Prevention or mitigation of adverse effects related to T cell engagers

Abstract

The present invention relates to the prevention or mitigation of adverse effects associated with T cell engagers such as cytokine release syndrome. Specifically, the present invention relates to the prevention or mitigation of such side effects using an inhibitor of NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

Description

Technical Field

[0001] The present invention relates to the prevention or reduction of adverse effects associated with T cell engagers such as cytokine release syndrome. Specifically, the present invention relates to the prevention or reduction of such side effects using an inhibitor of NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

Background Art

[0002] T cell engagers such as T cell bispecific antibodies (TCBs) or T cells expressing chimeric antigen receptors (CAR-T cells) are highly expected as cancer immunotherapeutic agents. T cell bispecific antibodies (TCBs) activate T cells by inducing the CD3ε chain of the T cell receptor and simultaneously bind to tumor-associated antigens on target cells. They enable the formation of an immunological synapse in which the release of pore-forming cytolytic proteins (perforin) and cytotoxic granules (granzyme B) induces target cell death. Several TCBs have been reported, such as CEA-TCB (sibisatamab; Bacac et al. (2016) Clin Cancer Res 22, 3286-97), CD20-TCB (glofitamab; Bacac et al. (2018) Clin Cancer Res 24, 4785-97), WT1-TCB (Augsberger et al. (2021) Blood 138, 2655-69) and BCMA-TCB (Seckinger et al. (2017) Cancer Cell 31, 396-410). Among these, glofitamab (CD20-TCB) has shown strong activity against diffuse large B cell lymphoma (DLBCL) and non-Hodgkin lymphoma (NHL) (Hutchings et al. (2021) J Clin Oncol 39, 1959-70).

[0003] Despite their promising activities, on-target activation of T cells is associated with the inherent risk of cytokine release syndrome (CRS), one of the most common safety hazards associated with treatment by T cell-induced therapies (see, for example, Shimabukuro-Vornhagen et al. (2018) J Immunother Cancer 6, 56). CRS is characterized by a cytokine storm resulting from the overactivation of T cells and innate immune cells, causing symptoms such as fever, hypotension, and respiratory failure, and in the worst cases, multiple organ failure. The ASTCT consensus classifies CRS into different grades based on clinical symptoms (fever, hypotension, and hypoxia). Regarding the management of grade 2 or higher CRS, this consensus recommends hospitalization of patients and the use of vasopressors and oxygen flow. High-dose glucocorticoids and / or blockade of IL-6R can also be utilized to alleviate symptoms (Lee et al. (2019), Biol Blood Marrow Transplant, 25, 625-38). In the specific case of CD3 bispecific antibodies, escalating or divided dosing schedules are widely used clinically to reduce the risk of first infusion cytokine storm that can be observed after uniform dosing. The pre-target cell depletion approach can also be applied to reduce the amount of circulating and tissue-resident CD20-expressing B cells, and thus suppress systemic on-target cytokine release by T cell-induced therapy against B cell malignancies. A clinically relevant example is pretreatment with obinutuzumab (Gazyva (登録商標) ) in combination with escalating dosing of glofitamab (CD20-TCB), which reduces the rate and severity of CRS. Anti-IL-6 treatment (e.g., with tocilizumab) and glucocorticoids are also used in the management of CRS. However, some patients are resistant to these approaches, and there is a growing need to develop new approaches for CRS reduction.

[0004] CRS symptoms are clinically managed, but these remain frequent dose-limiting safety hazards associated with the on-target activity of T cell engagers, putting patient safety at risk. Approaches to mitigate these life-threatening toxicities, particularly mitigation strategies that reduce cytokine release while maintaining treatment efficacy, are highly needed. SUMMARY OF THE INVENTION

[0005] The inventors have found that NLRP3 inhibitors can be used to reduce CRS by T cell-inducing therapies. NLRP3 inhibitors such as MCC950 have been found to reduce TCB-induced cytokine release while maintaining TCB-mediated T cell activation and target cell killing. These results suggest that NLRP3 inhibitors are an attractive and promising superior alternative or complement to currently used strategies such as steroids or IL-6 / IL-6R blockade for the reduction of CRS associated with T cell-inducing therapies.

[0006] Accordingly, in a first aspect, the present invention provides a T cell engager for use in the treatment of a disease in an individual, wherein the treatment comprises (a) administration of a T cell engager to the individual, and (b) administration of an NLRP3 inhibitor to the individual and.

[0007] The present invention further provides the use of a T cell engager in the manufacture of a medicament for the treatment of a disease in an individual, wherein the treatment comprises (a) administration of a T cell engager to the individual, and (b) administration of an NLRP3 inhibitor to the individual and.

[0008] The present invention also provides a method for the treatment of a disease in an individual, wherein the method comprises (a) administration of a T cell engager to the individual, and (b) administration of an NLRP3 inhibitor to the individual and.

[0009] According to any of the above aspects, administration of an NLRP3 inhibitor may be for preventing or reducing adverse effects associated with administration of a T cell engager.

[0010] In another aspect, the present invention provides an NLRP3 inhibitor for use in preventing or reducing adverse effects associated with administration of a T cell engager to an individual.

[0011] The present invention further provides the use of an NLRP3 inhibitor in the manufacture of a medicament for preventing or reducing adverse effects associated with administration of a T cell engager.

[0012] The present invention also provides a method for preventing or reducing adverse effects associated with administration of a T cell engager to an individual, the method comprising administration of an NLRP3 inhibitor to the individual.

[0013] The T cell engager for use, the NLRP3 inhibitor for use, the use or method described above and herein may incorporate any of the features described below, alone or in combination (unless the context otherwise indicates).

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

[0015] 「NLRP3」 refers to NACHT, LRR and PYD domain-containing protein 3 (also known as NOD, LRR and pyrin domain-containing protein 3, NLR (NOD-like receptor) family pyrin domain-containing 3, NOD-like receptor protein 3, or cryopyrin). NLRP3 is a three-element protein containing an amino-terminal pyrin domain (PYD), a central NACHT domain (the domain present in NAIP, CIITA, HET-E and TP1), and a carboxy-terminal leucine-rich repeat domain (LRR domain). The NACHT domain has ATPase activity essential for the self-association and function of NLRP3, while the LRR domain is thought to induce self-inhibition by folding back on the NACHT domain. NLRP3 is a component of the innate immune system and is mainly expressed in macrophages. It functions as an intracellular sensor (pattern recognition receptor (PRR)) that detects various pathogens and other damage-related signals, leading to the formation and activation of the NLRP3 inflammasome. The core of the NLRP3 inflammasome consists of NLRP3 as a signal sensor element, an adaptor (ASC, also known as PYCARD), and an effector pro-inflammatory caspase (caspase 1). The construction of the NLRP3 inflammasome results in the release of pro-inflammatory cytokines and pyroptotic cell death. For reviews, see, for example, Swanson et al. (2019) Nat Rev Immunol 19, 477-489. Human NLRP3 is also described in UniProt entry number Q96P20 (version 213), including its sequence.

[0016] An NLRP3 inhibitor is a compound that inhibits the NLRP3 inflammasome, particularly a small molecule pharmacological inhibitor. For example, an NLRP3 inhibitor can interfere with the NLRP3-NLRP3 interaction or the NLRP3-ASC interaction, or can be directed against the ATP-binding domain of NLRP3 that results in the blockade of its ATPase activity. For reviews, see, for example, Zahid et al. (2019) Frontiers Immunol 10, Art 2538.

[0017] The exemplary NLRP3 inhibitor is MCC950 (also known as CP-456,773), a direct inhibitor of NLRP3, which has been reported to target the NLRP3 ATP hydrolysis motif (Coll et al. (2019) Nat Chem Biol 15:556-9).

[0018] The chemical structure and CAS number of MCC950 are shown below:

Chem.

[0019] In some embodiments, the NLRP3 inhibitor is a small molecule NLRP3 inhibitor. In some embodiments, the NLRP3 inhibitor is a direct inhibitor of NLRP3. In some embodiments, the NLRP3 inhibitor blocks the ATPase domain of NLRP3. In some embodiments, the NLRP3 inhibitor is MCC950 or a derivative thereof (retaining NLRP3 inhibitory activity). In some embodiments, the NLRP3 inhibitor is MCC950, or a salt or ester thereof (e.g., the sodium salt of MCC950).

[0020] In some embodiments, the NLRP3 inhibitor is Cernoflast (recommended INN: List 87; WHO Drug Information, Vol. 36, No. 1, 2022, page 203), or a salt or ester thereof (e.g., the sodium salt of Cernoflast).

[0021] The chemical structure and CAS number of Cernoflast are shown below:

Chem.

[0022] In some embodiments, the NLRP3 inhibitor is emrenofrat (recommended INN: List 87; WHO Drug Information, Vol. 36, No. 1, 2022, page 104), or a salt or ester thereof (e.g., the sodium salt of emrenofrat).

[0023] The chemical structure and CAS number of emrenofrat are shown below:

Chemical formula

[0024] In some embodiments, the NLRP3 inhibitor is CY-09, or a salt or ester thereof (e.g., the sodium salt of CY-09).

[0025] The chemical structure and CAS number of CY-09 are shown below:

Chemical formula

[0026] Other exemplary NLRP3 inhibitors that may be useful in the present invention include IZD334 (also known as Somalix; Inflazome), IZD174 (also known as Inzomeride; Inflazome), DFV890 (also known as IFM-2427; IFM Therapeutics), IFM-632 (IFM Therapeutics), IFM-514 (IFM Therapeutics), JT194 (Jecure Therapeutics), JT349 (Jecure Therapeutics), NT-0167 (NodThera), NT-0796 (NodThera), NT-0249 (NodThera), VENT-01 (Ventus Therapeutics), VTX3232 (Ventyx Biosciences), VTX2735 (Ventyx Biosciences), BT032 (Bacainn Therapeutics), BT132 (Bacainn Therapeutics), OLT1177 (Olatec Therapeutics), ADS-032 (Adiso Therapeutics), ZYIL1 (Zydus Cadila), HY209 (Shaperon), AC-201 (TWi Biotechnology), AC-203 (TWi Biotechnology), and tranilast (also known as Rizaben; Kissei Pharmaceuticals), but are not limited thereto.

[0027] In some embodiments, the NLRP3 inhibitor is selected from the group consisting of MCC950, Cerenoflutamide, Emrenoflutamide, CY-09, IZD334, IZD174, DFV890, IFM-632, IFM-514, JT194, JT349, NT-0167, NT-0796, NT-0249, VENT-01, VTX3232, VTX2735, BT032, BT132, OLT1177, ADS-032, ZYIL1, HY209, AC-201, AC-203, and Tranilast. In some embodiments, the NLRP3 inhibitor is selected from the group consisting of MCC950, Cerenoflutamide, Emrenoflutamide, CY-09, IZD334, and IZD174. In some embodiments, the NLRP3 inhibitor is selected from the group consisting of MCC950, Cerenoflutamide, and Emrenoflutamide.

[0028] In some embodiments, administration of the NLRP3 inhibitor causes inhibition of the activity of the T cell engager. In some embodiments, administration of the NLRP3 inhibitor does not cause inhibition of another activity of the T cell engager. In some embodiments, administration of the NLRP3 inhibitor causes inhibition of a first activity of the T cell engager but does not cause inhibition of a second activity of the T cell engager. In some of these embodiments, the inhibition is complete inhibition. In some of these embodiments, the inhibition is partial inhibition. In some embodiments, administration of the NLRP3 inhibitor causes partial inhibition of a first activity of the T cell engager but does not cause inhibition of a second activity of the T cell engager.

[0029] In some embodiments, administration of the NLRP3 inhibitor causes inhibition of a first activity of the T cell engager and inhibition of a second activity of the T cell engager, where the inhibition of the first activity is stronger than the inhibition of the second activity. In some embodiments, administration of the NLRP3 inhibitor causes inhibition of a first activity of the T cell engager and inhibition of a second activity of the T cell engager, where the inhibition of the first activity is complete inhibition and the inhibition of the second activity is partial inhibition.

[0030] The "activity" of a T cell engager refers to the reaction in an individual's body caused by the T cell engager. Such activity includes cell responses of T cells, particularly CD4+ T cells and / or CD8+ T cells, such as proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers, and / or effects on target cells, particularly target cells (e.g., tumor cells) that express the target cell antigen of the T cell engager, such as lysis of the target cells.

[0031] In some embodiments, administration of an NLRP3 inhibitor causes inhibition of cytokine secretion by immune cells, particularly T cells (induced by a T cell engager). In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-1β, IL-6, and IL-8. The immune cells can include various immune cell types such as T cells, macrophages, monocytes, NK cells, etc. In some embodiments, the T cell is a CD8+ T cell or a CD4+ cell. In some embodiments, the inhibition is complete inhibition. In some embodiments, the inhibition is partial inhibition.

[0032] In some embodiments, administration of an NLRP3 inhibitor does not cause inhibition of activation of T cells (induced by a T cell engager). In some embodiments, the inhibition is complete inhibition. In some embodiments, administration of an NLRP3 inhibitor causes inhibition of activation of T cells (induced by a T cell engager), where the inhibition is partial inhibition.

[0033] As used herein, "activation of T cells" or "T cell activation" refers to one or more cellular responses selected from proliferation, differentiation, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers of T lymphocytes, particularly CD4+ or CD8+ T cells. Suitable assays for measuring T cell activation are known in the art and are described herein. In certain embodiments, T cell activation is the expression of activation markers, particularly the expression of CD25 and / or CD69 (optionally measured by flow cytometry). In certain embodiments, T cell activation is determined by measuring the expression of CD25 and / or CD69 on T cells, for example, by flow cytometry.

[0034] In some embodiments, administration of an NLRP3 inhibitor does not cause inhibition of the cytotoxic activity of T cells (induced by a T cell engager). In some embodiments, the inhibition is complete inhibition. In some embodiments, administration of an NLRP3 inhibitor causes inhibition of the cytotoxic activity of T cells (induced by a T cell engager), where the inhibition is partial inhibition.

[0035] The "cytotoxic activity" of T cells refers to the induction of lysis (i.e., death) of target cells by T lymphocytes, particularly CD4+ or CD8+ T cells. Cytotoxic activity typically involves degranulation of T lymphocytes associated with the release of cytotoxic effector molecules such as granzyme B and / or perforin from T lymphocytes.

[0036] In some embodiments, administration of an NLRP3 inhibitor causes inhibition of cytokine secretion by T cells (induced by a T cell engager), but does not cause inhibition of activation and / or cytotoxic activity of T cells (induced by a T cell engager). In some of these embodiments, the inhibition is complete inhibition. In some embodiments, the inhibition is partial inhibition. In some embodiments, administration of an NLRP3 inhibitor causes partial inhibition of cytokine secretion by T cells (induced by a T cell engager), but does not cause inhibition of activation and / or cytotoxic activity of T cells (induced by a T cell engager).

[0037] In some embodiments, administration of an NLRP3 inhibitor causes inhibition of cytokine secretion by T cells (induced by a T cell engager), and inhibition of activation and / or cytotoxic activity of T cells (induced by a T cell engager), where the inhibition of cytokine secretion is stronger than the inhibition of activation and / or cytotoxic activity. In some embodiments, administration of an NLRP3 inhibitor causes inhibition of cytokine secretion by T cells (induced by a T cell engager), and inhibition of activation and / or cytotoxic activity of T cells (induced by a T cell engager), where the inhibition of cytokine secretion is complete inhibition and the inhibition of activation and / or cytotoxic activity is partial inhibition.

[0038] Inhibition as used herein can be partial inhibition or complete inhibition. Complete inhibition is stronger inhibition than partial inhibition. Partial inhibition in some embodiments is inhibition of 30% or less, 40% or less, 50% or less, 60% or less, or 70% or less. In some embodiments, partial inhibition is inhibition of 30% or less. In some embodiments, partial inhibition is inhibition of 40% or less. In some embodiments, partial inhibition is inhibition of 50% or less. In some embodiments, partial inhibition is inhibition of 60% or less. In some embodiments, partial inhibition is inhibition of 70% or less. Complete inhibition in some embodiments is inhibition of at least 80%, at least 90%, or 100%. In some embodiments, complete inhibition is inhibition of at least 80%. In some embodiments, complete inhibition is inhibition of at least 90%. In some embodiments, complete inhibition is inhibition of 100%. In some embodiments, partial inhibition is inhibition of 50% or less and complete inhibition is inhibition of at least 80%. In some embodiments, complete inhibition is clinically meaningful and / or statistically significant and / or partial inhibition is not clinically meaningful and / or not statistically significant.

[0039] In some embodiments, administration of an NLRP3 inhibitor causes a decrease in the serum level of one or more cytokines in an individual. In some embodiments, administration of an NLRP3 inhibitor causes a decrease in the secretion of one or more cytokines by immune cells, particularly T cells, in an individual. In some embodiments, the one or more cytokines are selected from the group consisting of IL-1β, IL-6, and IL-8. Immune cells can include various immune cell types such as T cells, macrophages, monocytes, NK cells, and the like.

[0040] In some embodiments, the decrease persists after the NLRP3 inhibitor has not been administered to an individual for a given amount of time. In some embodiments, the amount 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 decrease also persists after subsequent administration of the T cell engager. In particular, the decrease persists even when administration of the NLRP3 inhibitor is stopped / no further administration of the NLRP3 inhibitor is performed. The decrease in serum levels / cytokine secretion is in particular compared to serum levels / cytokine secretion in individuals (including the same individual) not receiving the NLRP3 inhibitor (i.e., in such cases, serum levels / cytokine secretion is decreased compared to serum levels / cytokine secretion without / prior to administration of the NLRP3 inhibitor). The decrease in serum levels / cytokine secretion is in particular compared to serum levels / cytokine secretion in individuals (including the same individual) receiving administration of the T cell engager (in particular the first administration) but not receiving the NLRP3 inhibitor (i.e., in such cases, serum levels / cytokine secretion is decreased compared to serum levels / cytokine secretion with / after administration of the T cell engager but without / prior to administration of the NLRP3 inhibitor). Without the decrease, serum levels / cytokine secretion may increase / rise particularly in relation to administration of the T cell engager. In some embodiments, the decrease is clinically meaningful and / or statistically significant. In some embodiments, the decrease is at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%. In some embodiments, the decrease is at least 30%. In some embodiments, the decrease is at least 40%. In some embodiments, the decrease is at least 50%. In some embodiments, the decrease is at least 60%. In some embodiments, the decrease is at least 70%.

[0041] In some embodiments, administration of an NLRP3 inhibitor causes inhibition of adverse effects associated with administration of a T cell engager. In some embodiments, administration of an NLRP3 inhibitor does not cause inhibition of desired effects associated with administration of a T cell engager. In some embodiments, administration of an NLRP3 inhibitor causes inhibition of adverse effects associated with administration of a T cell engager, but does not cause inhibition of desired effects associated with administration of a T cell engager. In some of these embodiments, the above inhibition is complete inhibition. In some of these embodiments, the above inhibition is partial inhibition. In some embodiments, administration of an NLRP3 inhibitor causes partial inhibition of adverse effects associated with administration of a T cell engager, but does not cause inhibition of desired effects associated with administration of a T cell engager. In some of these embodiments, the above inhibition is clinically meaningful and / or statistically significant.

[0042] In some embodiments, administration of an NLRP3 inhibitor causes inhibition of adverse effects associated with administration of a T cell engager and inhibition of desired effects associated with administration of a T cell engager, where the inhibition of adverse effects is stronger than the inhibition of desired effects. In some embodiments, administration of an NLRP3 inhibitor causes inhibition of adverse effects associated with administration of a T cell engager and inhibition of desired effects associated with administration of a T cell engager, where the inhibition of adverse effects is complete inhibition and the inhibition of beneficial effects is partial inhibition. In some embodiments, administration of an NLRP3 inhibitor causes inhibition of adverse effects associated with administration of a T cell engager and inhibition of desired effects associated with administration of a T cell engager, where the above inhibition of adverse effects is clinically meaningful and / or statistically significant inhibition and the above inhibition of beneficial effects is not clinically meaningful and / or statistically significant inhibition.

[0043] As used herein, the "desired effect" is a beneficial and desired effect obtained from drug therapy in the treatment of an individual using a T cell engager, i.e., for example, a therapeutic or preventive effect such as the death of tumor cells, a decrease or delay in tumor growth, a decrease in tumor volume, a decrease or prevention of tumor metastasis, no disease progression or an increase in overall survival, or alleviation of disease symptoms.

[0044] The "adverse effect" may also be referred to as a "side effect" or "adverse event" (especially in clinical trials), and as used herein is an adverse and undesirable effect obtained from drug therapy in the treatment of an individual using a T cell engager.

[0045] According to the present invention, the adverse effect is related to the administration of the T cell engager. In some embodiments, the adverse effect is associated with the first administration of the T cell engager. In some embodiments, the adverse effect occurs at the time of the first administration of the T cell engager. In some embodiments, the adverse effect occurs primarily, or only at the time of the first administration of the T cell engager. In some embodiments, the adverse effect occurs within 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours of the administration of the T cell engager, particularly the first administration. In some embodiments, particularly when only a single administration of the T cell inducer is performed (during the course of treatment with the T cell engager), the adverse effect occurs within 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, or 21 days of the administration of the T cell engager.

[0046] In some embodiments, the adverse effect described above is cytokine release syndrome (CRS).

[0047] "Cytokine Release Syndrome" (abbreviated as "CRS") refers to an increase in the levels of cytokines such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-1β (IL-1β), interleukin-8 (IL-8) in the blood of a subject during or immediately after (e.g., within 1 day) administration of a therapeutic agent (e.g., a T cell engager), which causes harmful symptoms. CRS is an adverse reaction to a therapeutic agent and is timely related to the administration of the therapeutic agent. Typically, it occurs during or immediately after administration of the therapeutic agent, i.e., typically within 24 hours after administration (typically infusion), mainly at the first administration. In some cases, for example, after administration of CAR-T cells, CRS may also occur only during the proliferation of CAR-T cells, e.g., several days after administration. Usually, the incidence and severity decrease with subsequent administrations. The symptoms can range from symptomatic discomfort to fatal events and may include fever, chills, dizziness, hypertension, hypotension, hypoxia, dyspnea, restlessness, sweating, flushing, skin rash, tachycardia, tachypnea, headache, tumor pain, nausea, vomiting, and / or organ failure. CRS may be graded according to the modified cytokine release syndrome grading system established by Lee et al., Blood (2014) 124:188-195 or Lee et al., Biol Blood Marrow Transplant (2019) 25(4):625-638 (each incorporated herein by reference in its entirety). For a review of CRS, see, for example, Shimabukuro-Vornhagen et al., Journal for ImmunoTherapy of Cancer (2018) 6:56 (incorporated herein by reference in its entirety).

[0048] In some embodiments, the adverse effects are fever, hypotension, and / or hypoxia.

[0049] In some embodiments, the adverse effect is an elevated serum level of one or more cytokines. The elevated serum level is in particular compared to the serum level of a healthy individual and / or an individual who has not received a T cell engager (including the same individual) (i.e., in such a case, the serum level is elevated compared to the serum level in the absence of administration of the T cell engager). In some embodiments, the one or more cytokines are selected from the group consisting of IL-1β, IL-6 and IL-8.

[0050] In some embodiments, the administration of the NLRP3 inhibitor is at the time of (clinical) manifestation of the adverse effect (in the 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 manifestation of the adverse effect (i.e., the occurrence of clinical symptoms of side effects such as fever). In some embodiments, the administration of the NLRP3 inhibitor is in response to the (clinical) manifestation of the adverse effect (in the individual).

[0051] The administration of the NLRP3 inhibitor can be before, at the same time as and / or after the administration of the T cell engager. In some embodiments, the administration of the NLRP3 inhibitor is before the administration of the T cell engager. In some embodiments, the administration of the NLRP3 inhibitor is carried out at the same time as the administration of the T cell engager. In some embodiments, the administration of the NLRP3 inhibitor is after the administration of the T cell engager. When the administration of the NLRP3 inhibitor is before or after the administration of the T cell engager, such administration of the NLRP3 inhibitor 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 respectively before or after the administration of the T cell engager. The administration of the NLRP3 inhibitor can be intermittent or continuous. In some embodiments, the administration of the NLRP3 inhibitor is oral. In some embodiments, the administration of the NLRP3 inhibitor is parenteral, particularly intravenous.

[0052] In some embodiments, the administration of an NLRP3 inhibitor is at a dosage sufficient to cause inhibition of the activity of a T cell engager. In some embodiments, the administration of an NLRP3 inhibitor is at a dosage insufficient to cause inhibition of the activity of a T cell engager. In some embodiments, the administration of an NLRP3 inhibitor is at a dosage sufficient to cause inhibition of a first activity of a T cell engager but insufficient to cause inhibition of a second activity of the T cell engager. In some of these embodiments, the inhibition is complete inhibition. In some of these embodiments, the inhibition is partial inhibition. In some embodiments, the administration of an NLRP3 inhibitor is at a dosage sufficient to cause partial inhibition of a first activity of a T cell engager but insufficient to cause inhibition of a second activity of the T cell engager.

[0053] In some embodiments, the administration of an NLRP3 inhibitor is at a dosage sufficient to cause inhibition of cytokine secretion by immune cells, particularly T cells (induced by a T cell engager). In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-1β, IL-6, and IL-8. The immune cells can include various immune cell types such as T cells, macrophages, monocytes, NK cells, etc. In some embodiments, the T cell is a CD8+ T cell or a CD4+ cell. In some embodiments, the inhibition is complete inhibition. In some embodiments, the inhibition is partial inhibition.

[0054] In some embodiments, the administration of an NLRP3 inhibitor is at a dosage insufficient to cause inhibition of the activation of T cells (induced by a T cell engager). In some embodiments, the inhibition is complete inhibition. In some embodiments, the inhibition is partial inhibition.

[0055] In some embodiments, the administration of an NLRP3 inhibitor is at a dosage insufficient to cause inhibition of the cytotoxic activity of T cells (induced by a T cell engager). In some embodiments, the inhibition is complete inhibition. In some embodiments, the inhibition is partial inhibition.

[0056] In some embodiments, administration of an NLRP3 inhibitor is at a dose sufficient to cause inhibition of cytokine secretion by T cells (induced by a T cell engager), but is at a dose insufficient to cause inhibition of activation and / or cytotoxic activity of T cells (induced by a T cell engager). In some of these embodiments, the inhibition is complete inhibition. In some embodiments, the inhibition is partial inhibition. In some embodiments, administration of an NLRP3 inhibitor is at a dose sufficient to cause partial inhibition of cytokine secretion by T cells (induced by a T cell engager), but is at a dose insufficient to cause inhibition of activation and / or cytotoxic activity of T cells (induced by a T cell engager).

[0057] In some embodiments, administration of an NLRP3 inhibitor is at a dose sufficient to cause a decrease in the serum level of one or more cytokines in an individual. In some embodiments, administration of an NLRP3 inhibitor is at a dose sufficient to cause a decrease in the secretion of one or more cytokines by immune cells, particularly T cells, in an individual. In some embodiments, the one or more cytokines are selected from the group consisting of IL-1β, IL-6, and IL-8. The immune cells can include various immune cell types such as T cells, macrophages, monocytes, NK cells, and the like.

[0058] In some embodiments, the administration of an NLRP3 inhibitor is at a dosage sufficient to cause inhibition of the adverse effects associated with the administration of a T cell engager. In some embodiments, the administration of an NLRP3 inhibitor is at a dosage insufficient to cause inhibition of the desired effects associated with the administration of a T cell engager. In some embodiments, the administration of an NLRP3 inhibitor is at a dosage sufficient to cause inhibition of the adverse effects associated with the administration of a T cell engager, but at a dosage insufficient to cause inhibition of the desired effects associated with the administration of a T cell engager. In some of these embodiments, the inhibition is complete inhibition. In some of these embodiments, the inhibition is partial inhibition. In some embodiments, the administration of an NLRP3 inhibitor is at a dosage sufficient to cause partial inhibition of the adverse effects associated with the administration of a T cell engager, but at a dosage insufficient to cause inhibition of the desired effects associated with the administration of a T cell engager. In some of these embodiments, the inhibition is clinically meaningful and / or statistically significant.

[0059] In some embodiments, the administration of an NLRP3 inhibitor is at an effective dosage.

[0060] An “effective amount” or “effective dosage” of an agent, e.g., an NLRP3 inhibitor or a T cell engager, refers to an amount effective at dosages and for periods necessary to achieve the desired therapeutic or prophylactic result.

[0061] In some embodiments, the administration of an NLRP3 inhibitor is at a dosage equal to the dosage strength available for the NLRP3 inhibitor. Typically, for a given NLRP3 inhibitor, several dosage strengths (i.e., dosage forms such as tablets or capsules containing a specific amount of the active ingredient) are available. It would be most convenient to administer the NLRP3 inhibitor at such (commercially) available dosage strengths.

[0062] In some embodiments, the administration of the NLRP3 inhibitor is daily. In some embodiments, the administration of the NLRP3 inhibitor is once a day. In some embodiments, the administration of the NLRP3 inhibitor is once a day at a dosage as referred to herein. In some embodiments, the administration of the NLRP3 inhibitor is over a period during which the adverse effect persists (i.e., the administration of the NLRP3 inhibitor is from the onset of the adverse effect to the reduction or disappearance of the adverse effect). In some embodiments, the administration of the NLRP3 inhibitor is stopped after the adverse effect has been prevented or alleviated. In some embodiments, the administration of the NLRP3 inhibitor 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 NLRP3 inhibitor is one, two, three, four, five, six, seven, eight, nine, or ten times, particularly one, two, three, four, five, six, seven, eight, nine, or ten times during the treatment of an individual with a T cell bispecific antibody. In some embodiments, the administration of the NLRP3 inhibitor is for one, two, three, four, five, six, seven, eight, nine, or ten days. In some embodiments, the administration of the NLRP3 inhibitor is once a day for one, two, three, four, five, six, seven, eight, nine, or ten days. The administration of the NLRP3 inhibitor is generally concomitant with the administration of a T cell engager. In some embodiments, the administration of the NLRP3 inhibitor is related to the first administration of a T cell engager. The first administration is particularly the first administration of a T cell engager during the treatment of an individual with a T cell engager. The administration of the NLRP3 inhibitor can be before, simultaneous with, and / or after the first administration of the T cell engager. In some embodiments, the administration of the NLRP3 inhibitor is performed simultaneously with the first administration of the T cell engager. In some embodiments, the administration of the NLRP3 inhibitor is before the first administration of the T cell engager. In some embodiments, the administration of the NLRP3 inhibitor is after the first administration of the T cell engager. In some embodiments, the administration of the NLRP3 inhibitor is after the first administration of the T cell engager and before the second administration of the T cell engager.If the administration of the NLRP3 inhibitor is before or after the (first) administration of the T cell engager, such administration of the NLRP3 inhibitor 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 the administration of the T cell engager, respectively.

[0063] In some embodiments, the administration of the T cell engager is for a longer period than the administration of the NLRP3 inhibitor. In some embodiments, the administration of the T cell engager continues even after the administration of the NLRP3 inhibitor has been stopped. In some embodiments, the administration of the T cell engager is a single administration or repeated administrations. During the process of treating an individual with a T cell engager, the T cell engager can be administered one or more times. For example, the treatment of an individual with a T cell engager can include multiple treatment cycles each including one or more administrations of the T cell engager. In some embodiments, the administration of the T cell engager includes a first and a second administration.

[0064] For use in the present invention, the T cell engager will be prepared, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard 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 dosing schedule, and other factors known to the medical practitioner.

[0065] In some embodiments, the administration of the T cell engager is at an effective dose. In the case of systemic administration, the effective dose can first be estimated from in vitro assays such as cell culture assays. Then, the IC as determined in cell culture 50To achieve a blood concentration range that includes [the specific content], the dosage may be formulated in an animal model. Using such information, a useful dosage in humans can be determined more accurately. Also, the initial dosage can be estimated from in vivo data, for example, from an animal model, using techniques well known in the art. The dosage and dosing interval can be adjusted individually to provide a plasma level of the T cell engager sufficient to maintain a therapeutic effect. The normal patient dosage for administration by injection ranges from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. A therapeutically effective plasma concentration may be achieved by administering multiple doses each day. The level in plasma can be measured, for example, by HPLC.

[0066] For the prevention or treatment of a disease, an effective amount of a T cell engager can be administered. The appropriate route and dosage of the T cell engager can be determined based on the type of disease to be treated, the type of T cell engager, the severity and course of the disease, the clinical symptoms of the individual, the individual's clinical history and response to treatment, and the discretion of the attending physician. The dosing can be by any suitable route, for example, by injection such as intravenous injection or subcutaneous injection, depending in part on whether the administration is short-term or chronic. Various dosing schedules are contemplated herein, including single or multiple administrations at various time points, bolus administration, and pulse infusion, but are not limited thereto.

[0067] The T cell engager and the NLRP3 inhibitor can be administered by any suitable route and can be administered by the same or different administration routes. In some embodiments, the administration of the T cell engager is parenteral, particularly intravenous.

[0068] In some embodiments, the administration of the T cell engager is the first administration of the T cell engager to the individual, particularly the first administration of the T cell engager in the course of treating the individual with the T cell engager.

[0069] In some embodiments, administration of the T cell engager induces (i.e., causes or increases) activation of T cells. In some embodiments, administration of the T cell engager induces cytotoxic activity of T cells. In some embodiments, administration of the T cell engager induces cytokine secretion by T cells. In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-2, IL-6, IL-1β, IL-8, IFN-γ, IP-10, TNF-α, IL-1Ra, MCP-1 and MIP-1α. In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-1β, IL-6 and IL-8. In some embodiments, the T cells are CD8+ T cells or CD4+ cells.

[0070] In some embodiments, administration of the T cell engager results in activation of T cells, particularly cytotoxic T cells, at the site of cancer, e.g., within a solid tumor cancer. Activation can include proliferation of T cells, differentiation of T cells, cytokine secretion by T cells, release of cytotoxic effector molecules from T cells, cytotoxic activity of T cells, and expression of activation markers by T cells. In some embodiments, administration of the T cell engager results in an increase in the number of T cells, particularly cytotoxic T cells, at the site of cancer, e.g., within a solid tumor cancer.

[0071] A "T cell engager" refers to an immunotherapeutic agent that exerts its effect through the activity of T cells, particularly cytotoxic T cells. Such activity of T cells can include cellular responses of T cells, particularly CD4+ T cells and / or CD8+ T cells, such as proliferation, differentiation, expression of activation markers, cytokine secretion, release of cytotoxic effector molecules, and / or cytotoxic activity. T cell engagers contemplated herein typically include an antigen-binding portion that enables binding to a target cell antigen on a target cell, such as a tumor cell. Such T cell engagers exert their effect on those target cells, such as lysis of the target cells, through the activity of T cells. Exemplary T cell engagers include T cell bispecific antibodies, chimeric antigen receptor (CAR)-expressing T cells (CAR-T cells), and ImmTAC (‘‘Immune mobilising monoclonal T-cell receptors Against Cancer’’, a bispecific fusion protein of an engineered TCR and an antibody fragment that can bind to T cells and target cells) or TCR-modified T cells (TCR-T cells) characterized by an engineered T cell receptor that can bind to a specific antigenic determinant on a target cell, such as TCR-based approaches.

[0072] In certain embodiments of the invention, the T cell engager is a T cell bispecific antibody.

[0073] In other embodiments, the T cell engager is a CAR-T cell. In some embodiments, the T cell engager is an ImmTAC. In some embodiments, the T cell engager is a TCR-T cell.

[0074] The T cell bispecific antibodies that can be used in the present invention are described below.

[0075] The term "T cell bispecific antibody" means an antibody that is capable of binding, including simultaneously binding to T cells (usually via an antigen determinant expressed on T cells such as CD3) and target cells (usually via an antigen determinant expressed on target cells such as CD20).

[0076] In a preferred embodiment according to the present invention, the T cell bispecific antibody is capable of simultaneously binding to an antigen determinant on a T cell (i.e., a first antigen such as CD3) and an antigen determinant on a target cell (i.e., a second antigen such as CD20). In some embodiments, the T cell bispecific antibody is capable of bridging T cells and target cells by simultaneous binding to CD3 and the target cell antigen. In an even more preferred embodiment, such simultaneous binding results in lysis of the target cell, particularly a tumor cell expressing the target cell antigen (e.g., CD20). In some embodiments, such simultaneous binding results in activation of the T cells. In some embodiments, such simultaneous binding results in a cellular response of the T cells selected from the group consisting of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In some embodiments, binding of the T cell bispecific antibody to CD3 without simultaneous binding to the target cell antigen does not result in T cell activation. In some embodiments, the T cell bispecific antibody is capable of redirecting the cytotoxic activity of the T cells to the target cells. In a preferred embodiment, the above redirection is independent of MHC-mediated peptide antigen presentation by the target cells and / or the specificity of the T cells.

[0077] The term "bispecific" means that the antibody is capable of binding to at least two different antigen determinants. Typically, a bispecific antibody contains two antigen-binding sites, each of which is specific for a different antigen determinant. In some embodiments, the bispecific antibody is capable of binding together two antigen determinants, particularly two antigen determinants expressed on two separate cells.

[0078] 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 three-dimensional structure composed of a contiguous stretch of amino acids or different regions of non-contiguous amino acids) to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-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).

[0079] 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, the antigen-binding moiety can direct the entity to which it is attached (e.g., a second antigen-binding moiety) to a target site, such as a particular type of tumor cell having an antigenic determinant. In further embodiments, the antigen-binding moiety can activate signal transduction via its target antigen, such as a T cell receptor complex antigen. The antigen-binding moiety includes antibodies and fragments thereof as further defined herein. A particular antigen-binding moiety includes the antigen-binding domain of an antibody, which includes the variable region of the antibody heavy chain and the variable region of the antibody light chain. In some embodiments, the antigen-binding moiety can include an antibody constant region known in the art, as further defined below. Useful heavy chain constant regions include any of the five isotypes of α, δ, ε, γ, or μ. Useful light chain constant regions include any of the two isotypes of κ and λ.

[0080] "Specifically bind" means that the binding is antigen - selective and can be distinguished from unwanted or non - specific interactions. In this specification, the terms "bind" or "binding" generally refer to "specific binding". The binding ability of an antigen - binding portion to a specific antigen determinant can be measured by enzyme - linked immunosorbent assay (ELISA) or other techniques well - known to those skilled in the art, such as surface plasmon resonance (SPR) technology (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 portion to an irrelevant protein is less than about 10% of the binding of the antigen - binding portion to the antigen measured, for example, by SPR. In some embodiments, an antigen - binding portion that binds to an antigen, or an antibody containing the antigen - binding portion, has a dissociation constant (K -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M) of D .

[0081] "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). Unless otherwise indicated, the "binding affinity" used in this specification refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of the binding pair (e.g., an antigen - binding portion and an antigen, or a receptor and its ligand). The affinity of molecule X for binding partner Y is generally represented by the dissociation constant (K D ), which is related to the dissociation rate constant and the association rate constant (k off and k on) ratio. Therefore, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains 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).

[0082] "CD3" refers to any native CD3 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 indicated. This term encompasses "full-length" untreated CD3, as well as any form of CD3 resulting from intracellular processing. This term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In some embodiments, CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in UniProt (www.uniprot.org) accession number P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. See also SEQ ID NO: 1. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank accession number BAB71849.1. See also SEQ ID NO: 2.

[0083] As used herein, "target cell antigen" refers to an antigenic determinant presented on the surface of a target cell, such as a cell in a tumor, e.g., a cancer cell or a cell of the tumor stroma (in which case, "tumor cell antigen"). Preferably, the target cell antigen is not CD3 and / or is expressed on a cell different from CD3. In some embodiments, the target cell antigen is CD20, particularly human CD20.

[0084] As used herein, terms such as "first," "second," or "third" with respect to antigen-binding portions, etc. are used for convenience in distinguishing when more than one of each type of portion is present. The use of these terms is not intended to impart a particular order or orientation to bispecific antibodies, unless otherwise indicated.

[0085] As used herein, the term "valence" means the presence of a specified number of antigen-binding sites in an antibody. Thus, the term "monovalent binding to an antigen" means the presence of one (and no more than one) antigen-binding site specific for the antigen in the antibody.

[0086] The term "antibody" as used herein is used in the broadest sense and encompasses various 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.

[0087] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure that is substantially similar to a native antibody structure.

[0088] "Antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an intact antibody that binds to an 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 are included. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). Also see International Publication No. 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. For the description of Fab and F(ab') 2 fragments containing salvage receptor binding epitope residues and having an extended in vivo half-life, see U.S. Patent No. 5,869,046. A diabody is an antibody fragment having two antigen-binding sites that can be bivalent or bispecific. See, for example, EP404,097, International Publication No. 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). A single-domain antibody is an antibody fragment that includes all or part of the heavy-chain variable domain or all or part of the light-chain variable domain of an antibody. In some embodiments, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA, for example, U.S. Patent No. 6,248,516). Antibody fragments can be made by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies as described herein and production by recombinant host cells (e.g., E. coli or phage).

[0089] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or antibody light chain that is involved in the binding of an antibody to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of a natural antibody generally have a similar structure, and each domain contains four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6 th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. "Kabat numbering" as used herein with respect to variable region sequences refers to the numbering system defined by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0090] As used herein, the amino acid positions of all constant regions and domains of heavy and light chains are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), which is referred to herein as "Kabat numbering" or "Kabat numeration." Specifically, 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 kappa and lambda isotype light chain constant domains CL, and the Kabat EU index numbering system (see pages 661 - 723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3), and in this case, is further clarified herein by referring to "numbering according to the Kabat EU index."

[0091] As used herein, the terms "hypervariable region" or "HVR" refer to each region of the antibody variable domain where the sequence is hypervariable and determines antigen - binding specificity, e.g., "complementary - determining region" ("CDR"). Typically, an antibody contains six CDRs, i.e., three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include the following. (a) Hypervariable loops occurring 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 present 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)); and (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)).

[0092] Unless otherwise specified, CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the designation of CDRs may also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature.

[0093] "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 VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0094] The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by the antibody or immunoglobulin heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these are further subdivided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA2 It can be further divided. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0095] A "Fab molecule" refers to a protein composed of the VH and CH1 domains of the heavy chain of an immunoglobulin ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain").

[0096] A "crossover" Fab molecule (also referred to as "Crossfab") means a Fab molecule in which the variable or constant domains of the Fab heavy chain and light chain are exchanged (i.e., replaced with each other). That is, a crossover Fab molecule includes a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (VL-CH1 in the direction from the N-terminus to the C-terminus), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL in the direction from the N-terminus to the C-terminus). For the sake of clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and Fab heavy chain are exchanged, the peptide chain containing the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light chain and Fab heavy chain are exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0097] In contrast, a "conventional" Fab molecule means a Fab molecule in its native format, that is, a Fab molecule including a heavy chain composed of a heavy chain variable domain and a constant domain (VH-CH1 in the direction from the N-terminus to the C-terminus) and a light chain composed of a light chain variable domain and a constant domain (VL-CL in the direction from the N-terminus to the C-terminus).

[0098] 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 immunoglobulins can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which are subtypes, e.g., γ 1 (IgG 1 ), γ 2 (IgG 2 ), γ 3 (IgG 3 ), γ 4 (IgG 4 ), α 1 )(IgA 1 ) and α 2 )(IgA 2 The light chains of immunoglobulins 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 and an Fc domain connected through an immunoglobulin hinge region.

[0099] The terms "Fc domain" or "Fc region" as used herein are used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. The boundaries of the Fc region of an IgG heavy chain may vary slightly, but the human IgG heavy chain Fc region is typically defined as extending from Cys226, or from Pro230, to the carboxy terminus of the heavy chain. However, an antibody produced by a host cell may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell upon expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain either the full-length heavy chain or a cleaved 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, numbered 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 within the Fc region or constant region follows the EU numbering system, also called the EU index, described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (also incorporated by reference above). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that includes the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, the subunits of an IgG Fc domain include the IgG CH2 and IgG CH3 constant domains.

[0100] "Modifications that facilitate the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the formation of homodimers by the association of a polypeptide containing an Fc domain subunit with an identical polypeptide. As used herein, the association-facilitating modifications specifically include separate modifications made to each of two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, and these modifications are complementary to each other to facilitate the association of the two Fc domain subunits. For example, the association-facilitating modifications can change the structure or charge of one or both of the Fc domain subunits to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, and these may not be identical in that the additional components (e.g., antigen-binding portions) fused to each of the subunits are not the same. In some embodiments, the association-facilitating modifications include amino acid mutations within the Fc domain, specifically amino acid substitutions. In certain embodiments, the association-facilitating modifications include separate amino acid mutations, specifically amino acid substitutions, to each of the two subunits of the Fc domain.

[0101] The term "effector function" refers to biological activities resulting from the Fc region of an antibody that vary depending on the isotype of the antibody. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and activation of B cells.

[0102] The "percent identity (%) of amino acid sequence" 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 achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignments for determining the percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. One of ordinary skill in the art can determine appropriate parameters for alignment of sequences, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes here, the value of the percent amino acid sequence identity is generated using the ggsearch program of the FASTA package version 36.3.8c or subsequently using the BLOSUM50 comparison matrix. The FASTA program package was written by W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, W.R. Pearson (1996), "Effective protein sequence comparison" Meth. Enzymol. 266:227-258, and Pearson et al. (1997) Genomics 46:24-36, and is publicly available from http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml.Alternatively, the sequences can be compared using the ggsearch (global protein:protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) using the public server accessible at http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi to ensure a global rather than local alignment is performed. The percent amino acid identity is indicated in the output alignment header.

[0103] "Activating Fc receptor" is an Fc receptor that, following binding to the Fc domain of an antibody, induces signaling events that stimulate receptor-bearing cells to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0104] "Decreased binding", e.g., decreased binding to an Fc receptor, refers to a decrease in the affinity for each respective interaction, as measured, for example, by SPR. For clarity, this term includes reducing the affinity to zero (or below the detection limit of the assay method), i.e., complete loss of the interaction. Conversely, "increased binding" refers to an increase in the binding affinity for each respective interaction.

[0105] "Fused" means that the components (e.g., Fab molecules and Fc domain subunits) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0106] In certain embodiments, the T cell bispecific antibody binds to CD3 and a target cell antigen. Thus, in some embodiments, the T cell bispecific antibody comprises an antigen-binding portion that binds to CD3 and an antigen-binding portion that binds to the target cell antigen.

[0107] In some embodiments, the first and / or second antigen-binding portions are Fab molecules. In some embodiments, the first antigen-binding portion is a crossover Fab molecule in which the variable or constant regions of the Fab light chain and Fab heavy chain are exchanged. In such embodiments, the second antigen-binding portion is preferably a conventional Fab molecule.

[0108] In some embodiments where both the first and second antigen-binding portions of the T cell bispecific antibody are Fab molecules and, in one of the antigen-binding portions (particularly the first antigen-binding portion), the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other, i) the amino acid at position 124 in the constant domain CL of the first antigen-binding portion is replaced by a positively charged amino acid (numbering according to Kabat), and the amino acid at position 147 or 213 in the constant domain CH1 of the first antigen-binding portion is replaced by a negatively charged amino acid (numbering according to the Kabat EU index), or ii) the amino acid at position 124 in the constant domain CL of the second antigen-binding portion is replaced by a positively charged amino acid (numbering according to Kabat), and the amino acid at position 147 or 213 in the constant domain CH1 of the second antigen-binding portion is replaced by a negatively charged amino acid (numbering according to the Kabat EU index).

[0109] The T cell bispecific antibody does not contain both modifications mentioned in i) and ii). The constant domains CL and CH1 of the antigen-binding portion having VH / VL exchange are not replaced with each other (i.e., remain unexchanged).

[0110] In more specific embodiments, i) The amino acid at position 124 in the constant domain CL of the first antigen-binding portion is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and the amino acid at position 147 or 213 in the constant domain CH1 of the first antigen-binding portion is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index), or ii) The amino acid at position 124 in the constant domain CL of the second antigen-binding portion is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and the amino acid at position 147 or 213 in the constant domain CH1 of the second antigen-binding portion is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0111] In some embodiments, in the constant domain CL of the second antigen-binding portion, the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding portion, the amino acid at position 147 or 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0112] In a further embodiment, in the constant domain CL of the second antigen-binding portion, the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding portion, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0113] In a preferred embodiment, the amino acid at position 124 in the constant domain CL of the second antigen-binding portion is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), the amino acid at position 123 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), the amino acid at position 147 in the constant domain CH1 of the second antigen-binding portion is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0114] In some embodiments, in the constant domain CL of the second antigen-binding portion, the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), in the constant domain CH1 of the second antigen-binding portion, the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the Kabat EU index).

[0115] In some embodiments, in the constant domain CL of the second antigen-binding portion, the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), in the constant domain CH1 of the second antigen-binding portion, the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the Kabat EU index).

[0116] In certain embodiments, when the amino acid substitutions according to the above embodiments are made in the constant domain CL and the constant domain CH1 of the second antigen-binding portion, the constant domain CL of the second antigen-binding portion is the kappa isotype.

[0117] In some embodiments, the first antigen-binding portion and the second antigen-binding portion are optionally fused to each other via a peptide linker.

[0118] In some embodiments, each of the first and second antigen-binding portions is a Fab molecule, and (i) the second antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion at the C-terminus of the Fab heavy chain, or (ii) the first antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the second antigen-binding portion at the C-terminus of the Fab heavy chain.

[0119] In some embodiments, the T cell bispecific antibody provides monovalent binding to CD3.

[0120] In certain embodiments, the T cell bispecific antibody comprises a single antigen-binding portion that binds to CD3 and two antigen-binding portions that bind to a target cell antigen. Thus, in some embodiments, the T cell bispecific antibody comprises a third antigen-binding portion that binds to the target antigen, particularly a Fab molecule, more particularly a conventional Fab molecule. The third antigen-binding portion can incorporate all of the features described herein with respect to the second antigen-binding portion (e.g., CDR sequences, variable region sequences, and / or amino acid substitutions in the constant region), alone or in combination. In some embodiments, the third antigen portion is identical to the first antigen-binding portion (e.g., is also a conventional Fab molecule and contains the same amino acid sequence).

[0121] In certain embodiments, the T cell bispecific antibody further comprises an Fc domain composed of a first subunit and a second subunit. In some embodiments, the Fc domain is an IgG Fc domain. In certain embodiments, the Fc domain is IgG 1 Fc domain. In other embodiments, the Fc domain is IgG 4It is an Fc domain. Further, in certain embodiments, the Fc domain is an IgG comprising an amino acid substitution at position S228 (Kabat EU indexing numbering), particularly the amino acid substitution S228P. 4 It is an Fc domain. This amino acid substitution is in IgG 4 that reduces Fab arm exchange of the antibody in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In further particular embodiments, the Fc domain is a human Fc domain. In a particularly preferred embodiment, the Fc domain is human IgG 1 It is an Fc domain. Human IgG 1 An exemplary sequence of the Fc region is shown in SEQ ID NO: 3.

[0122] In some embodiments where each of the first, second, and third antigen-binding portions, if present, is a Fab molecule, (a)(i) the second antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion at the C-terminus of the Fab heavy chain, and the first antigen-binding portion is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, or (ii) the first antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the second antigen-binding portion at the C-terminus of the Fab heavy chain, and the second antigen-binding portion is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, (b) if present, the third antigen-binding portion is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

[0123] In some embodiments, the T cell bispecific antibody consists essentially of the first, second, and third antigen-binding portions (particularly Fab molecules), the Fc domain consisting of the first subunit and the second subunit, and optionally one or more peptide linkers.

[0124] The components of the T cell bispecific antibody can be fused directly to each other or, preferably, via one or more suitable peptide linkers. If the fusion of the Fab molecule is to the N-terminus of the subunit of the Fc domain, it is typically via the immunoglobulin hinge region.

[0125] The antigen-binding portion may be fused to the Fc domain or to each other directly or via a peptide linker comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G 4 S) n , (SG 4 ) n , (G 4 S) n , G 4 (SG 4 ) n or (G 4 S) n G 5 peptide linkers are included. "n" is generally an integer from 1 to 10, typically 2 to 4. In some embodiments, the peptide linker has a length of at least 5 amino acids, in some embodiments a length of 5 to 100, and in further embodiments a length of 10 to 50 amino acids. In some embodiments, the peptide linker is (GxS) n or (GxS) n G m , where G = glycine, S = serine, and (x = 3, n = 3, 4, 5 or 6, and m = 0, 1, 2 or 3) or (x = 4, n = 1, 2, 3, 4 or 5 and m = 0, 1, 2, 3, 4 or 5), and in some embodiments, x = 4 and n = 2 or 3, in further embodiments, x = 4 and n = 2, and in even further embodiments, x = 4, n = 1 and m = 5. In some embodiments, the peptide linker is (G 4 S) 2 . In other embodiments, the peptide linker is G 4 SG 5In addition, the linker may include (a part of) an immunoglobulin hinge region. In particular, when the Fab molecule is fused to the N-terminus of the Fc domain subunit, it may be fused via the immunoglobulin hinge region or a part thereof, with or without an additional peptide linker.

[0126] In certain embodiments, the Fc domain includes a modification that promotes the association of the first and second subunits of the Fc domain. The site where the protein-protein interaction between the two subunits of the human IgG Fc domain extends most extensively is in the CH3 domain. Thus, in some embodiments, the modification is in the CH3 domain of the Fc domain.

[0127] In certain embodiments, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which includes a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain. The knob-into-hole technique is described, for example, in U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, Ridgway et al., Prot Eng 9,617-621 (1996), and Carter, J Immunol Meth 248,7-15 (2001). Generally, this method involves introducing a protrusion ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") at the interface of the second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing small amino acid side chains from the contact surface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0128] Accordingly, in some embodiments, the amino acid residues within the CH3 domain of the first subunit of the Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit that can be disposed within the cavity within the CH3 domain of the second subunit, and the amino acid residues within the CH3 domain of the second subunit of the Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit that can accommodate the protrusion within the CH3 domain of the first subunit. Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and the cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0129] In certain such embodiments, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index). In further embodiments, in the first subunit of the Fc domain, further, the serine residue at position 354 is replaced with a cysteine residue (S354C), or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in the second subunit of the Fc domain, further, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the Kabat EU index). In preferred embodiments, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the Kabat EU index).

[0130] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or decrease effector function.

[0131] In certain embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In certain embodiments, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In some embodiments, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and cytokine secretion. In certain embodiments, the effector function is ADCC.

[0132] Typically, the same one or more amino acid substitutions are present in each of the two subunits of the Fc domain. In some embodiments, the one or more amino acid substitutions reduce the binding affinity of the Fc domain for the Fc receptor. In some embodiments, the one or more amino acid substitutions reduce the binding affinity of the Fc domain for the Fc receptor by at least one-half, at least one-fifth, or at least one-tenth.

[0133] In some embodiments, the Fc domain comprises an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331, and P329 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group consisting of L234, L235, and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In some such embodiments, the Fc domain is IgG 1 Fc domain, particularly human IgG 1It is an Fc domain. In some embodiments, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the Kabat EU index). In some embodiments, the Fc domain comprises additional amino acid substitutions at position P329 and at positions selected from E233, L234, L235, N297 and P331 (numbering according to the Kabat EU index). In a more specific embodiment, the additional amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D or P331S. In certain embodiments, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”). Specifically, in a preferred embodiment, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e., in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to the Kabat EU index). In some such embodiments, the Fc domain is IgG 1 an Fc domain, particularly human IgG 1 It is an Fc domain.

[0134] In some embodiments, the target cell antigen of the T cell bispecific antibody is CD20.

[0135] "CD20", also known as "B lymphocyte antigen B1", refers to any native CD20 derived 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. This term encompasses "full-length", untreated CD20, and any form of CD20 resulting from intracellular processing. This term also encompasses naturally occurring variants of CD20, such as splice variants or allelic variants. In some embodiments, CD20 is human CD20. Human CD20 is described in UniProt (www.uniprot.org) accession number P11836 (entry version 200), and the amino acid sequence of human CD20 is also shown in SEQ ID NO: 24.

[0136] Useful T cell bispecific antibodies that bind to CD20 in the present invention are described, for example, in International Publication No. WO 2016 / 020309, which is hereby incorporated by reference in its entirety.

[0137] In some embodiments, the T cell bispecific antibody comprises a first antigen-binding portion that binds to CD3 and a second antigen-binding portion that binds to CD20.

[0138] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region comprising heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, HCDR2 of SEQ ID NO: 5, and HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising light chain CDR (LCDR) 1 of SEQ ID NO: 7, LCDR2 of SEQ ID NO: 8, and LCDR3 of SEQ ID NO: 9.

[0139] In some embodiments, the second antigen-binding portion comprises a heavy chain variable region comprising heavy chain CDR (HCDR) 1 of SEQ ID NO: 12, HCDR2 of SEQ ID NO: 13, and HCDR3 of SEQ ID NO: 14, and a light chain variable region comprising light chain CDR (LCDR) 1 of SEQ ID NO: 15, LCDR2 of SEQ ID NO: 16, and LCDR3 of SEQ ID NO: 17.

[0140] In some embodiments, the T cell bispecific antibody is (i) a first antigen-binding portion comprising a heavy-chain variable region that binds to CD3 and comprises heavy-chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 4, HCDR2 of SEQ ID NO: 5, and HCDR3 of SEQ ID NO: 6, and a light-chain variable region that comprises light-chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 7, LCDR2 of SEQ ID NO: 8, and LCDR3 of SEQ ID NO: 9; (ii) a second antigen-binding portion that binds to CD20 and comprises a heavy-chain variable region that comprises heavy-chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 12, HCDR2 of SEQ ID NO: 13, and HCDR3 of SEQ ID NO: 14, and a light-chain variable region that comprises light-chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 15, LCDR2 of SEQ ID NO: 16, and LCDR3 of SEQ ID NO: 17.

[0141] In some embodiments, the first antigen-binding portion 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: 10 and 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: 11.

[0142] In some embodiments, the first antigen-binding portion comprises the heavy-chain variable region sequence of SEQ ID NO: 10 and the light-chain variable region sequence of SEQ ID NO: 11.

[0143] In some embodiments, the second antigen-binding portion 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: 18 and 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: 19. In some embodiments, the second antigen-binding portion comprises the heavy-chain variable region sequence of SEQ ID NO: 18 and the light-chain variable region sequence of SEQ ID NO: 19.

[0144] In some embodiments, the T cell bispecific antibody comprises a third antigen-binding portion that binds to CD20 and / or an Fc domain composed of a first subunit and a second subunit as described herein.

[0145] In a preferred embodiment, the T cell bispecific antibody is (i) a first antigen-binding portion that binds to CD3, comprising a heavy chain variable region comprising heavy chain complementarity-determining regions (HCDRs) 1 of SEQ ID NO: 4, HCDR2 of SEQ ID NO: 5, and HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising light chain CDRs (LCDRs) 1 of SEQ ID NO: 7, LCDR2 of SEQ ID NO: 8, and LCDR3 of SEQ ID NO: 9, and being a crossover Fab molecule in which either the variable or constant regions of the Fab light and heavy chains, particularly the variable regions, are exchanged, the first antigen-binding portion; (ii) a second antigen-binding portion and a third antigen-binding portion that bind to CD20, each comprising a heavy chain variable region comprising HCDRs 1 of SEQ ID NO: 12, HCDR2 of SEQ ID NO: 13, and HCDR3 of SEQ ID NO: 14, and a light chain variable region comprising LCDRs 1 of SEQ ID NO: 15, LCDR2 of SEQ ID NO: 16, and LCDR3 of SEQ ID NO: 17, and each of the second antigen-binding portion and the third antigen-binding portion being a Fab molecule, particularly a conventional Fab molecule, the second antigen-binding portion and the third antigen-binding portion; (iii) an Fc domain composed of a first subunit and a second subunit, wherein the second antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion at the C-terminus of the Fab heavy chain, the first antigen-binding portion is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and the third antigen-binding portion is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

[0146] In some embodiments, the first antigen-binding portion of the T cell bispecific antibody (which binds to CD20 and CD3) is a crossover Fab molecule in which the variable regions of the Fab light chain and Fab heavy chain are exchanged, and the second and (if present) third antigen-binding portions of the T cell bispecific antibody are such that in the constant domain CL, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), in the constant domain CH1, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index), and they are conventional Fab molecules.

[0147] In particular, in the above embodiment, in the constant domain CL of the second and third Fab molecules of (ii), the amino acid at position 124 may be substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 may be substituted by lysine (K) or arginine (R), particularly arginine (R) (numbering according to Kabat), in the constant domain CH1 of the second and third Fab molecules of (ii), the amino acid at position 147 may be substituted by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 may be substituted by glutamic acid (E) (numbering according to the Kabat EU index).

[0148] In some embodiments, the first antigen-binding portion of a T-cell bispecific antibody (that binds CD20 and CD3) 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: 10, and 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: 11. In some embodiments, the first antigen-binding portion comprises the heavy-chain variable region sequence of SEQ ID NO: 10 and the light-chain variable region sequence of SEQ ID NO: 11.

[0149] In some embodiments, the second and (if present) third antigen-binding portions of a T-cell bispecific antibody (that binds CD20 and CD3) comprise 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: 18, and 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: 19. In some embodiments, the second and (if present) third antigen-binding portions comprise the heavy-chain variable region of SEQ ID NO: 18 and the light-chain variable region of SEQ ID NO: 19.

[0150] The Fc domain according to the above embodiments can include all of the features described above in connection with the Fc domain, either alone or in combination.

[0151] In some embodiments, the Fc domain of a T-cell bispecific antibody (that binds CD20 and CD3) comprises a modification that promotes the association of the first and second subunits of the Fc domain, and / or the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector functions.

[0152] In some embodiments, the antigen-binding portion and the Fc region are fused to each other by a peptide linker, particularly a peptide linker such as SEQ ID NO: 21 and SEQ ID NO: 23.

[0153] In some embodiments, the T-cell bispecific antibody (which binds to CD20 and CD3) comprises a polypeptide (in particular two polypeptides) comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 20, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 21, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 22, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 23. In some embodiments, the T-cell bispecific antibody (which binds to CD20 and CD3) comprises a polypeptide (in particular two polypeptides) comprising the sequence of SEQ ID NO: 20, a polypeptide comprising the sequence of SEQ ID NO: 21, a polypeptide comprising the sequence of SEQ ID NO: 22, and a polypeptide comprising the sequence of SEQ ID NO: 23.

[0154] In a preferred embodiment, the T-cell bispecific antibody is glofitamab (WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Recommended INN: List 83, 2020, Volume 34, Number 1, p. 39).

[0155] In some embodiments, the disease (treated by the T-cell engager) is cancer.

[0156] As used herein, "treatment" (and its grammatical variations, e.g., "treat" or "treating") refers to a clinical intervention in an attempt to alter the natural course of a disease in an individual being treated, which can be performed for prophylaxis or during the course of clinical pathology. Desired effects of treatment include preventing the onset or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, remission or palliation of the disease state, and recovery or improved prognosis.

[0157] The term "cancer" refers to a physiological condition in mammals typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. 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, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eyes, head and neck, (central and peripheral) nervous system, lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital system. Precancerous conditions or lesions and cancer metastases are also included.

[0158] In some embodiments, the cancer is a cancer that expresses a target cell antigen of a T cell engager (e.g., a T cell bispecific antibody).

[0159] In some embodiments, the cancer is a CD20-expressing cancer (particularly in embodiments where the target cell antigen of a T cell engager, e.g., a T cell bispecific antibody, is CD20). "CD20-positive cancer" or "CD20-expressing cancer" means a cancer characterized by the expression or overexpression of CD20 in cancer cells. The expression of CD20 can be determined, for example, by quantitative real-time PCR (measuring CD20 mRNA levels), flow cytometry, immunohistochemistry (IHC), or Western blot assay. In some embodiments, the cancer expresses CD20. In some embodiments, the cancer expresses CD20 in 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.

[0160] In some embodiments, the cancer is a B cell cancer, particularly a CD20-positive B cell cancer (particularly in embodiments where the target cell antigen of a T cell engager, such as a T cell bispecific antibody, is CD20). In some embodiments, the cancer is selected from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), high-grade B cell lymphoma (HGBCL), primary mediastinal large B cell lymphoma (PMBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), or Hodgkin lymphoma (HL). In certain embodiments, the cancer is selected from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), high-grade B cell lymphoma (HGBCL), primary mediastinal large B cell lymphoma (PMBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL). In some embodiments, the cancer is NHL. In some embodiments, the NHL is relapsed or refractory (r / r) NHL. In some embodiments, the NHL is indolent NHL (iNHL) or aggressive NHL (aNHL). In some embodiments, the cancer is DLBCL. In some embodiments, the DLBCL is Richter's transformation. In some embodiments, the cancer is high-grade B cell lymphoma (HGBCL). In some embodiments, the cancer is primary mediastinal large B cell lymphoma (PMBCL). In some embodiments, the cancer is FL. In some embodiments, the FL is relapsed or refractory (r / r) FL. In some embodiments, the FL is transformed FL. In some embodiments, the cancer is MCL. In some embodiments, the MCL is relapsed or refractory (r / r) MCL. In some embodiments, the cancer is MZL.

[0161] In some embodiments, the cancer is treatable by a T cell engager. In some embodiments, the T cell engager is adapted for the treatment of the cancer.

[0162] In some embodiments, a T cell engager (particularly, a T cell bispecific antibody that binds to CD20 and CD3 as described earlier herein) is administered to an individual in a dosing regimen that includes at least a first dosing cycle and a second dosing cycle, where (a) the first dosing cycle includes a first dose (C1D1) and a second dose (C1D2) of the T cell engager, C1D1 is 2.5 mg, C1D2 is 10 mg, and (b) the second dosing cycle includes a single dose (C2D1) of the T cell engager of either 16 mg or 30 mg. In some embodiments, the single dose of the second dosing cycle includes 30 mg of the T cell engager. In some embodiments, the first dose (C1D1) is administered on day 1 of the first dosing cycle and the second dose (C1D2) is administered on day 8 of the first dosing cycle. In some embodiments, the single dose (C2D1) of the second dosing cycle is administered on day 1 of the second dosing cycle. In some embodiments, the dosing regimen includes 1 to 10 additional dosing cycles (C3 - C12). In some such embodiments, the 1 to 10 additional dosing cycles (C3 - C12) include a single dose (C3D1 - C12D1) of the T cell engager of either 16 mg or 30 mg. In some embodiments, the single dose of the additional dosing cycles (C3D1 - C12D1) includes 30 mg of the T cell engager. In some embodiments, the single dose of the additional dosing cycles (C3D1 - C12D1) is administered on day 1 of each respective additional dosing cycle. In some embodiments, the dosing regimen includes a total of 12 dosing cycles. In some embodiments, one treatment cycle consists of 14 days or 21 days. In some embodiments, one treatment cycle consists of 21 days. In some embodiments, treatment with the T cell engager is stopped after 12 dosing cycles.

[0163] As used herein, an "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. In some embodiments, the individual has a disease, particularly a disease that is treatable or should be treated by a T cell engager. In some embodiments, the individual has cancer, particularly cancer that is treatable or should be treated by a T cell engager. In particular, an individual herein is any single human subject eligible for treatment who has experienced or has experienced one or more signs, symptoms, or other indicators of cancer. In some embodiments, the individual has cancer or is diagnosed with cancer, particularly any of the cancers described above. In some embodiments, the individual has locally advanced cancer or metastatic cancer or is diagnosed with locally advanced cancer or metastatic cancer. The individual may or may not have been previously treated with a T cell engager (e.g., a T cell bispecific antibody) or another drug. In certain embodiments, the patient has not been previously treated with a T cell engager (e.g., a T cell bispecific antibody). The patient may have been treated with a therapy comprising one or more drugs other than a T cell engager (e.g., other than a T cell bispecific antibody) prior to the initiation of T cell engager therapy.

[0164] In some embodiments, the individual has elevated serum levels of one or more cytokines. In some embodiments, the elevated serum levels are associated with the administration of a T cell engager to the individual. The elevated serum levels are, in particular, compared to the serum levels of healthy individuals and / or individuals (including the same individual) who have not been administered a T cell engager (i.e., in such cases, the serum levels are elevated compared to the serum levels in the absence of administration of a T cell engager). In some embodiments, the one or more cytokines are selected from the group consisting of IL-1β, IL-6, and IL-8.

[0165] Cytokines according to any of the aspects of the invention can be one or more cytokines selected from the group consisting of interleukin (IL)-1β, IL-6, IL-8, interferon (IFN)-γ, IL-2, tumor necrosis factor (TNF)-α, IP-10 (also known as IFNγ-induced protein 10; C-X-C motif chemokine ligand 10 (CXCL10)), monocyte chemoattractant protein (MCP)-1, interleukin 1 receptor antagonist (IL-1Ra), macrophage inflammatory protein (MIP)-1α (also known as CCL3) and MIP-1β (also known as CCL4). In some aspects, the cytokine is one or more cytokines selected from the group consisting of IL-1β, IL-6 and IL-8. In some aspects, the cytokine is IL-1β. In some aspects, the cytokine is IL-6. In some aspects, the cytokine is IL-8. In some aspects, the cytokine is IFN-γ. In some aspects, the cytokine is IL-2. In some aspects, the cytokine is TNF-α. In some aspects, the cytokine is IP-10. In some aspects, the cytokine is MCP-1. In some aspects, the cytokine is IL-1Ra. In some aspects, the cytokine is MIP-1α. In some aspects, the cytokine is MIP-1β.

[0166] Preferably, the T cells according to any of the aspects of the invention are cytotoxic T cells. In some aspects, the T cells are CD4 + or CD8 + T cells. In some aspects, the T cells are CD8 + T cells. In some aspects, the T cells are CD4 + T cells.

[0167] In some embodiments, treatment with a T cell engager or administration of a T cell engager can result in a response in an individual. In some embodiments, the response can be a complete remission. In some embodiments, the response can be a durable response after discontinuation of treatment. In some embodiments, the response can be a complete remission that persists after discontinuation of treatment. In other embodiments, the response can be a partial remission. In some embodiments, the response can be a partial remission that persists after discontinuation of treatment. In some embodiments, treatment with a T cell engager and an NLRP3 inhibitor or administration thereof can improve the response as compared to treatment or administration with the T cell engager alone (i.e., without an NLRP3 inhibitor). In some embodiments, treatment or administration of a T cell engager and an NLRP3 inhibitor can increase the response rate in a patient population as compared to a corresponding patient population treated with the T cell engager alone (i.e., without an NLRP3 inhibitor).

[0168] A T cell engager can be used alone in therapy or in combination with other agents. For example, a T cell engager can be co-administered with at least one additional therapeutic agent. In some 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.

[0169] An NLRP3 inhibitor can be used alone or in combination with one or more other agents for preventing or reducing adverse effects associated with administration of a T cell engager, particularly CRS. An NLRP3 inhibitor can be used, for example, in combination with an IL-6R antagonist (e.g., tocilizumab), a steroid (e.g., a corticosteroid such as methylprednisolone, prednisone, and / or dexamethasone), or a TNF-α antagonist (e.g., etanercept).

[0170] In some embodiments, the NLRP3 inhibitor can be used in combination with a B cell depleting agent such as obinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453). As used herein, obinutuzumab is synonymous with GA101. The trade name is Gazyva (登録商標) or Gazyvaro (登録商標) . In some embodiments, obinutuzumab is administered as a pretreatment (the "Gazyva (登録商標) pretreatment") prior to the administration of the T cell engager, particularly prior to the first administration of the T cell engager (e.g., a T cell bispecific antibody that binds to CD20 and CD3 as described earlier herein).

[0171] In some embodiments, obinutuzumab is administered 1 to 15 days, particularly 1 to 10 days, prior to the (first) administration of the T cell engager. In certain embodiments, obinutuzumab is administered 7 days prior to the (first) administration of the T cell engager.

[0172] In some embodiments, obinutuzumab is administered at a dose of 1000 - 2000 mg. In some embodiments, obinutuzumab is administered as a single dose of 1000 mg. In some embodiments, obinutuzumab is administered as a single dose of 2000 mg. In some embodiments, obinutuzumab is administered at doses of 1000 mg for the first and second doses, respectively. In some embodiments, the first and second doses of obinutuzumab are administered on the same day as the (first) administration of the T cell engager, particularly 7 days prior to the administration. In certain embodiments, 2000 mg of obinutuzumab is administered as a single dose of 2000 mg or at doses of 1000 mg for the first and second doses, respectively, 7 days prior to the (first) administration of the T cell engager. In some embodiments, the first and second doses of obinutuzumab are administered on different days. In certain embodiments, the first dose of obinutuzumab is administered 7 days prior to the (first) administration of the T cell engager, and the second dose of obinutuzumab is administered 1 day prior to the above-mentioned administration of the T cell engager.

[0173] The administration of obinutuzumab can be continued during the course of treatment with a T cell engager (particularly a T cell bispecific antibody that binds to CD20 and CD3 as described earlier herein). In some embodiments, obinutuzumab is administered with a T cell engager on the first day of the second dosing cycle (C2) and on the first day of any subsequent dosing cycles (e.g., the 3rd (C3) - 12th (C12) dosing cycles). In some such embodiments, obinutuzumab is administered at a dose of 1000 mg. In some embodiments, treatment is stopped after a total of 12 dosing cycles with the T cell engager.

[0174]

Table 1

Brief Description of the Drawings

[0175]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Example

[0176] The following are examples of the methods and compositions of the present invention. It is understood that various other aspects can be implemented given the general description provided above.

[0177] Example 1. NLRP3 inhibitor prevents TCB-mediated cytokine release while minimizing its impact on T cell activation and T cell cytotoxicity To evaluate the effect of NLRP3 inhibitor on TCB-mediated target cell death and T cell activation, the inventors performed whole blood assays using fresh whole blood from three healthy donors incubated with increasing concentrations of CD20-TCB (SEQ ID NOs: 4-23) in combination with 10 μM NLRP3 inhibitor (MCC950, Sigma-Aldrich). At 24 hours, CD19+ B cell death was measured in whole blood by flow cytometry together with the expression of CD25 on CD4+ and CD8+ T cells.

[0178] The NLRP3 inhibitor minimally impaired CD20-TCB-induced B cell depletion, as indicated by the percentage of CD19+ B cells among CD45+ cells (Figure 1A, Figure 2A, and Figure 3A). Consistent with this, the NLRP3 inhibitor did not prevent the expression of the activation marker CD25 on CD4+ and CD8+ T cells and did not prevent T cell activation induced by CD20-TCB. (Figure 1B and Figure 1C, Figure 2B and Figure 2C, and Figure 3B and Figure 3C).

[0179] To determine the effect of NLRP3 inhibitors on cytokine release, cytokine levels were measured by Luminex in the sera of whole blood treated with CD20-TCB in the presence and absence of NLRP3 inhibitors. As a result, the NLRP3 inhibitor decreased the levels of IL-1β and IL-8 in all three donors and decreased the level of IL-6 in two of the three donors. However, it did not affect the levels of IFN-γ, IL-2, TNF-α, IP-10, IL-1Ra, MCP-1, MIP-1α, and MIP-1β (Figures 1D–1N, 2D–2N, and 3D–3N).

[0180] In summary, the NLRP3 inhibitor decreased the release of IL-6, IL-1β, and IL-8 while maintaining T cell cytotoxicity and T cell activation after stimulation with CD20-TCB. Considering the contribution of these cytokines in the pathophysiology of CRS, this data suggests that NLRP3 inhibitors may be attractive compounds for the reduction of CRS from the perspective of T cell-inducing therapies.

[0181] Example 2. NLRP3 inhibitors retain in vivo efficacy while preventing TCB-mediated cytokine release and weight loss. The effect of NLRP3 inhibitors on the anti-tumor effect and cytokine release mediated by CD20-TCB in vivo was evaluated using a diffuse large B cell lymphoma (DLBCL) tumor model in humanized NSG mice. Briefly, humanized NSG mice were engrafted with OCI-LY18 tumor cells. When the tumor volume reached 200 mm 3 the mice were randomly divided into 14 groups based on tumor size. They were then treated with 30 mg / kg of obinutuzumab (Gazyva (登録商標)After pretreatment with GpT, on day 7 after pretreatment, the mice were treated with CD20-TCB to deplete peripheral B cells. As shown in Figure 4A, the mice were intravenously injected with 0.5 mg / kg, 1 mg / kg, and 2 mg / kg of CD20-TCB weekly. The NLRP3 inhibitor was administered 1 hour before, 4 hours after, and 24 hours after the first CD20-TCB treatment, and 1 hour before and 4 hours after the second CD20-TCB treatment.

[0182] To evaluate the effect of the NLRP3 inhibitor on tumor growth, the tumor volume was calculated from vernier caliper measurements taken two or three times a week. Additionally, the number of CD8+ T cells was measured by flow cytometry in tumors harvested 24 hours after the third treatment with CD20-TCB. As a result, the NLRP3 inhibitor did not interfere with the anti-tumor effect mediated by CD20-TCB (Figure 4A) and did not prevent CD8+ T cell infiltration in tumors (Figure 4B).

[0183] To examine the effect of the NLRP3 inhibitor on CD20-TCB-mediated cytokine release in vivo, cytokine levels in serum collected by tail vein bleeding 3 hours after the first treatment with CD20-TCB were measured. Consistent with the previous in vitro findings, the NLRP3 inhibitor decreased the levels of IL-6, IL-8, and IL-1β induced by CD20-TCB while maintaining IL-2, IFN-γ, and TNF-α (Figures 5A - 5E, Figure 6). Furthermore, the NLRP3 inhibitor modified the cytokine profile induced by CD20-TCB treatment, as shown by a heatmap indicating the levels of IFN-γ, IL-2, TNF-α, IP-10, IL-1β, IL-1Ra, IL-6, IL-8, MCP-1, MIP-1α, and MIP-1β measured 3 hours after the first treatment with CD20-TCB (Figure 6). Finally, the inventors measured the body weight of each animal before and after the first treatment with CD20-TCB and calculated the change in body weight. The inventors found that the NLRP3 inhibitor prevented the weight loss induced by CD20-TCB, suggesting that the NLRP3 inhibitor may prevent CRS clinical symptoms (Figure 7).

[0184] In summary, the NLRP3 inhibitor maintained the anti-tumor activity of CD20-TCB while reducing the levels of IL-1β and IL-6. This prevented the weight loss induced by the initial treatment with CD20-TCB. Overall, this data supports the use of NLRP3 inhibitors for the reduction of CRS symptoms that can be induced by treatment with TCB.

[0185] For the purpose of clarity of understanding, the above invention has been described in some detail by way of explanation and examples, but the explanation and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific documents cited herein are hereby expressly incorporated by reference in their entirety.

Claims

1. A pharmaceutical agent for treating a disease of an individual, comprising a T cell bispecific antibody, wherein a NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inhibitor is administered to the individual. Pharmaceuticals.

2. (a) a T cell bispecific antibody; (b) comprising an NLRP3 inhibitor, Medicine used to treat diseases in individuals.

3. The pharmaceutical product according to claim 1 or 2, wherein the NLRP3 inhibitor is administered to prevent or mitigate adverse effects associated with the administration of the T cell bispecific antibody.

4. A pharmaceutical agent for preventing or mitigating adverse effects associated with the administration of a T cell bispecific antibody to an individual, comprising an NLRP3 inhibitor.

5. The pharmaceutical product according to any one of claims 1, 2, and 4, wherein the NLRP3 inhibitor is MCC950 or a derivative thereof.

6. The pharmaceutical product according to any one of claims 1, 2, and 4, wherein the NLRP3 inhibitor is selected from the group consisting of MCC950, cernoflast, emrenoflast, CY-09, IZD334, IZD174, DFV890, IFM-632, IFM-514, JT194, JT349, NT-0167, NT-0796, NT-0249, VENT-01, VTX3232, VTX2735, BT032, BT132, OLT1177, ADS-032, ZYIL1, HY209, AC-201, AC-203, and tranilast.

7. The NLRP3 inhibitor inhibits the adverse effects associated with the administration of the T cell bispecific antibody. The aforementioned adverse effects are (i) Cytokine release syndrome (CRS), (ii) fever, hypotension and / or hypoxia, and / or (iii) Increased serum levels of one or more cytokines, particularly one or more cytokines selected from the group consisting of IL-1β, IL-6, and IL-8. That is, A pharmaceutical product according to any one of claims 1, 2, and 4.

8. The pharmaceutical product according to claim 7, wherein the inhibition is complete inhibition, or clinically meaningful and / or statistically significant inhibition.

9. The administration of the NLRP3 inhibitor mentioned above (a) When adverse effects (clinically) manifest in the individual, (b) (i) Before, simultaneously with, and / or after administration of the T cell bispecific antibody, (ii) Intermittent or continuous, and / or (iii) Oral or parenteral, especially intravenous, and / or (c) relating to the first administration of the T cell bispecific antibody, and optionally before, simultaneously with, and / or after the first administration of the T cell bispecific antibody. The pharmaceutical product according to claim 1 or 4.

10. The administration of the T cell bispecific antibody described above (i) The effective dose, (ii) Parenteral, especially intravenous, and / or (iii) The first administration of the T cell bispecific antibody to the individual, A pharmaceutical product according to any one of claims 1, 2, and 4.

11. The T cell bispecific antibody binds to CD3 and the target cell antigen, and / or The T cell bispecific antibody comprises an antigen-binding portion that binds to CD3 and an antigen-binding portion that binds to a target cell antigen. A pharmaceutical product according to any one of claims 1, 2, and 4.

12. The pharmaceutical product according to claim 11, wherein the target cell antigen is CD20.

13. The T cell bispecific antibody is (i) A first antigen-binding region comprising a heavy chain variable region that binds to CD3 and includes heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, HCDR 2 of SEQ ID NO: 5, and HCDR 3 of SEQ ID NO: 6, and a light chain variable region that includes light chain CDR (LCDR) 1 of SEQ ID NO: 7, LCDR 2 of SEQ ID NO: 8, and LCDR 3 of SEQ ID NO: 9, (ii) A second antigen-binding region that binds to CD20 and includes a heavy chain variable region containing heavy chain CDR (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and a light chain variable region containing light chain CDR (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO:

17. The pharmaceutical product according to claim 12, including the above.

14. The T cell bispecific antibody is (i) A first antigen-binding moiety that binds to CD3, comprising a heavy chain variable region including heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, HCDR 2 of SEQ ID NO: 5, and HCDR 3 of SEQ ID NO: 6, and a light chain variable region including light chain CDR (LCDR) 1 of SEQ ID NO: 7, LCDR 2 of SEQ ID NO: 8, and LCDR 3 of SEQ ID NO: 9, and being a crossover Fab molecule in which either the variable region or the constant region of the Fab light chain and Fab heavy chain are exchanged, (ii) A second antigen-binding portion and a third antigen-binding portion that bind to CD20, comprising a heavy chain variable region including heavy chain CDR (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and a light chain variable region including light chain CDR (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17, wherein each of the second antigen-binding portion and the third antigen-binding portion is a Fab molecule, particularly a conventional Fab molecule, (iii) Fc domain consisting of a first subunit and a second subunit and Includes, The pharmaceutical product according to claim 12, wherein the second antigen-binding portion is fused to the N-terminus of the first antigen-binding portion of the Fab heavy chain at the C-terminus of the Fab heavy chain, the first antigen-binding portion is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and the third antigen-binding portion is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

15. The T cell bispecific antibody comprises a first antigen-binding portion that binds to CD3 and a second antigen-binding portion that binds to CD20, The pharmaceutical product according to claim 12, wherein the first antigen-binding portion of the T cell bispecific antibody comprises a heavy chain variable region sequence which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain variable region sequence which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11, and / or the second antigen-binding portion and (if present) the third antigen-binding portion of the T cell bispecific antibody comprises a heavy chain variable region sequence which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18, and a light chain variable region sequence which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

19.

16. The T cell bispecific antibody comprises a first antigen-binding portion that binds to CD3 and a second antigen-binding portion that binds to CD20, The first antigen-binding portion of the T cell bispecific antibody is a crossover Fab molecule in which the variable regions of the Fab light chain and Fab heavy chain are exchanged, and the second antigen-binding portion and (if present) the third antigen-binding portion of the T cell bispecific antibody are such that, in the constant domain CL, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), in the constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat The pharmaceutical product according to claim 12, which is a conventional Fab molecule (numbered according to the EU index).

17. The pharmaceutical product according to claim 12, wherein the T cell bispecific antibody comprises an Fc domain, the Fc domain of the T cell bispecific antibody comprises a modification that promotes the association of a first subunit and a second subunit of the Fc domain, and / or the Fc domain comprises one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

18. The pharmaceutical product according to any one of claims 1, 2, and 4, wherein the T cell bispecific antibody is grofitamab.

19. The pharmaceutical product according to any one of claims 1, 2, and 4, wherein the disease is cancer, in particular cancer that expresses the target cell antigen of the T cell bispecific antibody.

20. The aforementioned cancer, (i) CD20-expressing cancer, (ii) B-cell carcinoma and / or (iii) Select from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL). The pharmaceutical product according to claim 19.

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