8X, 0.7X, 0.6X, 0.5X, 0.4X, 0.3X, 0.2X, 0.1X, 0.05X, or 0.01X.
The presence and/or expression level/quantification system of a variety of biomarkers in a sample can be analyzed by a number of methods, many of which are known in the art and understood by the skilled artisan, including but not limited to immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, blood-based quantitative assays (e.g., serum-ELISA), biochemical enzyme activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction ("PCR") (which includes quantitative real-time PCR ("qRT-PCR")) and other amplification-type detection methods, e.g., branched DNA, SISBA, TMA, etc.), RNA-Seq, FISH, microarray analysis, gene expression profiling, and/or continuous analysis of gene expression ("SAGE"), as well as any of a variety of types of assays that can be performed by protein, gene, and/or tissue array analysis. For example, there are common Protocols for assessing the status of genes and gene products In Ausubel et al, 1995, Current Protocols In Molecular Biology, Unit 2(Northern blot), Unit 4 (Souther blot), Unit 15 (immunoblot) and Unit 18(PCR analysis). Multiplex immunoassays, such as those available from rules based Medicine or Meso Scale Discovery ("MSD"), may also be used.
In some embodiments, the presence and/or expression level/amount of a biomarker is determined using a method comprising: (a) subjecting a sample (e.g., a cancer sample from a subject) to gene expression profiling, PCR (e.g., rtPCR), RNA-seq, microarray analysis, SAGE, MassARRAY technique or FISH; and b) determining the presence and/or expression level/amount of the biomarker in the sample. In some embodiments, microarray methods include the use of microarray chips having one or more nucleic acid molecules that can hybridize under stringent conditions to nucleic acid molecules encoding the above-mentioned genes or having one or more polypeptides (e.g., peptides or antibodies) that can bind to one or more proteins encoded by the above-mentioned genes. In one embodiment, the PCR method is qRT-PCR. In one embodiment, the PCR method is multiplex PCR. In some embodiments, gene expression is measured by microarray. In some embodiments, gene expression is measured by qRT-PCR. In some embodiments, expression is measured by multiplex PCR.
Methods for assessing mRNA in a cell are well known and include, for example, hybridization assays using complementary DNA probes (e.g., in situ hybridization using labeled riboprobes specific for one or more genes, Northern blotting, and related techniques) and various nucleic acid amplification assays (e.g., RT-PCR using complementary primers specific for one or more genes, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA, and the like).
The mRNA can be conveniently determined from a sample from a mammal using Northern blot, dot blot or PCR analysis. In addition, such methods can include one or more steps that allow for the determination of the level of a target mRNA in a biological sample (e.g., by simultaneously examining the level of a comparative control mRNA sequence for a "housekeeping" gene, such as an actin family member). Optionally, the sequence of the amplified target cDNA can be determined.
Optional methods include protocols for examining or detecting mRNA (e.g., target mRNA) in a tissue sample or a cell sample by microarray technology. Test and control mRNA samples from the test tissue sample and the control tissue sample are reverse transcribed and labeled using a nucleic acid microarray to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, gene selections whose expression correlates with increased or decreased clinical benefit from anti-angiogenic therapy can be arranged on a solid support. Hybridization of a labeled probe to a particular member of the array indicates that the sample from which the probe is derived expresses the gene.
According to some embodiments, the presence and/or expression level/amount is measured by observing the protein expression level of the aforementioned genes. In certain embodiments, the method comprises: contacting the biological sample with an antibody directed against a biomarker described herein under conditions that allow binding of the biomarker, and detecting whether a complex is formed between the antibody and the biomarker. Such methods may be in vitro or in vivo. In one embodiment, the antibodies are used to select subjects eligible for BTK inhibitor therapy, e.g., to select biomarkers for individuals.
In certain embodiments, the sample is examined for the presence and/or expression level/amount of a biomarker protein using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable method of determining or detecting the presence of proteins in a sample. In some embodiments of any of the methods, assays, and/or kits, the plasmablast biomarker is selected from one or more of IgJ, Mzb1, and
IHC may be performed in combination with additional techniques such as morphological staining and/or fluorescence in situ hybridization. Two general approaches to IHC are available; direct assays and indirect assays. According to a first assay, the binding of an antibody to a target antigen is determined directly. This direct assay uses a labeled reagent, such as a fluorescent or enzyme-labeled primary antibody, which can be visualized without further antibody interaction. In a common indirect assay, an unconjugated primary antibody binds to the antigen and subsequently a labeled secondary antibody binds to the primary antibody. Wherein the secondary antibody is conjugated to an enzyme label, and a chromogenic or fluorogenic substrate is added to provide antigen visualization. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.
The first and/or second antibody used in IHC will generally be labeled with a detectable moiety. The numerous markers available can generally be grouped into the following categories: (a) radioisotopes, e.g.35S、14C、125I、3H and131i; (b) colloidal gold particles; (c) fluorescent labels including, but not limited to, rare earth element chelates (europium chelates), texas red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycoerythrin (phycerytherin), phycocyanin, or commercially available fluorophores such as SPECTRUMORANGE7 and SPECTRUM GREEN7 and/or derivatives of any one or more of the foregoing; (d) various enzyme-substrate markers are available and U.S. Pat. No. 4,275,149 provides a review of some of these markers. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferases; U.S. Pat. No. 4,737,456), luciferin, 2, 3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) and hydroperoxide as substrates; alkaline Phosphatase (AP) and p-nitrophenyl phosphate as chromogenic substrates; and β -D-galactosidase (β -D-Gal) with either a chromogenic substrate (e.g., p-nitrophenyl- β -D-galactosidase) or a fluorogenic substrate (e.g., 4-methylumbelliferyl- β -D-galactosidase). For a general review of these combinations, see U.S. Pat. nos. 4,275,149 and 4,318,980.
In some embodiments of any of the methods, the plasmablast biomarker is detected by immunohistochemistry using a diagnostic antibody (i.e., a primary antibody). In some embodiments, the tissue to be analyzed is kidney tissue. In some embodiments, the diagnostic antibody specifically binds to IgJ, Mzb1, or
In an alternative method, the sample may be contacted with an antibody specific for the biomarker under conditions sufficient to form an antibody-biomarker complex and the complex subsequently detected. The presence of biomarkers can be detected in a number of ways, such as by Western blotting and ELISA methods for analyzing various tissues and samples, including plasma or serum. A wide range of immunoassay techniques using this assay format are available, see, for example, U.S. Pat. nos. 4,016,043, 4,424,279 and 4,018,653. These methods include the non-competitive type as well as single-and two-site or "sandwich" assays in traditional competitive binding assays. These assays also include direct binding of labeled antibodies to the target biomarkers.
The tissue or cell sample may also be examined for the presence and/or expression level/amount of a selected biomarker by functional or activity-based assays. For example, if the biomarker is an enzyme, assays known in the art can be performed to determine or detect the presence of a given enzyme activity in a tissue or cell sample.
In certain embodiments, the sample is normalized for variability in the amount of biomarker analyzed and variability in the mass of the sample used and variability between assay runs. Such normalization can be achieved by detecting and incorporating the expression of certain normalization biomarkers, including well-known housekeeping genes, such as ACTB. Alternatively, normalization can be based on the average or median signal of all analyzed genes or a large subset thereof (global normalization scheme). The measured normalized amount of mRNA or protein in the subject sample is compared to the amount present in the reference collection based on the gene x gene. The normalized expression level of each mRNA or protein per subject per test sample can be expressed as a percentage of the expression levels measured in the reference set. The presence and/or expression level/amount measured in a particular subject sample to be analyzed will fall within some percentile within this range, which can be determined by methods well known in the art.
In certain embodiments, the relative expression levels of the genes are determined as follows:
relative expressed
Reference RNA 2exp (Ct housekeeping gene-Ct gene 1) relative to expressed
Normalized
Ct is the threshold cycle. Ct is the cycle number at which the fluorescence generated inside the reaction crosses the threshold line.
All experiments were normalized to a reference RNA that was a comprehensive mix of its RNAs from various tissue sources (e.g., reference RNA #636538 from Clontech, Mountain View, CA). The same reference RNA was included in each qRT-PCR run, allowing comparison of results between different experimental runs.
In one embodiment, the sample is a clinical sample. In another embodiment, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a tissue. Tissue biopsies are often used to obtain representative pieces of tissue. Alternatively, tumor cells may be obtained indirectly in the form of a tissue or fluid that is known or believed to contain the cells of interest. The genes or gene products can be detected from tissue or from other body samples such as urine, sputum, serum or plasma. By screening such body samples, the progress of therapy can be more easily monitored by testing such body samples for target genes or gene products.
In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combined multiplex from the same subject or individual obtained at one or more different time points when the test sample is obtained. For example, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject or individual at a time point prior to the time at which the test sample is obtained. Such a reference sample, reference cell, reference tissue, control sample, control cell or control tissue may be useful if the reference sample is obtained during the initial diagnosis of the disease and the test sample is obtained later when the disease has progressed.
In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled multiplex sample from one or more healthy individuals that are not the subject or the individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled multiplex sample from one or more individuals not the subject or the individual having the disease or disorder. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from normal tissue or a pooled plasma or serum sample from one or more individuals that are not the subject or the individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from a tissue or a pooled plasma or serum sample from one or more individuals not the subject or the individual having the disease or disorder.
In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a sample cell line. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is blood.
In some embodiments, the sample is a tissue sample from an individual. In some embodiments, the tissue sample is a blood or urine sample. In some embodiments, the tissue sample is a blood sample.
In some embodiments of any of the methods, the BTK inhibitor is a small molecule BTK inhibitor. In some embodiments, the small molecule BTK inhibitor is compound (a) or a pharmaceutically acceptable salt thereof.
In some embodiments of any of the methods, the individual or patient according to any of the above embodiments may be a human.
In yet another embodiment, provided herein are methods of treating SLE. In one embodiment, the method comprises administering to an individual having SLE an effective amount of a small molecule BTK inhibitor. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described below. In some embodiments, the individual may be a human.
The BTK inhibitors described herein can be used alone or in combination with other pharmaceutically active agents in therapy. For example, the additional therapeutic agent can be an anti-inflammatory agent, an immunomodulatory agent, a chemotherapeutic agent, an apoptosis enhancer, a neurotropic factor, an agent for treating a hepatic cardiovascular disease, an agent for treating a hepatic disease, an antiviral agent, an agent for treating a hematologic disorder, an agent for treating diabetes, and an agent for treating an immunodeficiency disorder. The second therapeutic agent may be an NSAID anti-inflammatory agent. The second therapeutic agent may be a chemotherapeutic agent. The second compound of the pharmaceutical combination or dosing regimen preferably has complementary activities to compound (I) such that they do not adversely affect each other.
In some embodiments, the additional therapeutic agent is selected from: corticosteroids (e.g., prednisone, prednisolone (prednisolone), methylprednisolone, and hydrocortisone); disease modifying anti-rheumatic drugs ("DMARDs," e.g., immunosuppressive or anti-inflammatory agents); antimalarial agents (e.g., hydroxychloroquine and chloroquine); immunosuppressive agents (e.g., cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate); anti-inflammatory agents (e.g., aspirin, NSAIDs (e.g., ibuprofen, naproxen, indomethacin, nabumetone, celecoxib)); antihypertensive agents (e.g., calcium channel blockers (e.g., amlodipine, nifedipine) and diuretics (e.g., furosemide)); statins (e.g., atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin); anti-B cell agents (e.g., anti-CD 20 (e.g., rituximab), anti-CD 22); anti-B lymphocyte stimulators ("anti-BLyS", e.g., belimumab, blinibimod);
The combination therapy may be administered in a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. Co-administration includes co-administration using separate formulations or a single pharmaceutical formulation, and sequential administration in any order, wherein preferably there is a period of time during which both (or all) active agents exert their biological activity simultaneously. Any suitable dosage of the aforementioned co-administered active agents are those presently used and may be reduced by the combined action (synergy) of the additional therapeutic agents.
Combination therapy may be "synergistic" such that the effect achieved when the active ingredients are used together is greater than the sum of the effects resulting from the separate use of the compounds. Mixing the active ingredients: (1) simultaneous administration or delivery; (2) alternating or parallel administration; or (3) when applied by some other protocol, a synergistic effect may be achieved. When delivered in alternation therapy, a synergistic effect may be achieved when the compounds are administered or delivered sequentially. Typically, during alternation therapy, an effective dose of each active ingredient is administered sequentially, i.e., sequentially, whereas in combination therapy, an effective dose of two or more active ingredients are administered together.
In combination therapy, a kit may comprise (a) a first container having a dosage form composition of the present disclosure, and optionally (b) a second container containing a second pharmaceutical formulation for co-administration with a dosage form composition of the present disclosure. In these aspects, the kit may comprise containers for containing the individual compositions, such as a split bottle (split foil packet) or a split foil packet (split foil packet). However, the separate compositions may also be contained within a single, undivided container. Typically, the kit will contain instructions for administration of the individual components. The kit form is particularly advantageous when the individual components are preferably administered in different (e.g., oral and parenteral) dosage forms, are administered at different dosage intervals, or when the prescribing physician desires to titrate the individual components of the combination.
The BTK inhibitor can be administered by any suitable means, including oral, parenteral, intrapulmonary and intranasal and, if desired for topical treatment, intralesional administration. In a preferred embodiment, the BTK inhibitor is administered orally.
Oral dosage forms comprising a BTK inhibitor include, but are not limited to, tablets or capsules comprising a BTK inhibitor or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients. In some embodiments, a tablet or capsule comprising a BTK inhibitor can be administered once or twice daily according to the methods provided herein. In certain embodiments provided herein, the oral dosage form is a tablet comprising compound (a) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.
Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.
The BTK inhibitors described herein can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disease or condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease or condition, the site of active agent delivery, the method of administration, the administration schedule and other factors known to the medical practitioner. BTTK inhibitors need not be formulated with, but are optionally formulated with, one or more active agents currently used to prevent or treat the disease or condition in question. The effective amount of such other active agents depends on the amount of BTK inhibitor present in the formulation, the type or treatment of the disease or disorder, and other factors discussed above. These agents are generally used at the same dosages and using the routes of administration described herein, or at about 1% to 99% of the dosages described herein, or at any dosage and any route empirically/clinically determined to be appropriate.
Therapeutic compositions comprising a BTK inhibitor
Small molecule BTK inhibitors are provided in the compositions, methods, and kits herein. Small molecule BTK inhibitors as provided herein are preferably organic molecules other than binding polypeptides or antibodies, and can be identified and chemically synthesized using known methods. Binding organic small molecules are typically less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic small molecules capable of binding, preferably specifically binding, to BTK as described herein can be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for screening libraries of small organic molecules for molecules capable of binding to a polypeptide target are well known in the art (see, e.g., PCT publication Nos. WO 2000/00823 and WO 2000/39585). The binding organic small molecule may be, for example, an aldehyde, ketone, oxime, hydrazone, semicarbazone (semicarbazone), carbazide, primary amine, secondary amine, tertiary amine, N-substituted hydrazine, hydrazide, alcohol, ether, thiol, thioether, disulfide, carboxylic acid, ester, amide, urea, carbamate, carbonate, ketal, thioketal, acetal, thioacetal, aryl halide, aryl sulfonate, alkyl halide, alkyl sulfonate, aromatic compound, heterocyclic compound, aniline, alkene, alkyne, diol, aminoalcohol, oxazolidine, oxazoline, thiazolidine, thiazoline, enamine, sulfonamide, epoxide, aziridine, isocyanate, sulfonyl chloride, diazo compound, acid chloride, and the like.
In some embodiments of any of the methods, the BTK inhibitor is selected from: ibrutinib, Acerabritinib (acallabasttinib), spebrutinib, BIIB068(Biogen), BMS-986195(Bristol-Myers Squibb), BMS-986142(Bristol-Myers Squibb), BMS-935177(Bristol-Myers Squibb), M2951 (MerckkKGaA), PRN-1008 (Princidia Biopharma), HM71224/LY3337641 (Hami/Lilly), ONO-4059/GS-4059(Gilead/Ono), AC0058(ACEA Biosciences), AC0025(ACEA Biosciences), ABBV-599(AbbVie), ABBV-105(AbbVie), PfPF-303 (PfPF-1 (Abekingner Biophyceae), CelbBg-90008), Ash-Biophycex-42486 (BGR-4255), Ash-BTiI-4243 (Bionice-4255), and Ab-4235 (Rabbit-K-BTE), and SAG-4235 (Bionice-4217), SAG-BTiex-05) TAX-020(Takeda), WX486/WXFL-10230486 (WuXiApTec/Humanwell) and X-022(X-Rx Discovery).
In some embodiments, the BTK inhibitor is compound (a) or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of the BTK inhibitors provided herein can be used in the methods herein. As used herein, the term "pharmaceutically acceptable salt" is intended to include salts of the active compounds, wherein the salts are prepared with relatively non-toxic acids or bases, depending on the particular substituents present on the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, base addition salts are obtained by contacting such compounds in neutral form with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc and the like. Salts derived from pharmaceutically acceptable organic bases include the following: primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, and the like, such as arginine, betaine, caffeine, choline, N' -dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucamine (glucamine), glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. When the compounds of the present invention contain relatively basic functional groups, acid addition salts are obtained by contacting such compounds in neutral form with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochlorides, hydrobromides, nitrates, carbonates, bicarbonates, phosphates, hydrogenphosphates, dihydrogenphosphates, sulfates, hydrogensulfates, hydroiodides or phosphites and the like, as well as those derived from relatively nontoxic organic acids such as acetates, propionates, isobutyrates, malonates, benzoates, succinates, suberates, fumarates, mandelates, phthalates, benzenesulfonates, p-toluenesulfonates, citrates, tartrates, methanesulfonates and the like. Also included are Salts of amino acids such as arginine Salts and the like, and Salts of organic acids such as glucuronic acid or galacturonic acid and the like (see, e.g., Berge, S.M. et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science,1977,66, 1-19). Certain specific compounds of the invention contain both basic and acidic functional groups that allow the compounds to be converted into base addition salts or acid addition salts.
The neutral form of the compound may be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form differs from the various salt forms in certain physical properties (e.g. solubility in polar solvents), but for the purposes of the present invention the salts are equivalent to the parent form.
In addition to salt forms, the present invention provides compounds in prodrug form. As used herein, the term "prodrug" refers to those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention in an ex vivo environment by chemical or biochemical means. For example, prodrugs can be slowly converted into the compounds of the invention upon placement in a transdermal reservoir patch (transdermal patch reservoir) containing the appropriate enzyme or chemical agent.
Prodrugs of the invention include compounds wherein an amino acid residue or a polypeptide chain having two or more (e.g., two, three, or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxyl, or carboxylic acid group of a compound of the invention. Amino acid residues include, but are not limited to, the 20 naturally occurring amino acids commonly designated by three letter symbols, and also include phosphoserine, phosphothreonine, phosphotyrosine, 4-hydroxyproline, hydroxylysine, desmosine (demosine), isodesmosine (isodesmosine), gamma-carboxyglutamic acid, hippuric acid, octahydroindole-2-carboxylic acid, statin, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, methyl-alanine, p-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, methionine sulfone, and t-butylglycine.
Other types of prodrugs are also contemplated. For example, the free carboxyl groups of the compounds of the present invention may be derivatized as amides or alkyl esters. As another example, compounds of the present invention containing a free hydroxyl group may be derivatized into prodrugs by converting the hydroxyl group to a group such as, but not limited to, phosphate esters, hemisuccinate esters, dimethyl glycinate or hydroxyphosphoryloxymethoxycarbonyl, such as Fleisher, D. et al, (1996) Improved organic Drug Delivery as outlined in the methods of solubility assays over come by the use of drugs of Advanced Drug Delivery Reviews,19: 115. Also included are carbamate prodrugs having hydroxyl and amino groups, and also carbonate prodrugs, sulfonates, and sulfates having hydroxyl groups. Derivatization of the hydroxyl group into (acyloxy) methyl and (acyloxy) ethyl ethers is also contemplated, wherein the acyl group may be an alkyl ester optionally substituted with groups including, but not limited to, ether, amine, and carboxylic acid functionalities, or wherein the acyl group is an amino acid ester as described above. Prodrugs of this type are described in J.Med.chem., (1996),39: 10. More specific examples include replacing the hydrogen atom of the alcohol group with a group such as (C)1-6) Alkanoyloxymethyl, 1- ((C)1-6) Alkanoyloxy) ethyl, 1-methyl-1- ((C)1-6) Alkanoyloxy) ethyl group, (C)1-6) Alkoxycarbonyloxymethyl, N- (C)1-6)Alkoxycarbonylaminomethyl, succinyl, (C)1-6) Alkanoyl, alpha-amino acid (C)1-4) Alkanoyl, arylacyl and α -aminoacyl, or α -aminoacyl- α -aminoacyl, wherein each α -aminoacyl is independently selected from the group consisting of a naturally occurring L-amino acid, p (o) (oh)2、-P(O)(O(C1-6) Alkyl radical)2Or a sugar group (the radical resulting from removal of the hydroxyl group of the hemiacetal form of the sugar).
For additional examples of prodrug derivatives, see, e.g., a) Design of produgs, H.Bundgaard, eds (Elsevier,1985) and Methods in Enzymology, Vol.42, p.309-396, K.Widder et al (Academic Press, 1985); b) a Textbook of Drug Design and Development, Krogsgaard-Larsen and H.Bundgaard,
Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in a variety of crystalline or amorphous forms. In general, all physical forms are equivalent for the purposes of the inventive concepts and are intended to be within the scope of the present inventions.
Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, positional isomers and individual isomers (e.g., individual enantiomers) are intended to be encompassed within the scope of the present invention.
Pharmaceutical preparations
Pharmaceutical formulations of BTK inhibitors are provided in the methods and kits herein.
In some embodiments of any of the methods, the BTK inhibitor (e.g., compound (a) or a pharmaceutically acceptable salt thereof) is administered at a dose of about 0.1 mg/kg/day to about 100 mg/kg/day, about 0.5 mg/kg/day to about 20 mg/kg/day, or about 1 mg/kg/day to about 10 mg/kg/day, based on the weight of the patient. In some embodiments, compound (a) or a pharmaceutically acceptable salt thereof is administered as a tablet at a dose of about 10 to 800 mg. In some embodiments, compound (a) is administered as the free base in a tablet at a dose of about 25 to 300 mg. In some embodiments, the tablet comprises 25 to 300mg of compound (a) as the free base and fumaric acid, wherein the weight ratio of compound (a)/fumaric acid is about 1:5 to about 3: 1; or about 1:2 to about 2: 1; or about 1:1.5 to about 1.5: 1. In some embodiments, the tablet comprises 25 to 300mg of compound (a) as the free base and fumaric acid, and wherein the fumaric acid content is about 5 wt.% to about 50 wt.%, about 5 wt.% to about 40 wt.%, about 5 wt.% to about 30 wt.%, about 10 wt.% to about 30 wt.%, about 20 wt.% to about 25 wt.%, about 5 wt.% to about 15 wt.%, or about 10 wt.% to about 15 wt.%. In certain of the above embodiments, the tablet weight is about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, or about 1000 mg. In some embodiments, the tablet further comprises at least one pharmaceutically acceptable excipient selected from the group consisting of fillers, binders, disintegrants, lubricants, and glidants. In some embodiments, the tablet comprises lactose and microcrystalline cellulose.
The tablet compositions of the present disclosure may further suitably comprise one or more pharmaceutically acceptable excipients selected from, but not limited to, fillers (diluents), disintegrants, binders, glidants, and lubricants. Fillers (or diluents) can be used to increase the bulk of the powdered drug constituting the tablet. Disintegrants may be used to support the tablet breaking into small pieces, ideally individual drug particles, as it is ingested and thus facilitate rapid dissolution and absorption of the drug. Binders may be used to ensure that granules and tablets can be formed with the required mechanical strength and brought together after the tablets have been compressed, preventing it from breaking into its component powders during packaging, distribution and routine handling. Glidants can be used to improve the flowability of the powder making up a tablet during production. Lubricants may be used to ensure that tableting powders do not stick to the equipment used to compress the tablet during manufacture, to improve powder flow during mixing and compression, and to minimize friction and breakage when the finished tablet is ejected from the equipment.
Fillers and binders may include dibasic calcium phosphate, microcrystalline cellulose
Disintegrants may be included in the disclosed formulations to facilitate separation of the particles from each other within the compact and to maintain the released particles separate from each other. Disintegrants may be present as intragranular components and/or as extragranular components. Disintegrants may include any suitable disintegrant such as, for example, cross-linked polymers such as cross-linked polyvinylpyrrolidone and cross-linked sodium carboxymethyl cellulose or cross-linked sodium carboxymethyl cellulose. In some particular aspects, the disintegrant is croscarmellose sodium. The disintegrant content is suitably about 1 wt.%, about 1.5 wt.%, about 2 wt.%, about 2.5 wt.%, about 3 wt.%, about 3.5 wt.%, about 4 wt.%, about 4.5 wt.%, or about 5 wt.% and ranges thereof, such as from about 1 wt.% to about 5 wt.%, or from about 2 wt.% to about 4 wt.%.
Glidants may include, for example, colloidal silicon dioxide, including highly dispersed silica (silica)
Lubricants may be used to compress the particles in the pharmaceutical composition. Lubricants may, for example, include polyethylene glycol (e.g., having a molecular weight of about 1000 to about 6000), magnesium and calcium stearate, sodium stearyl fumarate, talc, or any other suitable lubricant. In some particular aspects, the lubricant is magnesium stearate and/or sodium stearyl fumarate. The lubricant may be present as an intragranular component and/or as an extragranular component. The lubricant content is suitably about 0.5 wt.%, about 1 wt.%, about 1.5 wt.%, about 2 wt.%, about 2.5 wt.%, about 3 wt.%, about 3.5 wt.%, about 4 wt.%, about 4.5 wt.%, or about 5 wt.% and ranges thereof, such as from about 0.5 wt.% to about 5 wt.%, from about 1 wt.% to about 4 wt.%, from about 1 wt.% to about 3 wt.%, or from about 1 wt.% to about 2 wt.%.
A coating (e.g., a film coating) may be applied to the tablets of the present disclosure. Film coatings may be used, for example, to facilitate easy swallowing of tablets. Film coatings may also be used to improve taste and appearance. The film coating may be an enteric coating, if desired. The film coat may comprise polymeric film-forming materials such as hydroxypropyl methylcellulose, hydroxypropyl cellulose, acrylate or methacrylate copolymers, and polyvinyl alcohol-polyethylene glycol graft copolymers such as Opadry and Kollicoat IR. The film coat may contain, in addition to the film-forming polymer, plasticizers, e.g. polyethylene glycol, surfactants, e.g.
The formulations herein may also contain more than one active ingredient, preferably those having complementary activities that do not adversely affect each other, as required by the particular indication being treated. Such active ingredients are present in suitable combinations in amounts effective for the intended purpose.
The active ingredient may be embedded in microcapsules prepared, for example by coacervation techniques or interfacial polymerization (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's
Sustained release articles can be prepared. Suitable examples of sustained-release articles include solid hydrophobic polymeric semipermeable matrices containing the BTK inhibitor, which matrices are in the form of shaped articles (e.g., films or microcapsules).
The formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.
V. article of manufacture
In another embodiment, an article of manufacture is provided that contains a material described above that is useful for treating, preventing, and/or diagnosing a disorder. The article of manufacture comprises a container and a label or package insert either on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition that is effective, by itself or in combination with another composition, in the treatment, prevention and/or diagnosis of a condition and may have a sterile access port (e.g., the container may be an intravenous bag or a vial having a stopper penetrable by a hypodermic injection needle). At least one active agent in the composition is a BTK inhibitor as described herein. The label or package insert indicates that the composition is for use in treating a selected condition. Moreover, an article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises a BTK inhibitor; and (b) a second container having a composition therein, wherein the composition comprises an additional cytotoxic agent or an additional therapeutic agent.
In some embodiments, the article of manufacture comprises a container, a label on the container, and a composition contained within the container; wherein the composition comprises one or more reagents (e.g., a first antibody that binds to one or more biomarkers (e.g., a B-9Santa Cruz Biotechnology antibody) or probes and/or primers directed to one or more biomarkers described herein), a label on the container indicating that the composition can be used to assess the presence of one or more biomarkers in a sample, and instructions for using the reagents to assess the presence of one or more biomarkers in a sample. The article of manufacture can also contain a set of instructions and information for preparing samples and using reagents. In some embodiments, the article of manufacture can include reagents such as first and second antibodies, where the second antibody is conjugated to a label (e.g., an enzymatic label). In some embodiments, an article of manufacture comprises one or more probes and/or primers directed to one or more of the biomarkers described herein.
The article of manufacture in this embodiment may also comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose (dextrose) solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
Other optional components in the article of manufacture include one or more buffers (e.g., blocking buffers, wash buffers, substrate buffers, etc.), other reagents such as substrates (e.g., a chromogen chemically altered by an enzymatic label), epitope retrieval solutions, control samples (positive and/or negative controls), control sections, and the like.
Examples
The following are examples of methods and compositions. It should be understood that various other embodiments may be implemented in view of the general description provided above.
Blood sample analysis: 3-Gene plasmablast tag expression assay. Blood was collected in PAXgene RNA tubes (PreAnalytiX); total RNA was extracted using a commercially available kit according to the manufacturer's instructions (Qiagen).
The biomarker is as follows: IgJ, TXNDC5,
Expression of candidate biomarker genes in blood samples was assessed by human genome U133 Plus 2.0 array (Affymetrix inc., Santa Clara, CA). Microarray hybridization was performed by Asuragen Inc (Austin, TX). Raw CEL file data were summarized and normalized using Robust multi-array averaging (RMA) and analyzed using R and Bioconductor.
Alternatively, expression of the candidate biomarker genes in the blood sample is characterized by the Fluidigm qPCR assay. The 3 gene score was calculated from the average of IgJ, TXNDC5, and MZB1 and normalized using the reference gene TMEM 55B. Blood samples were evaluated using this assay developed on the Cobas 4800 platform (Roche Molecular Systems).
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