WO2024245577A1

WO2024245577A1 - Therapeutic combinations of an irreversible btk inhibitor and a reversible btk inhibitor

Info

Publication number: WO2024245577A1
Application number: PCT/EP2023/064843
Authority: WO
Inventors: Erik ENSING; Rogier Christian Buijsman; Martine Berendina Wilhemina PRINSEN; Winfried Robert Mulder; Michelle MULLER; Yvonne Gertruda Theodora Hendrika VAN MIL; Adrianus Petrus Antonius De Man
Original assignee: Netherlands Translational Research Center BV
Current assignee: Netherlands Translational Research Center BV
Priority date: 2023-06-02
Filing date: 2023-06-02
Publication date: 2024-12-05
Anticipated expiration: 2025-12-02

Abstract

The present invention relates to therapeutic combinations of an irreversible BTK inhibitor and a reversible BTK inhibitor and relates to a method for treating a subject having a hyperproliferative disease, in particular a B-cell hematological malignancy, which subject receives or has received a Bruton's tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, using said therapeutic combinations. The present invention further relates to method treating a subject diagnosed with or at risk of a recurrent or refractory form of a hyperproliferative disease, in particular a B-cell hematological malignancy, which subject has previously been treated with an irreversible BTK inhibitor, using said therapeutic combinations. In particular embodiments the method comprises: a) monitoring the patient over the course of therapy to determine whether the subject has a C481 mutation in BTK and b) co-administering to the patient an irreversible and a reversible BTK inhibitor if the patient has a C481 mutation in BTK. The invention further relates to a method for treating a subject having a hyperproliferative disease, in particular a B-cell hematological malignancy, wherein the treatment comprises administering said therapeutic combinations.

Description

Therapeutic combinations of an irreversible BTK inhibitor and a reversible BTK inhibitor

Field of the invention

The present invention relates to therapeutic combinations of an irreversible BTK inhibitor and a reversible BTK inhibitor and relates to a method for treating a subject having a hyperproliferative disease, in particular a B-cell hematological malignancy, which subject receives or has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, using said therapeutic combinations. The present invention further relates to method treating a subject diagnosed with or at risk of a recurrent or refractory form of a hyperproliferative disease, in particular a B-cell hematological malignancy, which subject has previously been treated with an irreversible BTK inhibitor, using said therapeutic combinations. In particular embodiments the method comprises: a) monitoring the patient over the course of therapy to determine whether the subject has a C481 mutation in BTK and b) co-administering to the patient an irreversible and a reversible BTK inhibitor if the patient has a C481 mutation in BTK. The invention further relates to a method for treating a subject having a hyperproliferative disease, in particular a B-cell hematological malignancy, wherein the treatment comprises administering said therapeutic combinations.

Background of the invention

Kinases are enzymes that transfer a phosphate group from ATP to a protein. Kinases play an important role in regulating cellular functions such as cell proliferation, subcellular translocation, apoptosis, inflammation and metabolism (Attwood M.M. et al (2021) Nat Rev Drug Discov). The human kinome is composed of over 500 kinases. The development of smallmolecule kinase inhibitors for the treatment of diverse types of cancer has proven successful in clinical therapy.

Bruton’s tyrosine kinase (BTK) is a member of the Src-related Tec family of protein kinases constituting a large subset of kinases, which play a central role in the regulation of a wide variety of cellular signaling processes. BTK plays a key role in B-cell receptor (BCR) signaling and a critical role in the regulation of survival, proliferation, activation and differentiation of B-lineage cells. Targeting of BTK with small molecule inhibitors such as the FDA approved covalent, irreversible, BTK inhibitors ibrutinib, acalabrutinib, zanubrutinib and tirabrutinib has proven to be efficacious in several B-cell malignancies including Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma (MCL), Waldenstrom’s Macroglobulinemia (WM) and Small Lymphocytic Lymphoma SLL. Where tumors have a strong oncogenic drive from one particular kinase, specific amino acid mutations have been observed as acquired resistance mechanisms of targeted drug molecules. These mutations may naturally occur in very low levels but become more prevalent upon drug treatment (pre-existing mutations), or these can also be produced by random mutation within tumors (acquired mutations) (Barouch-Bentov et al. 2011). Where such mutations have a clear advantage upon drug treatment (/.e. a mutation where the drug is no longer able to bind to the target) then resistance may rapidly develop and patients can relapse quickly. In these cases, the understanding of the impact and mechanism of these mutations has enabled additional drug discovery efforts to develop subsequent “generations” of drug molecules to target such resistance forms.

A drawback of the first generation BTK inhibitor, ibrutinib, is that drug resistance in B- cell malignancies can develop when BTK acquires mutations at the cysteine at position C481 of the kinase domain. This mutation abrogates the covalent binding of ibrutinib hampering its efficacy. Quinquenel et al. performed a ‘snapshot’ screening to determine the prevalence of resistance mutations and found that the presence of the BTK mutation was significantly associated with subsequent CLL progression. The correlation between disease progression, and the emergence and temporal dynamics of the most common resistance inducing C481 S BTK mutation have been determined for CLL patients receiving single-agent ibrutinib treatment (Bbdbr et al. British Journal of Haematology 2021). Using digital droplet PCR the authors have been able to show that in 72.7% of the patients emergence of the BTK C481 S mutation preceded the symptoms of clinical relapse with a median of nine months. Woyach et al. showed that that detection of the C481 mutation in patients being treated with covalent, irreversible, BTK inhibitors is an indication for imminent relapse, usually within 12-18 months after detection (Woyach et al. (2017) J. Clin. Oncol. 35, 1437-1443). Since ibrutinib resistance confers poor survival, early detection of resistance provides clinically relevant information for the transition of affected patients to alternative treatment strategies.

Second-generation covalent, irreversible, BTK inhibitors, which include acalabrutinib, zanubrutinib, and tirabrutinib, offer greater BTK selectivity and therefore limited off-target toxicity. These inhibitors, however, do not overcome resistance by C481 mutation. To treat patients with relapsed CLL having C481 BTK mutations non-covalent, reversible, BTK inhibitors have been developed including LOXO-305 (pirtobrutinib) and ARQ-531 (nemtabrutinib) and vecabrutinib. These agents do not require covalent binding to the BTK C481 residue and effectively inhibit both wild type and mutant BTK with C481 substitutions. In preclinical studies, non-covalent BTK inhibitors including pirtobrutinib (LOXO-305), ARQ-351 and vecabrutinib, inhibited B-cell- receptor signaling in BTK C481 -mutant cell and animal models. Moreover, the phase 1-2 clinical trial of pirtobrutinib showed promising efficacy for patients with B-cell cancer who had previously been treated with covalent BTK inhibitors (with 62% of patients with CLL having a response), including patients with or without BTK C481 mutations (with a response occurring in 71% and 66% of the patients, respectively) (Wang et al. (2022) N. Engl. J. Med. 386, 735-43). Pirtobrutinib was first disclosed in WO2017/103611 , ARQ-531 was disclosed in WO2017/111787 and vecabrutinib is disclosed in WO2013/185084. Further reversible BTK inhibitors are disclosed in WO2017/046604, , W02020/015735, WO2020/239124, WO2021/093839, W02020/043638, WO2013/067274, WO2018097234, WO2013/010380, W02016/161570, WO2016/161571 , WO2016/106624, WO2016/106625, WO2016/106626, WO2016106623, WO2016/106628 and WO2016/109222. These reversible BTK inhibitors are being developed not only for B-cell hematological malignancies, but also for immunological and inflammatory diseases.

Furthermore other reversible BTK inhibitors in development are fenebrutinib (Crawford et al. 2018 and Reiff et al. 2018), BMS-986142 (Watterson et al. 2016), luxeptinib (Thieme et al. 2022 and WO2014/104757, AS-0871 (Wataru et al. 2018 and W02015/012149), AS-1763 (Wataru et al. 2021 and WO2018/097234) and BIIB091 (Hopkins et al. 2021 and WO2018/191577).

Despite the successful treatment of patients with a C481 mutation with pirtobrutinib new acquired mutations have emerged after treatment with this non-covalent inhibitor. Mutations were identified in these patients that were clustered in the kinase domain of BTK (V416L, A428D, M437R, T474I, and L528W) and that conferred resistance to pirtobrutinib and other non- covalent, reversible, inhibitors.

The most well-known clinical documented BTK mutations for ibrutinib, acalabrutinib, zanubrutinib and pirtobrutinib are given in Figure 1 mutants (Nakoda et al. 2023).

A general desire of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance (Al-Lazikani et al. 2012; Yap et al. 2013 and Li et al. 2014) Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. Cancer cell lines are an attractive model to investigate new drug combinations because they can be used to determine whether new combinations are truly synergistic, as opposed to additive (Zhao et al. 2004 and Chou 2010). Moreover, cancer cell lines provide a good representation of the diversity of genetic changes that drive human cancers (Garnett et al. 2012 and Barretina et al. 2013).

There are several ways to determine synergy between two compounds in an in vitro assay. From this, two basic methods have evolved, the curve shift analysis (Chou 2010 and Straetemans et al. 2005) and the combination matrix experiment with Bliss-scoring (Borisy et al. 2003; Cokol et al. 2011 and Mathews et al. 2014). Summary of the invention

In a first aspect of the invention, there is provided a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, the method comprising the steps: a) monitoring the subject, preferably at predetermined intervals of time, over the course of the therapy for treatment of the hyperproliferative disease to determine whether the subject has a mutation in an endogenous gene encoding BTK that results in or provides a risk of resistance of the subject to the therapy, preferably resistance of the subject to the irreversible BTK inhibitor of the (mono) therapy, preferably the subject has a mutation in an endogenous gene encoding BTK that results in expression of a BTK protein with a modification at an amino acid position corresponding to amino acid position 481 of the amino acid sequence according to SEQ ID NO: I; and

(b) if the subject has shown said mutation, administering to the subject a therapeutically combinatory amount of an irreversible BTK inhibitor and a reversible BTK inhibitor, in particular if the subject has the BTK modification corresponding to amino acid position 481 identified in step a).

In another aspect of the invention, there is provided a method for treating a subject diagnosed with or at risk of a recurrent or refractory form of a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has previously been treated with an irreversible BTK inhibitor, and wherein the method comprises administering to said subject a therapeutic combinatory amount of an irreversible BTK inhibitor and a reversible BTK inhibitor.

In another aspect of the invention, there is provided a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, the method comprising: administering to the subject a therapeutic combinatory amount of an irreversible BTK inhibitor and a reversible BTK inhibitor.

In other aspects of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method according to the invention.

In a particular aspect of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, the method comprising the steps: a) monitoring the subject, preferably at predetermined intervals of time, over the course of the therapy for treatment of the hyperproliferative disease to determine whether the subject has a mutation in an endogenous gene encoding BTK that results in or provides a risk of resistance of the subject to the therapy, preferably resistance of the subject to the irreversible BTK inhibitor of the (mono) therapy, preferably the subject has a mutation in an endogenous gene encoding BTK that results in expression of a BTK protein with a modification at an amino acid position corresponding to amino acid position 481 of the amino acid sequence according to SEQ ID NO: I; and

(b) if the subject has shown said mutation, administering to the subject a therapeutically combinatory amount of said irreversible BTK inhibitor and said reversible BTK inhibitor, in particular if the subject has the BTK modification corresponding to amino acid position 481 identified in step a).

In a particular aspect of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject diagnosed with or at risk of a recurrent or refractory form of a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has previously been treated with an irreversible BTK inhibitor, and wherein the method comprises administering to said subject a therapeutic combinatory amount of said irreversible BTK inhibitor and said reversible BTK inhibitor.

In a particular aspect of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, the method comprising: administering to the subject a therapeutic combinatory amount of said irreversible BTK inhibitor and said reversible BTK inhibitor.

In other aspects of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor according to the invention.

In other aspects of the invention, there is provided a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor according to the invention for use as a medicament.

In other aspects of the invention, there is provided use of a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor according to the invention in the manufacture of a medicament for treating a subject having a hyperproliferative disease, preferably treating a subject having a B-cell hematological malignancy

The present inventors have surprisingly found that a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor, in particular embodiments combinations according to the invention, is more effective than an irreversible or reversible BTK inhibitor alone. In particular, it has been shown that a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor, is more effective than an irreversible or reversible BTK inhibitor alone in inhibiting B-cell lymphoma cells comprising two separate cell pools each containing a different mutation in BTK.

Moreover, combinations of a reversible BTK inhibitor compound according to the invention and an irreversible, covalent, inhibitor is synergistically effective in inhibiting B-cell lymphoma cells. In specific examples, a synergistic effect of inhibition has been shown in a B-cell lymphoma cell line in which BTK has at least a C481 S modification.

The present invention will be illustrated further by means of the following non-limiting examples.

Definitions

The term "pharmaceutical composition” as used herein has its conventional meaning and refers to a composition which is pharmaceutically acceptable.

The term "pharmaceutically acceptable” as used herein has its conventional meaning and refers to compounds, material, compositions and/or dosage forms, which are, within the scope of sound medical judgment suitable for contact with the tissues of mammals, especially humans, without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.

The term "effective amount' as used herein, refers to an amount of the compound of the invention, and/or an additional therapeutic agent, or a composition thereof, that is effective in producing the desired therapeutic, ameliorative, inhibitory or preventive effect when administered to a subject suffering from a BTK-mediated disease or disorder. In the combination therapies of the present invention, an effective amount can refer to each individual agent or to the combination as a whole, wherein the amounts of all agents administered are together effective, but wherein the component agents of the combination may not be present individually in an effective amount.

The term "therapeutically combinatory amount” as used herein has its meaning in the context of combination therapy of the compounds according to the invention, or a composition thereof, that is effective in producing the desired therapeutic, ameliorative, inhibitory or preventive effect when administered to a subject suffering from a hyperproliferative disease, in particular a B-cell hematological malignancy . In particular embodiments, the therapeutically combinatory amount is the total amount of the two (or more) compounds selected of an irreversible BTK inhibitor and a reversible BTK inhibitor used for treating the hyperproliferative disease, in particular a B-cell hematological malignancy.

The term “combination" as used herein, means a product that results from the mixing or combining of an irreversible BTK inhibitor and a reversible BTK inhibitor (and any additional therapeutic agents) and includes both fixed and non-fixed combinations. The term “fixed combination” means that the irreversible BTK inhibitor and the reversible BTK inhibitor are both administered in a single entity or dosage form. The term “non-fixed combination” means that the irreversible BTK inhibitor and the reversible BTK inhibitor are administered as separate entities or dosage forms either simultaneously, concurrently or sequentially with no specific intervening time limits, wherein such administration provides effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more active ingredients.

A "subject" is a human or non-human mammal. In one embodiment, a subject is a human.

The term "controlling” is intended to referto all processes wherein there may be a slowing, interrupting, arresting or stopping of the progression of the diseases and conditions affecting the mammal. However, "controlling” does not necessarily indicate a total elimination of all disease and condition symptoms, and is intended to include prophylactic treatment.

The term "excipient” as used herein has its conventional meaning and refers to a pharmaceutically acceptable ingredient, which is commonly used in the pharmaceutical technology for preparing a granulate, solid or liquid oral dosage formulation.

The term "salt” as used herein has its conventional meaning and includes the acid addition and base salts of the compound of the invention.

The term "solvate” as used herein has its conventional meaning. One or more compounds of the invention or the pharmaceutically acceptable salts thereof may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. "Solvate" means a physical association of a compound of this invention with one or more solvent molecules. This physical association Involves varying degrees of ionic and covalent bonding. Including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Examples of suitable solvates include ethanolates, methanolates, and the like. "Hydrate" is a solvate wherein the solvent molecule is H2O and includes any hydrate of the compound or the salt of said compound.

The term "treatment” as used herein has its conventional meaning and refers to curative, disease controlling, palliative and prophylactic treatment.

The term "unit dosage form” has its conventional meaning and refers to a dosage form which has the capacity of being administered to a subject, preferably a human, to be effective, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising the therapeutic agent, i.e. the compound of the invention.

The term "BTK” as used herein has its conventional meaning and refers to Bruton's Tyrosine Kinase. Bruton's tyrosine kinase (BTK) is a member of the Src-related Tec family of protein kinases which are a large subset of kinases which play a central role in the regulation of a wide variety of cellular signaling processes. BTK plays a key role in the B-cell receptor signaling and a critical role in the regulation of survival, proliferation, activation and differentiation of B- lineage cells. Targeting of BTK with small molecule inhibitors such as the FDA approved irreversible BTK inhibitors ibrutinib, acalabrutinib, zanubrutinib and tirabrutinib has proven to be efficacious in several B-cell malignancies including Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma (MCL), Waldenstrom’s Macroglobulinemia (WM) and Small Lymphocytic Lymphoma SLL. Combinations of BTK inhibitors with other novel drugs or regimens results in more profound responses and much higher rates of minimal residual disease negativity.

The term "BTK inhibitoT’ as used herein has its conventional meaning and refers to an inhibitor for BTK. A BTK inhibitor may be a small molecule inhibitor. Inhibitors may be irreversible inhibitors, such as by forming a covalent bond, and may be reversible inhibitors, which may form a reversible interaction with BTK.

The term “covalent BTK inhibitor” as used herein has its conventional meaning and refers to a BTK inhibitor that reacts with its target protein (BTK) to form a covalent complex in which the protein has lost its function. Covalent inhibitors can be reversible or irreversible, depending on the rate of the reverse reaction. The terms 'covalent inhibitor' and 'irreversible inhibitor' are often used and are considered the same for the purpose of this patent application .

The term "irreversible BTK inhibitoT’ as used herein has its conventional meaning and refers to a BTK inhibitor that, after formation of a covalent complex with the protein, possesses an off-rate that is slow relative to the rate of re-synthesis of the target protein (BTK) in vivo, so that once the target protein is inhibited, it does not regain activity.

The term “rate of re-synthesis” as used herein has its conventional meaning and refers to the rate at which a cell and/or organism replaces a protein target with freshly synthesized functional protein. The re-synthesis rate defines the rate at which an irreversibly inhibited protein target will recover activity in vivo, once the inhibitor is no longer present.

The term "reversible BTK inhibitoT’ as used herein has its conventional meaning and refers to a BTK inhibitorthat inactivates the BTK enzyme through non-covalent, transcient, interactions. Unlike an irreversible inhibitor, a reversible inhibitor can dissociate from the enzyme.

The term "mutant-BTK” as used herein has its conventional meaning and refers to mutations of BTK. Mutations of BTK may be referred to by an altered or modified amino acid target (such as C as single-letter amino acid code for cysteine) at a certain position of the BTK structure (such as 481). Additionally, the amino acid substitution at the modification position may be referred to by an additional amino acid single-letter amino acid code, such as C481 S for serine substitution and C481T for threonine substitution of cysteine at the 481 position.

The term “modification" as used herein has its conventional meaning and refers to modification of a sequence of amino acids of a polypeptide, protein or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively.

The term "resistance to BTK inhibitoT’ as used herein has its conventional meaning and refers to a BTK inhibitor, which shows a reduction in effectiveness, after being effective initially in treating a hyperproliferative disease, in particular a B-cell hematological malignancy. A drawback of the currently approved irreversible inhibitors is that patients treated with these inhibitors can develop drug resistance when BTK proteins with variations at the catalytic site are not able to bind efficiently to irreversible inhibitors in patients. This is a rather common event in patients treated with irreversible inhibitors and who experience relapse. A major mechanism for the acquired resistance is the emergence of BTK cysteine 481 (C481) mutations. These mutations hamper binding of irreversible inhibitors such as ibrutinib, acalabrutinib, zanubrutinib and tirabrutinib which form a covalent bond with this amino acid. Other mutations I modifications that can result in acquired resistance to both irreversible covalent and reversible non-covalent BTK inhibitors are valine 416 (V416), alanine 428 (A428), methionine 437 (M437), threonine 474 (T474) and leucine 528 (L528) modifications, which can reduce BTK inhibitor binding to BTK.

By the term “drug resistance" as used herein is meant the major cause of cancer treatment failure. While a treatment may be effective initially, the heterogeneity of cancer and its ability to adapt can allow the cancer to become resistant to the treatment and regrow.

The term ’’relapse” as used herein has the conventional meaning and refers to evidence of disease progression in a patient who has previously achieved criteria of a complete response or partial remission.

The term "disease progression” as used herein has the conventional meaning and refers to a measured increase in tumor size or tumor burden.

The term “ recurrent or refractory form of a hyperproliferative disease” as used herein has the conventional meaning and refers to a particular cancer that is resistant, or non-responsive to therapy with a particular therapeutic agent. A cancer can be refractory to therapy with a particular therapeutic agent either from the onset of treatment (i.e., non-responsive to initial exposure to the therapeutic agent), or as a result of developing resistance to the therapeutic agent, either over the course of a first treatment period with the therapeutic agent or during a subsequent treatment period with the therapeutic agent.

The term "acquired drug resistance” as used herein has the conventional meaning and refers to resistance of a drug caused by mutations in the target protein following treatment. The mutations in the target protein hamper the binding of drug resulting in a regrowth of the tumor.

The term "clinical drug resistance” as used herein has the conventional meaning and refers to growth of a tumour while the patient is on treatment, that develops following after an initial clinical benefit (a clinical response or prolonged stable disease).

The term "wt-BTK” or “WT-BTK” or “BTK ¹" as used herein has its conventional meaning and refers to wild-type Bruton’s Tyrosine Kinase. A wild-type BTK has the regular meaning of a phenotype of the typical form of BTK as it occurs in nature. Originally, the wild-type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "mutant" allele. The term “IC₅o” as used herein has its conventional meaning and refers to the concentration of a substance that results in a 50% effect on some measure of biochemical and/or cellular function or substance-target binding interaction.

The term “plC₅o" as used herein has its conventional meaning and refers to the negative logarithm of the IC50 in molar concentration.

The term “G/₅o” as used herein has its conventional meaning and refers to the concentration of a substance that inhibits cell growth by 50%.

The term “LD50" as used herein refers to the concentration of a substance that results in 50% cell death.

The term “efficacy” as used herein has its conventional meaning and refers to the effect that is reached at a particular concentration of a substance.

The terms first, second, third and the like in the description and in the claims, are used for distinguishing between for example similar elements, compositions, constituents in a composition, or separate method steps, and not necessarily for describing a sequential or chronological order. The terms are interchangeable under appropriate circumstances and the embodiments of the invention can operate in other sequences than described or illustrated herein, unless specified otherwise.

Furthermore, the various embodiments, although referred to as “preferred” or “e.g." or “for example” or “in particular” and the like are to be construed as exemplary manners in which the invention may be implemented rather than as limiting the scope of the invention.

The term “comprising”, used in the claims, should not be interpreted as being restricted to for example the elements or the method steps or the constituents of a compositions listed thereafter; it does not exclude other elements or method steps or constituents in a certain composition. It needs to be interpreted as specifying the presence of the stated features, integers, (method) steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a method comprising steps A and B” should not be limited to a method consisting only of steps A and B, rather with respect to the present invention, the only enumerated steps of the method are A and B, and further the claim should be interpreted as including equivalents of those method steps. Thus, the scope of the expression “a composition comprising components A and B” should not be limited to a composition consisting only of components A and B, rather with respect to the present invention, the only enumerated components of the composition are A and B, and further the claim should be interpreted as including equivalents of those components.

In addition, reference to an element or a component by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element or component are present, unless the context clearly requires that there is one and only one of the elements or components. The indefinite article "a" or "an" thus usually means "at least one". Legend to the figures

Figure 1 : Map of clinically documented BTK mutants.

Figure 2: Proliferation assay of single compounds (monotherapy) in wt-BTK TMD8 cells.

Figure 3: Proliferation assay of single compounds (monotherapy) in BTK C481S TMD8 cells.

Figure 4: Proliferation assay of single compounds (monotherapy) in BTK T474I TMD8 cells.

Figure 5: Proliferation assay of single compounds (monotherapy) in BTK T474I/C481S TMD8 cells.

Figure 6: Proliferation assay of single compounds (monotherapy) in BTK V416L TMD8 cells.

Figure 7: Proliferation assay of single compounds (monotherapy) in BTK L528W TMD8 cells.

Figure 8: Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK V416L TMD8 cells.

Figure 9: Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK T474I TMD8 cells.

Figure 10: Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK L528WTMD8 cells.

Figure 11 : Proliferation assay of ibrutinib and pirtobrutinib (combination therapy) in a mixture of BTK C481 S TMD8 cells and BTK V416L TMD8 cells.

Figure 12: Proliferation assay of ibrutinib and pirtobrutinib (combination therapy) in a mixture of BTK C481 S TMD8 cells and BTK T474I TMD8 cells.

Figure 13: Proliferation assay of acalabrutinib and pirtobrutinib (combination therapy) in a mixture of BTK C481 S TMD8 cells and BTK T474I TMD8 cells.

Figure 14: Proliferation assay of acalabrutinib and pirtobrutinib (combination therapy) in a mixture of BTK C481 S TMD8 cells and BTK L528W TMD8 cells.

Figure 15: Ibrutinib resistance mutations can be detected before clinical relapse. For 20 patients with a detectable mutation in BTK or PLCG2 at time of relapse, samples before relapse were analyzed retrospectively to determine the interval of time between mutation detection and clinical relapse. An initial clone could be detected at an estimated median of 9.3 months before relapse. UPN, unique patient number (Woyach et al. 2017).

Detailed description of the invention

The inventors have established that a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor is more effective than an irreversible or reversible BTK inhibitor alone. In particular, it has now been shown that a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor is more effective than an irreversible or reversible BTK inhibitor alone in inhibiting B-cell lymphoma cells comprising two separate cell pools each containing a different mutation in BTK. Moreover, combinations of a reversible BTK inhibitor and an irreversible, covalent, BTK inhibitor is synergistically effective in inhibiting B-cell lymphoma cells comprising two separate cell pools each containing a different mutation in BTK.

Embodiments

Reversible BTK inhibitors

In preferred embodiments the reversible BTK inhibitor of the combination is selected from the group consisting of: pirtobrutinib, nemtabrutinib, fenebrutinib, BMS-986142, luxeptinib, AS- 0871 , AS-1763 and BIIB091 , preferably pirtobrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

Irreversible BTK inhibitors

In preferred embodiments, the irreversible BTK inhibitor of the combination is selected from the group consisting of: ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, spebrutinib, branebrutinib, evobrutinib, remibrutinib, tolebrutinib, orelabrutinib, elsubrutinib, edralbrutinib, ACP-5862, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

In a preferred embodiment, the irreversible BTK inhibitor of the combination is ibrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

In a preferred embodiment, the irreversible BTK inhibitor of the combination is acalabrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

In a preferred embodiment, the irreversible BTK inhibitor of the combination is zanubrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

In a preferred embodiment, the irreversible BTK inhibitor of the combination is tirabrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

BTK Protein Sequence

SEQ ID NO: 1 tyrosine-protein kinase BTK [Homo sapiens]

10 20 30 40 50 60

MAAVILESI F LKRSQQKKKT SPLNFKKRLF LLTVHKLSYY EYDFERGRRG SKKGSIDVEK

70 80 90 100 110 120

ITCVETWPE KNPPPERQI P RRGEESSEME QI SI IERFPY PFQWYDEGP LYVFSPTEEL

130 140 150 160 170 180

RKRWIHQLKN VIRYNSDLVQ KYHPCFWIDG QYLCCSQTAK NAMGCQILEN RNGSLKPGSS

190 200 210 220 230 240

HRKTKKPLPP TPEEDQILKK PLPPEPAAAP VSTSELKKW ALYDYMPMNA NDLQLRKGDE

250 260 270 280 290 300

YFILEESNLP WWRARDKNGQ EGYI PSNYVT EAEDSIEMYE WYSKHMTRSQ AEQLLKQEGK

310 320 330 340 350 360

EGGFIVRDSS KAGKYTVSVF AKSTGDPQGV IRHYWCSTP QSQYYLAEKH LFSTI PELIN 370 380 390 400 410 420

YHQHNSAGLI SRLKYPVSQQ NKNAPSTAGL GYGSWEIDPK DLTFLKELGT GQFGWKYGK

430 440 450 460 470 480

WRGQYDVAIK MIKEGSMSED EFIEEAKVMM NLSHEKLVQL YGVCTKQRPI FI ITEYMANG

490 500 510 520 530 540

CLLNYLREMR HRFQTQQLLE MCKDVCEAME YLESKQFLHR DLAARNCLVN DQGWKVSDF

550 560 570 580 590 600

GLSRYVLDDE YTSSVGSKFP VRWSPPEVLM YSKFSSKSDI WAFGVLMWEI YSLGKMPYER

610 620 630 640 650 660

FTNSETAEHI AQGLRLYRPH LASEKVYTIM YSCWHEKADE RPTFKILLSN ILDVMDEES*

Mutant BTK

In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is C481 S.

In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of cysteine to an amino acid selected from leucine, isoleucine, valine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 481 of the BTK protein.

In a more preferred embodiment, the modification is a substitution of cysteine to serine at amino acid position 481 of the BTK protein.

In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 416 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is V416L.

In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 416 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of valine to an amino acid selected from leucine, isoleucine, cysteine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 416 of the BTK protein.

In a more preferred embodiment, the modification is a substitution of valine to leucine at amino acid position 416 of the BTK protein.

In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 428 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is A428D.

In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 428 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of alanine to an amino acid selected from leucine, isoleucine, valine, cysteine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 428 of the BTK protein.

In a more preferred embodiment, the modification is a substitution of alanine to aspartic acid at amino acid position 428 of the BTK protein.

In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 437 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is M437R.

In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 437 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of methionine to an amino acid selected from leucine, isoleucine, valine, alanine, glycine, cysteine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 437 of the BTK protein.

In a more preferred embodiment, the modification is a substitution of methionine to arginine at amino acid position 437 of the BTK protein.

In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 474 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is T474I.

In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 474 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of threonine to an amino acid selected from leucine, isoleucine, valine, alanine, glycine, methionine, serine, cysteine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 474 of the BTK protein.

In a more preferred embodiment, the modification is a substitution of threonine to isoleucine at amino acid position 474 of the BTK protein. In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 528 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is L528W.

In a preferred embodiment, the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 528 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of leucine to an amino acid selected from cysteine, isoleucine, valine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 528 of the BTK protein.

In a more preferred embodiment, the modification is a substitution of leucine to tryptophane at amino acid position 528 of the BTK protein.

Prior therapy

In preferred embodiments, prior therapy has been given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received a Bruton’s tyrosine kinase (BTK) inhibitorfor treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, ibrutinib before starting a new treatment option for managing the same condition.

In preferred embodiments, prior therapy has been given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received a Bruton’s tyrosine kinase (BTK) inhibitorfor treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, acalabrutinib before starting a new treatment option for managing the same condition.

In preferred embodiments, prior therapy has been given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received a Bruton’s tyrosine kinase (BTK) inhibitorfor treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, zanubrutinib before starting a new treatment option for managing the same condition.

In preferred embodiments, prior therapy has been given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received a Bruton’s tyrosine kinase (BTK) inhibitorfor treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, tirabrutinib before starting a new treatment option for managing the same condition.

Maintenance Therapy In preferred embodiments, maintenance therapy is given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received and still is receiving a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, ibrutinib.

In preferred embodiments, maintenance therapy is given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received and still is receiving a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, acalabrutinib. In preferred embodiments, maintenance therapy is given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received and still is receiving a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, zanubrutinib.

In preferred embodiments, maintenance therapy is given to a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, where the subject has received and still is receiving a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, more preferably, tirabrutinib.

Provided herein are embodiments of methods including maintenance therapy of subject having a B-cell hematological malignancy. In some embodiments, the methods for maintenance therapy comprise treating a B-cell hematological malignancy with a covalent and/or irreversible BTK inhibitor for an initial treatment period, followed by a maintenance therapy regimen. In some embodiments, the methods for maintenance therapy comprise treating a B-cell hematological malignancy with a covalent and/or irreversible BTK inhibitor for a period of six months or longer, such as, for example, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or longer.

Monitoring subject

In preferred embodiments, the subject is monitored over the course of the therapy with a BTK inhibitor for treatment of the hyperproliferative disease to determine whether the subject has a mutation in an endogenous gene encoding BTK that results in or provides a risk of resistance of the subject to the therapy, preferably resistance of the subject to the irreversible BTK inhibitor. In preferred embodiments, the subject is monitored at predetermined intervals of time over the course of the therapy for treatment of the hyperproliferative disease, wherein the predetermined interval of time is every week, every 2 weeks, every 3 weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every year following the first administration of the covalent and/or irreversible BTK inhibitor. The treatment which is monitored is in particular a treatment of administration of an irreversible BTK inhibitor.

Methods for monitoring subject

In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 481 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 481 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 481 of the BTK protein.

In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 481 ; and (b) does not bind to nucleic acids encoding the wild-type BTK having cysteine at amino acid position 481. In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 416 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 416 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 416 of the BTK protein.

In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 416; and (b) does not bind to nucleic acids encoding the wild-type BTK having valine at amino acid position 416. In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 428 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 428 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 428 of the BTK protein.

In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 428; and (b) does not bind to nucleic acids encoding the wild-type BTK having alanine at amino acid position 428. In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 437 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 437 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 437 of the BTK protein.

In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 437; and (b) does not bind to nucleic acids encoding the wild-type BTK having methionine at amino acid position 437. In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 474 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 474 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 474 of the BTK protein.

In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 474; and (b) does not bind to nucleic acids encoding the wild-type BTK having threonine at amino acid position 474. In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe. In some embodiments of the methods, testing comprises performing polymerase chain reaction (PCR) amplification of nucleic acids that encode amino acid position 528 of the BTK protein. In some embodiments, PCR amplification comprises using a pair of oligonucleotide primers that flank the region encoding amino acid position 528 of the BTK protein. In some embodiments, the method comprises sequencing the amplified nucleic acids. In some embodiments of the methods, testing comprises performing deep sequencing of nucleic acids that encode amino acid position 528 of the BTK protein.

In some embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 528; and (b) does not bind to nucleic acids encoding the wild-type BTK having tryptophane at amino acid position 528. In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

In some embodiments, the sample for use in the methods contains one or more tumor cells from the subject. In some embodiments, the sample for use in the methods contains cell free DNA (cfDNA). In some embodiments, the sample for use in the methods contains circulating tumor DNA (ctDNA). In some embodiments, the sample for use in the methods contains tumor derived extracellular vesicles (EV). In some embodiments, the sample is a tumor biopsy sample, a blood sample, a serum sample, a plasma sample, a saliva sample, a urinary sample, a lymph sample, or a bone marrow aspirate.

In some embodiments of the methods, the nucleic acids used in the method is isolated from a tumor cell sample from the subject. In some embodiments of the methods, the nucleic acids used in the method are isolated from extracellular vesicles (EV) isolated from the subject. In some embodiments of the method the nucleic acids used in the method are isolated from a tumor biopsy sample, a blood sample, a serum sample, a plasma sample, a saliva sample, a urinary sample, a lymph sample, or a bone marrow aspirate from the subject.

In some embodiments of the methods, the nucleic acids for use in the assay are RNA or DNA. In some embodiments of the methods, the nucleic acids for use in the assay are genomic DNA. In some embodiments of the methods, the nucleic acids for use in the assay are total RNA. In some embodiments of the methods, the nucleic acids for use in the assay are messenger RNA (mRNA).

In some embodiments of the methods, the nucleic acids for use in the assay are complementary DNA (cDNA).

In some embodiments of the methods, the method further comprises isolating mRNA from the RNA sample. In some embodiments of the methods, the method further comprises reverse transcription of the total RNA, or mRNA, into cDNA. In preferred embodiments of the methods, testing comprises contacting the nucleic acids with a sequence specific nucleic acid probe, wherein the sequence specific nucleic acid probe: (a) binds to nucleic acids encoding a modified BTK that is modified at amino acid position 481 ; and (b) does not bind to nucleic acids encoding the wild-type BTK having cysteine at amino acid position 481 . In some embodiments of the methods, testing comprises PCR amplification using the sequence specific nucleic acid probe.

In preferred embodiments, the sample for use in the methods contains one or more tumor cells from the subject. In preferred embodiments, the sample for use in the methods contains circulating tumor DNA (ctDNA). In preferred embodiments, the sample is a tumor biopsy sample, a blood sample, a serum sample, a plasma sample, a saliva sample, a urinary sample, a lymph sample, or a bone marrow aspirate.

In preferred embodiments of the methods, the nucleic acids used in the method is isolated from a tumor cell sample from the subject. In preferred embodiments of the method the nucleic acids used in the method are isolated from a tumor biopsy sample, a blood sample, a serum sample, a plasma sample, a saliva sample, a urinary sample, a lymph sample, or a bone marrow aspirate from the subject.

In preferred embodiments of the methods, the nucleic acids for use in the assay is genomic DNA.

Definition subject - recurrent or refractory form of a hyperproliferative disease

In preferred embodiments, the subject is diagnosed with or at risk of a recurrent or refractory form of a hyperproliferative disease, which subject has previously been treated with an irreversible BTK inhibitor.

At risk of a recurrent or refractory form of a hyperproliferative disease in the context of this invention is defined as the subject having a mutation in a gene that results in expression of a mutated BTK protein that is unable to bind a BTK inhibitor, preferably an irreversible BTK inhibitor.

In preferred embodiments, the subject is diagnosed with recurrent or refractory form of a hyperproliferative disease, preferably a B-cell hematological malignancy that is resistant, or non-responsive to therapy with an irreversible BTK inhibitor.

In preferred embodiments, the subject is identified as having a mutation in a gene that results in expression of a mutated BTK protein that is unable to bind a BTK inhibitor, preferably an irreversible BTK inhibitor, which provides a risk of a recurrent or refractory form of a hyperproliferative disease, preferably a B-cell haematological malignancy. Definition relapse or refractory of disease

In preferred embodiments, the combination therapy is started when the B-cell hematological malignancy of the subject is relapsed or refractory, preferably wherein CLL/SLL is relapsed or refractory.

In preferred embodiments, the combination therapy is started when the B-cell hematological malignancy of the subject is relapsed or refractory and the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 , of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the mutant modification is C481 S.

Combination therapy

Combination - selection irreversible inhibitor

In preferred embodiments, the irreversible BTK inhibitor of the combination therapy is the same as the irreversible BTK inhibitor of a prior (maintenance) therapy. In a particular example, the irreversible BTK inhibitor of the combination therapy is ibrutinib, while the irreversible BTK inhibitor of a prior (maintenance) therapy is also ibrutinib.

In alternative embodiments, the irreversible BTK inhibitor of the combination therapy is different from the irreversible BTK inhibitor of a prior (maintenance) therapy. In a particular example, the irreversible BTK inhibitor of the combination therapy is acalabrutinib, while the irreversible BTK inhibitor of a prior (maintenance) therapy is ibrutinib.

Dose - therapeutically combinatory amount

The term "therapeutically combinatory amount” in preferred embodiments, is the total amount of the two (or more) compounds selected of an irreversible BTK inhibitor and a reversible BTK inhibitor, in particular the total amount of the irreversible BTK inhibitor and the reversible BTK inhibitor, used fortreating the hyperproliferative disease, in particular a B-cell hematological malignancy.

In preferred embodiments, the irreversible BTK inhibitor of the combination is administered in an amount in the range of 70 - 750 mg / day and / or wherein the irreversible BTK inhibitor of the combination is administered using one or more unit doses having an amount in the range of 70 - 750 mg / unit dose.

In preferred embodiments, the irreversible BTK inhibitor of the combination is administered daily at a dose selected from the group consisting of 70 mg, 100 mg, 140 mg, 160 mg, 200 mg, 280 mg, 320 mg, 420 mg, 480 mg, and 560 mg per day and preferably wherein the reversible BTK inhibitor is administered at a dose in accordance to said therapeutically combinatory amount. In preferred embodiments, the irreversible BTK inhibitor of the combination is administered twice daily using a unit dose of 80 mg, 100 mg or 160 mg and preferably wherein the reversible BTK inhibitor is administered at a dose in accordance to said therapeutically combinatory amount.

In preferred embodiments, the dose amount of the irreversible BTK inhibitor of the therapeutically combinatory amount of the combination is lower than the dose amount of the same irreversible BTK inhibitor administered during the prior therapy.

In preferred embodiments, the therapeutically combinatory amount of the dose of the irreversible BTK inhibitor and the dose of the reversible BTK inhibitor of the combination is lower than the dose of the irreversible BTK inhibitor, which was administered during the prior therapy.

Dosage forms

In preferred embodiments, the irreversible BTK inhibitor and the reversible BTK inhibitor of the combination are in a combined dosage form.

In alternative preferred embodiments, the irreversible BTK inhibitor and the reversible BTK inhibitor of the combination are in separate dosage forms.

Pharmaceutical composition

In a preferred embodiment, the pharmaceutical composition comprises a combination of an irreversible BTK inhibitor and a reversible BTK inhibitor, wherein the reversible BTK inhibitor is selected from the group consisting of: pirtobrutinib, nemtabrutinib, fenebrutinib, BMS- 986142, luxeptinib, AS-0871 , AS-1763 and BIIB091 , preferably pirtobrutinib.

Pharmaceutical compositions in accordance with the present invention may comprise, as one of the active ingredients (‘API’), selected from the group consisting of: pirtobrutinib, nemtabrutinib, fenebrutinib, BMS-986142, luxeptinib, AS-0871 , AS-1763 and BIIB091 , preferably pirtobrutinib.

As used herein, “a pharmaceutically acceptable salt” includes any salt that retains the activity of the active agent(s) and is acceptable for pharmaceutical use. A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt. Preferably, the pharmaceutically acceptable salt is the HCI-salt of the compound of the invention. The pharmaceutically acceptable salt of the disclosed compounds may be prepared by methods of pharmacy well known to those skilled in the art.

Furthermore, the compositions can comprise compounds according to the invention in the form of a solvate, comprising a pharmaceutically acceptable solvent, such as water (‘hydrate’), ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

As used herein, the term “pharmaceutical composition” refers to a composition comprising a compound according to the invention or a salt or solvate thereof and, as the case may be, one or more additional, non-toxic ingredients, which composition is in a form suitable for administration to a (human) subject, through any route of administration, and which composition is physiologically tolerated upon such administration.

The compositions of the invention may thus comprise one or more additional ingredients. In a preferred embodiment, the composition comprises one or more carriers and/or excipients. As is known by those of average skill in the art, the appropriate choice of excipients is dependent on multiple factors, including the physicochemical properties of the API, the preferred pharmaceutical form, the preferred route of administration, the desired rate of release, etc. The compositions of the invention can be formulated for a variety of routes of administration, oral administration being particularly preferred. It is within the purview of those of average skill in the art to conceive and develop suitable formulations, relying on the common general knowledge as reflected in text books such as Remington’s Pharmaceutical Sciences (Meade Publishing Co., Easton, Pa., 20.sup.th Ed., 2000), the entire disclosure of which is herein incorporated by reference, and routine development efforts.

In accordance with the various aspects of the invention, the composition is preferably provided in a unit dosage form. The term ‘unit dosage form’ refers to a physically discrete unit suitable as a unitary dosage for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with any suitable pharmaceutical carriers) and/or excipient(s). Exemplary, non-limiting unit dosage forms include a tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip, gelcap as well as any metered volume of a solution, suspension, syrup or elixir or the like, which may be contained, for instance in a vial, syringe, applicator device, sachet, spray, micropump etc. In accordance with particularly preferred embodiments of the invention, the unit dosage form, is a unit dosage form that is suitable for oral administration. Most preferably, it is a solid unit dosage form, such as a tablet.

Besides the compound according to the invention as such, pharmaceutically acceptable salts thereof may also be used. Pharmaceutically acceptable salts of compounds of the invention include the acid addition and base salts thereof, such as preferably the calcium, potassium or sodium salts. For a review on suitable salts, reference is made “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

Pharmaceutically acceptable salts of compounds according to the invention may be readily prepared by mixing together solutions of compounds according to the invention and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the salt may vary from completely ionised to almost non-ionised.

Medical Use

The compounds and the pharmaceutical compositions of the present invention are useful as inhibitors of tyrosine kinases, in particular of BTK. In particular, compounds of this invention are useful as inhibitors of tyrosine kinases that are important in hyper-proliferative diseases, especially in cancer, such as a B-cell hematological malignancy, and in the process of angiogenesis.

The reversible BTK inhibitors selected from the group consisting of: pirtobrutinib, nemtabrutinib, fenebrutinib, BMS-986142, luxeptinib, AS-0871 , AS-1763 and BIIB091 , preferably pirtobrutinib, and pharmaceutical compositions comprising these, either alone or in combination with an irreversible BTK inhibitor, thereof can be used to treat or prevent a variety of conditions, diseases or disorders mediated by Bruton’s Tyrosine kinase (BTK).

Such conditions, diseases or disorders include: cancers or tumors, including alimentary/gastrointestinal tract cancer, colon cancer, liver cancer, skin cancer including mast cell tumor and squamous cell carcinoma, breast and mammary cancer, ovarian cancer, prostate cancer, B cell hematological malignancy, lymphoma and leukemia (including but not limited to acute myelogenous leukemia, chronic myelogenous leukemia, mantle cell lymphoma, NHL B- cell lymphomas (e.g. precursor B-ALL, marginal zone B-cell lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, mediastinal large B-cell lymphoma), Hodgkin lymphoma, NK and T cell lymphomas, TEL-Syk and ITK-Syk fusion driven tumors, myelomas including multiple myeloma, myeloproliferative disorders kidney cancer, lung cancer, muscle cancer, bone cancer, bladder cancer, brain cancer, melanoma including oral and metastatic melanoma, Kaposi’s sarcoma, proliferative diabetic retinopathy, and angiogenic- associated disorders including solid tumors, and pancreatic cancer.

In preferred embodiments, the hyperproliferative disease is a B-cell hematological malignancy.

In particular embodiments, the B-cell hematological malignancy is any one of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma (GCB DLBCL), primary mediastinal B-cell lymphoma (PMBL), nonHodgkin lymphoma, Burkitt’s lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell leukemia, mantle cell lymphoma, B-cell prolymphocytic leukemia, lym- phoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

In preferred embodiments, the B-cell hematological malignancy is mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B- cell lymphoma (DLBCL), Waldenstrom macroglobulinemia (WM), follicular lymphoma (FL) and marginal zone lymphoma (MZL).

In preferred embodiments, the B-cell malignancy is Mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL).

Composition/Formulation

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration by injection, e.g. bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly or by intramuscular injection). Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

An example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD cosolvent system (VPD:5W) consists of VPD diluted 1 :1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Many of the compounds of the invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.

Specific embodiments

The following preferred embodiments are included:

• wherein the irreversible BTK inhibitor of the combination is selected from the group consisting of: ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, spebrutinib, branebrutinib, evobrutinib, remibrutinib, tolebrutinib, orelabrutinib, elsubrutinib, edralbrutinib, ACP-5862, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

• wherein the subject has been administered a BTK inhibitor for treatment of the hyperproliferative disease, preferably the B-cell hematological malignancy, preferably before recurrence or relapse of the hyperproliferative disease.

• wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is C481 S.

• wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of cysteine to an amino acid selected from leucine, isoleucine, valine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 481 of the BTK protein.

• wherein the modification is a substitution of cysteine to serine at amino acid position 481 of the BTK protein.

• wherein the reversible BTK inhibitor of the combination is selected from the group consisting of: pirtobrutinib, nemtabrutinib, fenebrutinib, BMS-986142, luxeptinib, AS- 0871 , AS-1763 and BIIB091 , preferably pirtobrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

• wherein the irreversible BTK inhibitor of the combination is selected from the group consisting of: ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

• wherein the irreversible BTK inhibitor of the combination is ibrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

• wherein the irreversible BTK inhibitor of the combination is acalabrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

• wherein the irreversible BTK inhibitor of the combination is zanubrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof. • wherein the irreversible BTK inhibitor of the combination is tirabrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

• wherein the subject is identified as having one or more mutations in a gene that results in expression of a BTK protein with a modification at one or more amino acid positions selected from the group consisting of: 416, 428, 437, 474, 481 and 528.

• wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 416 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the mutant modification is V416L.

• wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 428 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the mutant modification is A428D.

• wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 437 of the amino acid sequence set forth in SEQ ID NO: I, more preferably wherein the mutant modification is M437R.

• wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 474 of the amino acid sequence set forth in SEQ ID NO: I, more preferably wherein the mutant modification is T474I.

• wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein at amino acid position 528 of the amino acid sequence set forth in SEQ ID NO: I, more preferably wherein the mutant modification is L528W.

• wherein the subject is monitored at predetermined intervals of time over the course of the therapy for treatment of the hyperproliferative disease, wherein the predetermined interval of time is every week, every 2 weeks, every 3 weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every year following the first administration of the irreversible BTK inhibitor.

• wherein the irreversible BTK inhibitor of the combination is administered in an amount in the range of 70 - 750 mg / day and I or wherein the irreversible BTK inhibitor of the combination is administered using one or more unit doses having an amount in the range of 70 - 750 mg I unit dose.

• wherein the irreversible BTK inhibitor of the combination is administered daily at a dose selected from the group consisting of 70 mg, 100 mg, 140 mg, 160 mg, 200 mg, 280 mg, 320 mg, 420 mg and, 480 mg, and 560 mg per day and preferably wherein the reversible BTK inhibitor is administered at a dose in accordance to said therapeutically combinatory amount.

• wherein the irreversible BTK inhibitor of the combination is administered twice daily using a unit dose of 80 mg, 100 mg or, 160 mg and preferably wherein the reversible BTK inhibitor is administered at a dose in accordance to said therapeutically combinatory amount.

• wherein the dose amount of the irreversible BTK inhibitor of the therapeutically combinatory amount of the combination is lower than the dose amount of the same irreversible BTK inhibitor administered during the prior therapy.

• wherein the therapeutically combinatory amount of the dose of the irreversible BTK inhibitor and the dose of the reversible BTK inhibitor of the combination is lower than the dose of the irreversible BTK inhibitor, which was administered during the prior therapy.

• wherein the hyperproliferative disease is a B cell hematological malignancy.

• wherein the B-cell hematological malignancy is any one of chronic lymphocytic leukemia

(CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma (GCB DLBCL), primary mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma, Burkitt’s lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell leukemia, mantle cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

• wherein the B-cell hematological malignancy is any one of mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), Waldenstrom macroglobulinemia (WM), follicular lymphoma (FL) and marginal zone lymphoma (MZL).

• wherein the B-cell malignancy is Mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL).

• wherein the B-cell hematological malignancy of the subject is relapsed or refractory, preferably wherein CLL/SLL is relapsed or refractory.

• wherein the B-cell hematological malignancy of the subject is relapsed or refractory and the subject is identified as having a mutation in a gene that results in expression of a BTK protein with modification at amino acid position 481 , of the amino acid sequence set according to SEQ ID NO: I, more preferably wherein the mutant modification is C481 S. • wherein the irreversible BTK inhibitor and the reversible BTK inhibitor of the combination are in a combined dosage form.

• wherein the irreversible BTK inhibitor and the reversible BTK inhibitor of the combination are in separate dosage forms.

Examples

Cell culture

TMD8 diffuse large B-cell lymphoma cells were purchased from Tokyo Medical and Dental University. TMD8 cells expressing mutant BTK were created at Synthego Corporation. TMD8 cell lines expressing BTK C481 S, BTK T474I, BTK T474I/C481 S, BTK V416L and BTK L528W were generated via CRISPR/Cas9. BTK T474I/C481 S expressing cells were generated by re- editing BTK C481 S cells. TMD8 mutant cell pools were enriched to obtain a 100% mutant genotype. The mutation status of all cell lines was confirmed via sequencing. Cells were cultured in MEM-alpha cell culture medium (cat. no. 22571038, Life Technologies), supplemented with 10% (v/v) heat-inactivated fetal bovine calf serum (Avantor, cat. no. 97068085) and 1 % pe n ici 11 i n/st re pto my ci n .

Proliferation assays

Inhibition of proliferation in response to compound was measured using the ATPIite 1 Step™ assay (cat. no. 6016736, Perkin Elmer). The assay utilizes ATP as a marker for metabolically active cells. A decline in ATP concentration indicates that compounds exert anti-proliferative effects on the cells under investigation, compared to vehicle treated cells. Measurement of ATP is based on the production of light caused by the reaction of ATP with added luciferase and D- luciferin. Luminescence was recorded using an Envision multilabel reader (Perkin Elmer). The ATP signal of the cells at the start of incubation (t=0) was recorded separately to distinguish between cell growth and cell death. Maximum growth was determined via vehicle treated cells, which indicates 100% viability (vehicle control). Percentage viability was used as the main y-axis signal. IC50S were fitted by non-linear regression using IDBS XLfit ™5 using a 4-parameter logistic curve, yielding a maximum signal, minimum signal, hill-parameter and IC50. In addition to the IC50, efficacy, GI50 and LD50 were also determined.

Proliferation assays on BTK single mutant cell lines

WT-BTK, BTK C481 S, BTK T474I, BTK T474I/C481 S, BTK V416L and BTK L528W TMD8 cell lines were seeded at 800 cells per well in a white 384-well culture plate (cat. no. 781080, Greiner Bio-One) and allowed to rest for at least 4 hours at 37 °C, 95 % humidity, and 5 % CO2. Pirtobrutinib, ibrutinib, acalabrutinib and nemtabrutinb were diluted in 3.16-fold dilution steps in 100% DMSO and further diluted in 20 mM Hepes, generating a dose response range from 316 nM to 0.0316 nM in the assay. DMSO was used as a vehicle control. Luminescence was recorded at 96 hours.

Combination experiments with BTK C481 S and BTK-mutant TMD8 cells

The BTK C481 S TMD8 cell line was combined with either BTK T474I, BTK V416L or BTK L528W TMD8 cell lines and seeded at 800 cells per well for each cell line (1600 cells/well total) in white 384-well culture plates (cat. no. 781080, Greiner Bio-One). Cells were allowed to rest for at least 4 hours at 37 °C, 95 % humidity and 5 % CO2. Ibrutinib or acalabrutinib were diluted in 3.16-fold dilution steps in 100% DMSO and further diluted in 20 mM Hepes, generating a 9-point dose response range from 316 nM to 0.0316 nM in the assay. Pirtobrutinib was added in a fixed concentration to each concentration of ibrutinib or acalabrutinib, ranging from 316 nM to 0.316 nM. DMSO was used as a vehicle control. Luminescence was recorded at 96 hours. Antiproliferative responses were assessed 1) calculating efficacy of the single compounds and the combination compounds, compared to vehicle control at 96h, and 2) calculating the gain in efficacy of the combination compared to the single compounds.

Results

Proliferation assay of single compounds (monotherapy) in wt-BTK TMD8 cells (Figure 2)

Viability of pirtobrutinib and other compounds in wt-BTK TMD8 cells. All compounds inhibit proliferation with <10 nM IC50S and >90% efficacy, nemtabrutinib excluded. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

Table T. Proliferation data for wt-BTK TMD8 cells

Proliferation assay of single compounds (monotherapy) in BTK C481S TMD8 cells. (Figure 3)

Viability of pirtobrutinib and other compounds in BTK C481 S TMD8 cells. Pirtobrutinib and nemtabrutinib inhibit proliferation. Ibrutinib and acalabrutinib do not inhibit proliferation at the tested concentrations. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

Table 2: Proliferation data for BTK C481 S TMD8 cells

Proliferation assay of single compounds (monotherapy) in BTK T474I TMD8 cells. (Figure

4)

Viability of pirtobrutinib and other compounds in BTK T474I TMD8 cells. Ibrutinib and acalabrutinib inhibit proliferation. Pirtobrutinib and nemtabrutinib do not inhibit proliferation at the tested concentrations. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

Table 3: Proliferation data for BTK T474I TMD8 cells

Proliferation assay of single compounds (monotherapy) in BTK T474I/C481S TMD8 cells.

(Figure 5)

Viability of pirtobrutinib and other compounds in BTK T474I/C481S TMD8 cells. All compounds are unable to inhibit proliferation at the tested concentrations. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

Table 4: Proliferation data for BTK T474I/C481S TMD8 cells

Proliferation assay of single compounds (monotherapy) in BTK V416L TMD8 cells. (Figure 6)

Viability of pirtobrutinib and other compounds in BTK V416L TMD8 cells. Ibrutinib and acalabrutinib inhibit proliferation. Pirtobrutinib and nemtabrutinib do not inhibit proliferation at the tested concentrations. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

Table 5’. Proliferation data for BTK V416L TMD8 cells

Proliferation assay of single compounds (monotherapy) in BTK L528W TMD8 cells. (Figure 7)

Viability of pirtobrutinib and other compounds in BTK L528W TMD8 cells. Acalabrutinib inhibits the proliferation. Ibrutinib and pirtobrutinib show low efficacy and nemtabrutinib does not inhibit proliferation at the tested concentrations. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability at the highest tested concentration from vehicle treated cells. Dotted line indicates the cell population at the start of the experiment (t=0).

Table 6: Proliferation data for BTK L528WTMD8 cells

Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK V416L TMD8 cells (Figure 8)

Viability of ibrutinib, acalabrutinib and pirtobrutinib in a 1 :1 mixture of BTK C481S TMD8 cells + BTK V416L TMD8 cells after 96h incubation. Graphs represent efficacy of compounds used in the standard of care. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the highest tested concentration from vehicle treated cells. The dotted line indicates the cell population at the start of the experiment (t=0).

Table 7: Proliferation data for compounds in a mixture of BTK C481S TMD8 cells + BTK V416L TMD8 cells.

Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK T474I TMD8 cells (Figure 9)

Viability of ibrutinib, acalabrutinib and pirtobrutinib in a 1 :1 mixture of BTK C481 S TMD8 cells + BTK T474I TMD8 cells after 96h incubation. Graphs represent efficacy of compounds used in the standard of care. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the highest tested concentration from vehicle treated cells. The dotted line indicates the cell population at the start of the experiment (t=0).

Table 8: Proliferation data for compounds in a mixture of BTK C481 S TMD8 cells + BTK T474I TMD8 cells.

Proliferation assay of single compounds (monotherapy) in a mixture of BTK C481S TMD8 cells and BTK L528W TMD8 cells (Figure 10)

Viability of ibrutinib, acalabrutinib and pirtobrutinib in a 1 :1 mixture of BTK C481 S TMD8 cells + BTK L528W TMD8 cells after 96h incubation. Graphs represent efficacy of compounds used in the standard of care. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the highest tested concentration from vehicle treated cells. The dotted line indicates the cell population at the start of the experiment (t=0).

Table 9: Proliferation data for compounds in a mixture of BTK C481 S TMD8 cells + BTK L528W TMD8 cells.

COMBINATION THERAPY - IBRUTINIB - pirtobrutinib

Proliferation assay of ibrutinib and pirtobrutinib (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK V416L TMD8 cells (Figure 11)

Viability of ibrutinib, 100 nM pirtobrutinib and the combination of ibrutinib and 100 nM pirtobrutinib in a 1 :1 mixture of BTK C481 S TMD8 cells + BTK V416L TMD8 cells after 96h incubation. In the combination, each concentration of ibrutinib contained the indicated concentration of pirtobrutinib. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the indicated concentrations from vehicle treated cells. Individual efficacies, theoretical efficacy of the combination and measured efficacy of the combination were determined at 100 nM ibrutinib (log M: -7). The dotted line indicates the cell population at the start of the experiment (t=0).

Table 10 Proliferation data for a combination of ibrutinib and pirtobrutinib in a mixture of BTK C481 S TMD8 cells + BTK V416L TMD8 cells.

Proliferation assay of ibrutinib and pirtobrutinib (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK T474I TMD8 cells (Figure 12)

Viability of ibrutinib, 100 nM pirtobrutinib, and the combination of ibrutinib and 100 nM pirtobrutinib in a 1 :1 mixture of BTK C481 S TMD8 cells + BTK T474I TMD8 cells after 96h incubation. In the combination, each concentration of ibrutinib contained the indicated concentration of pirtobrutinib. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the indicated concentrations from vehicle treated cells. Individual efficacies, theoretical efficacy of the combination and measured efficacy of the combination were determined at 100 nM ibrutinib (log M: -7). The dotted line indicates the cell population at the start of the experiment (t=0).

Table 1T. Proliferation data for a combination of ibrutinib and pirtobrutinib in a mixture of BTK C481S TMD8 cells + BTK T474I TMD8 cells.

COMBINATION THERAPY - Acalabrutinib - pirtobrutinib

Proliferation assay of acalabrutinib and pirtobrutinib (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK T474I TMD8 cells (Figure 13)

Viability of acalabrutinib, 100 nM pirtobrutinib, and the combination of acalabrutinib and 100 nM pirtobrutinib in a 1 :1 mixture of BTK C481S TMD8 cells + BTK T474I TMD8 cells after 96h incubation. In the combination, each concentration of acalabrutinib contained the indicated concentration of pirtobrutinib. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the indicated concentrations from vehicle treated cells. Individual efficacies, theoretical efficacy of the combination and measured efficacy of the combination were determined at 100 nM acalabrutinib (log M: -7). The dotted line indicates the cell population at the start of the experiment (t=0).

Table 12 Proliferation data for a combination of acalabrutinib and pirtobrutinib in a mixture of BTK C481S TMD8 cells + BTK T474I TMD8 cells.

Proliferation assay of acalabrutinib and pirtobrutinib (combination therapy) in a mixture of BTK C481S TMD8 cells and BTK L528W TMD8 cells (Figure 14)

Viability of acalabrutinib, 100 nM pirtobrutinib, and the combination of acalabrutinib and 100 nM pirtobrutinib in a 1 :1 mixture of BTK C481S TMD8 cells + BTK L528W TMD8 cells after 96h incubation. In the combination, each concentration of acalabrutinib contained the indicated concentration of pirtobrutinib. Vehicle treated cells represent 100% viability. Efficacy was calculated by subtracting % viability of the indicated concentrations from vehicle treated cells. Individual efficacies, theoretical efficacy of the combination and measured efficacy of the combination were determined at 100 nM acalabrutinib (log M: -7). The dotted line indicates the cell population at the start of the experiment (t=0). Table 13 Proliferation data for a combination of acalabrutinib and pirtobrutinib in a mixture of

BTK C481S TMD8 cells + BTK L528WTMD8 cells.

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Claims

1. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has received a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of the hyperproliferative disease, preferably an irreversible BTK inhibitor, the method comprising the steps:

(a) monitoring the subject, preferably at predetermined intervals of time, over the course of the therapy for treatment of the hyperproliferative disease to determine whether the subject has a mutation in an endogenous gene encoding BTK that results in resistance or provides a risk of resistance of the subject to the therapy, preferably resistance of the subject to the irreversible BTK inhibitor of the (mono) therapy, preferably the subject has a mutation in an endogenous gene encoding BTK that results in expression of a BTK protein with a modification at an amino acid position corresponding to amino acid position 481 of the amino acid sequence according to SEQ ID NO: I; and

(b) if the subject has shown said mutation, administering to the subject a therapeutically combinatory amount of said irreversible BTK inhibitor and said reversible BTK inhibitor, in particular if the subject has the BTK modification corresponding to amino acid position 481 identified in step (a).

2. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject diagnosed with a recurrent or refractory form of a hyperproliferative disease, preferably a B-cell hematological malignancy, which subject receives or has previously been treated with an irreversible BTK inhibitor, and wherein the method comprises administering to said subject a therapeutic combinatory amount of said irreversible BTK inhibitor and said reversible BTK inhibitor.

3. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method for treating a subject having a hyperproliferative disease, preferably a B-cell hematological malignancy, the method comprising: administering to the subject a therapeutic combinatory amount of an irreversible BTK inhibitor and a reversible BTK inhibitor.

4. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of claims 1-3, wherein the irreversible BTK inhibitor of the combination is selected from the group consisting of: ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, spebrutinib, branebrutinib, evobrutinib, remibrutinib, tolebrutinib, orelabrutinib, elsubrutinib, edralbrutinib, ACP-5862, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

5. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims 3 or 4, wherein the subject has been administered a BTK inhibitor for treatment of the hyperproliferative disease, preferably the B-cell hematological malignancy, preferably before recurrence or relapse of the hyperproliferative disease.

6. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the protein modification is C481 S.

7. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 481 of the amino acid sequence according to SEQ ID NO: I, wherein the modification is a substitution of cysteine to an amino acid selected from leucine, isoleucine, valine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 481 of the BTK protein.

8. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of claim 7, wherein the modification is a substitution of cysteine to serine at amino acid position 481 of the BTK protein.

9. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the reversible BTK inhibitor of the combination is selected from the group consisting of: pirtobrutinib, nemtabrutinib, fenebrutinib, BMS-986142, luxeptinib, AS-0871 , AS-1763 and BIIB091 , preferably pirtobrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

10. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is selected from the group consisting of: ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

11. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is ibrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

12. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is acalabrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

13. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is zanubrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

14. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the irreversible BTK inhibitor of the combination is tirabrutinib, and pharmaceutically acceptable salts, cocrystals, hydrates, solvates, and prodrugs thereof.

15. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the subject is identified as having one or more mutations in a gene that results in expression of a BTK protein with a modification at one or more amino acid positions selected from the group consisting of: 416, 428, 437, 474, 481 and 528.

16. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 416 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the mutant modification is V416L.

17. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 428 of the amino acid sequence according to SEQ ID NO: I, more preferably wherein the mutant modification is A428D.

18. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 437 of the amino acid sequence set forth in SEQ ID NO: I, more preferably wherein the mutant modification is M437R.

19. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein with a modification at amino acid position 474 of the amino acid sequence set forth in SEQ ID NO: I, more preferably wherein the mutant modification is T474I.

20. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of preceding claims, wherein the subject is identified as having a mutation in a gene that results in expression of a BTK protein at amino acid position 528 of the amino acid sequence set forth in SEQ ID NO: I, more preferably wherein the mutant modification is L528W.

21. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the subject is monitored at predetermined intervals of time over the course of the therapy for treatment of the hyperproliferative disease, wherein the predetermined interval of time is every week, every 2 weeks, every 3 weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every year following the first administration of the irreversible BTK inhibitor.

22. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the irreversible BTK inhibitor of the combination is administered in an amount in the range of 70 - 750 mg / day and I or wherein the irreversible BTK inhibitor of the combination is administered using one or more unit doses having an amount in the range of 70 - 750 mg I unit dose.

23. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the irreversible BTK inhibitor of the combination is administered daily at a dose selected from the group consisting of 70 mg, 100 mg, 140 mg, 160 mg, 200 mg, 280 mg, 320 mg, 420 mg and, 480 mg, and 560 mg per day and preferably wherein the reversible BTK inhibitor is administered at a dose in accordance to said therapeutically combinatory amount.

24. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the irreversible BTK inhibitor of the combination is administered twice daily using a unit dose of 80 mg, 100 mg or, 160 mg and preferably wherein the reversible BTK inhibitor is administered at a dose in accordance to said therapeutically combinatory amount.

25. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the dose amount of the irreversible BTK inhibitor of the therapeutically combinatory amount of the combination is lower than the dose amount of the same irreversible BTK inhibitor administered during the prior therapy.

26. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the therapeutically combinatory amount of the dose of the irreversible BTK inhibitor and the dose of the reversible BTK inhibitor of the combination is lower than the dose of the irreversible BTK inhibitor, which was administered during the prior therapy.

27. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the hyperproliferative disease is a B- cell hematological malignancy.

28. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the B-cell hematological malignancy is any one of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC- DLBCL), germinal center diffuse large B-cell lymphoma (GCB DLBCL), primary mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma, Burkitt’s lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell leukemia, mantle cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, mediastinal (thymic) large B- cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

29. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the B-cell hematological malignancy is any one of mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), Waldenstrom macroglobulinemia (WM), follicular lymphoma (FL) and marginal zone lymphoma (MZL).

30. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the B-cell malignancy is Mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL).

31. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the claim 27 - 30, wherein the B-cell hematological malignancy of the subject is relapsed or refractory, preferably wherein CLL/SLL is relapsed or refractory.

32. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the claim 27 - 31 , wherein the B-cell hematological malignancy of the subject is relapsed or refractory and the subject is identified as having a point mutation in a gene that results in expression of a BTK protein with modification at amino acid position 481 , of the amino acid sequence set according to SEQ ID NO: I, more preferably wherein the mutant modification is C481 S.

33. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the irreversible BTK inhibitor and the reversible BTK inhibitor of the combination are in a combined dosage form.

34. Combination of an irreversible BTK inhibitor and a reversible BTK inhibitor for use in a method of any one of the preceding claims, wherein the irreversible BTK inhibitor and the reversible BTK inhibitor of the combination are in separate dosage forms.