WO2024037594A1 - Combination treatment with orelabrutinib and tafasitamab - Google Patents

Combination treatment with orelabrutinib and tafasitamab Download PDF

Info

Publication number
WO2024037594A1
WO2024037594A1 PCT/CN2023/113559 CN2023113559W WO2024037594A1 WO 2024037594 A1 WO2024037594 A1 WO 2024037594A1 CN 2023113559 W CN2023113559 W CN 2023113559W WO 2024037594 A1 WO2024037594 A1 WO 2024037594A1
Authority
WO
WIPO (PCT)
Prior art keywords
tafasitamab
orelabrutinib
lenalidomide
ibrutinib
cells
Prior art date
Application number
PCT/CN2023/113559
Other languages
French (fr)
Inventor
Xuesong OUYANG
Hongjuan ZHANG
Ruixia LIANG
Original Assignee
Beijing Innocare Pharma Tech Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Innocare Pharma Tech Co., Ltd. filed Critical Beijing Innocare Pharma Tech Co., Ltd.
Publication of WO2024037594A1 publication Critical patent/WO2024037594A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • the present invention relates to the use of a Bruton’s tyrosine kinase (BTK) inhibitor orelabrutinib and an anti-CD19 antibody such as tafasitamab, optionally in combination with lenalidomide, for treating CD19 antigen-positive B cell tumors.
  • BTK tyrosine kinase
  • Lymphoma is a malignancy of the lymphatic system, with the incidence increasing year over year in recent years.
  • the pathological types of lymphoma are complex, and the treatment methods are diverse.
  • B cell lymphomas are usually treated with rituximab and combined chemotherapy CHOP (R-CHOP) , or with other chemotherapy regimens such as CVAD with cyclophosphamide, vincristine, doxorubicin, and dexamethasone.
  • R-CHOP combined chemotherapy
  • CVAD with cyclophosphamide, vincristine, doxorubicin, and dexamethasone.
  • refractory NHL such as diffuse large cell lymphoma (DLBCL) , mantle cell lymphoma (MCL) , and follicular lymphoma (FL) still have unmet medical needs, and new treatment regimens need to be developed.
  • DLBCL diffuse large cell lymphoma
  • MCL mantle cell lymphoma
  • Bruton's tyrosine kinase (BTK) inhibitor has shown promising efficacy in the treatment of B cell lymphoma.
  • Orelabrutinib is the new-generation highly selective Bruton’s tyrosine kinase (BTK) inhibitor.
  • Orelabrutinib irreversibly inhibits BTK activity by covalently binding to BTK with high specificity, thereby inhibiting the activation of BCR signaling pathway, inducing cell apoptosis, and therefore reducing the proliferation of malignant B cells, thus playing a vital role in the treatment of B cell malignancies.
  • Orelabrutinib has been approved for the treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) /small lymphocytic lymphoma (SLL) , and relapsed/refractory mantle cell lymphoma (MCL) in China.
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • MCL mantle cell lymphoma
  • multiple clinical trials of orelabrutinib are being carried out for the treatment of B cell lymphomas including marginal zone lymphoma (MZL) , central nervous system lymphoma (CNSL) , Waldenstrom's macroglobulinemia (WM) and diffuse large B cell lymphoma (DLBCL) .
  • MZL marginal zone lymphoma
  • CNSL central nervous system lymphoma
  • WM Waldenstrom's macroglobulinemia
  • DLBCL diffuse large B cell lymphoma
  • CD19 Another important drug target for B cell malignancies is CD19, which is abnormally over-expressed on malignant B cells, such as most pre-B acute lymphoblastic leukemia (ALL) , non-Hodgkin's lymphoma (NHL) and B cell chronic lymphocytic leukemia (CLL) .
  • ALL pre-B acute lymphoblastic leukemia
  • NHL non-Hodgkin's lymphoma
  • CLL B cell chronic lymphocytic leukemia
  • ADC antibody-drug conjugate
  • CAR-T chimeric antigen receptor T cell therapy
  • Tafasitamab is a humanized Fc-enhanced monoclonal antibody targeting CD19.
  • tafasitamab After binding to CD19, tafasitamab mediates B cell lysis through several mechanisms, including: 1) binding to immune effector cells such as NK cells, ⁇ T cells and macrophages for tumor eradication; and 2) directly inducing cell death (apoptosis) .
  • immune effector cells such as NK cells, ⁇ T cells and macrophages for tumor eradication
  • apoptosis directly inducing cell death
  • the Fc domain of tafasitamab includes 2 amino acid substitutions S239D and I332E (Lazar et al., 2006) which enhances the binding affinity to activating Fc ⁇ receptors on effector cells, especially Fc ⁇ RIIIa, and significantly enhances the antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) functions of tafasitamab.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • Lenalidomide activates T cells to release the cytokines interferon ⁇ (IFN- ⁇ ) and interleukin-2 (IL-2) , and thus stimulates NK cell activity and induce NK cell proliferation (Gribben et al., 2015; Kotla et al. al. al., 2009) .
  • lenalidomide also promotes expression of Fc ⁇ RIII in NK cells, which is the receptor with high-binding affinity to tafasitamab (Lapalombella et al., 2008) .
  • the enhancement of tafasitamab mediated NK cell activation and ADCC activity by lenalidomide was demonstrated in previously published in vitro data (Awan et al., 2010) .
  • lenalidomide can directly induce tumor cell death by G0-G1 cell cycle arrest; or by increasing p21 Cip1/Waf1 and AP-1 transcription and promoting the NF- ⁇ B cell pathway activation, etc. (Gribben et al., 2015) .
  • FIG. 1 shows the verification of CD19 expression levels across different B cell tumor lines.
  • FIG. 2A shows ADCC activity of tafasitamab combined with orelabrutinib or ibrutinib determined by reporter assays.
  • FIG. 2B shows the synergy score distribution diagram of tafasitamab and orelabrutinib combination.
  • FIG. 2C shows the synergy score distribution diagram of tafasitamab and ibrutinib combination.
  • FIG. 2D shows the synergy score curve of combinations of tafasitamab and orelabrutinib or ibrutinib.
  • FIG. 3A shows the ADCC activity of tafasitamab and lenalidomide combined with orelabrutinib or ibrutinib determined by reporter assays.
  • FIG. 3B shows the synergy score distribution diagram of combination of tafasitamab, lenalidomide and orelabrutinib.
  • FIG. 3C shows the synergy score distribution diagram of combination of tafasitamab, lenalidomide and ibrutinib.
  • FIG. 3D shows the synergy score curve of tafasitamab, lenalidomide combined with orelabrutinib or ibrutinib.
  • FIG. 4A shows Western blot images indicating the effects of the tafasitamab alone or tafasitamab combined with orelabrutinib/ibrutinib on the phosphorylation levels of BTK and ITK proteins in co-cultured TMD-8 and Jurkat cells.
  • FIG. 4B shows the quantified ratios of phosphorylated BTK protein /total BTK protein in co-cultured TMD-8 and Jurkat cells following treatment of tafasitamab alone or tafasitamab combined with orelabrutinib or ibrutinib.
  • FIG. 4A shows Western blot images indicating the effects of the tafasitamab alone or tafasitamab combined with orelabrutinib/ibrutinib on the phosphorylation levels of BTK and ITK proteins in co-cultured TMD-8 and Jurkat cells.
  • FIG. 4B shows the quantified ratios of
  • 4C shows the quantified ratios of phosphorylated ITK protein /total ITK protein in co-cultured TMD-8 and Jurkat cells following treatment of tafasitamab alone or tafasitamab combined with orelabrutinib or ibrutinib.
  • FIG. 5A shows the primary NK cells mediated ADCC activity (donor 1) of tafasitamab combined with orelabrutinib or ibrutinib.
  • FIG. 5B shows the synergy score distribution diagram of tafasitamab and orelabrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells (donor 1) .
  • FIG. 5C shows the synergy score distribution diagram of tafasitamab and ibrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells (donor 1) .
  • FIG. 5A shows the primary NK cells mediated ADCC activity (donor 1) of tafasitamab combined with orelabrutinib or ibrutinib.
  • FIG. 5B shows the synergy score distribution diagram of tafasitamab and orelabrutinib combination indicating
  • FIG. 5D shows the synergy score curve of combinations of tafasitamab and orelabrutinib or ibrutinib indicating ADCC activity of tafasitamab driven by primary NK cells (donor 1) .
  • FIG. 5E shows the primary NK cells mediated ADCC activity (donor 2) of tafasitamab combined with orelabrutinib or ibrutinib.
  • FIG. 5F shows the synergy score distribution diagram of tafasitamab and orelabrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells (donor 2) .
  • FIG. 5G shows the synergy score distribution diagram of tafasitamab and ibrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells (donor 2) .
  • FIG. 5H shows the synergy score curve of combinations of tafasitamab and orelabrutinib or ibrutinib indicating ADCC activity of tafasitamab driven by primary NK cells (donor 2) .
  • FIG. 6A shows the primary NK cells mediated ADCC activity of tafasitamab and lenalidomide combined with orelabrutinib or ibrutinib.
  • FIG. 6B shows the synergy score distribution diagram of tafasitamab, lenalidomide and orelabrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells.
  • FIG. 6C shows the synergy score distribution diagram of tafasitamab, lenalidomide and ibrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells.
  • FIG. 6A shows the primary NK cells mediated ADCC activity of tafasitamab and lenalidomide combined with orelabrutinib or ibrutinib.
  • FIG. 6B shows the synergy score distribution diagram of tafasitamab, lenalidom
  • 6D shows the synergy score curve of combinations of tafasitamab, lenalidomide and orelabrutinib or ibrutinib indicating ADCC activity of tafasitamab driven by primary NK cells.
  • FIG. 7A shows the effects of direct tumor killing of RS4; 11 cells by tafasitamab, lenalidomide, orelabrutinib alone, and combinations.
  • FIG. 7B shows the effects of direct tumor killing of TMD-8 cells by tafasitamab, lenalidomide, orelabrutinib alone, and combinations.
  • FIG. 8A shows the effects of direct and immune cell-dependent tumor killing of REC-1 cells by tafasitamab, lenalidomide, orelabrutinib alone, and combinations.
  • FIG. 8B shows the synergy score distribution diagram of tafasitamab, lenalidomide and orelabrutinib combination indicating the effects of direct and immune cell-dependent tumor killing of REC-1 cells.
  • FIG. 8C shows the synergy score curve of combination of tafasitamab, lenalidomide and orelabrutinib indicating the effects of direct and immune cell-dependent tumor killing of REC-1 cells.
  • FIG. 9A shows the efficacy of orelabrutinib, tafasitamab-lenalidomide, and tafasitamab-lenalidomide-orelabrutinib in the REC-1 (MCL) xenograft tumor model.
  • FIG. 9B shows the relative body weight changes of the REC-1 tumor bearing mice during the treatment with orelabrutinib, tafasitamab-lenalidomide, and tafasitamab-lenalidomide-orelabrutinib.
  • FIG. 9A shows the efficacy of orelabrutinib, tafasitamab-lenalidomide, and tafasitamab-lenalidomide-orelabrutinib in the REC-1 (MCL) xenograft tumor model.
  • FIG. 9B shows the relative body weight changes of the REC-1 tumor bearing mice during the treatment with orelab
  • 9C shows the survival curves of the REC-1 tumor bearing mice treated with orelabrutinib, tafasitamab-lenalidomide, and tafasitamab-lenalidomide-orelabrutinib.
  • FIG. 10 shows the protocol for an open-label, multi-cohort study of tafasitamab and lenalidomide combined with orelabrutinib in patients with relapsed or refractory B-cell non-Hodgkin’s lymphoma (B-NHL) .
  • Tafasitamab is an Fc-enhanced humanized monoclonal antibody (mAb) belonging to the immunoglobulin (Ig) G1 subclass. It binds to the CD19 antigen on the surface of B lymphocytes, resulting in B cell exhaustion.
  • mAb humanized monoclonal antibody
  • Ig immunoglobulin G1 subclass. It binds to the CD19 antigen on the surface of B lymphocytes, resulting in B cell exhaustion.
  • Tafasitamab is an anti-neoplastic drug with ATC classification code L01XC35.
  • Tafasitamab can be produced by recombinant DNA technology in Chinese hamster ovary (CHO) cells, in the form of a disulfide-linked glycosylated tetramer comprising two identical 451 amino acid heavy chains and two identical 219 amino acid ⁇ light chains.
  • Tafasitamab has an N-linked glycosylation site at position 301 of the heavy chain sequence.
  • the molecular weight of tafasitamab is about 150 kDa.
  • a monosaccharide is connected to each of the two heavy chains.
  • Tafasitamab is disclosed in US Patent No. 10,617,691 as MOR00208; all the disclosure relating to Tafasitamab in the ‘691 Patent is incorporated herein by reference.
  • Tafasitamab comprises a variable heavy chain VH of the following sequence (SEQ ID NO: 1) with CDR1 ( SYVMH, SEQ ID NO: 2) , CDR2 ( NPYNDG, SEQ ID NO: 3) , and CDR3 ( GTYYYGTRVFDY, SEQ ID NO: 4) underlined:
  • variable light chain VL of the following sequence (SEQ ID NO: 5) with CDR1 ( RSSKSLQNVNGNTYLY, SEQ ID NO: 6) , CDR2 ( RMSNLNS, SEQ ID NO: 7) , and CDR3 ( MQHLEYPIT, SEQ ID NO: 8) underlined:
  • Tafasitamab contains a heavy chain constant domain CH of the following sequence:
  • Orelabrutinib has the chemical name of 2- (4-phenoxyphenyl) -6- [1- (prop-2-enoyl) piperidin-4-yl] pyridine-3-carboxamide, with molecular formula of C26H25N3O3, and molecular weight of 427.5. Its structure is shown as
  • SynergyFinder for the calculation of synergy coefficient, the web version (www. synergyfinderplus. org) of SynergyFinder is used to analyze the synergistic effect of the pharmaceutical composition of the disclosure.
  • SynergyFinder includes four mathematical models: Bliss, Loewe, HSA and ZIP.
  • HSA model assumes that the pharmaceutical effect of the pharmaceutical composition with the synergistic effect is greater than the maximum pharmaceutical effect of single drug
  • Bliss model assumes each drug takes effect independently, i.e., multiplication effect of independent action of single drug
  • Loewe model assumes the drugs do not interact with each other and each drug has the same maximum pharmaceutical effect
  • ZIP model assumes non-interacting drugs with minimal changes in their dose-response curves when combined ( Paltun et al., 2021) .
  • the calculation methods of the four models were described by Ianevski et al. (2020) .
  • the Loewe model is used to analyze the synergistic effect of the pharmaceutical composition of the disclosure, and the synergy score is defined as follows: ⁇ -10 considered as strong antagonism, -10 to about 0 considered as antagonism, 0 considered as no effect, 0 to about 10 considered as additive effect and >10 considered as synergistic effect.
  • the present disclosure is directed to a method for treating CD19 antigen-positive B cell tumors.
  • the method comprises the step of administering a BTK inhibitor orelabrutinib and an anti-CD19 antibody, or an antigen-binding fragment thereof, to a subject in need thereof.
  • the antigen-binding fragment thereof is an Fab, Fab', F (ab’ ) 2 , or single-chain variable fragment (scFv) .
  • the anti-CD19 antibody or an antigen-binding fragment thereof has a VH that comprises CDR1 having the amino acid sequence of SEQ ID NO: 2, CDR2 having the amino acid sequence of SEQ ID NO: 3, and CDR3 having the amino acid sequence of SEQ ID NO: 4; and has a VL that comprises CDR1 having the amino acid sequence of SEQ ID NO: 6, CDR2 having the amino acid sequence of SEQ ID NO: 7, and CDR3 having the amino acid sequence of SEQ ID NO: 8.
  • the anti-CD19 antibody or an antigen-binding fragment thereof has a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 5.
  • the anti-CD19 antibody is Tafasitamab, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity thereof, provided that the sequence variation is in the non-CDR framework region and/or constant region.
  • the sequence variation i.e., the amino acid changes, are preferably of a minor amino acid change such as a conservative amino acid substitution.
  • a conservative amino acid substitution is well-known to a person skilled in the art.
  • Non-Hodgkin's lymphoma includes diseases such as follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma, mantle cell lymphoma, and marginal zone lymphoma.
  • CD19 and BTK are two critical pathogenic targets in B cell malignancies.
  • the present method targets both CD19 with an anti-CD19 antibody such as tafasitamab and BTK with orelabrutinib.
  • an anti-CD19 antibody such as tafasitamab and BTK with orelabrutinib.
  • tafasitamab and orelabrutinib in combination induces CD19-dependent and BTK-dependent direct tumor killing and have a combined tumor killing effect.
  • orelabrutinib Due to its high kinase selectivity, orelabrutinib does not inhibit the phosphorylation of interleukin-2-inducible T-cell kinase (ITK) . Therefore, use of tafasitamab in combination with orelabrutinib retains the ADCC function of tafasitamab. Again without wishing to be bound by any particular theory, the combination of orelabrutinib and tafasitamab promotes BTK-dependent B cell killing and CD19-dependent B cell tumor killing.
  • ITK interleukin-2-inducible T-cell kinase
  • orelabrutinib can be administered orally.
  • Orelabrutinib may be administered orally with a dosage between 50-250 mg/day or 100-200 mg/day.
  • orelabrutinib may be administered orally with a dosage of about 150 mg once a day in a 28-day treatment cycle.
  • Orelabrutinib may be administered at the same time or substantially the same time every day, for example on an empty stomach or after meals.
  • an anti-CD19 antibody such as tafasitamab can be administered intravenously (IV) based, for example, on the body weight, with a dosage of 8 mg/kg/day to 30 mg/kg/day.
  • IV intravenously
  • tafasitamab may be administered about 12 mg/kg intravenously (IV) on Days 1, 4, 8, 15, 22 of Cycle 1, Days 1, 8, 15, 22 of Cycles 2-3, Day 1 and 15 of Cycles 4-12.
  • patients without disease progression may receive continuous orelabrutinib with or without tafasitamab until disease progression.
  • Orelabrutinib and an anti-CD19 antibody such as tafasitamab can be administered simultaneously, or sequentially.
  • orelabrutinib can be administered before an anti-CD19 antibody such as tafasitamab, or orelabrutinib and an anti-CD19 antibody such as tafasitamab can be administered at the same or substantially the same time; or orelabrutinib can be administered after an anti-CD19 antibody such as tafasitamab.
  • the present method further comprises administering lenalidomide to the subject.
  • Lenalidomide is an immunomodulator and has a chemical name of 3- (7-amino-3-oxo-1H-isoindol-2-yl) piperidine-2, 6-dione.
  • Lenalidomide is used to regulate the immune response of a patient and it also has direct tumor killing effect.
  • Lenalidomide can be orally administered in a fixed dose between 5-25 mg/day.
  • lenalidomide can be orally administered at about 5 mg/day, 10 mg/day, 15 mg/day, 20 mg/day and 25 mg/day.
  • orelabrutinib is administered before an anti-CD19 antibody such as tafasitamab and lenalidomide.
  • orelabrutinib is administered together with an anti-CD19 antibody such as tafasitamab and lenalidomide at the same or substantially the same time.
  • orelabrutinib is administered after an anti-CD19 antibody such as tafasitamab and lenalidomide.
  • B cell malignancies are highly heterogeneous; individual patients may have dramatically different responses to mono-therapeutics due to their unique pathogenesis.
  • the combined three-drug treatment regimen of the present disclosure can target patients with different clinical sensitivities to orelabrutinib, tafasitamab, and lenalidomide, by treating tumors with multiple mechanism of actions (MOAs) additively or synergistically.
  • MOAs mechanism of actions
  • the combination of the three-drug treatment can enhance the tumor killing effect.
  • tafasitamab-insensitive tumor cells the combination of the three-drug treatment can enhance the tumor killing.
  • the present disclosure provides a new treatment regimen for CD19-positive B cell malignancies.
  • the present disclosure further provides a kit comprising orelabrutinib and an anti-CD19 antibody such as tafasitamab.
  • the kit may further comprise lenalidomide.
  • orelabrutinib is in a tablet or a capsule form.
  • tafasitamab is in a form of lyophilized powder.
  • tafasitamab formulations are described in WO2018002031 (US20190322742) .
  • lenalidomide is in a tablet or capsule form.
  • orelabrutinib is provided in a bottle containing 50 mg orelabrutinib tablets.
  • kits of the present disclosure can optionally comprise instructions for administering the anti-CD19 antibody, orelabrutinib and/or lenalidomide.
  • orelabrutinib is administered orally in a fixed dose between 50 mg/day to 150 mg/day
  • tafasitamab is intravenously administered from 8 mg/kg to 30 mg/kg according to the body weight
  • lenalidomide is administered in a fixed dose, between 5 mg/day to 25 mg/day.
  • orelabrutinib in a 28-day treatment cycle, can be orally administered at 150 mg/day at the same time period every day continuously; tafasitamab can be intravenously (IV) administered on Days 1, 4, 8, 15, 22 of Cycle 1, Days 1, 8, 15, 22 of Cycles 2-3, Day 1 and 15 of Cycles 4-12; lenalidomide can be orally administered at 25 mg/day, on Days 1-21 of each cycle of Cycles 1-12.
  • Ibrutinib is a small molecule drug that inhibits B-cell proliferation and survival by irreversibly binding to BTK. Ibrutinib is the first approved BTK inhibitor and is in the same class as orelabrutinib. The inventors have discovered that orelabrutinib provides advantages over ibrutinib, when used in combination treatment with tafasitamab.
  • the present disclosure compares the ADCC activity of tafasitamab when combined with orelabrutinib or ibrutinib. Orelabrutinib fully retains the ADCC function of tafasitamab. In contrast, ibrutinib inhibits the ADCC function of tafasitamab, and the combination of ibrutinib with tafasitamab in certain of the experiments shown results in an antagonistic effect.
  • the present disclosure also compares the ADCC activity of tafasitamab when combined with lenalidomide, plus either orelabrutinib or ibrutinib. Orelabrutinib in combination with lenalidomide completely retains the ADCC function of tafasitamab. In contrast, ibrutinib in combination with lenalidomide inhibits the ADCC function of tafasitamab.
  • orelabrutinib is superior to ibrutinib in retaining the ADCC function of tafasitamab in both 2-drug (with tafasitamab) and 3-drug (with tafasitamab and lenalidomide) combinations, especially at a dose of 400 nM, which resembles the plasma exposure of orelabrutinib and ibrutinib and is a clinically relevant dose.
  • Example 1 Verification of CD19 antigen expression in B cell tumor lines, including TMD-8, RS4; 11 and REC-1
  • TMD-8 is a human diffuse large B cell lymphoma (DLBCL) cell line, RS4; 11 is a human acute lymphoma (ALL) cell line and REC-1 is a human mantle cell lymphoma (MCL) cell line.
  • DLBCL human diffuse large B cell lymphoma
  • ALL human acute lymphoma
  • MCL human mantle cell lymphoma
  • the expression levels of CD19 antigen on the cell membrane were quantified by flowcytometry.
  • the cells were incubated with 10 ⁇ g/ml of tafasitamab or isotype control antibody for 20 minutes at room temperature. Following washing off the unbound antibody, the cells were stained with 1 ⁇ L of PE fluorescent-labeled anti-human IgG-Fc antibody in the dark at room temperature for 20 minutes.
  • the binding ratio of CD19 was detected by NovoCyte Quanteon flow cytometer (Agilent) . As shown in FIG. 1 and Table 1, robust expression of CD19 antigen is detected on the plasma membrane of nearly 100%of TMD-8, RS4; 11 and REC-1 tumor cells, with the expression intensity of RS4; 11>REC-1>TMD-8.
  • Example 2 Orelabrutinib retains the ADCC activity of tafasitamab in the reporter assays
  • ADCC Antibody-dependent cellular cytotoxicity
  • MOA mechanism of action
  • the Fc-domain of tafasitamab contains 2 amino acid substitutions, i.e., S239D and I332E (Lazar et al., 2006) , which leads to its improved affinity to the activating Fc ⁇ R receptors on the effector cells, especially the affinity to Fc ⁇ receptor IIIa (Fc ⁇ RIIIa) .
  • Fc ⁇ RIIIa Fc ⁇ receptor IIIa
  • the Fab end of tafasitamab bound to CD19 antigen on the tumor cells its Fc-domain binds to Fc ⁇ RIIIa on the membrane of NK cells and triggers NK cell activation.
  • ITK and BTK belong to the TEC kinase family with high homology. Dubovsky et al.
  • Jurkat-NFAT-Luc2-CD16a-V158 reporter cell line constructed by Beijing KYinno Biotechnology Co., Ltd. was used as substitute effector cells.
  • These effector cells with extracellular over-expression of a high affinity Fc ⁇ RIIIa variant CD16a-V158 were incubated with target TMD-8 cells for 4 hours at 1: 1 effector-target ratio, and different concentrations of tafasitamab (starting from 500 ng/mL, 5-fold dilution) , with or without BTK inhibitors and/or lenalidomide.
  • the NFAT-Luc2 luciferase reporter was activated, and the bioluminescent signal was detected by a Plus multi-function microplate reader (BMG Labtech) to reflect the ADCC activity of tafasitamab.
  • IC 50 of tafasitamab alone or its combination with 400 nM or 4000 nM of orelabrutinib was: 3.02 ng/mL, 2.93 ng/mL and 2.18 ng/mL, respectively.
  • IC50 values are not statistically significant, there is a trend of improvement in ADCC activity of tafasitamab when combined with orelabrutinib compared to tafasitamab alone.
  • the Emax values reflecting the maximal ADCC activity, were at 20.85-fold for tafasitamab alone; 24.61-fold for tafasitamab and 400 nM of orelabrutinib combination; and 27.83-fold for tafasitamab and 4000 nM of orelabrutinib combination.
  • the increases in Emax values were statistically significant (p ⁇ 0.01@400 nM, p ⁇ 0.0001@4000 nM, Oneway ANOVA) .
  • N/A non-applicable. **p ⁇ 0.01 vs. Tafasitamab group, ****p ⁇ 0.0001 vs. Tafasitamab group.
  • lenalidomide leads to phosphorylation of CD28 costimulatory receptor and enhances the activation of T cells and NK cells; it also directly regulates the secretion of cytokines, i.e., promoting the release of IL-2 and IFN- ⁇ , and inhibiting the release of TNF ⁇ ; lenalidomide can also directly induce the apoptosis of tumor cells (Kotla et al., 2009) .
  • the combination of tafasitamab and lenalidomide has been granted accelerated approval by the FDA and EMA for the treatment of adult patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL) unsuitable to autologous stem cell transplantation.
  • DLBCL diffuse large B cell lymphoma
  • N/A non-applicable. **p ⁇ 0.01 vs. Tafasitamab + lenalidomide group, ****p ⁇ 0.0001 vs. Tafasitamab +lenalidomide group.
  • Example 3 Orelabrutinib has no off-target effect on ITK phosphorylation in Jurkat effector cells, confers its ability to retain the ADCC activity of tafasitamab.
  • ITK is a tyrosine kinase with high homology to BTK in the TEC kinase family.
  • the activation signal of Fc ⁇ R on the surface of NK cell membrane leads to ITK phosphorylation, promotes the downstream calcium signal transduction and the release of granzymes to kill target cells bound by antibody.
  • ITK is a key kinase mediating ADCC signals. Therefore, the effects of orelabrutinib and ibrutinib on ITK phosphorylation in Jurkat cells were compared in co-culture experiment of TMD-8 and Jurkat cells.
  • Control group (Veh, Vehicle) was DMSO.
  • the cells in the medicated 6-well plate were placed at 37°C and 5%CO2 conditions and cultured for 6 hours.
  • the cells were collected and resuspended in 70 ⁇ L of RIPA lysis solution, set on the ice for 30 minutes.
  • the samples were then centrifuged at 12,000 rpm for 10 minutes.
  • the supernatant was collected and transferred to a 1.5 mL sterile centrifuge tube.
  • After mixing with 4 ⁇ loading buffer and denaturing for 10 minutes, the samples were loaded to the 4-12%Tris-Glycine mini gel (Life Technologies, Carlsbad, CA) for electrophoresis, followed by protein transferred to PVDF membrane with the semi-dry transfer electrophoresis apparatus (Hoefer, MA) .
  • the PVDF membrane was incubated at 4°C overnight with the primary antibody diluted to an appropriate concentration. Following incubation, the PVDF membrane was washed with 1 ⁇ TBST for 5 times and subjected to image scanning on CLx imaging system (LI-COR, Lincoln, NE) .
  • ibrutinib treatment leads to significantly reduced phosphorylation levels of ITK by an average of 53.7%compared to the control group (FIG. 4A, 4C) .
  • Off-target inhibition of ITK phosphorylation is only associated with ibrutinib, but not orelabrutinib.
  • BTK inhibitors ibrutinib and orelabrutinib both lead to effective inhibition of BTK phosphorylation in the TMD-8 and Jurkat co-culture assay.
  • these two compounds show strikingly different activities on ITK, i.e, ibrutinib moderately inhibits ITK phosphorylation, which at least in part explains its antagonism in ADCC activity of tafasitamab;
  • orelabrutinib is a highly selective BTK inhibitor with no cross-activity on ITK, which confers its ability to retain the ADCC activity of tafasitamab.
  • Example 4 Orelabrutinib retains tafasitamab-mediated killing of CD19+ tumor cells by peripheral blood-derived NK cells
  • the ADCC reporter assays have provided strong evidence that highly selective BTK inhibitor orelabrutinib modestly enhances, or well retains the ADCC activity of tafasitamab in the 2-drug or 3-drug combinations, while ibrutinib consistently suppresses the ADCC activity of tafasitamab due to its off-target activity on ITK.
  • PBMCs peripheral blood mononuclear cells
  • ADCC killing experiment using primary NK cells as effector cells, and the CD19+ RS4; 11 (ALL) and REC-1 (MCL) tumor cell lines as target cells.
  • Human PBMCs usually contain 60%CD3+ T cells, 10%to 20%CD56+ NK cells, and 10%CD14+ monocytes.
  • PBMCs peripheral blood mononuclear cells
  • ADCC activity of tafasitamab was determined by the lysis rate of target cells, detected by NovoCyteQuanteon flow cytometer (Agilent) .
  • FIG. 5A showed that tafasitamab had a dose dependent ADCC activity mediated by primary NK cells from Donor 1.
  • Low concentration (400 nM) of ibrutinib did not affect the ADCC activity of tafasitamab, while high concentration (4000 nM) of ibrutinib completely inhibited the ADCC activity of tafasitamab.
  • orelabrutinib enhanced the ADCC activity of tafasitamab at the low concentration of 400 nM, while no effect was observed at the high concentration of 4000 nM.
  • SynergyFinder was used to calculate the synergy scores of the combined medicament (results shown in FIG. 5B-D) .
  • orelabrutinib and ibrutinib were 5.51 and -5.81, respectively.
  • orelabrutinib had no effect on the ADCC function of tafasitamab at the dose of 4000 nM, while additive to synergistic effect was achieved at the dose of 400 nM;
  • ibrutinib presented strong antagonistic effect at the dose of 4000 nM, and no effect at the dose of 400 nM.
  • FIG. 5E and Table 4 show strong ADCC activity of tafasitamab on RS4; 11 cells with NK cells derived from Donor 2.
  • Tafasitamb single agent led to maximal lysis rate (Emax) of 72.24%in the 10 ⁇ g/mL treatment group.
  • ADCC activity of tafasitamab is severely suppressed when combined with ibrutinib at either 400 nM or 4000 nM doses, with non-applicable IC50 values and Emax of 23.18%and 6.44%respectively.
  • FIG. 5F-H when synergy scores were calculated by SynergyFinder, orelabrutinib and ibrutinib have average synergy scores of -8.61 and -45.6, respectively.
  • N/A non-applicable.
  • the target tumor cells (REC-1) were labeled with 1.6 ⁇ M of CFSE, and treated with test drugs.
  • Orelabrutinib or ibrutinib were tested at two concentrations: 400 nM and 4000 nM; lenalidomide was tested at 1000 nM; and tafasitamab was tested at a total of 10 doses with 5-fold serial dilution starting from 10 ⁇ g/mL.
  • PBMCs carrying the high-affinity Fc ⁇ RIIIa 158 V/V were used as effector cells at effector-target ratio of 30: 1. After incubation at 37°C for 16 hours in the 96-U well plate, the cells were washed twice with PBS, and stained with 7-AAD at room temperature for 10 minutes in the dark. ADCC activity of tafasitamab was determined by the lysis rate of target cells, detected by NovoCyteQuanteon flow cytometer (Agilent) .
  • orelabrutinib at low concentration showed no effect on ADCC activity of tafasitamab, while high concentration (4000 nM) of orelabrutinib had moderate inhibition.
  • 400 nM of ibrutinib already inhibited the ADCC function of tafasitamab, while 4000 nM of ibrutinib completely abolished the activity.
  • This data is consistent with our findings in the ADCC reporter assays that in the 3-drug combinations, orelabrutinib retains the ADCC activity of tafasitamab better than ibrutinib.
  • orelabrutinib is superior to ibrutinib in retaining the ADCC function of tafasitamab in both 2-drug and 3-drug combinations, especially at low dose of 400 nM.
  • 400 nM dose of BTK inhibitors is also clinically relevant, which closely resembles the plasma exposure of orelabrutinib or ibrutinib in patients.
  • Example 5 Combination of orelabrutinib with tafasitamab and lenalidomide improves direct tumor lysis activity
  • Tafasitamab can inhibit proliferation and induce direct cytotoxicity against CD19-expressing B cell tumors. This tumor killing activity is further enhanced by Fc ⁇ R-mediated antibody cross-linking when the high affinity Fc domain of tafasitamab binds to Fc ⁇ R.
  • lenalidomide has also been reported to have direct tumor-killing effects by degradation of key transcription factors required by tumor growth (Kim et al., 2013) ; orelabrutinib inhibits the BTK activity, thus leads to BCR signaling blockade and stalled tumor cell growth.
  • test cells 3000 of test cells (RS4; 11 or TMD-8) were inoculated in each well of the 96-U well plate, followed by addition of test compounds to the final concentration of 1000 nM for lenalidomide and/or 400 nM for orelabrutinib. After incubation at 37°C for 72 hours, 50 ⁇ L of CTG-Glo reagent (Promega, Madison, WI) was applied for luminescent signal detection by the multi-function microwell plate detector (Perkin Elmer, Waltham, MA) to determine the tumor cell lysis rate.
  • CTG-Glo reagent Promega, Madison, WI
  • TMD-8 cells are moderately responsive to monotherapies of all 3 tested drugs.
  • the tumor cell lysis rates were at 45%for lenalidomide, 42%for orelabrutinib, and 14%to 29%for tafasitamab from low to high doses.
  • Two-drug combinations of tafasitamab/lenalidomide and tafasitamab/orelabrutinib both improved tumor killing with 52%to 71%cell lysis rate and 47%to 55%cell lysis rate, respectively.
  • Three-drug combination of tafasitamab/lenalidomide/orelabrutinib further improved the tumor killing with 91%to 94%cell lysis rate.
  • Example 6 Combination of orelabrutinib with tafasitamab and lenalidomide enhances tumor lysis activity when co-cultured with PBMC
  • tafasitamab and lenalidomide can promote the tumor cell killing through different mechanism of actions: 1) lenalidomide enhances the ADCC activity of tafasitamab mediated by NK cells and ⁇ T cells; 2) lenalidomide enhances the CD4+, CD8+effector T cell activities, strengthening the CD19+ tumor cell killing effect; 3) both tafasitamab and lenalidomide can promote tumor cell apoptosis and lead to direct tumor killing.
  • orelabrutinib can improve tumor killing activity of tafasitamab and lenalidomide with the presence of immune cells, we performed tumor lysis assays by co-culturing target tumor cells with PBMCs.
  • REC-1 (MCL) cell line was used as the target cells and co-cultured with PBMCs for 72 hours at the effector-target ratio of 1: 1.
  • the REC-1 cells were labeled with 1.6 ⁇ M of CFSE and then treated with test drugs.
  • Orelabrutinib was tested at two concentrations: 50 nM and 400 nM; lenalidomide was tested at 1000 nM; and tafasitamab was tested at a total of 10 doses with 5-fold serial dilution starting from 10 ⁇ g/mL.
  • the 96-U well plate was incubated at 37°C for 72 hours, followed by 2-times washing with PBS and staining with 7-AAD at room temperature for 10 minutes in the dark. Tumor lysis was detected by the NovoCyte Quanteon flow cytometer (Agilent) .
  • Example 7 Combination of tafasitamab and lenalidomide with orelabrutinib improves the in vivo therapeutic efficacy in the treatment of mice bearing human REC-1 xenograft tumors
  • mice 45 6 ⁇ 8-week-old female CB17 SCID mice (supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd) were each inoculated subcutaneously on the right flank with 1 x 10 ⁇ 6 REC-1 cells for tumor formation.
  • 40 tumor-bearing mice were randomly assigned into 4 groups (10 mice per group) according to body weight.
  • the test articles were administrated according to predetermined regimens as shown in Table 5.
  • PO oral administration
  • IV intravenous injection
  • QD once per day
  • BID twice per day
  • BIW twice per week.
  • mice were monitored 3 times per week. Animals with tumor volume exceeding 2,000 mm 3 were euthanized, which was also our tumor volume cutoff for survival analysis.
  • this in vivo efficacy study of REC-1 xenograft tumor model further demonstrates the treatment benefit of the combined medicament of tafasitamab, lenalidomide and orelabrutinib in an animal model.
  • Example 8 Clinical safety summary of orelabrutinib and tafasitamab combined with lenalidomide in marginal zone lymphoma (MZL) and diffuse large B cell lymphoma (DLBCL)
  • ICP-CL-00104 In a multicenter, open phase II clinical registration study (ICP-CL-00104) for evaluating the safety and efficacy of orelabrutinib in the treatment of relapsed or refractory marginal zone lymphoma (r/r MZL) , as of the data statistics to date, a total of 111 subjects received orelabrutinib treatment and all of them were included in the safety analysis set.
  • the safety endpoints were set as adverse event, vital sign and abnormal laboratory result evaluated according to CTCAE V4.
  • the median duration of treatment was 11.27 months (range: 0.1 -35.9 months) . 76.6%of the subjects were treated for more than 5.5 months, and 53.2%of the subjects were treated for at least 11.0 months.
  • the overall subject compliance was good, with a median drug relative exposure of 99.40%.
  • the safety analysis was performed in the safety analysis set of 111 cases.
  • Safety indicators include adverse event, laboratory test, vital sign and electrocardiogram, etc.
  • the results of ECG, clinical laboratory tests and other indicators, as well as their changes from baseline were summarized according to the visit, and all completed tests, cases and percentage were listed in the form of a cross-tab (according to the normal value range or the investigator's judgment of clinical significance) .
  • the data of vital signs including blood pressure, body temperature, heart rate and respiration, was described, including the values at each time point and the amount of change from the baseline.
  • Atrial fibrillation and atrial flutter are important adverse events of ibrutinib, and are related to its off-target effects.
  • the reported incidence of grade ⁇ 3 atrial fibrillation and/or atrial flutter is 3.7%in connection with ibrutinib.
  • no study drug-associated atrial fibrillation event of grade ⁇ 3 was reported in this study.
  • the second primary malignant tumor was an adverse event that required special attention for BTK inhibitors.
  • One case of the second primary malignant tumor was observed in this trial, which was gastric adenocarcinoma and was related to the study drug.
  • the second primary malignancies were observed with a minimum incidence of 10%in other BTK inhibitors.
  • orelabrutinib The observed adverse events of orelabrutinib were mainly blood system toxicity and infection with the low overall incidence, and most of them recovered after clinical observation and symptomatic treatment, and no dose adjustment of orelabrutinib was required (as shown in Table 8) . In conclusion, the treatment risks caused by orelabrutinib are all within the controllable range, it has excellent safety and overall tolerance.
  • Table 8 List of common hematology related laboratory test abnormalities in treatment of relapsed and refractory MZL by BTK inhibitors
  • the median duration of exposure in the study was 9.2 months (range: 0.2 -32.1 months) ; 30 (37.0%) patients received the treatment with the combination of tafasitamab and lenalidomide (12 cycles) . 34 (42.0%) patients continued to receive the treatment by the tafasitamab monotherapy starting from Cycle 13. Most TEAEs were CTCAE grade 1 (743/1464 case times) or 2 (428/1464 case times) .
  • the most commonly reported grade ⁇ 3 hematologic TEAEs were neutropenia (39 [48.1%] patients) , thrombocytopenia (14 [17.3%] patients) , febrile neutropenia (10 [12.3%] patients) , leukopenia (7 [8.6%] patients) and anemia (6 [7.4%] patients) .
  • the most commonly reported grade ⁇ 3 nonhematologic TEAEs were pneumonia (5 [6.2%] patients) and hypokalemia (5 [6.2%] patients) .
  • the clinical data of orelabrutinib in marginal zone lymphoma shows that its safety is superior to that of ibrutinib, and the safety of tafasitamab combined with lenalidomide in diffuse large B cell lymphoma has also been demonstrated in L-MIND clinical trial.
  • the clinical data supports that the clinical safety of the combined medicament of 3 drugs: orelabrutinib, tafasitamab and lenalidomide is superior to the combined medicament of 3 drugs: ibrutinib, tafasitamab and lenalidomide.
  • Example 9 Clinical data summary on effectiveness of single drug of orelabrutinib in treatment of relapsed/refractory marginal zone lymphoma (r/r MZL)
  • ICP-CL-00104 In a phase II multicenter, open clinical trial (ICP-CL-00104) for evaluating the efficacy and safety of orelabrutinib in the treatment of relapsed/refractory marginal zone lymphoma (r/r MZL) , the dose of orelabrutinib was 150 mg, once daily (QD) . Up to the data statistics time, a total of 90 subjects diagnosed with MZL by the pathology center were included in the modified full analysis set (mFAS) .
  • mFAS modified full analysis set
  • the primary study endpoint was the overall response rate (ORR) evaluated by the Independent Review Committee (IRC) according to Lugano 2014 criteria, and the secondary endpoints were ORR, progression-free survival (PFS) , duration of response (DOR) , time to onset (TTR) , time to progression (TTP) and overall survival (OS) , etc.
  • ORR overall response rate evaluated by IRC was 58.9% (95%CI: 48.0, 69.2)
  • the best efficacy outcome was CR in 8 cases (8.9%) and PR in 45 cases (50.0%) .
  • 83 subjects had previously received the anti-CD20 monoclonal antibody treatment.
  • the ORR evaluated by IRC was 57.8% (95%CI: 46.5, 68.6) , the best efficacy outcome was CR in 8 cases (9.6%) and PR in 40 cases (48.2%) .
  • orelabrutinib achieved the expected efficacy results in treatment of relapsed or refractory marginal zone lymphoma (MZL) at the QD administration of 150.
  • MZL relapsed or refractory marginal zone lymphoma
  • the overall response rate was significantly increased compared to the historical data, and the clinical efficacy lasted for a long period.
  • the study results are encouraging.
  • mFAS modified full analysis set
  • Orelabrutinib is prepared as a solid dispersion preparation to improve the drug solubility and in vivo absorption.
  • the solid dispersions are then mixed with 200 mg mannitol 200SD, 10 mg hypromellose EXF, 20 mg croscarmellose sodium (dry granulation part) to prepare granules by dry granulation process, mixed with 10 mg croscarmellose sodium SD-711, 6 mg silicon dioxide and 4 mg magnesium stearate and compressed into 12 mm round tablets, and finally bottled and label induced to obtain a finished product.
  • Anti-CD19 antibody tafasitamab is a white to pale yellow freeze-dried powder, and is supplied in 20 mL disposable glass vial containing 200 mg of tafasitamab. It is resuspended in 5 mL of water for injection (WFI) to yield approximately 5.4 mL of preparation containing 40 mg/mL tafasitamab, 25 mM citrate buffer, 200 mM trehalose and 0.02% (w/v) polysorbate 20 (pH 6.0) . After resuspension, tafasitamab is transferred to a 250 mL infusion bag containing 0.9%sodium chloride injection for dilution, and then intravenously (i. v. ) administered for 2 hours continuously.
  • WFI water for injection
  • Example 12 Clinical protocol of tafasitamab and lenalidomide in combination with orelabrutinib to treat relapsed/refractory B cell non-Hodgkin’s lymphoma (r/r B-NHL)
  • This study is an open-label, single-arm, multi-cohort study to evaluate the safety and efficacy of tafasitamab and lenalidomide combined with orelabrutinib in patients with relapsed/refractory non-Hodgkin’s lymphoma (NHL) .
  • the study protocol is summarized in FIG. 10.
  • the doses of tafasitamab and lenalidomide are the same as approved, and the dose of orelabrutinib is 150 mg, once daily (QD) .
  • This study includes four cohorts of patients with relapsed/refractory B-NHL of different lymphoma histopathological types as follows:
  • the primary study endpoint is the overall response rate (ORR) evaluated by investigators according to Lugano 2014 criteria, and the secondary endpoints are duration of response (DOR) , progression-free survival (PFS) , time to response (TTR) , overall survival (OS) , safety, and PK, etc.
  • DOR overall response rate
  • PFS progression-free survival
  • TTR time to response
  • OS overall survival
  • PK safety, and PK, etc.
  • the efficacy results are qualitatively described with the number and percentage (95%confidence intervals) of the responders, who achieve complete response (CR) or partial response (PR) , using the Clopper-Pearson exact binomial distribution method.
  • the time-to-event indicators such as DOR, PFS, TTR, and OS are statistically described using the Kaplan-Meier (KM) method.
  • Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy. Haematologica 100, 77-86.
  • Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes. Blood 122, 2539-2549.
  • Lenalidomide induces apoptosis and alters gene expression in non-small cell lung cancer cells. Oncol Lett 5, 588-592.

Abstract

Provided is the use of a combined medicament of two drugs: Bruton's tyrosine kinase (BTK) inhibitor orelabrutinib and an anti-CD19 antibody such as tafasitamab, or a combined medicament of three drugs: Bruton's tyrosine kinase (BTK) inhibitor orelabrutinib, an anti-CD19 antibody such as tafasitamab, and immunomodulator lenalidomide, for treating B cell malignancies such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia, or acute lymphoblastic leukemia.

Description

COMBINATION TREATMENT WITH ORELABRUTINIB AND TAFASITAMAB FIELD OF INVENTION
The present invention relates to the use of a Bruton’s tyrosine kinase (BTK) inhibitor orelabrutinib and an anti-CD19 antibody such as tafasitamab, optionally in combination with lenalidomide, for treating CD19 antigen-positive B cell tumors.
BACKGROUND OF THE INVENTION
Lymphoma is a malignancy of the lymphatic system, with the incidence increasing year over year in recent years. The pathological types of lymphoma are complex, and the treatment methods are diverse. B cell lymphomas are usually treated with rituximab and combined chemotherapy CHOP (R-CHOP) , or with other chemotherapy regimens such as CVAD with cyclophosphamide, vincristine, doxorubicin, and dexamethasone. However, patients with relapsed or refractory NHL, such as diffuse large cell lymphoma (DLBCL) , mantle cell lymphoma (MCL) , and follicular lymphoma (FL) still have unmet medical needs, and new treatment regimens need to be developed.
In recent years, targeted therapies have become important therapeutic strategies. Bruton's tyrosine kinase (BTK) inhibitor has shown promising efficacy in the treatment of B cell lymphoma. Orelabrutinib is the new-generation highly selective Bruton’s tyrosine kinase (BTK) inhibitor. Orelabrutinib irreversibly inhibits BTK activity by covalently binding to BTK with high specificity, thereby inhibiting the activation of BCR signaling pathway, inducing cell apoptosis, and therefore reducing the proliferation of malignant B cells, thus playing a vital role in the treatment of B cell malignancies.
Orelabrutinib has been approved for the treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) /small lymphocytic lymphoma (SLL) , and relapsed/refractory mantle cell lymphoma (MCL) in China. In addition, multiple clinical trials of orelabrutinib are being carried out for the treatment of B cell lymphomas including marginal zone lymphoma (MZL) , central nervous system lymphoma (CNSL) , Waldenstrom's macroglobulinemia (WM) and diffuse large B cell lymphoma (DLBCL) .
Another important drug target for B cell malignancies is CD19, which is abnormally over-expressed on malignant B cells, such as most pre-B acute lymphoblastic leukemia (ALL) , non-Hodgkin's lymphoma (NHL) and B cell chronic lymphocytic leukemia (CLL) . There are 9 approved drugs specific to this target globally, including a monoclonal antibody,  a bi-specific antibody, an antibody-drug conjugate (ADC) , and a chimeric antigen receptor T cell therapy (CAR-T) . Tafasitamab is a humanized Fc-enhanced monoclonal antibody targeting CD19. After binding to CD19, tafasitamab mediates B cell lysis through several mechanisms, including: 1) binding to immune effector cells such as NK cells, γδT cells and macrophages for tumor eradication; and 2) directly inducing cell death (apoptosis) . Compared to unmodified Fc structure, the Fc domain of tafasitamab includes 2 amino acid substitutions S239D and I332E (Lazar et al., 2006) which enhances the binding affinity to activating Fcγreceptors on effector cells, especially FcγRIIIa, and significantly enhances the antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) functions of tafasitamab.
Lenalidomide activates T cells to release the cytokines interferon γ (IFN-γ) and interleukin-2 (IL-2) , and thus stimulates NK cell activity and induce NK cell proliferation (Gribben et al., 2015; Kotla et al. al. al., 2009) . In addition, lenalidomide also promotes expression of FcγRIII in NK cells, which is the receptor with high-binding affinity to tafasitamab (Lapalombella et al., 2008) . The enhancement of tafasitamab mediated NK cell activation and ADCC activity by lenalidomide was demonstrated in previously published in vitro data (Awan et al., 2010) . On the other hand, lenalidomide can directly induce tumor cell death by G0-G1 cell cycle arrest; or by increasing p21Cip1/Waf1 and AP-1 transcription and promoting the NF-κB cell pathway activation, etc. (Gribben et al., 2015) .
DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the verification of CD19 expression levels across different B cell tumor lines.
FIG. 2A shows ADCC activity of tafasitamab combined with orelabrutinib or ibrutinib determined by reporter assays. FIG. 2B shows the synergy score distribution diagram of tafasitamab and orelabrutinib combination. FIG. 2C shows the synergy score distribution diagram of tafasitamab and ibrutinib combination. FIG. 2D shows the synergy score curve of combinations of tafasitamab and orelabrutinib or ibrutinib.
FIG. 3A shows the ADCC activity of tafasitamab and lenalidomide combined with orelabrutinib or ibrutinib determined by reporter assays. FIG. 3B shows the synergy score distribution diagram of combination of tafasitamab, lenalidomide and orelabrutinib. FIG. 3C shows the synergy score distribution diagram of combination of tafasitamab, lenalidomide and ibrutinib. FIG. 3D shows the synergy score curve of tafasitamab, lenalidomide combined  with orelabrutinib or ibrutinib.
FIG. 4A shows Western blot images indicating the effects of the tafasitamab alone or tafasitamab combined with orelabrutinib/ibrutinib on the phosphorylation levels of BTK and ITK proteins in co-cultured TMD-8 and Jurkat cells. FIG. 4B shows the quantified ratios of phosphorylated BTK protein /total BTK protein in co-cultured TMD-8 and Jurkat cells following treatment of tafasitamab alone or tafasitamab combined with orelabrutinib or ibrutinib. FIG. 4C shows the quantified ratios of phosphorylated ITK protein /total ITK protein in co-cultured TMD-8 and Jurkat cells following treatment of tafasitamab alone or tafasitamab combined with orelabrutinib or ibrutinib.
FIG. 5A shows the primary NK cells mediated ADCC activity (donor 1) of tafasitamab combined with orelabrutinib or ibrutinib. FIG. 5B shows the synergy score distribution diagram of tafasitamab and orelabrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells (donor 1) . FIG. 5C shows the synergy score distribution diagram of tafasitamab and ibrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells (donor 1) . FIG. 5D shows the synergy score curve of combinations of tafasitamab and orelabrutinib or ibrutinib indicating ADCC activity of tafasitamab driven by primary NK cells (donor 1) . FIG. 5E shows the primary NK cells mediated ADCC activity (donor 2) of tafasitamab combined with orelabrutinib or ibrutinib. FIG. 5F shows the synergy score distribution diagram of tafasitamab and orelabrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells (donor 2) . FIG. 5G shows the synergy score distribution diagram of tafasitamab and ibrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells (donor 2) . FIG. 5H shows the synergy score curve of combinations of tafasitamab and orelabrutinib or ibrutinib indicating ADCC activity of tafasitamab driven by primary NK cells (donor 2) .
FIG. 6A shows the primary NK cells mediated ADCC activity of tafasitamab and lenalidomide combined with orelabrutinib or ibrutinib. FIG. 6B shows the synergy score distribution diagram of tafasitamab, lenalidomide and orelabrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells. FIG. 6C shows the synergy score distribution diagram of tafasitamab, lenalidomide and ibrutinib combination indicating ADCC activity of tafasitamab driven by primary NK cells. FIG. 6D shows the synergy score curve of combinations of tafasitamab, lenalidomide and orelabrutinib or ibrutinib indicating ADCC activity of tafasitamab driven by primary NK cells.
FIG. 7A shows the effects of direct tumor killing of RS4; 11 cells by tafasitamab,  lenalidomide, orelabrutinib alone, and combinations. FIG. 7B shows the effects of direct tumor killing of TMD-8 cells by tafasitamab, lenalidomide, orelabrutinib alone, and combinations.
FIG. 8A shows the effects of direct and immune cell-dependent tumor killing of REC-1 cells by tafasitamab, lenalidomide, orelabrutinib alone, and combinations. FIG. 8B shows the synergy score distribution diagram of tafasitamab, lenalidomide and orelabrutinib combination indicating the effects of direct and immune cell-dependent tumor killing of REC-1 cells. FIG. 8C shows the synergy score curve of combination of tafasitamab, lenalidomide and orelabrutinib indicating the effects of direct and immune cell-dependent tumor killing of REC-1 cells.
FIG. 9A shows the efficacy of orelabrutinib, tafasitamab-lenalidomide, and tafasitamab-lenalidomide-orelabrutinib in the REC-1 (MCL) xenograft tumor model. FIG. 9B shows the relative body weight changes of the REC-1 tumor bearing mice during the treatment with orelabrutinib, tafasitamab-lenalidomide, and tafasitamab-lenalidomide-orelabrutinib. FIG. 9C shows the survival curves of the REC-1 tumor bearing mice treated with orelabrutinib, tafasitamab-lenalidomide, and tafasitamab-lenalidomide-orelabrutinib.
FIG. 10 shows the protocol for an open-label, multi-cohort study of tafasitamab and lenalidomide combined with orelabrutinib in patients with relapsed or refractory B-cell non-Hodgkin’s lymphoma (B-NHL) .
DETAILED DESCRIPTION OF THE INVENTION
Tafasitamab
Tafasitamab is an Fc-enhanced humanized monoclonal antibody (mAb) belonging to the immunoglobulin (Ig) G1 subclass. It binds to the CD19 antigen on the surface of B lymphocytes, resulting in B cell exhaustion. Tafasitamab is an anti-neoplastic drug with ATC classification code L01XC35. Tafasitamab can be produced by recombinant DNA technology in Chinese hamster ovary (CHO) cells, in the form of a disulfide-linked glycosylated tetramer comprising two identical 451 amino acid heavy chains and two identical 219 amino acid κlight chains. Tafasitamab has an N-linked glycosylation site at position 301 of the heavy chain sequence. The molecular weight of tafasitamab is about 150 kDa. A monosaccharide is connected to each of the two heavy chains. Tafasitamab is disclosed in US Patent No. 10,617,691 as MOR00208; all the disclosure relating to Tafasitamab in the ‘691 Patent is incorporated herein by reference.
Tafasitamab comprises a variable heavy chain VH of the following sequence (SEQ ID NO: 1) with CDR1 (SYVMH, SEQ ID NO: 2) , CDR2 (NPYNDG, SEQ ID NO: 3) , and CDR3 (GTYYYGTRVFDY, SEQ ID NO: 4) underlined:
and a variable light chain VL of the following sequence (SEQ ID NO: 5) with CDR1 (RSSKSLQNVNGNTYLY, SEQ ID NO: 6) , CDR2 (RMSNLNS, SEQ ID NO: 7) , and CDR3 (MQHLEYPIT, SEQ ID NO: 8) underlined:
Tafasitamab contains a heavy chain constant domain CH of the following sequence:
and a light chain constant domain CL of the following sequence:
Orelabrutinib
Orelabrutinib has the chemical name of 2- (4-phenoxyphenyl) -6- [1- (prop-2-enoyl) piperidin-4-yl] pyridine-3-carboxamide, with molecular formula of C26H25N3O3, and molecular weight of 427.5. Its structure is shown as
Synergy Coefficient Calculation
In the present application, for the calculation of synergy coefficient, the web version (www. synergyfinderplus. org) of SynergyFinder is used to analyze the synergistic effect of the pharmaceutical composition of the disclosure. SynergyFinder includes four mathematical models: Bliss, Loewe, HSA and ZIP. The four models have different emphases: HSA model assumes that the pharmaceutical effect of the pharmaceutical composition with the synergistic effect is greater than the maximum pharmaceutical effect of single drug; Bliss model assumes each drug takes effect independently, i.e., multiplication effect of independent action of single drug; Loewe model assumes the drugs do not interact with each other and each drug has the same maximum pharmaceutical effect; ZIP model assumes non-interacting drugs with minimal changes in their dose-response curves when combined (Paltun et al., 2021) . The calculation methods of the four models were described by Ianevski et al. (2020) . The Loewe model is used to analyze the synergistic effect of the pharmaceutical composition of the disclosure, and the synergy score is defined as follows: < -10 considered as strong antagonism, -10 to about 0 considered as antagonism, 0 considered as no effect, 0 to about 10 considered as additive effect and >10 considered as synergistic effect.
The present disclosure is directed to a method for treating CD19 antigen-positive B cell tumors. The method comprises the step of administering a BTK inhibitor orelabrutinib and an anti-CD19 antibody, or an antigen-binding fragment thereof, to a subject in need thereof.
In certain embodiments, the antigen-binding fragment thereof is an Fab, Fab', F (ab’ ) 2, or single-chain variable fragment (scFv) .
In one embodiment, the anti-CD19 antibody or an antigen-binding fragment thereof has a VH that comprises CDR1 having the amino acid sequence of SEQ ID NO: 2, CDR2 having the amino acid sequence of SEQ ID NO: 3, and CDR3 having the amino acid sequence of SEQ ID NO: 4; and has a VL that comprises CDR1 having the amino acid sequence of SEQ ID NO: 6, CDR2 having the amino acid sequence of SEQ ID NO: 7, and CDR3 having the amino acid sequence of SEQ ID NO: 8.
In one embodiment, the anti-CD19 antibody or an antigen-binding fragment thereof has a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 5.
In a preferred embodiment, the anti-CD19 antibody is Tafasitamab, or a sequence  having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity thereof, provided that the sequence variation is in the non-CDR framework region and/or constant region. The sequence variation, i.e., the amino acid changes, are preferably of a minor amino acid change such as a conservative amino acid substitution. A conservative amino acid substitution is well-known to a person skilled in the art.
The present method is useful in treating CD19 antigen-positive B cell tumors such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic leukemia. Non-Hodgkin's lymphoma includes diseases such as follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma, mantle cell lymphoma, and marginal zone lymphoma.
CD19 and BTK are two critical pathogenic targets in B cell malignancies. The present method targets both CD19 with an anti-CD19 antibody such as tafasitamab and BTK with orelabrutinib. Without wishing to be bound by any particular theory, tafasitamab and orelabrutinib in combination induces CD19-dependent and BTK-dependent direct tumor killing and have a combined tumor killing effect.
Due to its high kinase selectivity, orelabrutinib does not inhibit the phosphorylation of interleukin-2-inducible T-cell kinase (ITK) . Therefore, use of tafasitamab in combination with orelabrutinib retains the ADCC function of tafasitamab. Again without wishing to be bound by any particular theory, the combination of orelabrutinib and tafasitamab promotes BTK-dependent B cell killing and CD19-dependent B cell tumor killing.
In the present method, orelabrutinib can be administered orally. Orelabrutinib may be administered orally with a dosage between 50-250 mg/day or 100-200 mg/day. For example, orelabrutinib may be administered orally with a dosage of about 150 mg once a day in a 28-day treatment cycle. Orelabrutinib may be administered at the same time or substantially the same time every day, for example on an empty stomach or after meals.
In the present method, an anti-CD19 antibody such as tafasitamab can be administered intravenously (IV) based, for example, on the body weight, with a dosage of 8 mg/kg/day to 30 mg/kg/day. For example, in a 28-day treatment cycle, tafasitamab may be administered about 12 mg/kg intravenously (IV) on Days 1, 4, 8, 15, 22 of Cycle 1, Days 1, 8, 15, 22 of Cycles 2-3, Day 1 and 15 of Cycles 4-12. Starting from cycle 13, patients without disease progression may receive continuous orelabrutinib with or without tafasitamab until disease progression.
“About” , as used herein, refers to ± 10%of the recited value.
Orelabrutinib and an anti-CD19 antibody such as tafasitamab can be administered simultaneously, or sequentially. For example, orelabrutinib can be administered before an anti-CD19 antibody such as tafasitamab, or orelabrutinib and an anti-CD19 antibody such as tafasitamab can be administered at the same or substantially the same time; or orelabrutinib can be administered after an anti-CD19 antibody such as tafasitamab.
In one embodiment, the present method further comprises administering lenalidomide to the subject. Lenalidomide is an immunomodulator and has a chemical name of 3- (7-amino-3-oxo-1H-isoindol-2-yl) piperidine-2, 6-dione. Lenalidomide is used to regulate the immune response of a patient and it also has direct tumor killing effect.
Lenalidomide can be orally administered in a fixed dose between 5-25 mg/day. For example, lenalidomide can be orally administered at about 5 mg/day, 10 mg/day, 15 mg/day, 20 mg/day and 25 mg/day.
In one embodiment, orelabrutinib is administered before an anti-CD19 antibody such as tafasitamab and lenalidomide. In another embodiment, orelabrutinib is administered together with an anti-CD19 antibody such as tafasitamab and lenalidomide at the same or substantially the same time. In yet another embodiment, orelabrutinib is administered after an anti-CD19 antibody such as tafasitamab and lenalidomide.
When an anti-CD19 antibody such as tafasitamab and lenalidomide are combined with orelabrutinib, all three drugs can have direct tumor killing activity. Further, when orelabrutinib and lenalidomide are combined with tafasitamab, the ADCC activity of tafasitamab is retained. The combination of three drugs has a synergistic effect and promotes the killing of tumor cells in an immune cell dependent manner.
B cell malignancies are highly heterogeneous; individual patients may have dramatically different responses to mono-therapeutics due to their unique pathogenesis. The combined three-drug treatment regimen of the present disclosure can target patients with different clinical sensitivities to orelabrutinib, tafasitamab, and lenalidomide, by treating tumors with multiple mechanism of actions (MOAs) additively or synergistically. In orelabrutinib-insensitive tumor cells, the combination of the three-drug treatment can enhance the tumor killing effect. Similarly, in tafasitamab-insensitive tumor cells, the combination of the three-drug treatment can enhance the tumor killing. The present disclosure provides a new treatment regimen for CD19-positive B cell malignancies.
The present disclosure further provides a kit comprising orelabrutinib and an anti-CD19 antibody such as tafasitamab. The kit may further comprise lenalidomide. In one  embodiment, orelabrutinib is in a tablet or a capsule form. In one embodiment, tafasitamab is in a form of lyophilized powder. In one embodiment, tafasitamab formulations are described in WO2018002031 (US20190322742) . In one embodiment, lenalidomide is in a tablet or capsule form. For example, orelabrutinib is provided in a bottle containing 50 mg orelabrutinib tablets. For example, tafasitamab is provided in a single dose vial containing 200 mg lyophilized powder vial ready for reconstitution. For example, lenalidomide is provided in a bottle containing 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, or 25 mg lenalidomide capsules. Kits of the present disclosure can optionally comprise instructions for administering the anti-CD19 antibody, orelabrutinib and/or lenalidomide.
In one embodiment, orelabrutinib is administered orally in a fixed dose between 50 mg/day to 150 mg/day, tafasitamab is intravenously administered from 8 mg/kg to 30 mg/kg according to the body weight, and lenalidomide is administered in a fixed dose, between 5 mg/day to 25 mg/day.
For example, in a 28-day treatment cycle, orelabrutinib can be orally administered at 150 mg/day at the same time period every day continuously; tafasitamab can be intravenously (IV) administered on Days 1, 4, 8, 15, 22 of Cycle 1, Days 1, 8, 15, 22 of Cycles 2-3, Day 1 and 15 of Cycles 4-12; lenalidomide can be orally administered at 25 mg/day, on Days 1-21 of each cycle of Cycles 1-12. Ibrutinib is a small molecule drug that inhibits B-cell proliferation and survival by irreversibly binding to BTK. Ibrutinib is the first approved BTK inhibitor and is in the same class as orelabrutinib. The inventors have discovered that orelabrutinib provides advantages over ibrutinib, when used in combination treatment with tafasitamab.
The present disclosure compares the ADCC activity of tafasitamab when combined with orelabrutinib or ibrutinib. Orelabrutinib fully retains the ADCC function of tafasitamab. In contrast, ibrutinib inhibits the ADCC function of tafasitamab, and the combination of ibrutinib with tafasitamab in certain of the experiments shown results in an antagonistic effect.
The present disclosure also compares the ADCC activity of tafasitamab when combined with lenalidomide, plus either orelabrutinib or ibrutinib. Orelabrutinib in combination with lenalidomide completely retains the ADCC function of tafasitamab. In contrast, ibrutinib in combination with lenalidomide inhibits the ADCC function of tafasitamab.
The inventors have shown that orelabrutinib is superior to ibrutinib in retaining the ADCC function of tafasitamab in both 2-drug (with tafasitamab) and 3-drug (with  tafasitamab and lenalidomide) combinations, especially at a dose of 400 nM, which resembles the plasma exposure of orelabrutinib and ibrutinib and is a clinically relevant dose.
The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.
EXAMPLES
Example 1. Verification of CD19 antigen expression in B cell tumor lines, including TMD-8, RS4; 11 and REC-1
TMD-8 is a human diffuse large B cell lymphoma (DLBCL) cell line, RS4; 11 is a human acute lymphoma (ALL) cell line and REC-1 is a human mantle cell lymphoma (MCL) cell line. In this example, the expression levels of CD19 antigen on the cell membrane were quantified by flowcytometry. The cells were incubated with 10 μg/ml of tafasitamab or isotype control antibody for 20 minutes at room temperature. Following washing off the unbound antibody, the cells were stained with 1 μL of PE fluorescent-labeled anti-human IgG-Fc antibody in the dark at room temperature for 20 minutes. The binding ratio of CD19 was detected by NovoCyte Quanteon flow cytometer (Agilent) . As shown in FIG. 1 and Table 1, robust expression of CD19 antigen is detected on the plasma membrane of nearly 100%of TMD-8, RS4; 11 and REC-1 tumor cells, with the expression intensity of RS4; 11>REC-1>TMD-8.
Table 1. CD19 expression intensity in different B cell tumor lines
Example 2. Orelabrutinib retains the ADCC activity of tafasitamab in the reporter assays
Antibody-dependent cellular cytotoxicity (ADCC) represents an important mechanism of action (MOA) for tafasitamab. The Fc-domain of tafasitamab was engineered  to improve its binding affinity to FcγR by up to 100 times relative to the original anti-CD19 antibody with native Fc-domain, thus significantly enhanced the ADCC functionality of tafasitamab. Specifically, compared to unmodified Fc-domain, the Fc-domain of tafasitamab contains 2 amino acid substitutions, i.e., S239D and I332E (Lazar et al., 2006) , which leads to its improved affinity to the activating FcγR receptors on the effector cells, especially the affinity to Fcγ receptor IIIa (FcγRIIIa) . While the Fab end of tafasitamab bound to CD19 antigen on the tumor cells, its Fc-domain binds to FcγRIIIa on the membrane of NK cells and triggers NK cell activation. This FcγR signaling in NK cells is in part dependent on ITK activation through phosphorylation, which in turn leads to calcium mobilization, granzyme release, and tumor lysis (Khurana et al., 2007) . ITK and BTK belong to the TEC kinase family with high homology. Dubovsky et al. has reported the irreversible binding of BTK inhibitor ibrutinib (PCI-32765) to ITK (Dubovsky et al., 2013) , which is at least in part accountable for ibrutinib-induced inhibition of ADCC activity of the anti-CD20 antibody rituximab (Da Roit et al., 2015; Yu et al., 2021) .
In the ADCC reporter assay, Jurkat-NFAT-Luc2-CD16a-V158 reporter cell line constructed by Beijing KYinno Biotechnology Co., Ltd. was used as substitute effector cells. These effector cells with extracellular over-expression of a high affinity FcγRIIIa variant CD16a-V158, were incubated with target TMD-8 cells for 4 hours at 1: 1 effector-target ratio, and different concentrations of tafasitamab (starting from 500 ng/mL, 5-fold dilution) , with or without BTK inhibitors and/or lenalidomide. When the Fab-domain of tafasitamab bound to CD19 antigen on the target cells and the Fc-domain of tafasitamab bound to CD16a-V158, the NFAT-Luc2 luciferase reporter was activated, and the bioluminescent signal was detected by aPlus multi-function microplate reader (BMG Labtech) to reflect the ADCC activity of tafasitamab. The data was analyzed by GraphPad Prism 7.0 software, and the nonlinear S-curve four-parameter fit Y=Bottom + (Top-Bottom) / (1+10^ ( (LogIC50-X) *HillSlope) ) regression was used to fit the data to a dose-effect curve, in which ‘Bottom’ indicates the lower limit of the bioluminescent signal, representing the lower asymptote of the S curve, ‘Top’ indicates the upper limit of the bioluminescent signal, representing the upper asymptote of the S curve. ‘IC50’ is the antibody concentration value at which the bioluminescent signal reaches 50%of ‘Top’ . HillSlope is the increase rate of absorbance, equivalent to the slope of the curve, and the IC50 value is calculated accordingly.
As shown in FIG. 2A and Table 2, the combination of orelabrutinib and tafasitamab results in modestly enhanced ADCC activity of tafasitamab. IC50 of tafasitamab alone or its  combination with 400 nM or 4000 nM of orelabrutinib was: 3.02 ng/mL, 2.93 ng/mL and 2.18 ng/mL, respectively. Although the differences of IC50 values are not statistically significant, there is a trend of improvement in ADCC activity of tafasitamab when combined with orelabrutinib compared to tafasitamab alone. Furthermore, at the maximum tafasitamb concentration of 500 ng/mL, the Emax values, reflecting the maximal ADCC activity, were at 20.85-fold for tafasitamab alone; 24.61-fold for tafasitamab and 400 nM of orelabrutinib combination; and 27.83-fold for tafasitamab and 4000 nM of orelabrutinib combination. The increases in Emax values were statistically significant (p < 0.01@400 nM, p < 0.0001@4000 nM, Oneway ANOVA) . On the contrary, 400 nM of ibrutinib significantly inhibited the ADCC activity of tafasitamab with Emax of 8.26-fold (p < 0.0001, Oneway ANOVA) ; while 4000 nM of ibrutinib completely abolished the ADCC activity of tafasitamab with non-applicable IC50 value and Emax down to 1.07-fold (p < 0.0001, Oneway ANOVA) .
As shown in FIG. 2B-D, when SynergyFinder was applied to calculate the additivity of the combined medicament, orelabrutinib and tafasitamab combination has an average synergy score of 3.72, while ibrutinib and tafasitamab combination has an average synergy score of -11.61.
In conclusion, our ADCC reporter assays have demonstrated that orelabrutinib modestly improves the ADCC activity of tafasitamab, and the combination has additive effects. In the meantime, ibrutinib compromises the ADCC function of tafasitamab, and the combination is antagonistic.
Table 2. ADCC activity of tafasitamab in combination with orelabrutinib or ibrutinib measured by reporter assays
N/A: non-applicable. **p < 0.01 vs. Tafasitamab group, ****p < 0.0001 vs. Tafasitamab group.
As an immunomodulator, lenalidomide leads to phosphorylation of CD28 costimulatory receptor and enhances the activation of T cells and NK cells; it also directly  regulates the secretion of cytokines, i.e., promoting the release of IL-2 and IFN-γ, and inhibiting the release of TNFα; lenalidomide can also directly induce the apoptosis of tumor cells (Kotla et al., 2009) . The combination of tafasitamab and lenalidomide has been granted accelerated approval by the FDA and EMA for the treatment of adult patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL) unsuitable to autologous stem cell transplantation. To elucidate the effects of BTK inhibitors on tafasitamab and lenalidomide combination, we further investigated the ADCC activity of tafasitamab combined with lenalidomide and orelabrutinib/ibrutinib by the reporter assays.
As shown in FIG. 3A and Table 3, when 400 nM or 4000 nM of orelabrutinib was combined with tafasitamab and lenalidomide, the ADCC activity of tafasitamab was well retained, with IC50 values of 2.36 ng/mL and 2.39 ng/mL respectively, not significantly different from tafasitamab alone, and its combination with lenalidomide (IC50 = 3.02 ng/mL and 2.17 ng/mL, respectively) . The Emax values, reflecting the maximal ADCC activity of tafasitamab at 500 ng/mL, were also comparable, except for modest reduction in the low dose orelabrutinib (400 nM) treated group (Emax = 18.9-fold, p < 0.01 vs. tafasitamab and lenalidomide combination group) . On the contrary, the 3-drug combination with 400 nM of ibrutinib resulted in much reduced ADCC activity of tafasitamab (IC50 = 2.04 ng/mL and Emax = 8.8-fold) , significantly lower than tafasitamab and lenalidomide combination group (p < 0.0001) ; at a higher dose, 4000 nM of ibrutinib further eliminated the ADCC function of tafasitamab with non-applicable IC50 value and Emax of 1.92-fold (p < 0.0001) .
When additivity of the combined medicament was calculated by SynergyFinder, orelabrutinib showed very weak antagonistic effect with tafasitamab and lenalidomide, which was not statistically significant (Average synergy score = -0.94, p = 0.13, FIG. 3B) ; however, as shown in FIG. 3C, ibrutinib had strong antagonistic effect with average synergy score of -11.63, p value of 0.024 (p < 0.05, statistically significant) . The dynamic change of synergy scores at different dose levels is shown in FIG. 3D. Clearly, in the 3-drug combinations, while orelabrutinib well retains the ADCC activity of tafasitamab, ibrutinib markedly inhibits its ADCC function.
Taken together, in the ADCC reporter assays, while orelabrutinib slightly enhances, or well retains the ADCC activity of tafasitamab in the 2-drug or 3-drug combinations, ibrutinib consistently suppresses the ADCC activity of tafasitamab.
Table 3. ADCC activity of tafasitamab in combination with lenalidomide and orelabrutinib or ibrutinib measured by reporter assays
N/A: non-applicable. **p < 0.01 vs. Tafasitamab + lenalidomide group, ****p < 0.0001 vs. Tafasitamab +lenalidomide group.
Example 3. Orelabrutinib has no off-target effect on ITK phosphorylation in Jurkat effector cells, confers its ability to retain the ADCC activity of tafasitamab.
To further elucidate the mechanistic basis leading to different effects of orelabrutinib and ibrutinib on ADCC activity of antibody when combined with tafasitamab, we investigated the pharmacodynamic changes of tyrosine kinases BTK and ITK phosphorylation in a cell co-culture assay. ITK is a tyrosine kinase with high homology to BTK in the TEC kinase family. In the process of ADCC signaling, the activation signal of FcγR on the surface of NK cell membrane leads to ITK phosphorylation, promotes the downstream calcium signal transduction and the release of granzymes to kill target cells bound by antibody. ITK is a key kinase mediating ADCC signals. Therefore, the effects of orelabrutinib and ibrutinib on ITK phosphorylation in Jurkat cells were compared in co-culture experiment of TMD-8 and Jurkat cells.
Cells in the logarithmic growth phase were harvested and counted with a platelet counter. Cell viability was detected by trypan blue exclusion method to ensure cell viability was above 90%. The effector and target cells were adjusted to a ratio of 1: 1, and a total of 2 x 10^6 cells/well was plated to 6-well plates, placed at 37℃, 5%CO2 and 95%humidity conditions and cultured for 1 hour. Orelabrutinib and ibrutinib solutions were prepared by adding 10 μL of drug solution to each well to the final concentration of 4000 nM. The treatment groups were as follows: 1. Single drug groups: tafasitamab (3 concentrations) , orelabrutinib (4000 nM) , or ibrutinib (4000 nM) ; 2. Combined medicament groups:
tafasitamab + orelabrutinib (4000 nM) , tafasitamab + ibrutinib (4000 nM) ; 3. Control group (Veh, Vehicle) was DMSO.
The cells in the medicated 6-well plate were placed at 37℃ and 5%CO2 conditions and cultured for 6 hours. The cells were collected and resuspended in 70 μL of RIPA lysis solution, set on the ice for 30 minutes. The samples were then centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and transferred to a 1.5 mL sterile centrifuge tube. After mixing with 4×loading buffer and denaturing for 10 minutes, the samples were loaded to the 4-12%Tris-Glycine mini gel (Life Technologies, Carlsbad, CA) for electrophoresis, followed by protein transferred to PVDF membrane with the semi-dry transfer electrophoresis apparatus (Hoefer, MA) . After 1 hour blocking in confining liquid (LI-COR, Lincoln, NE) , the PVDF membrane was incubated at 4℃ overnight with the primary antibody diluted to an appropriate concentration. Following incubation, the PVDF membrane was washed with 1×TBST for 5 times and subjected to image scanning onCLx imaging system (LI-COR, Lincoln, NE) .
The experimental results show that the single drug treatment of tafasitamab has no effect on the phosphorylated protein levels and total protein levels of BTK and ITK at all tested concentrations (FIG. 4A-C) . With the combinatory treatment of BTK inhibitors and tafasitamab, both orelabrutinib and ibrutinib can effectively reduce the phosphorylation protein levels of BTK (FIG. 4A-B) . The ratio of the phosphorylated BTK to total BTK protein decreases by 73.5%to 81.5%with little variation (FIG. 4B) . Interestingly, while the phosphorylated ITK protein is not changed in all orelabrutinib treated groups compared to vehicle group (FIG. 4A, 4C) , ibrutinib treatment leads to significantly reduced phosphorylation levels of ITK by an average of 53.7%compared to the control group (FIG. 4A, 4C) . Off-target inhibition of ITK phosphorylation is only associated with ibrutinib, but not orelabrutinib.
In summary, BTK inhibitors ibrutinib and orelabrutinib both lead to effective inhibition of BTK phosphorylation in the TMD-8 and Jurkat co-culture assay. However, these two compounds show strikingly different activities on ITK, i.e, ibrutinib moderately inhibits ITK phosphorylation, which at least in part explains its antagonism in ADCC activity of tafasitamab; on the other hand, orelabrutinib is a highly selective BTK inhibitor with no cross-activity on ITK, which confers its ability to retain the ADCC activity of tafasitamab.
Example 4. Orelabrutinib retains tafasitamab-mediated killing of CD19+ tumor cells by peripheral blood-derived NK cells
The ADCC reporter assays have provided strong evidence that highly selective BTK inhibitor orelabrutinib modestly enhances, or well retains the ADCC activity of tafasitamab in the 2-drug or 3-drug combinations, while ibrutinib consistently suppresses the ADCC activity of tafasitamab due to its off-target activity on ITK. To further verify if same effect can be driven by primary NK cells derived from human peripheral blood mononuclear cells (PBMCs) of different healthy donors, we performed ADCC killing experiment using primary NK cells as effector cells, and the CD19+ RS4; 11 (ALL) and REC-1 (MCL) tumor cell lines as target cells. Human PBMCs usually contain 60%CD3+ T cells, 10%to 20%CD56+ NK cells, and 10%CD14+ monocytes. Primary NK cells containing PBMCs were co-incubated with CD19+ RS4; 11 or REC-1 cells for 4 hours or 16 hours under the condition of effector-target ratio of E: T = 30: 1.
In the assays of 2-drug combinations with tafasitamab and orelabrutinib/ibrutinib, we used 1.6 μM of CFSE (Carboxyfluoresceinsuccinimidyl ester, 565082, BD) to label the target tumor cells and treated with test drugs. Two concentrations (400 and 4000 nM) of orelabrutinib or ibrutinib were combined with different concentrations of tafasitamab. A total of 9 concentrations of tafasitamab were tested starting from initial 10 μg/mL, serially diluted with the complete medium by 5 times down to 25.4 pg/mL. PBMCs were added and co-incubated at 37℃ for 4 hours. At the end of incubation, cells were washed twice with PBS, then stained with 7-AAD at room temperature for 10 minutes in the dark. ADCC activity of tafasitamab was determined by the lysis rate of target cells, detected by NovoCyteQuanteon flow cytometer (Agilent) .
FIG. 5A showed that tafasitamab had a dose dependent ADCC activity mediated by primary NK cells from Donor 1. Low concentration (400 nM) of ibrutinib did not affect the ADCC activity of tafasitamab, while high concentration (4000 nM) of ibrutinib completely inhibited the ADCC activity of tafasitamab. In contrast, orelabrutinib enhanced the ADCC activity of tafasitamab at the low concentration of 400 nM, while no effect was observed at the high concentration of 4000 nM. SynergyFinder was used to calculate the synergy scores of the combined medicament (results shown in FIG. 5B-D) . The mean synergy scores of orelabrutinib and ibrutinib were 5.51 and -5.81, respectively. In particular, orelabrutinib had no effect on the ADCC function of tafasitamab at the dose of 4000 nM, while additive to synergistic effect was achieved at the dose of 400 nM; ibrutinib presented strong antagonistic  effect at the dose of 4000 nM, and no effect at the dose of 400 nM.
FIG. 5E and Table 4 show strong ADCC activity of tafasitamab on RS4; 11 cells with NK cells derived from Donor 2. Tafasitamb single agent led to maximal lysis rate (Emax) of 72.24%in the 10 μg/mL treatment group. The combination of tafasitamab and 400 nM of orelabrutinib fully retained the ADCC activity of tafasitamab with the lysis rate curve completely merged; while high dose of 4000 nM orelabrutinib moderately inhibited the ADCC activity of tafasitamab (Emax = 46.34%) . In contrast, ADCC activity of tafasitamab is severely suppressed when combined with ibrutinib at either 400 nM or 4000 nM doses, with non-applicable IC50 values and Emax of 23.18%and 6.44%respectively. As shown in FIG. 5F-H, when synergy scores were calculated by SynergyFinder, orelabrutinib and ibrutinib have average synergy scores of -8.61 and -45.6, respectively. Ibrutinib had strong antagonistic effects with tafasitamab at both 400 nM and 4000 nM dose levels with statistical significance (p = 0.0046) . In the meantime, while orelabrutinib also showed strong antagonistic effect at the dose of 4000 nM, it showed no effect at the dose of 400 nM, and the overall effects were not statistically significant (p = 0.37) .
In conclusion, despite the variations caused by intrinsic activities of NK cells derived from different donors, our ADCC assays utilizing primary NK cells have verified our findings from the reporter assays, that in this 2-drug combination study, the ADCC function of tafasitamab is better supported by orelabrutinib than ibrutinib, especially at lower dose level of 400 nM.
Table 4. ADCC activity of tafasitamab in combination with orelabrutinib or ibrutinib driven by primary PBMC-derived NK cells
N/A: non-applicable.
Furthermore, we have applied this ADCC assay driven by primary NK cells to explore the effects of BTK inhibitors on the ADCC activity of tafasitamab combined with lenalidomide. The target tumor cells (REC-1) were labeled with 1.6 μM of CFSE, and treated  with test drugs. Orelabrutinib or ibrutinib were tested at two concentrations: 400 nM and 4000 nM; lenalidomide was tested at 1000 nM; and tafasitamab was tested at a total of 10 doses with 5-fold serial dilution starting from 10μg/mL. PBMCs carrying the high-affinity FcγRIIIa 158 V/V were used as effector cells at effector-target ratio of 30: 1. After incubation at 37℃ for 16 hours in the 96-U well plate, the cells were washed twice with PBS, and stained with 7-AAD at room temperature for 10 minutes in the dark. ADCC activity of tafasitamab was determined by the lysis rate of target cells, detected by NovoCyteQuanteon flow cytometer (Agilent) .
As shown in FIG. 6A, orelabrutinib at low concentration (400 nM) showed no effect on ADCC activity of tafasitamab, while high concentration (4000 nM) of orelabrutinib had moderate inhibition. Conversely, 400 nM of ibrutinib already inhibited the ADCC function of tafasitamab, while 4000 nM of ibrutinib completely abolished the activity. This data is consistent with our findings in the ADCC reporter assays that in the 3-drug combinations, orelabrutinib retains the ADCC activity of tafasitamab better than ibrutinib.
We also performed drug additivity analysis by SynergyFinder. As the results shown in FIG. 6B-D. The mean synergy scores of orelabrutinib and ibrutinib were -4.1 and -20.01, respectively. 400 nM of orelabrutinib had no effect or a weak additive effect on the combined medicament depending on the concentrations of tafasitamab, while 4000 nM of orelabrutinib showed an antagonistic effect. On the other hand, ibrutinib showed strong antagonistic effects at both 400 nM and 4000 nM dose levels.
Taken together, utilizing PBMC derived primary NK cells as effector cells, we have confirmed in this experiment that orelabrutinib is superior to ibrutinib in retaining the ADCC function of tafasitamab in both 2-drug and 3-drug combinations, especially at low dose of 400 nM. Notably, 400 nM dose of BTK inhibitors is also clinically relevant, which closely resembles the plasma exposure of orelabrutinib or ibrutinib in patients.
Example 5. Combination of orelabrutinib with tafasitamab and lenalidomide improves direct tumor lysis activity
Tafasitamab can inhibit proliferation and induce direct cytotoxicity against CD19-expressing B cell tumors. This tumor killing activity is further enhanced by FcγR-mediated antibody cross-linking when the high affinity Fc domain of tafasitamab binds to FcγR. In addition, lenalidomide has also been reported to have direct tumor-killing effects by degradation of key transcription factors required by tumor growth (Kim et al., 2013) ;  orelabrutinib inhibits the BTK activity, thus leads to BCR signaling blockade and stalled tumor cell growth.
To investigate the immunomodulation independent tumor-killing activity with combined medicament of tafasitamab, lenalidomide and orelabrutinib, 20 μg/mL of anti-human IgG Fc secondary antibody was coated to the 96-U well culture plates (100 μL/well) to model FcγR cross-linking and direct cytotoxicity of tafasitamab against RS4; 11 (ALL) and TMD-8 (DLBLC) cells. After over-night coating, 0.64 ng/mL or 16 ng/mL of tafasitamab was added to each well and incubated at 37℃ for 2 hours. Subsequently, 3000 of test cells (RS4; 11 or TMD-8) were inoculated in each well of the 96-U well plate, followed by addition of test compounds to the final concentration of 1000 nM for lenalidomide and/or 400 nM for orelabrutinib. After incubation at 37℃ for 72 hours, 50 μL of CTG-Glo reagent (Promega, Madison, WI) was applied for luminescent signal detection by the multi-function microwell plate detector (Perkin Elmer, Waltham, MA) to determine the tumor cell lysis rate.
As shown in FIG. 7A, in the single agent treatment groups, RS4; 11 cells were very sensitive to lenalidomide (84%cell lysis) ; modestly responsive to tafasitamab (2.4%cell lysis @0.64 ng/mL and 24%cell lysis @16 ng/mL) ; but non-responsive to orelabrutinib (0%cell lysis) . However, 2-drug combinations of tafasitamab and orelabrutinib resulted in significantly improved tumor killing with 58%to 66%cell lysis rates. In the other 2-drug or 3-drug combinations with lenalidomide, i.e., tafasitamab/lenalidomide or tafasitamab/lenalidomide/orelabrutinib, the responses were mostly driven by effective tumor killing of lenalidomide, resulted in tumor lysis rates of 83%to 87%, with little variations.
As shown in FIG. 7B, TMD-8 cells are moderately responsive to monotherapies of all 3 tested drugs. The tumor cell lysis rates were at 45%for lenalidomide, 42%for orelabrutinib, and 14%to 29%for tafasitamab from low to high doses. Two-drug combinations of tafasitamab/lenalidomide and tafasitamab/orelabrutinib both improved tumor killing with 52%to 71%cell lysis rate and 47%to 55%cell lysis rate, respectively. Three-drug combination of tafasitamab/lenalidomide/orelabrutinib further improved the tumor killing with 91%to 94%cell lysis rate.
Collectively, in our evaluation of direct tumor lysis activity with combined medicament of orelabrutinib with tafasitamab and lenalidomide, we have found that different B cell tumor lines, regardless of their responses to monotherapies of tested drugs, may all be effectively eradicated by 3-drug combination. This finding reflects a greatly appreciated clinical scenario that B cell malignancies are highly heterogeneous. Individual patients may  have dramatically different responses to mono-therapeutics due to their unique pathogenesis; nevertheless, the combination of orelabrutinib with tafasitamab and lenalidomide may still offer effective treatment by tackling tumors with multiple mechanism of actions additively or synergistically. This provides fundamental scientific rationale of testing combined medicament in clinical studies.
Example 6. Combination of orelabrutinib with tafasitamab and lenalidomide enhances tumor lysis activity when co-cultured with PBMC
The combination of tafasitamab and lenalidomide can promote the tumor cell killing through different mechanism of actions: 1) lenalidomide enhances the ADCC activity of tafasitamab mediated by NK cells and γδT cells; 2) lenalidomide enhances the CD4+, CD8+effector T cell activities, strengthening the CD19+ tumor cell killing effect; 3) both tafasitamab and lenalidomide can promote tumor cell apoptosis and lead to direct tumor killing. To investigate if orelabrutinib can improve tumor killing activity of tafasitamab and lenalidomide with the presence of immune cells, we performed tumor lysis assays by co-culturing target tumor cells with PBMCs.
REC-1 (MCL) cell line was used as the target cells and co-cultured with PBMCs for 72 hours at the effector-target ratio of 1: 1. The REC-1 cells were labeled with 1.6 μM of CFSE and then treated with test drugs. Orelabrutinib was tested at two concentrations: 50 nM and 400 nM; lenalidomide was tested at 1000 nM; and tafasitamab was tested at a total of 10 doses with 5-fold serial dilution starting from 10 μg/mL. After adding same numbers of PBMCs as REC-1 cells, the 96-U well plate was incubated at 37℃ for 72 hours, followed by 2-times washing with PBS and staining with 7-AAD at room temperature for 10 minutes in the dark. Tumor lysis was detected by the NovoCyte Quanteon flow cytometer (Agilent) .
As the results shown in FIG. 8A, single agent treatment of orelabrutinib or lenalidomide induced only baseline tumor lysis. Under the condition of low effector-target ratio (E: T=1: 1) , the tumor lysis activity of tafasitamab alone was also weak. While the combination of tafasitamab and 400 nM of orelabrutinib had no improvement in tumor killing, tafasimab and lenalidomide much effectively eradicated the tumor cells with maximal lysis rate (Emax) of 46% (tafasitamab @10 μg/mL) . When orelabrutinib was combined with tafasimab and lenalidomide, the tumor lysis rate was further enhanced with Emax of 61%when tafasitamab was at 10 μg/mL.
Drug additivity analysis was performed by SynergyFinder to compare 3-drug  combination of tafsitamab/lenalidomide/orelabrutinib with 2-drug combination of tafasitamab/lenalidomide. As shown in FIG. 8B-C, the mean synergy score was 16.31, indicating strong synergistic effect of the 3-drug combination. This synergy is more prominent at lower orelabrutinib dose of 50 nM than 400 nM. The overall synergistic effect is statistically significant (p = 0.018) , fully demonstrated the potential treatment benefit of the combined medicament of these 3 drugs.
Example 7. Combination of tafasitamab and lenalidomide with orelabrutinib improves the in vivo therapeutic efficacy in the treatment of mice bearing human REC-1 xenograft tumors
To further confirm the therapeutic benefit of combinatory treatment of tafasitamab and lenalidomide with orelabrutinib, we performed an in vivo efficacy study by treating the REC-1 xenograft tumor bearing mice with vehicle control, orelabrutinib alone, tafasitamab and lenalidomide, or 3-drug combinations.
In this study, 45 6～8-week-old female CB17 SCID mice (supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd) were each inoculated subcutaneously on the right flank with 1 x 10^6 REC-1 cells for tumor formation. On day 3 after tumor inoculation, 40 tumor-bearing mice were randomly assigned into 4 groups (10 mice per group) according to body weight. The test articles were administrated according to predetermined regimens as shown in Table 5.
Table 5. Dosing regimens for in vivo efficacy study of REC-1 xenografts with tafasitamab combined with lenalidomide and orelabrutinib
PO: oral administration, IV: intravenous injection, QD: once per day, BID: twice per day, BIW: twice per week.
In this study, tumor volumes and body weights of the mice were monitored 3 times per week. Animals with tumor volume exceeding 2,000 mm3 were euthanized, which was also our tumor volume cutoff for survival analysis.
As shown in FIG. 9A, 1 mg/kg of orelabrutinib treatment had very modest REC-1 tumor growth control; 3 mg/kg of tafasitamab and 30 mg/kg of lenalidomide combo showed moderate efficacy; while the 3-drug combinatory treatment of tafasitamab, lenalidomide and orelabrutinib significantly delayed the REC-1 tumor growth in CB17 SCID mice. There were also no abnormal animal body weight reductions in any of the treatment groups during the entire course of the study (FIG. 9B) . In the survival analysis, as the survival curves shown in FIG. 9C and the survival rates shown in Table 6, our data indicate the superior survival benefit of the 3-drug combinatory treatment, where it offered 100%protection of tumor bearing mice 38 days post grouping (PG-D38) , significantly improved compared with other treatment groups (p < 0.0001) .
Taken together, in line with our in vitro efficacy assays, this in vivo efficacy study of REC-1 xenograft tumor model further demonstrates the treatment benefit of the combined medicament of tafasitamab, lenalidomide and orelabrutinib in an animal model.
Table 6. Survival rates of the REC-1 tumor bearing mice treated with tafasitamab, lenalidomide and/or orelabrutinib
****p < 0.0001
Example 8. Clinical safety summary of orelabrutinib and tafasitamab combined with  lenalidomide in marginal zone lymphoma (MZL) and diffuse large B cell lymphoma (DLBCL)
In a multicenter, open phase II clinical registration study (ICP-CL-00104) for evaluating the safety and efficacy of orelabrutinib in the treatment of relapsed or refractory marginal zone lymphoma (r/r MZL) , as of the data statistics to date, a total of 111 subjects received orelabrutinib treatment and all of them were included in the safety analysis set. The safety endpoints were set as adverse event, vital sign and abnormal laboratory result evaluated according to CTCAE V4. The median duration of treatment was 11.27 months (range: 0.1 -35.9 months) . 76.6%of the subjects were treated for more than 5.5 months, and 53.2%of the subjects were treated for at least 11.0 months. The overall subject compliance was good, with a median drug relative exposure of 99.40%. The safety analysis was performed in the safety analysis set of 111 cases. Safety indicators include adverse event, laboratory test, vital sign and electrocardiogram, etc. The results of ECG, clinical laboratory tests and other indicators, as well as their changes from baseline were summarized according to the visit, and all completed tests, cases and percentage were listed in the form of a cross-tab (according to the normal value range or the investigator's judgment of clinical significance) . The data of vital signs, including blood pressure, body temperature, heart rate and respiration, was described, including the values at each time point and the amount of change from the baseline.
In this study, the number and incidence of adverse events, study drug-related adverse events, serious adverse events, and adverse events leading to discontinuation of the trial drug are described. The incidence of adverse events is summarized according to CTCAE grade, and the subjects with all adverse events are listed. Meanwhile, the safety data of Ibrutinib Label FDA 2022 is combined, and the summary is as shown in Table 7.
Atrial fibrillation and atrial flutter are important adverse events of ibrutinib, and are related to its off-target effects. The reported incidence of grade ≥3 atrial fibrillation and/or atrial flutter is 3.7%in connection with ibrutinib. In contrast, no study drug-associated atrial fibrillation event of grade ≥3 was reported in this study. The second primary malignant tumor was an adverse event that required special attention for BTK inhibitors. One case of the second primary malignant tumor was observed in this trial, which was gastric adenocarcinoma and was related to the study drug. The second primary malignancies were observed with a minimum incidence of 10%in other BTK inhibitors. Diarrhea was rarely observed in all 111 subjects in this trial, with an incidence of only 3.6%, and most of the  events were at grade 1-2, similar to the MZL population, and significantly lower than the incidence of ibrutinib (43.0%) . Hypertension was another common adverse event in connection with ibrutinib, occurring in 14.0%of ibrutinib-treated subjects, of which 5.0%events were at grade 3/4. In this trial, 3 cases (2.7%) of hypertension occurred, and 2 cases (1.8%) of grade 3 and above occurred.
Table 7. List of common ≥10%treatment related adverse events (TRAEs) in treatment of relapsed and refractory MZL with BTK inhibitors (Orelabrutinib vs. Ibrutinib)

NA: Not available. 1Ibrutinib Label FDA 2022
The observed adverse events of orelabrutinib were mainly blood system toxicity and infection with the low overall incidence, and most of them recovered after clinical observation and symptomatic treatment, and no dose adjustment of orelabrutinib was required  (as shown in Table 8) . In conclusion, the treatment risks caused by orelabrutinib are all within the controllable range, it has excellent safety and overall tolerance.
Table 8. List of common hematology related laboratory test abnormalities in treatment of relapsed and refractory MZL by BTK inhibitors
(Orelabrutinib vs. Ibrutinib)
NA: Not available. 1Ibrutinib Label FDA 2022
This trial has verified that orelabrutinib was better tolerated than ibrutinib in subjects with relapsed or refractory MZL, and the incidence of most adverse events was lower than the reported results of other BTK inhibitors such as ibrutinib.
On the other hand, in a phase II, single-arm, open, multi-center study (L-MIND, study No. NCT02399085) for evaluating the safety and efficacy of treatment of patients with relapsed or refractory diffuse large B cell lymphoma (r/r DLBCL) with the combination of tafasitamab (former name: MOR00208) and lenalidomide, the combination of tafasitamab and lenalidomide was well tolerated, and the observed treatment emergent adverse events (TEAEs) during the treatment period reflect the established safety characteristics of lenalidomide.
The median duration of exposure in the study was 9.2 months (range: 0.2 -32.1 months) ; 30 (37.0%) patients received the treatment with the combination of tafasitamab and lenalidomide (12 cycles) . 34 (42.0%) patients continued to receive the treatment by the tafasitamab monotherapy starting from Cycle 13. Most TEAEs were CTCAE grade 1 (743/1464 case times) or 2 (428/1464 case times) . The most commonly reported grade ≥3 hematologic TEAEs were neutropenia (39 [48.1%] patients) , thrombocytopenia (14 [17.3%] patients) , febrile neutropenia (10 [12.3%] patients) , leukopenia (7 [8.6%] patients) and  anemia (6 [7.4%] patients) . The most commonly reported grade ≥3 nonhematologic TEAEs were pneumonia (5 [6.2%] patients) and hypokalemia (5 [6.2%] patients) .
In conclusion, the clinical data of orelabrutinib in marginal zone lymphoma shows that its safety is superior to that of ibrutinib, and the safety of tafasitamab combined with lenalidomide in diffuse large B cell lymphoma has also been demonstrated in L-MIND clinical trial. The clinical data supports that the clinical safety of the combined medicament of 3 drugs: orelabrutinib, tafasitamab and lenalidomide is superior to the combined medicament of 3 drugs: ibrutinib, tafasitamab and lenalidomide.
Example 9. Clinical data summary on effectiveness of single drug of orelabrutinib in treatment of relapsed/refractory marginal zone lymphoma (r/r MZL) 
In a phase II multicenter, open clinical trial (ICP-CL-00104) for evaluating the efficacy and safety of orelabrutinib in the treatment of relapsed/refractory marginal zone lymphoma (r/r MZL) , the dose of orelabrutinib was 150 mg, once daily (QD) . Up to the data statistics time, a total of 90 subjects diagnosed with MZL by the pathology center were included in the modified full analysis set (mFAS) . The primary study endpoint was the overall response rate (ORR) evaluated by the Independent Review Committee (IRC) according to Lugano 2014 criteria, and the secondary endpoints were ORR, progression-free survival (PFS) , duration of response (DOR) , time to onset (TTR) , time to progression (TTP) and overall survival (OS) , etc. Up to the data statistics time, the overall response rate (ORR) evaluated by IRC was 58.9% (95%CI: 48.0, 69.2) , and the best efficacy outcome was CR in 8 cases (8.9%) and PR in 45 cases (50.0%) . In mFAS, 83 subjects had previously received the anti-CD20 monoclonal antibody treatment. The ORR evaluated by IRC was 57.8% (95%CI: 46.5, 68.6) , the best efficacy outcome was CR in 8 cases (9.6%) and PR in 40 cases (48.2%) .
As mentioned above, orelabrutinib achieved the expected efficacy results in treatment of relapsed or refractory marginal zone lymphoma (MZL) at the QD administration of 150. The overall response rate was significantly increased compared to the historical data, and the clinical efficacy lasted for a long period. The study results are encouraging. In the modified full analysis set (mFAS) of MZL reviewed and confirmed by the Pathology Center review, 74 subjects (82.2%) had lymphoma stage ≥ III at the baseline. In subjects at the more advanced stage, generally, orelabrutinib still achieved encouraging efficacy results. Up to the date of data collection (median follow-up period: 18.9 months) , the overall response rates (ORR, 58.9%) evaluated by IRC was evidently superior to that of ibrutinib (46%) , and  the subjects with the complete responses (CR) and partial responses (PR) were improved compared with ibrutinib. As shown in Table 9, orelabrutinib showed satisfactory efficacy in the treatment of MZL subjects.
Table 9: Comparison of overall optimum efficacy of orelabrutinib and ibrutinib in treatment of relapsed or refractory marginal zone lymphoma (r/r MZL)
NE: Not equal. 1 Ibrutinib Label FDA 2022.2 (Noy et al., 2020)
Example 10. Preparing a solid dispersion of orelabrutinib
Orelabrutinib is prepared as a solid dispersion preparation to improve the drug solubility and in vivo absorption.
50 mg tablet orelabrutinib bulk drug and 200 mg excipient hydroxypropyl methylcellulose acetate succinate 912G/AS-MG are dissolved in methanol; methanol is used as a solvent in the preparation of solid dispersions and then removed in the process. The solid dispersions are obtained by spray drying, with the dispersion ratio of 1: 4. The solid dispersions are then mixed with 200 mg mannitol 200SD, 10 mg hypromellose EXF, 20 mg croscarmellose sodium (dry granulation part) to prepare granules by dry granulation process, mixed with 10 mg croscarmellose sodium SD-711, 6 mg silicon dioxide and 4 mg magnesium stearate and compressed into 12 mm round tablets, and finally bottled and label induced to obtain a finished product.
Example 11. Preparing tafasitamab for infusion
Anti-CD19 antibody tafasitamab is a white to pale yellow freeze-dried powder, and is  supplied in 20 mL disposable glass vial containing 200 mg of tafasitamab. It is resuspended in 5 mL of water for injection (WFI) to yield approximately 5.4 mL of preparation containing 40 mg/mL tafasitamab, 25 mM citrate buffer, 200 mM trehalose and 0.02% (w/v) polysorbate 20 (pH 6.0) . After resuspension, tafasitamab is transferred to a 250 mL infusion bag containing 0.9%sodium chloride injection for dilution, and then intravenously (i. v. ) administered for 2 hours continuously.
Example 12. Clinical protocol of tafasitamab and lenalidomide in combination with orelabrutinib to treat relapsed/refractory B cell non-Hodgkin’s lymphoma (r/r B-NHL)
This study is an open-label, single-arm, multi-cohort study to evaluate the safety and efficacy of tafasitamab and lenalidomide combined with orelabrutinib in patients with relapsed/refractory non-Hodgkin’s lymphoma (NHL) . The study protocol is summarized in FIG. 10. The doses of tafasitamab and lenalidomide are the same as approved, and the dose of orelabrutinib is 150 mg, once daily (QD) .
This study includes four cohorts of patients with relapsed/refractory B-NHL of different lymphoma histopathological types as follows:
Cohort A: Diffuse large B-cell lymphoma (DLBCL)
Cohort B: Mantle cell lymphoma (MCL)
Cohort C: Follicular lymphoma (FL)
Cohort D: Marginal zone lymphoma (MZL) .
10 to 20 patients are enrolled in each cohort. Subjects who meet the inclusion criteria enter the corresponding cohorts according to the histopathological types of lymphoma. All subjects receive 12 cycles of the combinatory therapy, followed by the maintenance with orelabrutinib until disease progression. The detailed dosing strategy is shown in FIG 10.
The primary study endpoint is the overall response rate (ORR) evaluated by investigators according to Lugano 2014 criteria, and the secondary endpoints are duration of response (DOR) , progression-free survival (PFS) , time to response (TTR) , overall survival (OS) , safety, and PK, etc. The efficacy results are qualitatively described with the number and percentage (95%confidence intervals) of the responders, who achieve complete response (CR) or partial response (PR) , using the Clopper-Pearson exact binomial distribution method. The time-to-event indicators such as DOR, PFS, TTR, and OS are statistically described using the Kaplan-Meier (KM) method.
REFERENCES
Awan, F.T., Lapalombella, R., Trotta, R., Butchar, J.P., Yu, B., Benson, D.M., Jr., Roda, J. M., Cheney, C., Mo, X., Lehman, A., et al. (2010) . CD19 targeting of chronic lymphocytic leukemia with a novel Fc-domain-engineered monoclonal antibody. Blood 115, 1204-1213.
Da Roit, F., Engelberts, P.J., Taylor, R.P., Breij, E.C., Gritti, G., Rambaldi, A., Introna, M., Parren, P.W., Beurskens, F.J., and Golay, J. (2015) . Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy. Haematologica 100, 77-86.
Dubovsky, J.A., Beckwith, K.A., Natarajan, G., Woyach, J.A., Jaglowski, S., Zhong, Y., Hessler, J.D., Liu, T.M., Chang, B.Y., Larkin, K.M., et al. (2013) . Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes. Blood 122, 2539-2549.
Gribben, J.G., Fowler, N., and Morschhauser, F. (2015) . Mechanisms of Action of Lenalidomide in B cell Non-Hodgkin Lymphoma. J Clin Oncol 33, 2803-2811.
Paltun, B., Kaski, S., and Mamitsuka, H. (2021) . Machine learning approaches for drug combination therapies. Briefings in Bioinformatics 22.
Ianevski, A., Giri, A.K., and Aittokallio, T. (2020) . SynergyFinder 2.0: visual analytics of multi-drug combination synergies. Nucleic Acids Res 48, W488-W493.
Kim, K., An, S., Cha, H.J., Choi, Y.M., Choi, S.J., An, I.S., Lee, H.G., Min, Y.H., Lee, S. J., and Bae, S. (2013) . Lenalidomide induces apoptosis and alters gene expression in non-small cell lung cancer cells. Oncol Lett 5, 588-592.
Kotla, V., Goel, S., Nischal, S., Heuck, C., Vivek, K., Das, B., and Verma, A. (2009) . Mechanism of action of lenalidomide in hematological malignancies. J Hematol Oncol 2, 36.
Lapalombella, R., Yu, B., Triantafillou, G., Liu, Q., Butchar, J.P., Lozanski, G., Ramanunni, A., Smith, L.L., Blum, W., Andritsos, L., et al. (2008) . Lenalidomide down-regulates the CD20 antigen and antagonizes direct and antibody-dependent cellular cytotoxicity of rituximab on primary chronic lymphocytic leukemia cells. Blood 112, 5180-5189.
Lazar, G.A., Dang, W., Karki, S., Vafa, O., Peng, J.S., Hyun, L., Chan, C., Chung, H.S., Eivazi, A., Yoder, S.C., et al. (2006) . Engineered antibody Fc variants with enhanced effector function. Proceedings of the National Academy of Sciences of the United States of America 103, 4005-4010.
Noy, A., de Vos, S., Coleman, M., Martin, P., Flowers, C.R., Thieblemont, C., Morschhauser, F., Collins, G.P., Ma, S., Peles, S., et al. (2020) . Durable ibrutinib responses in relapsed/refractory marginal zone lymphoma: long-term follow-up and biomarker analysis. Blood Adv 4, 5773-5784.
Yu, H., Wang, X., Li, J., Ye, Y., Wang, D., Fang, W., Mi, L., Ding, N., Wang, X., Song, Y., and Zhu, J. (2021) . Addition of BTK inhibitor orelabrutinib to rituximab improved anti-tumor effects in B cell lymphoma. Mol Ther Oncolytics 21, 158-170.

Claims (7)

  1. A method for treating a CD19 antigen-positive B cell tumor, comprising administering orelabrutinib and an anti-CD19 antibody or an antigen-binding fragment thereof, to a subject in need thereof, wherein the VH of the anti-CD19 antibody comprises CDR1 having the amino acid sequence of SEQ ID NO: 2, CDR2 having the amino acid sequence of SEQ ID NO: 3, and CDR3 having the amino acid sequence of SEQ ID NO: 4; and the VL of the anti-CD19 antibody comprises CDR1 having the amino acid sequence of SEQ ID NO: 6, CDR2 having the amino acid sequence of SEQ ID NO: 7, and CDR3 having the amino acid sequence of SEQ ID NO: 8.
  2. The method of claim 1, wherein the VH has the amino acid sequence of SEQ ID NO: 1, and VL has the amino acid sequence of SEQ ID NO: 5.
  3. The method of claim 1, wherein the anti-CD19 antibody is tafasitamab.
  4. The method according to any one of claims 1-3, wherein orelabrutinib is administered orally with a dose between 50 mg/day to 150 mg/day, and tafasitamab is administered intravenously with a dose between 12 mg/kg to 24 mg/kg per day, according to the body weight.
  5. The method according to any one of claims 1-3, further comprising administering lenalidomide to the subject.
  6. The method according to claim 5, wherein orelabrutinib is administered orally with a dose between 50 mg/day to 150 mg/day, tafasitamab is administered intravenously with a dose between 12 mg/kg to 24 mg/kg per day, according to the body weight, and lenalidomide is administered between 5 mg/day to 25 mg/day.
  7. The method according to any one of claims 1-6, wherein the B cell tumor is non-Hodgkin's lymphoma, chronic lymphocytic leukemia, or acute lymphoblastic leukemia.
PCT/CN2023/113559 2022-08-19 2023-08-17 Combination treatment with orelabrutinib and tafasitamab WO2024037594A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202263373032P 2022-08-19 2022-08-19
US63/373,032 2022-08-19
US202363493143P 2023-03-30 2023-03-30
US63/493,143 2023-03-30

Publications (1)

Publication Number Publication Date
WO2024037594A1 true WO2024037594A1 (en) 2024-02-22

Family

ID=89940756

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/113559 WO2024037594A1 (en) 2022-08-19 2023-08-17 Combination treatment with orelabrutinib and tafasitamab

Country Status (1)

Country Link
WO (1) WO2024037594A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180153892A1 (en) * 2015-05-26 2018-06-07 Morphosys Ag Combination of an anti-cd19 antibody and a bruton's tyrosine kinase inhibitor and uses thereof
US20220184208A1 (en) * 2020-12-04 2022-06-16 Morphosys Ag Anti-cd19 combination therapy

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180153892A1 (en) * 2015-05-26 2018-06-07 Morphosys Ag Combination of an anti-cd19 antibody and a bruton's tyrosine kinase inhibitor and uses thereof
US20220184208A1 (en) * 2020-12-04 2022-06-16 Morphosys Ag Anti-cd19 combination therapy

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
SALLES GILLES, DŁUGOSZ-DANECKA MONIKA, GHESQUIÈRES HERVÉ, JURCZAK WOJCIECH: "Tafasitamab for the treatment of relapsed or refractory diffuse large B-cell lymphoma", EXPERT OPINION ON BIOLOGICAL THERAPY, INFORMA HEALTHCARE, vol. 21, no. 4, 3 April 2021 (2021-04-03), pages 455 - 463, XP093140040, ISSN: 1471-2598, DOI: 10.1080/14712598.2021.1884677 *
SALLES GILLES; DUELL JOHANNES; GONZáLEZ BARCA EVA; TOURNILHAC OLIVIER; JURCZAK WOJCIECH; LIBERATI ANNA MARINA; NAGY ZSOLT; OB: "Tafasitamab plus lenalidomide in relapsed or refractory diffuse large B-cell lymphoma (L-MIND): a multicentre, prospective, single-arm, phase 2 study", THE LANCET ONCOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 21, no. 7, 5 June 2020 (2020-06-05), AMSTERDAM, NL , pages 978 - 988, XP086205226, DOI: 10.1016/S1470-2045(20)30225-4 *
SHIRLEY MATT: "Bruton Tyrosine Kinase Inhibitors in B-Cell Malignancies: Their Use and Differential Features", TARGETED ONCOLOGY, SPRINGER INTERNATIONAL PUBLISHING, CHAM, vol. 17, no. 1, 14 December 2021 (2021-12-14), Cham, pages 69 - 84, XP037673162, ISSN: 1776-2596, DOI: 10.1007/s11523-021-00857-8 *
SONG YUQIN, XU WEI, SONG YONGPING, LIU LIHONG, LIN SONG, LI ZHIMING, LIU TING, YI SHUHUA, ZHOU DAOBIN, ZHANG MINGZHI, HU YU, JIN J: "Pooled Analysis of Safety Data from Clinical Trials of Orelabrutinib Monotherapy in Hematologic Malignancies", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 136, no. Supplement 1, 5 November 2020 (2020-11-05), US , pages 43 - 43, XP093140037, ISSN: 0006-4971, DOI: 10.1182/blood-2020-140172 *
YU HUI, WANG XING, LI JIAO, YE YINGYING, WANG DEDAO, FANG WEI, MI LAN, DING NING, WANG XIAOGAN, SONG YUQIN, ZHU JUN: "Addition of BTK inhibitor orelabrutinib to rituximab improved anti-tumor effects in B cell lymphoma", MOLECULAR THERAPY - ONCOLYTICS, vol. 21, 1 June 2021 (2021-06-01), pages 158 - 170, XP093140038, ISSN: 2372-7705, DOI: 10.1016/j.omto.2021.03.015 *
ZHANG HONGJUAN, LIANG RUIXIA, XU HAIPENG, LI XIAORONG, ZHAO RENBIN, ZHANG JASON BIN, OUYANG DAVY XUESONG: "Abstract 4013: Combination of BTK inhibitor orelabrutinib, anti-CD19 antibody tafasitamab, and IMiD lenalidomide for the treatment of B cell malignancies", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 83, no. 7_Supplement, 4 April 2023 (2023-04-04), US , pages 4013 - 4013, XP093140036, ISSN: 1538-7445, DOI: 10.1158/1538-7445.AM2023-4013 *

Similar Documents

Publication Publication Date Title
JP6788600B2 (en) A combination of PD-1 antagonist and VEGFR / FGFR / RET tyrosine kinase inhibitor for the treatment of cancer
US20230087443A1 (en) Enhancement of cd47 blockade therapy by proteasome inhibitors
US20190315877A1 (en) Anti-dr5 antibodies and methods of use thereof
EP4321171A2 (en) Sirp alpha-antibody fusion proteins
US11779631B2 (en) CD47 blockade therapy by HDAC inhibitors
CN108136025A (en) A kind of novel method using immune modulating treatment cancer
CN106413751A (en) Combination of an anti-CCR4 antibody and a 4-1BB agonist for treating cancer
TW200521142A (en) CD40 antibody formulation and methods
JP2020522498A (en) Bispecific antibody that binds to CD123 CD3
WO2022052874A1 (en) Use of chiauranib in combination with immune checkpoint inhibitor in antitumor therapy
KR20210006405A (en) Inhibition of the combination of PD-1/PD-L1, TGFβ and DNA-PK for the treatment of cancer
US20220160700A1 (en) Therapeutic combination of quinoline derivative and antibody
US8992915B2 (en) Combination of CD37 antibodies with ICE
EP2849783A1 (en) Combination of cd37 antibodies with further agents
TW200904469A (en) Methods of treating cancer by administering human IL-18 combinations
CA3149209A1 (en) Methods and compositions for reducing immunogenicity by non-depletional b cell inhibitors
US20150231242A1 (en) Combination of cd37 antibodies with bendamustine
US20210130464A1 (en) Methods and Compositions for Reducing Immunogenicity By Non-Depletional B Cell Inhibitors
CN109311972A (en) For treating the method and composition of the sensitive and intractable chylous diarrhea of chylous diarrhea, non-chylous diarrhea seitan
WO2024037594A1 (en) Combination treatment with orelabrutinib and tafasitamab
KR20210016426A (en) Dosing of bispecific antibodies that bind CD123 and CD3
TW202408575A (en) Combination treatment with orelabrutinib and tafasitamab
US20160106837A1 (en) Combination of cd37 antibodies with chlorambucil
CN112439060B (en) New use of PD-L1 immunotherapy
WO2018201794A1 (en) Chimeric antigen receptor against human cd19 antigen and application thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23854493

Country of ref document: EP

Kind code of ref document: A1