Preventing or reducing adverse effects associated with NK cell-engaging agents

Abstract

The present invention relates to preventing or reducing adverse effects associated with NK cell-engaging agents, such as cytokine-associated infusion reactions. In particular, the present invention relates to preventing or reducing such side effects using inhibitors of Src, JAK and / or mTOR.

Description

[Technical field]

[0001] The present invention relates to the prevention or reduction of adverse effects associated with natural killer (NK) cell engaging agents, such as cytokine-associated infusion reactions. In particular, the present invention relates to the prevention or reduction of such side effects using inhibitors of Src, JAK and / or mTOR. [Background technology]

[0002] Natural killer (NK) cell-engaging agents, such as effector-enhancing antibodies, show considerable promise as cancer immunotherapy drugs. However, treatment with natural killer (NK) cell-engaging agents, such as effector-enhancing antibodies, may be associated with safety concerns due to cytokine release. A common adverse effect reported for NK cell-engaging agents, such as the antibody obinutuzumab, is infusion-related reaction (IRR), which may be caused by cytokine release. Symptoms of IRR are diverse and include fever, chills, headache, nausea, hypotension, dyspnea, fatigue, and / or diarrhea, and may be life-threatening (see, e.g., Snowden et al., International Journal of Nursing Practice (2015) 21 (Suppl. 2), 15-27). Approaches to reduce these severe toxicities are greatly needed.

[0003] The Src kinase inhibitor dasatinib has been identified as a promising candidate to prevent or reduce adverse effects, especially cytokine release syndrome (CRS), caused by T cell-engaging agents such as CAR-T cells (Weber et al., Blood Advances (2019) 3, 711-7; Mestermann et al., Sci Transl Med (2019) 11, eaau5907) as well as T cell bispecific antibodies (TCBs) (Leclercq et al., J Immunother Cancer (2020) 8 (Suppl 3): A690 (abstract 653)). Without distinguishing between desirable and undesirable activities of these agents, dasatinib completely switches off the functionality of CAR-T cells and TCB-induced T cells.

[0004] Methods for preventing or reducing the adverse effects of NK cell-engaging agents while maintaining their therapeutic effectiveness are highly desirable. Summary of the Invention

[0005] We have found that inhibitors of Src kinase (Src), Janus kinase (JAK) and / or mammalian target of rapamycin (mTOR) signaling can be used to alleviate CRS due to NK cell-engaging therapy. Src inhibitors such as dasatinib, mTOR inhibitors such as temsirolimus, sirolimus and everolimus, and JAK inhibitors such as ruxolitinib were found to potently prevent cytokine release induced by NK cell-engaging antibodies while simultaneously maintaining target cell killing mediated by such antibodies. These results provide evidence that the mechanisms of cytokine release associated with the development of IRR and target cell killing mediated by NK cell-engaging agents can be uncoupled, suggesting the use of inhibitors of Src kinase (Src), mammalian target of rapamycin (mTOR) and / or Janus kinase (JAK) as an attractive strategy for alleviating IRR associated with NK cell-engaging therapy.

[0006] Thus, in a first aspect, the present invention provides a natural killer (NK) cell-engaging agent for use in the treatment of a disease in an individual, wherein said treatment comprises: (a) administering to the individual a NK cell-engaging agent; and (b) administering to the individual an inhibitor of Src kinase (Src), Janus kinase (JAK) and / or mammalian target of rapamycin (mTOR) signaling. Includes.

[0007] The invention further provides the use of a NK cell-engaging agent in the manufacture of a medicament for the treatment of a disease in an individual, wherein said treatment comprises: (a) administering to the individual a NK cell-engaging agent; and (b) administering to the individual an inhibitor of Src, JAK and / or mTOR signaling. Includes.

[0008] The present invention also provides a method for the treatment of a disease in an individual, said method comprising: (a) administering to the individual a NK cell-engaging agent; and (b) administering to the individual an inhibitor of Src, JAK and / or mTOR signaling. Includes.

[0009] According to any of the above aspects, administration of an inhibitor of Src, JAK and / or mTOR signaling may be for the prevention or reduction of adverse effects associated with administration of an NK cell-engaging agent.

[0010] In another aspect, the present invention provides an inhibitor of Src, JAK and / or mTOR signaling for use in preventing or reducing adverse effects associated with the administration of an NK cell-engaging agent to an individual.

[0011] The invention further provides the use of an inhibitor of Src, JAK and / or mTOR signaling in the manufacture of a medicament for preventing or reducing adverse effects associated with the administration of an NK cell-engaging agent.

[0012] The present invention also provides a method for preventing or reducing adverse effects associated with administration of an NK cell-engaging agent to an individual, the method comprising administration to the individual of an inhibitor of Src, JAK and / or mTOR signaling.

[0013] The NK cell-engaging agents for use, inhibitors of Src, JAK and / or mTOR signaling for use, uses or methods described above and herein may incorporate, alone or in combination, any of the features described below (unless the context dictates otherwise).

[0014] Unless specifically defined herein, terms are used herein as commonly used in the art.

[0015] In some embodiments, the inhibitor of Src, JAK and / or mTOR signaling is a Src inhibitor.In more specific embodiments, the inhibitor of Src, JAK and / or mTOR signaling is a Src kinase inhibitor, particularly a small molecule Src kinase inhibitor.In certain embodiments, the inhibitor of Src, JAK and / or mTOR signaling is dasatinib.

[0016] Dasatinib is a Src kinase inhibitor and is sold under the trade name Sprycel® (among other things) for the treatment of certain cases of chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). Its CAS number, IUPAC name and chemical structure are shown below. CAS number: 302962-49-8 IUPAC name: N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide monohydrate Chemical structure: TIFF2024514281000001.tif27170

[0017] In some embodiments, the inhibitor of Src, JAK and / or mTOR signaling is an mTOR inhibitor. In more specific embodiments, the inhibitor of Src, JAK and / or mTOR signaling is an mTOR kinase inhibitor, in particular a small molecule mTOR kinase inhibitor.

[0018] "mTOR" stands for mammalian target of rapamycin (also known as FK506-binding protein 12-rapamycin complex-associated protein 1 (FRAP1)), a serine / threonine-specific protein kinase that belongs to the family of phosphatidylinositol 3-kinase (PI3K)-related kinases. It serves as a core component of two distinct protein complexes, mTOR complex 1 (TORC1) and mTOR complex 2 (TORC2), that regulate different cellular processes. Human mTOR is described in UniProt entry P42345 (version 218). mTOR inhibitors are compounds that inhibit mTOR. The most established inhibitors of mTOR are the so-called rapalogs, which are derivatives of rapamycin. Rapalogs include sirolimus, temsirolimus, everolimus, and ridaforolimus. Second-generation mTOR inhibitors are ATP-competitive mTOR kinase inhibitors designed to compete with ATP at the catalytic site of mTOR.

[0019] Exemplary mTOR inhibitors contemplated as useful in the present invention are provided in Table 1 below. JPEG2024514281000002.jpg232170

[0020] In some embodiments, the mTOR inhibitor is a derivative of rapamycin (also known as a rapalog).

[0021] In some embodiments, the mTOR inhibitor is selected from the group consisting of sirolimus, temsirolimus, everolimus and ridaforolimus, particularly the group consisting of sirolimus, temsirolimus, and everolimus.

[0022] In a particular embodiment, the mTOR inhibitor is sirolimus. In a further particular embodiment, the mTOR inhibitor is temsirolimus. In a further particular embodiment, the mTOR inhibitor is everolimus.

[0023] In some embodiments, the inhibitor of Src, JAK and / or mTOR signaling is a JAK inhibitor. In more specific embodiments, the inhibitor of Src, JAK and / or mTOR signaling is a JAK kinase inhibitor, in particular a small molecule JAK kinase inhibitor.

[0024] "JAK" stands for Janus kinase and refers to a family of intracellular non-receptor tyrosine kinases that transmit cytokine-mediated signals through the JAK / STAT pathway. JAKs have two nearly identical phosphoryl transfer domains, one that exhibits kinase activity and the other that negatively regulates the first kinase activity. The four JAK family members are JAK1, JAK2, JAK3, and TYK2 (tyrosine kinase 2). In certain embodiments herein, the JAK is JAK1 and / or JAK2 (JAK1 / 2). Human JAK1 and JAK2 are described in UniProt entries P23458 (version 221) and P60674 (version 224), respectively. JAK inhibitors (also called jakinibs) are compounds that inhibit the activity of one or more of the JAK family of enzymes (JAK1, JAK2, JAK3, TYK2), thereby disrupting the JAK / STAT signaling pathway.

[0025] Exemplary JAK inhibitors believed to be useful in the present invention are provided in Table 2 below. JPEG2024514281000003.jpg233170JPEG2024514281000004.jpg255169JPEG2024514281000005.jpg255163

[0026] In some embodiments, the JAK inhibitor is a JAK1 and / or JAK2 (JAK1 / 2) inhibitor. In some embodiments, the JAK inhibitor is selected from the group consisting of ruxolitinib, baricitinib, momelotinib, upadacitinib, filgotinib, abrocitinib, itacitinib, solcitinib, oclacitinib, fedratinib, gandotinib, lestaurtinib, and pacritinib.

[0027] In certain embodiments, the JAK inhibitor is a JAK1 and JAK2 inhibitor. In certain such embodiments, the JAK inhibitor is selected from the group consisting of ruxolitinib, baricitinib, and momelotinib.

[0028] In some embodiments, the JAK inhibitor is a JAK1 inhibitor. In certain such embodiments, the JAK inhibitor is selected from the group consisting of upadacitinib, filgotinib, abrocitinib, itacitinib, solcitinib, and oclacitinib.

[0029] In some embodiments, the JAK inhibitor is a JAK2 inhibitor. In certain such embodiments, the JAK inhibitor is selected from the group consisting of fedratinib, gandotinib, lestaurtinib, and pacritinib.

[0030] In certain embodiments, the JAK inhibitor is ruxolitinib.

[0031] In certain embodiments, the inhibitor of Src, JAK and / or mTOR signaling is selected from the group consisting of dasatinib, sirolimus, temsirolimus, everolimus, and ruxolitinib. In further particular embodiments, the inhibitor of Src, JAK and / or mTOR signaling is selected from the group consisting of dasatinib, sirolimus, and ruxolitinib.

[0032] In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of an activity of the NK cell-engaging agent. In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling does not cause inhibition of another activity of the NK cell-engaging agent. In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of a first activity of the NK cell-engaging agent but not a second activity of the NK cell-engaging agent. In some of these embodiments, the inhibition is complete inhibition.

[0033] In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of a first activity of the NK cell-engaging agent and inhibition of a second activity of the NK cell-engaging agent, wherein said inhibition of the first activity is stronger than said inhibition of the second activity. In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of a first activity of the NK cell-engaging agent and inhibition of a second activity of the NK cell-engaging agent, wherein said inhibition of the first activity is complete inhibition and said inhibition of the second activity is partial inhibition.

[0034] The "activity" of an NK cell-engaging agent refers to a response in an individual's body caused by the NK cell-engaging agent. Such activity includes the activation of NK cells, particularly CD16 + This may include the cellular response(s) of the NK cell, such as proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers, and / or the effect on target cells, particularly target cells (e.g., tumor cells) that express a target cell antigen of the NK cell-engaging agent, such as lysis of the target cell.

[0035] In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of cytokine secretion by immune cells, particularly NK cells (induced by the NK cell-engaging agent). In some embodiments, said immune cells are CD16 +The immune cells are. In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-6, IFN-γ, IL-8, TNF-α, IL-2, IL-12, IL-1β, MCP-1 and IL-10, particularly the group consisting of IL-6, IFN-γ, IL-8 and TNF-α. The immune cells may include various immune cell types, such as NK cells, macrophages, monocytes, T cells, etc. In some embodiments, the T cells are γδ T cells. In some embodiments, the inhibition is complete inhibition.

[0036] In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling does not cause inhibition of NK cell activation (induced by the NK cell-engaging agent). In some embodiments, said inhibition is complete. In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of NK cell activation (induced by the NK cell-engaging agent), where said inhibition is partial.

[0037] As used herein, "NK cell activation" or "NK cell activation" refers to the activation of NK cells, particularly CD16 + It refers to one or more cellular responses selected from proliferation, differentiation, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers of NK cells. Suitable assays for measuring NK cell activation are known in the art and described herein. In certain embodiments, NK cell activation is the expression of activation markers, in particular the expression of CD25 and / or CD69 (optionally measured by flow cytometry). In certain embodiments, NK cell activation is determined by measuring the expression of CD25 and / or CD69 on NK cells, for example by flow cytometry.

[0038] In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling does not cause inhibition of NK cell cytotoxic activity (induced by NK cell-engaging agent). In some embodiments, said inhibition is complete. In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of NK cell cytotoxic activity (induced by NK cell-engaging agent), where said inhibition is partial inhibition.

[0039] The "cytotoxic activity" of NK cells is a function of NK cells, especially CD16 + It refers to the induction of lysis (i.e., killing) of target cells by NK cells. Cytotoxic activity is typically accompanied by NK cell degranulation, which is associated with the release of cytotoxic effector molecules such as granzyme B and / or perforin from the NK cell.

[0040] In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of cytokine secretion by NK cells (induced by the NK cell-engaging agent) but does not cause inhibition of NK cell activation and / or cytotoxic activity (induced by the NK cell-engaging agent). In some of these embodiments, said inhibition is complete.

[0041] In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of cytokine secretion by NK cells (induced by the NK cell-engaging agent) and inhibition of activation and / or cytotoxic activity of NK cells (induced by the NK cell-engaging agent), wherein said inhibition of cytokine secretion is stronger than said inhibition of activation and / or cytotoxic activity. In some embodiments, (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of cytokine secretion by NK cells (induced by the NK cell-engaging agent) and inhibition of activation and / or cytotoxic activity of NK cells (induced by the NK cell-engaging agent), wherein said inhibition of cytokine secretion is complete inhibition and said inhibition of activation and / or cytotoxic activity is partial inhibition.

[0042] Inhibition herein can be partial inhibition or complete inhibition. Complete inhibition is stronger than partial inhibition. Partial inhibition in some embodiments is 30% or less, 40% or less, 50% or less, 60% or less, or 70% or less inhibition. In some embodiments, partial inhibition is 30% or less inhibition. In some embodiments, partial inhibition is 40% or less inhibition. In some embodiments, partial inhibition is 50% or less inhibition. In some embodiments, partial inhibition is 60% or less inhibition. In some embodiments, partial inhibition is 70% or less inhibition. Complete inhibition in some embodiments is at least 80%, at least 90%, or 100% inhibition. In some embodiments, complete inhibition is at least 80% inhibition. In some embodiments, complete inhibition is at least 90% inhibition. In some embodiments, complete inhibition is 100% inhibition. In some embodiments, partial inhibition is 50% or less inhibition and complete inhibition is at least 80% inhibition. In some embodiments, complete inhibition is clinically meaningful and / or statistically significant and / or partial inhibition is not clinically meaningful and / or statistically significant.

[0043] In some embodiments, the administration of an inhibitor of Src, JAK and / or mTOR signaling causes a decrease in serum levels of one or more cytokines in an individual. In some embodiments, the administration of an inhibitor of Src, JAK and / or mTOR signaling causes a decrease in secretion of one or more cytokines by immune cells, particularly NK cells, in an individual. In some embodiments, the immune cells are CD16 + The immune cells are. In some embodiments, the one or more cytokines are selected from the group consisting of IL-6, IFN-γ, IL-8, TNF-α, IL-2, IL-12, IL-1β, MCP-1 and IL-10, in particular the group consisting of IL-6, IFN-γ, IL-8 and TNF-α. The immune cells may include various immune cell types, such as NK cells, macrophages, monocytes, T cells, etc. In some embodiments, the T cells are γδ T cells.

[0044] In some embodiments, the reduction persists after the inhibitor of Src, JAK and / or mTOR signaling has not been administered (to the individual) for a period of time. In some embodiments, the period of time is about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 96 hours. In some embodiments, the reduction persists after subsequent administration of the NK cell-engaging agent. In particular, the reduction persists after administration of the inhibitor of Src, JAK and / or mTOR signaling has ceased / no further administration of the inhibitor of Src, JAK and / or mTOR signaling has been administered. Said reduction in serum levels / cytokine secretion is in particular compared to serum levels / cytokine secretion in an individual (including the same individual) who has not been administered an inhibitor of Src, JAK and / or mTOR signaling (i.e. in such cases the serum levels / cytokine secretion is reduced compared to serum levels / cytokine secretion without / before administration of an inhibitor of Src, JAK and / or mTOR signaling). Said reduction in serum levels / cytokine secretion is in particular compared to serum levels / cytokine secretion in an individual (including the same individual) who has been administered an NK cell-engaging agent (in particular the first administration) but without administration of an inhibitor of Src, JAK and / or mTOR signaling (i.e. in such cases the serum levels / cytokine secretion is reduced with / after administration of an NK cell-engaging agent but compared to serum levels / cytokine secretion without / before administration of an inhibitor of Src, JAK and / or mTOR signaling). In the absence of said reduction, serum levels / cytokine secretion may be particularly elevated / increased in association with (administration of) an NK cell-engaging agent. In some embodiments, the reduction is clinically meaningful and / or statistically significant. In some embodiments, the reduction is at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%. In some embodiments, the reduction is at least 30%. In some embodiments, the reduction is at least 40%. In some embodiments, the reduction is at least 50%.In some embodiments, the reduction is at least 60%. In some embodiments, the reduction is at least 70%.

[0045] In some embodiments, the (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of adverse effects associated with administration of the NK cell-engaging agent. In some embodiments, the (administration of) an inhibitor of Src, JAK and / or mTOR signaling does not cause inhibition of desired effects associated with administration of the NK cell-engaging agent. In some embodiments, the (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of adverse effects associated with administration of the NK cell-engaging agent, but does not cause inhibition of desired effects associated with administration of the NK cell-engaging agent. In some of these embodiments, the inhibition is complete inhibition. In some of these embodiments, the inhibition is clinically meaningful and / or statistically significant.

[0046] In some embodiments, the (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of adverse effects associated with administration of the NK cell-engaging agent and inhibition of the desired effect associated with administration of the NK cell-engaging agent, wherein said inhibition of adverse effects is stronger than said inhibition of the desired effect. In some embodiments, the (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of adverse effects associated with administration of the NK cell-engaging agent and inhibition of the desired effect associated with administration of the NK cell-engaging agent, wherein said inhibition of adverse effects is complete inhibition and said inhibition of beneficial effects is partial inhibition. In some embodiments, the (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of adverse effects associated with administration of the NK cell-engaging agent and inhibition of the desired effect associated with administration of the NK cell-engaging agent, wherein said inhibition of adverse effects is clinically meaningful and / or statistically significant inhibition and said inhibition of beneficial effects is not clinically meaningful and / or statistically significant inhibition.

[0047] A "desired effect", as used herein, is a beneficial and desired effect resulting from drug therapy, particularly in treating an individual with an NK cell-engaging agent, i.e., a therapeutic or prophylactic effect, such as, for example, killing tumor cells, reducing or slowing tumor growth, reducing tumor volume, reducing or preventing tumor metastasis, increasing progression-free or overall survival, alleviating disease symptoms, and the like.

[0048] An "adverse effect", which may also be referred to as a "side effect" or "adverse event" (particularly in clinical trials), as used herein, is a harmful and unwanted effect resulting from drug therapy in the treatment of an individual, particularly with an NK cell-engaging agent.

[0049] According to the present invention, the adverse effect is related to administration of the NK cell-engaging agent. In some embodiments, the adverse effect is related to the first administration of the NK cell-engaging agent. In some embodiments, the adverse effect occurs primarily or only upon the first administration of the NK cell-engaging agent. In some embodiments, the adverse effect occurs within 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 96 hours of administration of the NK cell-engaging agent, particularly the first administration. In some embodiments, particularly when only a single administration of the NK cell-engaging agent is administered (during the course of treatment with the NK cell-engaging agent), the adverse effect occurs within 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, or 21 days of administration of the NK cell-engaging agent.

[0050] In some embodiments, the adverse effect is an infusion-related reaction (IRR), particularly an IRR associated with cytokine release.

[0051] "Infusion-related reactions" (abbreviated as "IRR") refer to adverse effects associated with the (intravenous) administration of a therapeutic agent (e.g., an NK cell-engaging agent). IRRs always involve the immune system and are timely related to the administration of the therapeutic agent. They typically occur during or shortly after administration of the therapeutic agent, i.e., typically within 24 hours after administration (typically intravenous infusion), mainly at the first dose. In some cases, IRRs can also occur only later, e.g., several days after administration of the therapeutic agent. Usually, the incidence and severity decreases with subsequent doses. Symptoms range from symptomatic discomfort to fatal events and may include fever, chills, pyrexia, hypotension, hypoxia, dyspnea, flushing, skin rash, muscle pain, tachycardia, headache, dizziness, nausea, vomiting and / or organ failure. IRRs can be graded according to severity from grade 1 (mild) to grade 4 (life-threatening). See, e.g., Snowden et al., International Journal of Nursing Practice (2015) 21 (Suppl. 2), 15-27; Vogel, Clinical Journal of Oncology Nursing (2010) 14, E10-21. In certain embodiments herein, an IRR is caused by increased levels of cytokines, such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-6 (IL-6), interleukin-8 (IL-8), and the like, in the blood of a subject during or shortly after (e.g., within one day) administration of a therapeutic agent (e.g., an NK cell-engaging agent), resulting in adverse symptoms.

[0052] In some embodiments, the adverse effects are fever, hypotension and / or dyspnea.

[0053] In some embodiments, the adverse effect is an elevated serum level of one or more cytokines, particularly as compared to the serum level of a healthy individual and / or to the serum level of an individual (including the same individual) not administered the NK cell-engaging agent (i.e., in such cases, the serum level is elevated compared to the serum level in the absence of administration of the NK cell-engaging agent). In some embodiments, the one or more cytokines are selected from the group consisting of IL-6, IFN-γ, IL-8, TNF-α, IL-2, IL-12, IL-1β, MCP-1 and IL-10, particularly the group consisting of IL-6, IFN-γ, IL-8 and TNF-α.

[0054] In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is performed at the (clinical) onset of adverse effects (in an individual).The administration can be, for example, within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours or 24 hours after the onset of adverse effects (i.e., the occurrence of clinical symptoms of side effects such as fever).In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is in response to the (clinical) onset of adverse effects (in an individual).

[0055] In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is prior to the administration of the NK cell-engaging agent. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is concurrent with the administration of the NK cell-engaging agent. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is subsequent to the administration of the NK cell-engaging agent. When the administration of the inhibitor of Src, JAK and / or mTOR signaling is prior to or subsequent to the administration of the NK cell-engaging agent, such administration of the inhibitor of Src, JAK and / or mTOR signaling can be, for example, within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours or 24 hours prior to or subsequent to the administration of the NK cell-engaging agent, respectively. The administration of the inhibitor of Src, JAK and / or mTOR signaling can be intermittent or continuous. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is oral. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is parenteral, in particular intravenous.

[0056] In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose sufficient to cause inhibition of the activity of the NK cell-engaging agent. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose insufficient to cause inhibition of another activity of the NK cell-engaging agent. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose sufficient to cause inhibition of a first activity of the NK cell-engaging agent, but insufficient to cause inhibition of a second activity of the NK cell-engaging agent. In some of these embodiments, the inhibition is complete inhibition.

[0057] In some embodiments, administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose sufficient to cause inhibition of cytokine secretion by immune cells, particularly NK cells (induced by the NK cell-engaging agent).+ The immune cells are. In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-6, IFN-γ, IL-8, TNF-α, IL-2, IL-12, IL-1β, MCP-1 and IL-10, particularly the group consisting of IL-6, IFN-γ, IL-8 and TNF-α. The immune cells may include various immune cell types, such as NK cells, macrophages, monocytes, T cells, etc. In some embodiments, the NK cells are CD16 + In some embodiments, the T cells are NK cells. In some embodiments, the T cells are γδ T cells. In some embodiments, the inhibition is complete inhibition.

[0058] In some embodiments, administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose that is insufficient to cause inhibition of NK cell activation (induced by the NK cell-engaging agent), hi some embodiments, said inhibition is complete.

[0059] In some embodiments, administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose that is insufficient to cause inhibition of NK cell cytotoxic activity (induced by the NK cell-engaging agent), which in some embodiments is complete inhibition.

[0060] In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose sufficient to cause inhibition of cytokine secretion by NK cells (induced by the NK cell-engaging agent), but insufficient to cause inhibition of NK cell activation and / or cytotoxic activity (induced by the NK cell-engaging agent). In some of these embodiments, said inhibition is complete.

[0061] In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose sufficient to cause a decrease in serum levels of one or more cytokines in an individual. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose sufficient to cause a decrease in the secretion of one or more cytokines by immune cells, particularly NK cells, in an individual. In some embodiments, the immune cells are CD16 + The immune cells are. In some embodiments, the one or more cytokines are selected from the group consisting of IL-6, IFN-γ, IL-8, TNF-α, IL-2, IL-12, IL-1β, MCP-1 and IL-10, in particular the group consisting of IL-6, IFN-γ, IL-8 and TNF-α. The immune cells may include various immune cell types, such as NK cells, macrophages, monocytes, T cells, etc. In some embodiments, the T cells are γδ T cells.

[0062] In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose sufficient to cause inhibition of adverse effects associated with administration of the NK cell-engaging agent. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose insufficient to cause inhibition of desired effects associated with administration of the NK cell-engaging agent. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose sufficient to cause inhibition of adverse effects associated with administration of the NK cell-engaging agent, but at a dose insufficient to cause inhibition of desired effects associated with administration of the NK cell-engaging agent. In some of these embodiments, the inhibition is complete inhibition. In some of these embodiments, the inhibition is clinically meaningful and / or statistically significant.

[0063] In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is an effective dose.

[0064] An "effective amount" or "effective dose" of an agent, e.g., an inhibitor of Src, JAK and / or mTOR signaling or an NK cell-engaging agent, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0065] In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is at a dose equal to the dosage strength available for the inhibitor of Src, JAK and / or mTOR signaling. Usually, for a particular inhibitor of Src, JAK and / or mTOR signaling, several dosage strengths (i.e., dosage forms such as tablets or capsules containing a specific amount of active ingredient) are available. It will be most convenient to administer the inhibitor of Src, JAK and / or mTOR signaling at such (commercially) available dosage strength. For example, if the inhibitor of Src, JAK and / or mTOR signaling is dasatinib, it can be preferably administered at a dose of 20mg, 50mg, 70mg, 80mg, 100mg or 140mg, in particular 100mg (administration is preferably oral administration). For example, if the inhibitor of Src, JAK and / or mTOR signaling is everolimus, it can be preferably administered at a dose of 2.5mg, 5mg, 7.5mg or 10mg (administration is preferably oral administration). For example, when the inhibitor of Src, JAK and / or mTOR signaling is sirolimus, it can be preferably administered at a dose of 0.5mg, 1mg or 2mg (administration is preferably oral administration).For example, when the inhibitor of Src, JAK and / or mTOR signaling is ruxolitinib, it can be preferably administered at a dose of 5mg, 10mg, 15mg, 20mg or 25mg (administration is preferably oral administration).When the inhibitor of Src, JAK and / or mTOR signaling is temsirolimus, it can be preferably administered at a dose of 12.5mg or 25mg (administration is preferably intravenous administration, particularly using a solution of 25mg / ml active ingredient).

[0066] In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is daily. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is once a day. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is once a day at the dose as mentioned herein. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is for the duration of the adverse effect (i.e., the administration of the inhibitor of Src, JAK and / or mTOR signaling is from the onset of the adverse effect to the reduction or disappearance of the adverse effect). In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is stopped after the adverse effect is prevented or reduced. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is stopped after the reduction or disappearance of the adverse effect. The reduction is particularly clinically meaningful and / or statistically significant. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, particularly 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times during the course of treatment of the individual with the NK cell-engaging agent. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is once a day for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. The administration of the inhibitor of Src, JAK and / or mTOR signaling is generally concomitant with the administration of the NK cell-engaging agent. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is concomitant with a first administration of the NK cell-engaging agent, particularly the first administration of the NK cell-engaging agent in the course of treating an individual with the NK cell-engaging agent. In some embodiments, the administration of the inhibitor of Src, JAK and / or mTOR signaling is simultaneous with the first administration of the NK cell-engaging agent.In some embodiments, administration of the inhibitor of Src, JAK and / or mTOR signaling is performed before the first administration of the NK cell-engaging agent. In some embodiments, administration of the inhibitor of Src, JAK and / or mTOR signaling is after the first administration of the NK cell-engaging agent. In some embodiments, administration of the inhibitor of Src, JAK and / or mTOR signaling is after the first administration of the NK cell-engaging agent and before the second administration of the NK cell-engaging agent. When administration of the inhibitor of Src, JAK and / or mTOR signaling is before or after the (first) administration of the NK cell-engaging agent, such administration of the inhibitor of Src, JAK and / or mTOR signaling can be, for example, within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 48 ​​hours or 72 hours before or after administration of the NK cell-engaging agent, respectively.

[0067] In some embodiments, the administration of the NK cell-engaging agent is for a longer period of time than the administration of the inhibitor of Src, JAK and / or mTOR signaling. In some embodiments, the administration of the NK cell-engaging agent continues after the administration of the inhibitor of Src, JAK and / or mTOR signaling has stopped. In some embodiments, the administration of the NK cell-engaging agent is a single administration or repeated administration. In the course of treating an individual with the NK cell-engaging agent, the NK cell-engaging agent can be administered once or several times. For example, the treatment of an individual with the NK cell-engaging agent can include multiple treatment cycles, each of which includes one or more administrations of the NK cell-engaging agent. In some embodiments, the administration of the NK cell-engaging agent includes a first and a second administration.

[0068] For use in the present invention, the NK cell-engaging agents will be prepared, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration scheduling, and other factors known to the physician.

[0069] In some embodiments, the administration of the NK cell-engaging agent is at an effective dose. For systemic administration, the effective dose can be estimated initially from in vitro assays, such as cell culture assays. The IC determined in cell culture can then be used to determine the effective dose. 50 Doses may be formulated in animal models to achieve a blood concentration range including 0.1 mg / kg / day, 10 mg / kg / day, 20 mg / kg / day, 30 mg / kg / day, 40 mg / kg / day, 50 mg / kg / day, 60 mg / kg / day, 70 mg / kg / day, 80 mg / kg / day, 90 mg / kg / day, 100 mg / kg / day, 15 mg / kg / day, 100 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 30 mg / kg / day, 50 mg / kg / day, 100 mg / kg / day, 15 ...

[0070] An effective amount of the NK cell-engaging agent can be administered to prevent or treat a disease. The appropriate route of administration and dosage of the NK cell-engaging agent can be determined based on the type of disease to be treated, the type of NK cell-engaging agent, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician. Dosing can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses over various time points, bolus doses, and pulse infusions.

[0071] The NK cell-engaging agent and the inhibitor of Src, JAK and / or mTOR signaling can be administered by any suitable route, and may be administered by the same or different routes of administration. In some embodiments, administration of the NK cell-engaging agent is parenteral, particularly intravenous.

[0072] In some embodiments, the administration of the NK cell-engaging agent is the first administration of the NK cell-engaging agent to the individual, and particularly the first administration of the NK cell-engaging agent in the course of treatment of the individual with the NK cell-engaging agent.

[0073] In some embodiments, the (administration of) NK cell engaging agent induces (i.e., causes or increases) activation of NK cells. In some embodiments, the (administration of) NK cell engaging agent induces cytotoxic activity of NK cells. In some embodiments, the (administration of) NK cell engaging agent induces cytokine secretion by NK cells. In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-6, IFN-γ, IL-8, TNF-α, IL-2, IL-12, IL-1β, MCP-1 and IL-10, particularly the group consisting of IL-6, IFN-γ, IL-8 and TNF-α. In some embodiments, the NK cells are CD16 + They are NK cells.

[0074] In some embodiments, administration of the NK cell-engaging agent results in activation of NK cells, particularly at the site of the cancer (e.g., within a solid tumor cancer). The activation can include proliferation of NK cells, differentiation of NK cells, cytokine secretion by NK cells, release of cytotoxic effector molecules from NK cells, cytotoxic activity of NK cells, and expression of activation markers by NK cells. In some embodiments, administration of the NK cell-engaging agent results in an increase in the number of NK cells at the site of the cancer (e.g., within a solid tumor cancer).

[0075] The "NK cell-engaging agent" refers to an agent that binds to NK cells, particularly CD16 +It refers to an immunotherapeutic agent that exerts its effect through the activity of NK cells. Such activity of NK cells refers to the effects of NK cells, particularly CD16, on the proliferation, differentiation, expression of activation markers, cytokine secretion, release of cytotoxic effector molecules and / or cytotoxic activity. + It may include a cellular response(s) of NK cells.

[0076] The NK cell-engaging agent may induce or enhance the activity of NK cells through stimulation of CD16, particularly CD16a, on NK cells. Thus, in some embodiments, the NK cell-engaging agent is a CD16 binding agent. In such embodiments, the NK cell-engaging agent comprises an antigen-binding portion that binds to CD16, particularly CD16a, such as an Fc region or an antigen-binding domain of an antibody that binds to CD16.

[0077] CD16 (also known as Fcγ receptor III, FcγRIII) is a cell surface antigen expressed on certain immune cells. It exists both as a transmembrane form (CD16a, Fcγ receptor IIIa) expressed, for example, on NK cells and activated macrophages, and as a glycosylphosphatidylinositol (GPI)-anchored form (CD16b, FcγRIIIb) expressed on neutrophils. As used herein, "CD16" refers specifically to CD16a, also known as Fcγ receptor IIIa (see UniProt accession number P08637 [entry version 215] and SEQ ID NO: 11 for the human protein). Thus, the term "CD16 positive cells" or "CD16 + "Cells" refers to cells that express CD16, particularly CD16a.

[0078] In certain embodiments, the NK cell-engaging agent comprises an Fc region. In some embodiments, the NK cell-engaging agent is an antibody comprising an Fc region, particularly an IgG antibody comprising an Fc region, most particularly an IgG1 antibody comprising an Fc region. In some embodiments, the Fc region comprised in the NK cell-engaging agent is an IgG1 Fc region, particularly a human IgG1 Fc region. In some embodiments, the Fc region comprised in the NK cell-engaging agent is an IgG1 Fc region, particularly a human IgG1 Fc region.

[0079] The Fc region contained in the NK cell-engaging agent is capable of binding to CD16, i.e., the Fc region binds to CD16 (also referred to as a CD16-binding Fc region). Such an Fc region is also an effector-competent Fc region, i.e., an Fc region capable of inducing effector functions, in particular antibody-dependent cell-mediated cytotoxicity (ADCC).

[0080] The term "effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); cytokine secretion; immune complex-mediated antigen uptake by antigen-presenting cells; downregulation of cell surface receptors (e.g., B cell receptor); and activation of B cells.

[0081] Antibody-dependent cellular cytotoxicity (ADCC) is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells, particularly NK cells. As used herein, the term "increased ADCC" or "enhanced ADCC" is defined as either an increase in the number of target cells lysed in a given time at a given concentration of antibody in the medium surrounding the target cells by the mechanism of ADCC defined above, and / or a decrease in the concentration of antibody in the medium surrounding the target cells required to achieve the lysis of a given number of target cells in a given time by the mechanism of ADCC. The increase in ADCC is an increase over ADCC mediated by the same antibody produced by the same type of host cell that is not engineered, but using the same standard production, purification, formulation and storage methods (known to those skilled in the art). For example, the increase in ADCC mediated by an antibody produced by a host cell engineered by the methods described herein to have an altered glycosylation pattern (e.g., to express a glycosyltransferase, GnTIII, or other glycosyltransferase) is an increase relative to the ADCC mediated by the same antibody produced by a non-engineered host cell of the same type.

[0082] The assay for evaluating the ADCC activity of antibody is known in the art.The example of the in vitro assay for evaluating the ADCC activity of antibody is described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166,1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the antibody may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0083] The NK cell-engaging agents contemplated herein typically further comprise an antigen-binding portion that allows binding to a target cell antigen on a target cell, such as a tumor cell. Thus, in some embodiments, the NK cell-engaging agent binds to a target cell antigen. Such NK cell-engaging agents exert an effect on their target cells, such as lysis of the target cells, through the activity of NK cells.

[0084] As used herein, a "target cell antigen" refers to an antigenic determinant presented on the surface of a target cell, e.g., a cell in a tumor, such as a cancer cell or a cell of the tumor stroma (in which case a "tumor cell antigen"). Preferably, the target cell antigen is not CD16 and / or is expressed on a different cell than CD16. In some embodiments, the target cell antigen is CD20, particularly human CD20.

[0085] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a contiguous stretch of amino acids or a conformational configuration composed of distinct regions of non-contiguous amino acids) to which an antigen-binding moiety binds, forming an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virally infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM).

[0086] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that binds (including specifically binds) to an antigenic determinant. In some embodiments, an antigen-binding moiety can direct the entity to which it is attached (e.g., a second antigen-binding moiety) to a target site, e.g., a particular type of tumor cell bearing the antigenic determinant. Antigen-binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen-binding moieties include the antigen-binding domain of an antibody, including an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, an antigen-binding moiety may include an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of five isotypes: alpha, delta, epsilon, gamma, or mu. Useful light chain constant regions include any of two isotypes: kappa and lambda.

[0087] "Specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. As used herein, the term "bind" or "binding" generally refers to "specific binding". The ability of an antigen-binding moiety to bind to a specific antigenic determinant can be measured by either enzyme-linked immunosorbent assay (ELISA) or other techniques known to those skilled in the art, such as surface plasmon resonance (SPR) techniques (e.g., analyzed with a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In some embodiments, the degree of binding of the antigen-binding moiety to an unrelated protein is less than about 10% of the binding of the antigen-binding moiety to the antigen, as measured, for example, by SPR. In certain embodiments, an antigen-binding portion that binds to an antigen, or an antibody comprising the antigen-binding portion thereof, has a specific affinity to an antigen of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M to 10 -13 Up to M, for example 10 -9 M to 10 -13 Dissociation constant (K D ).

[0088] In certain embodiments, the NK cell engaging agent can simultaneously bind to an antigenic determinant on an NK cell (e.g., CD16, particularly CD16a) and an antigenic determinant on a target cell (e.g., a target cell antigen such as CD20). In some embodiments, the NK cell engaging agent can crosslink the NK cell and the target cell by simultaneous binding to CD16 and the target cell antigen. In some embodiments, such simultaneous binding results in lysis of the target cell, particularly a target cell antigen (e.g., CD20)-expressing tumor cell. In some embodiments, such simultaneous binding results in activation of the NK cell. In some embodiments, such simultaneous binding results in a cellular response of the NK cell selected from the group of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. In some embodiments, binding of the NK cell engaging agent to CD16 without simultaneous binding to the target cell antigen does not result in NK cell activation. In some embodiments, the NK cell engaging agent can direct the cytotoxic activity of the NK cell to the target cell.

[0089] Exemplary NK cell-engaging agents include antibodies, particularly effector-enhancing antibodies such as obinutuzumab, imgatuzumab, margetuximab, mogamulizumab, etc. These exemplary NK cell-engaging agents bind to CD16 (particularly CD16A) through (engineered) Fc domains. Further exemplary NK cell-engaging agents include antibodies that bind to CD16 (particularly CD16A) through the antigen-binding domain of the antibody, particularly bi / multispecific antibodies that bind to CD16 and a target cell antigen (e.g., a tetravalent bispecific antibody based on the ROCK® (Redirected Optimized Cell Killing; Affimed) platform that binds to CD16A and a target cell antigen through the antigen-binding domain). The NK cell engaging agent may also be a trispecific / trifunctional antibody that binds CD16 (particularly CD16A) and a second NK cell antigen, and a target cell antigen (e.g., an antibody based on the TriNKET™ (Trispecific NK Cell Engager Therapeutic; Dragonfly) platform that binds CD16A (through the Fc domain), NKG2D and a target cell antigen (through the antigen binding domain), or an antibody based on a trifunctional NK cell engager (NKCE; Innate Pharma) that binds CD16A (through the Fc domain), NKp46 and a target cell antigen (through the antigen binding domain)).

[0090] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0091] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure.

[0092] An "antibody fragment" is a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single domain antibodies. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments that have two antigen binding sites that can be bivalent or bispecific. See, for example, EP 404,097, WO 1993 / 01161, Hudson et al., Nat Med 9, 129-134 (2003), and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1). Antibody fragments may be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0093] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The "Kabat numbering" used herein with respect to variable region sequences refers to the numbering system described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0094] As used herein, amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system, referred to herein as "Kabat numbering" or "Kabat numbering", as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). In particular, the Kabat numbering system (see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3), which is further clarified herein by reference to "Kabat EU index numbering."

[0095] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence and determines antigen binding specificity, e.g., a "complementarity determining region" ("CDR"). Typically, antibodies contain six CDRs; three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include the following: (a) hypervariable loops located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs located at amino acid residues 24–34 (L1), 50–56 (L2), 89–97 (L3), 31–35b (H1), 50–65 (H2), and 95–102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact sites located at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262;732-745 (1996)).

[0096] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the designations of CDRs can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0097] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the HVR and FR sequences are generally represented in the following order in a VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0098] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by the antibody or immunoglobulin heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the various classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0099] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called the variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called the variable light domain or light chain variable region, followed by a constant light (CL) domain, also called the light chain constant region. The heavy chains of an immunoglobulin may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which may be further classified into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chains of an immunoglobulin may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Immunoglobulins essentially consist of two Fab molecules connected via an immunoglobulin hinge region and an Fc domain.

[0100] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, antibodies produced by a host cell may undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, an antibody produced by a host cell may contain a full-length heavy chain or a truncated variant of the full-length heavy chain. This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (K447) may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also supra).

[0101] In a particularly preferred embodiment, the Fc domain herein is a human IgG1 Fc domain. An exemplary sequence of a human IgG1 Fc region is given in SEQ ID NO:1.

[0102] In certain aspects of the invention, the NK cell-engaging agent is an antibody, particularly an effector-enhancing antibody.

[0103] Antibodies with enhanced effector functions, especially enhanced ADCC capabilities, are an emerging species in the field of cancer therapy. It has been recognized that the effector functions of antibodies mediated by their Fc regions are an important mechanism of action in antibody-based cancer therapy. Of particular importance in this regard is antibody-dependent cellular cytotoxicity (ADCC), i.e., the destruction of antibody-coated target cells (e.g., tumor cells) by NK cells (natural killer cells) and other immune effector cells, which is triggered when antibodies bound to the surface of the cells interact with activating Fc receptors on the effector cells.

[0104] Therefore, enhancing the ADCC activity of therapeutic antibodies has become of great interest, and various methods for enhancing ADCC have been described. For example, Shields et al. (J Biol Chem 9(2), 6591-6604(2001)) showed that amino acid substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues) improve ADCC. Alternatively, increased Fc receptor binding and effector function can be obtained by modifying the glycosylation of the antibody. IgG1 type antibodies, which are the most commonly used antibodies in cancer immunotherapy, have a conserved N-linked glycosylation site at Asn297 in each CH2 domain of the Fc region. The two complex biantennary oligosaccharides attached to Asn297 are embedded between the CH2 domains and form extensive contacts with the polypeptide backbone, and their presence is essential for antibodies to mediate effector functions, including ADCC (Lifely et al., Glycobiology 5, 813-822 (1995); Jefferis et al., Immunol Rev 163, 59-76 (1998); Wright and Morrison, Trends Biotechnol 15, 26-32 (1997)). Umana et al. (Nat Biotechnol 17, 176-180 (1999) and U.S. Pat. No. 6,602,684 (WO 99 / 54342), the entire contents of which are incorporated herein by reference, showed that overexpression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisected oligosaccharides, in Chinese hamster ovary (CHO) cells significantly increases the in vitro ADCC activity of antibodies produced in those cells. Overexpression of GnTIII in production cell lines generally results in antibodies that are nonfucosylated and rich in bisected oligosaccharides that are hybrid type. When mannosidase II (ManII) is overexpressed in production cell lines in addition to GnTIII, antibodies that are rich in bisected, nonfucosylated oligosaccharides of complex type are obtained (Ferrara et al., Biotechn Bioeng93,851-861(2006)).Both types of antibodies show a significant increase in ADCC compared to antibodies with unmodified glycans, but only antibodies with a majority of the N-glycans of the complex type are able to induce significant complement-dependent cytotoxicity (Ferrara et al., Biotechn Bioeng 93, 851-861 (2006)). Removal of fucose from the innermost N-acetylglucosamine residues of the oligosaccharide core appears to be a key factor for increased ADCC activity (Shinkawa et al., J Biol Chem 278, 3466-3473 (2003)). Therefore, further methods for producing antibodies with reduced fucosylation have been developed, including expression in α(1,6)-fucosyltransferase-deficient host cells (Yamane-Ohnuki et al., Biotech Bioeng 87, 614-622 (2004); Niwa et al., J Immunol Methods 306, 151-160 (2006)).

[0105] Several effector-enhanced antibodies have shown promising results in the clinic, including the glycoengineered anti-EGFR antibody imgatuzumab, as well as the glycoengineered anti-CD20 antibody obinutuzumab, which is marketed under the trade name Gazyva® / Gazyvaro® for the treatment of certain forms of follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL).

[0106] An "effector-enhanced antibody" as defined herein with respect to various embodiments of the invention is an antibody engineered to have increased effector function, particularly increased ADCC activity and / or increased CD16 (particularly CD16a) binding, compared to the corresponding non-engineered antibody. In some embodiments, the effector-enhanced antibody has an effector function that is increased by at least 2-fold, at least 10-fold, or even at least 100-fold compared to the corresponding non-engineered antibody. In particular embodiments, the increased effector function is increased binding to CD16, particularly CD16a, most particularly human CD16a. In some such embodiments, the binding affinity to CD16 is increased by at least 2-fold, particularly at least 10-fold, compared to the binding affinity of the corresponding non-engineered antibody. In some embodiments, the increased effector function is increased ADCC. In some such embodiments, the ADCC is increased by at least 2-fold, particularly at least 10-fold, compared to the ADCC mediated by the corresponding non-engineered antibody. In some embodiments, the increased effector function is increased binding to activating Fc receptors and increased ADCC.

[0107] Increased effector function can result, for example, from glycoengineering of the Fc region or introduction of amino acid mutations into the Fc region of an antibody. In some embodiments, the effector-enhanced antibody is engineered by introduction of one or more amino acid mutations in the Fc region. In some such embodiments, the amino acid mutation is an amino acid substitution. In certain such embodiments, the amino acid substitution is at position 298, 333, and / or 334 (EU numbering of residues) of the Fc region. Further suitable amino acid mutations are described, for example, in Shields et al., J Biol Chem 9(2), 6591-6604 (2001); U.S. Patent No. 6,737,056; WO 2004 / 063351 and WO 2004 / 099249. The mutant Fc region can be prepared by deletion, substitution, insertion or modification of amino acids using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The exact nucleotide changes can be confirmed, for example, by sequencing.

[0108] In some embodiments, the effector-enhanced antibody is engineered by modifying glycosylation in the Fc region. In certain embodiments, the effector-enhanced antibody is engineered to increase the proportion of non-fucosylated oligosaccharides in the Fc region compared to a non-engineered antibody. Increasing the proportion of non-fucosylated oligosaccharides in the Fc region of the antibody results in an antibody with increased effector function, particularly increased ADCC.

[0109] In certain embodiments, the effector-enhanced antibody is a glycoengineered antibody that comprises an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a non-glycoengineered antibody. In some such embodiments, the antibody is produced in a host cell engineered to have increased β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity compared to a non-engineered host cell. In more specific embodiments, the host cell is further engineered to have increased α-mannosidase II (ManII) activity compared to a non-engineered host cell. The host cell can be engineered to have increased β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity by overexpression of one or more polypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. Similarly, the host cell can be engineered to have increased α-mannosidase II (ManII) activity by overexpression of one or more polypeptides having α-mannosidase II (ManII) activity. This glycoengineering methodology is described in more detail in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540, WO 03 / 011878, the entire contents of each of which are incorporated herein by reference in their entirety.

[0110] In an alternative embodiment, the effector-enhanced antibody is a glycoengineered antibody that comprises an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a non-glycoengineered antibody, and the antibody is produced in a host cell with reduced α(1,6)-fucosyltransferase activity. The host cell with reduced α(1,6)-fucosyltransferase activity can be a cell in which the α(1,6)-fucosyltransferase gene has been disrupted or otherwise inactivated, e.g., knocked out (see Yamane-Ohnuki et al., Biotech Bioeng 87, 614 (2004); Kanda et al., Biotechnol Bioeng, 94(4), 680-688 (2006); Niwa et al., J Immunol Methods 306, 151-160 (2006)).

[0111] Other examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al., Arch Biochem Biophys 249, 533-545 (1986); U.S. Patent Application No. 2003 / 0157108; and WO 2004 / 056312, especially Example 11). Alternatively, the antibodies useful in the invention can be glycoengineered to reduce fucose residues in the Fc region, e.g., by reducing or abolishing the activity of GDP-fucose transporter protein in the host cell used for antibody production, according to the techniques disclosed in EP 1 176 195 A1, WO 03 / 084570, WO 03 / 085119, and U.S. Patent Application Publication Nos. 2003 / 0115614, 2004 / 093621, 2004 / 110282, 2004 / 110704, 2004 / 132140, U.S. Pat. No. 6,946,292 (Kyowa).

[0112] Glycoengineered antibodies useful in the present invention can also be produced in expression systems that produce modified glycoproteins, such as those taught in WO 03 / 056914 (GlycoFi, Inc.) or WO 2004 / 057002 and WO 2004 / 024927 (Greenovation).

[0113] In some embodiments, the effector-enhanced antibody is engineered to have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to a non-engineered antibody. In some embodiments, at least about 20%, about 40%, about 60% or about 80%, preferably at least about 40%, of the N-linked oligosaccharides in the Fc region of the effector-enhanced antibody are nonfucosylated. In some embodiments, about 40% to about 80% of the N-linked oligosaccharides in the Fc region of the effector-enhanced antibody are nonfucosylated. The nonfucosylated oligosaccharides may be hybrid or complex type.

[0114] In some embodiments, the effector-enhanced antibody is engineered to have an increased percentage of bisected oligosaccharides in the Fc region compared to a non-engineered antibody. In some embodiments, at least about 20%, about 40%, about 60% or about 80%, preferably at least about 40%, of the N-linked oligosaccharides in the Fc region of the effector-enhanced antibody are bisected. In some embodiments, about 40% to about 80% of the N-linked oligosaccharides in the Fc region of the effector-enhanced antibody are bisected. The bisected oligosaccharides can be hybrid or complex type.

[0115] In some embodiments, the effector-enhanced antibody is engineered to have an increased proportion of bisected, nonfucosylated oligosaccharides in the Fc region compared to a non-engineered antibody. In some embodiments, at least about 20%, about 40%, about 60% or about 80%, preferably at least about 40%, of the N-linked oligosaccharides in the Fc region of the effector-enhanced antibody are bisected and nonfucosylated. In some embodiments, about 40% to about 80% of the N-linked oligosaccharides in the Fc region of the effector-enhanced antibody are bisected and nonfucosylated. The bisected, nonfucosylated oligosaccharides can be hybrid or complex type.

[0116] In some embodiments, an effector-enhanced antibody is an antibody having at least about 20%, about 40%, about 60% or about 80% nonfucosylated oligosaccharides in its Fc region. In some embodiments, an effector-enhanced antibody is an antibody having at least about 40% nonfucosylated oligosaccharides in its Fc region. In some embodiments, an effector-enhanced antibody is an antibody having at least about 20%, about 40%, about 60% or about 80% bisected oligosaccharides in its Fc region. In some embodiments, an effector-enhanced antibody is an antibody having at least about 40% bisected, nonfucosylated oligosaccharides in its Fc region.

[0117] The oligosaccharide structure of the antibody Fc region can be analyzed by methods well known in the art, for example, MALDI TOF mass spectrometry as described in Umana et al., Nat Biotechnol 17, 176-180 (1999) or Ferrara et al., Biotechn Bioeng 93, 851-861 (2006). The percentage of nonfucosylated oligosaccharides is the amount of oligosaccharides lacking fucose residues relative to all oligosaccharides (e.g., complex, hybrid, high mannose structures) attached to Asn297, identified in N-glycosidase F treated samples by MALDI TOF MS. Asn297 refers to an asparagine residue located at about position 297 of the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located upstream or downstream of position 297, i.e., about ±3 amino acids between positions 294 and 300, due to minor sequence variations of the antibody. The percentage of bisected or bisected nonfucosylated oligosaccharides is determined similarly.

[0118] As used herein, the terms "engineer, engineered, engineering" are intended to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modification of the amino acid sequence, glycosylation pattern, or side groups of individual amino acids, and combinations of these approaches. In particular, "engineer" with the "glyco-" prefix, as well as the term "glycosylation engineering" includes metabolic engineering of the glycosylation machinery of a cell, including genetic engineering of the oligosaccharide synthesis pathway to achieve altered glycosylation of glycoproteins expressed in the cell. Additionally, glycosylation engineering includes the effects of mutations and the cellular environment on glycosylation. In some aspects, glycosylation engineering is a change in glycosyltransferase activity. Glycosyltransferases include β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), β(1,4)-galactosyltransferase (GalT), β(1,2)-N-acetylglucosaminyltransferase I (GnTI), β(1,2)-N-acetylglucosaminyltransferase II (GnTII) and α(1,6)-fucosyltransferase. In certain embodiments, the engineering results in altered glucosaminyltransferase activity and / or changes in fucosyltransferase activity (e.g., as described above).

[0119] "Increased binding", e.g., increased binding to CD16, refers to increased affinity for the respective interaction, e.g., as measured by SPR.

[0120] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). As used herein, unless otherwise specified, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D), and the dissociation rate constant and the association rate constant (k off and k on ) is the ratio of the rate constants. Thus, equivalent affinities may contain different rate constants, as long as the ratio of the rate constants is the same. Affinity can be measured by well-established methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0121] The binding affinity to CD16 can be easily determined, for example, by surface plasmon resonance (SPR) using standard equipment, such as a BIAcore instrument (GE Healthcare), and such CD16 may be obtained by recombinant expression. In some embodiments, the binding affinity to CD16 (particularly CD16a) is measured by surface plasmon resonance at 25°C.

[0122] In some embodiments, the effector-enhanced antibody is a full-length antibody. In some embodiments, the effector-enhanced antibody is an IgG antibody. In some embodiments, the effector-enhanced antibody is an IgG1 antibody. The effector-enhanced antibody comprises an Fc region, particularly an IgG Fc region, more particularly an IgG1 Fc region. In some embodiments, the Fc region is a human Fc region, particularly a human IgG Fc region, more particularly a human IgG1 Fc region.

[0123] Effector enhancing antibodies bind to target cell antigens on target cells, such as tumor cells.

[0124] In some embodiments, the effector enhancing antibody binds to CD20, particularly human CD20 (ie, the effector enhancing antibody is an anti-CD20 antibody, particularly an anti-human CD20 antibody).

[0125] "CD20", also known as "B-lymphocyte antigen B1", refers to any native CD20 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed CD20 as well as any form of CD20 resulting from processing of cells. The term also encompasses naturally occurring variants of CD20, such as splice variants or allelic variants. In some embodiments, the CD20 is human CD20. Human CD20 is described in UniProt (www.uniprot.org) accession number P11836 (entry version 202), and the amino acid sequence of human CD20 is also set forth in SEQ ID NO: 10.

[0126] In some embodiments, the NK cell-engaging agent is an effector-enhancing anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is an IgG antibody, particularly an IgG1 antibody. In some embodiments, the anti-CD20 antibody is a full-length antibody. In some embodiments, the anti-CD20 antibody comprises an Fc region, particularly an IgG Fc region, more particularly an IgG1 Fc region. In some embodiments, the anti-CD20 antibody comprises a human Fc region, particularly a human IgG Fc region, more particularly a human IgG1 Fc region. In some embodiments, the anti-CD20 antibody is engineered to have an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to an unengineered antibody. In some embodiments, at least about 40% of the N-linked oligosaccharides in the Fc region of the anti-CD20 antibody are non-fucosylated.

[0127] In some embodiments, the anti-CD20 antibody comprises a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3, and an HCDR3 of SEQ ID NO: 4, and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO: 5, an LCDR2 of SEQ ID NO: 6, and an LCDR3 of SEQ ID NO: 7. In some embodiments, the anti-CD20 antibody comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8, and / or a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD20 antibody comprises a heavy chain variable region sequence of SEQ ID NO: 8 and / or a light chain variable region sequence of SEQ ID NO: 9.

[0128] In a particular embodiment, the anti-CD20 antibody is obinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453). As used herein, obinutuzumab is synonymous with GA101. Trade names include GAZYVA® or GAZYVARO®.

[0129] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished using a variety of methods within the skill of the art, such as publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the ggsearch program in the FASTA package version 36.3.8c or followed by the BLOSUM50 comparison matrix. The FASTA program package is written by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et. al. (1997) Genomics 46:24-36, and is publicly available at http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml.Alternatively, sequences can be compared using the public server accessible at http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the program ggsearch(global protein:protein) and default options (BLOSUM50; open:-10; ext:-2; Ktup=2), ensuring global rather than local alignments. Percent amino acid identity is given in the output alignment header.

[0130] In some embodiments, the disease (treated by the NK cell-engaging agent) is cancer.

[0131] As used herein, "treatment" (and its grammatical variants, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed for prophylaxis or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, attenuation of any direct or indirect pathological consequences of the disease, prevention of metastasis, reducing the rate of disease progression, remission or alleviation of symptoms, and recovery or improved prognosis.

[0132] The term "cancer" refers to a physiological condition in mammals that is typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Non-limiting examples of cancer include blood cancers such as leukemia, bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, bile duct cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, skin cancer, squamous cell carcinoma, sarcoma, bone cancer, and kidney cancer. Other cell proliferative disorders include, but are not limited to, tumors located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, breast, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included.

[0133] In some embodiments, the cancer is one that expresses a target cell antigen of the NK cell engaging agent (e.g., an effector enhancing antibody).

[0134] In some embodiments, the cancer is a CD20-expressing cancer (in certain embodiments where the target cell antigen of the NK cell-engaging agent, e.g., effector-enhancing antibody, is CD20). By "CD20-positive cancer" or "CD20-expressing cancer" is meant a cancer characterized by expression or overexpression of CD20 on cancer cells. Expression of CD20 can be determined, for example, by quantitative real-time PCR (measuring CD20 mRNA levels), immunohistochemistry (IHC), or Western blot assay. In some embodiments, the cancer expresses CD20. In some embodiments, the cancer expresses CD20 on at least 20%, preferably at least 50% or at least 80% of tumor cells, as determined by immunohistochemistry (IHC) using an antibody specific for CD20.

[0135] In some embodiments, the cancer is a B-cell cancer, particularly a CD20 positive B-cell cancer. In some embodiments, the cancer is selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM) or Hodgkin's lymphoma (HL). In a particular embodiment, the cancer is selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL). In a more detailed embodiment, the cancer is FL. In some embodiments, the cancer is CLL.

[0136] In some embodiments, the cancer is treatable by the NK cell-engaging agent. In some embodiments, the NK cell-engaging agent is indicated for the treatment of cancer.

[0137] An "individual" or "subject" herein is a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. In some embodiments, the individual has a disease, particularly a disease treatable or to be treated by an NK cell-engaging agent. In some embodiments, the individual has a cancer, particularly a cancer treatable or to be treated by an NK cell-engaging agent. In particular, an individual herein is any single human subject eligible for treatment who is experiencing or has experienced one or more signs, symptoms, or other indicators of cancer. In some embodiments, the individual has cancer or has been diagnosed with cancer, particularly any of the cancers listed above. In some embodiments, the individual has locally advanced or metastatic cancer or has been diagnosed with locally advanced or metastatic cancer. The individual may have been previously treated with an NK cell-engaging agent (e.g., an effector-enhancing antibody) or another drug, or may have not received such treatment. In certain embodiments, the patient has not been previously treated with an NK cell-engaging agent (e.g., an effector-enhancing antibody). The patient may have been treated with a therapy including one or more drugs other than an NK cell-engaging agent (e.g., other than an effector-enhancing antibody) before NK cell-engaging agent therapy is initiated.

[0138] In some embodiments, the individual has elevated serum levels of one or more cytokines. In some embodiments, the elevated serum levels are associated with administration of an NK cell-engaging agent to the individual. The elevated serum levels are in particular compared to serum levels in healthy individuals and / or in individuals (including the same individual) to which the NK cell-engaging agent has not been administered (i.e., in such cases, the serum levels are elevated compared to serum levels in the absence of administration of the NK cell-engaging agent). In some embodiments, the one or more cytokines are selected from the group consisting of IL-6, IFN-γ, IL-8, TNF-α, IL-2, IL-12, IL-1β, MCP-1 and IL-10, in particular the group consisting of IL-6, IFN-γ, IL-8 and TNF-α.

[0139] The cytokine according to any of the embodiments of the present invention may be one or more cytokines selected from the group consisting of interleukin (IL)-6, interferon (IFN)-gamma, IL-8, tumor necrosis factor (TNF)-alpha, IL-2, monocyte chemoattractant protein (MCP)-1, IL-12, IL-1beta, and IL-10. In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-6, IFN-gamma, IL-8, and TNF-alpha, IL-2, and MCP-1. In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-6, IFN-gamma, IL-8, TNF-alpha, and MCP-1. In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-6, IFN-gamma, IL-8, and TNF-alpha. In some embodiments, the cytokine is IL-6. In some embodiments, the cytokine is IFN-gamma. In some embodiments, the cytokine is IL-8. In some embodiments, the cytokine is TNF-alpha. In some embodiments, the cytokine is MCP-1. In some embodiments, the cytokine is IL-1β. In some embodiments, the cytokine is IL-10. In some embodiments, the cytokine is IL-12. In some embodiments, the cytokine is IL-2.

[0140] Preferably, the NK cells according to any of the aspects of the invention are CD16 + They are NK cells.

[0141] In some embodiments, treatment with or administration of an NK cell-engaging agent may result in a response in an individual. In some embodiments, the response may be a complete response. In some embodiments, the response may be a response that persists after cessation of treatment. In some embodiments, the response may be a complete response that persists after cessation of treatment. In some embodiments, the response may be a partial response. In some embodiments, the response may be a partial response that persists after cessation of treatment. In some embodiments, treatment or administration with an NK cell-engaging agent and an inhibitor of Src, JAK and / or mTOR signaling may improve the response compared to treatment or administration of an NK cell-engaging agent alone (i.e., without an inhibitor of Src, JAK and / or mTOR signaling). In some embodiments, treatment or administration of an NK cell-engaging agent and an inhibitor of Src, JAK and / or mTOR signaling may increase the response rate in a patient population compared to a corresponding patient population treated with an NK cell-engaging agent alone (i.e., without an inhibitor of Src, JAK and / or mTOR signaling).

[0142] The NK cell-engaging agent may be used alone or together with other agents in treatment. For example, the NK cell-engaging agent may be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-cancer agent, such as a chemotherapeutic agent, an inhibitor of tumor cell proliferation, or an activator of tumor cell apoptosis. In particular, in a specific embodiment where the NK cell-engaging agent is an effector-enhancing anti-CD20 antibody (e.g., obinutuzumab), the additional therapeutic agent is selected from cyclophosphamide, doxorubicin, vincristine, prednisone or prednisolone, chlorambucil, or bendamustine. In some such embodiments, the additional therapeutic agent is a combination of chemotherapeutic agents, in particular a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone or prednisolone (CHOP), or a combination of cyclophosphamide, vincristine, and prednisone or prednisolone (CVP).

[0143] Inhibitors of Src, JAK and / or mTOR signaling can be used alone or together with one or more other agents to prevent or reduce adverse effects associated with administration of NK cell-engaging agents, particularly CRS. Inhibitors of Src, JAK and / or mTOR signaling can be used, for example, together with IL-6R antagonists (e.g., tocilizumab), steroids (e.g., corticosteroids such as methylprednisolone or dexamethasone) or TNF-α antagonists (e.g., etanercept).

[0144] TIFF2024514281000006.tif240170 [Brief description of the drawings]

[0145] [Figure 1]Effect of 100 nM ruxolitinib (ruxo), 100 nM sirolimus (siro), and 100 nM dasatinib (dasa) on obinutuzumab-induced CD19+ B cell depletion in a whole blood assay. Fresh whole blood was incubated with 100, 10, 1, and 0.1 μg / mL obinutuzumab or PGLALA IgG in the presence and absence of 100 nM ruxolitinib, 100 nM sirolimus, and 100 nM dasatinib for 48 hours. PGLALA IgG has a silent Fc region and is a negative control. At 48 hours, blood was collected, lysed, and the percentage of CD19+ B cells among live cells was measured by flow cytometry. n=mean of 2 donors. [Figure 2A-C] Effect of 100 nM ruxolitinib (ruxo), 100 nM sirolimus (siro), and 100 nM dasatinib (dasa) on obinutuzumab-induced IFN-γ (A), IL-2 (B), and TNF-α (C) for obinutuzumab at 100, 10, 1, and 0.1 μg / mL in a whole blood assay. Fresh whole blood was incubated with 100, 10, 1, and 0.1 μg / mL obinutuzumab or PGLALA IgG for 48 hours in the presence and absence of 100 nM ruxolitinib, 100 nM sirolimus, and 100 nM dasatinib. PGLALA IgG has a silent Fc region and is a negative control. At 24 hours, serum was collected and cytokines were analyzed by Luminex. n = mean of two donors. [Fig. 2D-F]Effect of 100 nM ruxolitinib (ruxo), 100 nM sirolimus (siro), 100 nM dasatinib (dasa) on obinutuzumab-induced IL-6 (D), IL-8 (E), and MCP-1 (F) for obinutuzumab at 100, 10, 1, and 0.1 μg / mL in a whole blood assay. Fresh whole blood was incubated with obinutuzumab or PGLALA IgG at 100, 10, 1, and 0.1 μg / mL in the presence and absence of 100 nM ruxolitinib, 100 nM sirolimus, and 100 nM dasatinib for 48 hours. PGLALA IgG has a silent Fc region and is a negative control. At 24 hours, serum was collected and cytokines were analyzed by Luminex. n = mean of two donors. EXAMPLES

[0146] The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0147] Example 1. The JAK1 / 2 inhibitor ruxolitinib, the mTOR inhibitor sirolimus, and the Src inhibitor dasatinib can prevent FcγR-mediated infusion reactions. The CD20-targeting effector-enhancing antibody obinutuzumab (Gazyva®) depletes B cells via FcγR signaling and may therefore be associated with the risk of infusion reactions characterized by cytokine release induced via FcγR signaling. To evaluate whether the mTOR inhibitor sirolimus, the JAK1 / 2 inhibitor ruxolitinib and the Src inhibitor dasatinib can prevent cytokine release induced via FcγR signaling, whole blood assays were performed using increasing doses of obinutuzumab in the presence and absence of sirolimus, ruxolitinib and dasatinib. The corresponding anti-CD20 IgG with silent Fc moiety (containing the "PGLALA" mutations L234A, L235A, P329G (Kabat EU numbering)) was used as a negative control ("PGLALA IgG"). In this assay, fresh whole blood is incubated with obinutuzumab at concentrations ranging from 100 μg / mL to 0.1 μg / mL in the presence and absence of 100 nM sirolimus, 100 nM ruxolitinib and 100 nM dasatinib. To evaluate the effect of different kinase inhibitors on cytokine release, serum was collected after 24 h and cytokines were analyzed by Luminex. To evaluate the effect of different kinase inhibitors on B cell depletion, blood was lysed after 48 h and B cell depletion was measured by flow cytometry.

[0148] As a result, treatment with 100 nM sirolimus, 100 nM ruxolitinib, and 100 nM dasatinib did not prevent B cell depletion induced by obinutuzumab at 48 hours (Figure 1). However, treatment with 100 nM dasatinib significantly reduced the levels of IFN-γ, IL-2, TNF-α, IL-6, IL-6, and MCP-1 (Figure 2A-F). Treatment with 100 nM sirolimus and 100 nM ruxolitinib also reduced the levels of IFN-γ, TNF-α, IL-6, IL-6, and MCP-1, and to a lesser extent, IL-2 (Figure 19A-F).

[0149] The mTOR inhibitor sirolimus, the JAK1 / 2 inhibitor ruxolitinib, and the Src inhibitor dasatinib reduced cytokine release, with the Src inhibitor being the most potent in reducing cytokine release among the three classes of kinase inhibitors. However, surprisingly, kinase inhibitors (including dasatinib) did not interfere with obinutuzumab-induced B cell depletion. Therefore, we propose that these kinase inhibitors may be used to reduce infusion reactions after treatment with antibody signaling via FcγR.

[0150] The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but these illustrations and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

Claims

1. 1. A medicament comprising a natural killer (NK) cell-engaging agent for use in the treatment of a disease in an individual, said treatment comprising: (a) administering to an individual an NK cell-engaging agent; and (b) administering to the individual an inhibitor of Src kinase (Src), Janus kinase (JAK) and / or mammalian target of rapamycin (mTOR) signaling. A pharmaceutical comprising:

2. The method of claim 1, wherein administration of an inhibitor of Src, JAK and / or mTOR signaling is for the prevention or reduction of adverse effects associated with administration of an NK cell-engaging agent.

3. A medicament comprising an inhibitor of Src, JAK and / or mTOR signalling for use in preventing or reducing adverse effects associated with the administration of an NK cell-engaging agent to an individual.

4. 4. The medicament of any one of claims 1 to 3, wherein the inhibitor of Src, JAK and / or mTOR signaling is an Src inhibitor, optionally dasatinib.

5. The pharmaceutical of any one of claims 1 to 3, wherein the inhibitor of Src, JAK and / or mTOR signaling is an mTOR inhibitor, optionally selected from the group consisting of sirolimus, temsirolimus, and everolimus.

6. 4. The medicament of any one of claims 1 to 3, wherein the inhibitor of Src, JAK and / or mTOR signaling is a JAK inhibitor, optionally a JAK1 and / or JAK2 inhibitor, optionally ruxolitinib.

7. 4. The pharmaceutical of any one of claims 1 to 3, wherein (administration of) an inhibitor of Src, JAK and / or mTOR signaling causes inhibition of adverse effects associated with administration of an NK cell-engaging agent, optionally wherein said inhibition is complete or is clinically meaningful and / or statistically significant.

8. The pharmaceutical of any one of claims 1 to 3, wherein (administration of) an inhibitor of Src, JAK and / or mTOR signaling does not cause inhibition of the desired effect associated with administration of an NK cell-engaging agent, optionally wherein said inhibition is complete inhibition or is clinically meaningful and / or statistically significant inhibition.

9. Adverse effects include: (i) Cytokine release syndrome (CRS); (ii) fever, hypotension and / or hypoxia; and / or (iii) elevated serum levels of one or more cytokines, in particular one or more cytokines selected from the group consisting of IL-6, IFN-γ, IL-8, TNF-α, IL-2, IL-12, IL-1β, MCP-1 and IL-10, in particular IL-6, IFN-γ, IL-8 and TNF-α. The pharmaceutical composition according to claim 2 or 3,

10. The method of claim 2 or 3, wherein the administration of the inhibitor of Src, JAK and / or mTOR signaling is performed upon (clinical) manifestation of adverse effects (in an individual).

11. Administration of an inhibitor of Src, JAK and / or mTOR signaling (i) before, concomitant with, or after administration of an NK cell-engaging agent; (ii) intermittent or continuous; and / or (iii) Oral or parenteral, particularly intravenous The pharmaceutical composition according to any one of claims 1 to 3,

12. 4. The pharmaceutical composition of claim 1, wherein administration of an inhibitor of Src, JAK and / or mTOR signaling is concomitant with a first administration of an NK cell-engaging agent, optionally prior to, simultaneously with or following the first administration of an NK cell-engaging agent.

13. Administration of a NK cell-engaging agent (i) at an effective dose; (ii) parenterally, especially intravenously; and / or (iii) A first administration of an NK cell-engaging agent to the individual. The pharmaceutical composition according to any one of claims 1 to 3,

14. The medicament according to any one of claims 1 to 3, wherein the NK cell-engaging agent is a CD16 binding agent and / or the NK cell-engaging agent comprises an Fc region, in particular an IgG Fc region, more particularly an IgG1 Fc region.

15. The method according to any one of claims 1 to 3, wherein the NK cell-engaging agent is an antibody, in particular an effector-enhancing antibody.

16. The method of claim 14, wherein the NK cell-engaging agent binds to a target cell antigen.

17. The pharmaceutical composition of claim 16, wherein the target cell antigen is CD20.

18. The pharmaceutical of any one of claims 1 to 3, wherein the NK cell-engaging agent is an effector-enhancing anti-CD20 antibody.

19. the anti-CD20 antibody comprises a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO:2, a HCDR2 of SEQ ID NO:3, and a HCDR3 of SEQ ID NO:4, and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO:5, a LCDR2 of SEQ ID NO:6, and a LCDR3 of SEQ ID NO:7; and / or The pharmaceutical of claim 18, wherein the anti-CD20 antibody comprises a heavy chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8, and / or a light chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

9.

20. The pharmaceutical of claim 18, wherein the anti-CD20 antibody is engineered to have an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a non-engineered antibody.

21. The pharmaceutical composition of claim 18, wherein the anti-CD20 antibody is obinutuzumab.

22. The pharmaceutical of any one of claims 1 to 3, wherein the disease (treated by the NK cell-engaging agent) is cancer.

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