Prevention or mitigation of adverse reactions associated with T cell cement

Abstract

The present invention relates to the prevention or mitigation of adverse reactions associated with T cell cement, such as cytokine release syndrome. In particular, the present invention relates to the use of inhibitors of Protein 3 (NLRP3) containing NACHT, LRR and PYD domains to prevent or ameliorate such side effects.

Description

Technical Field

[0001] The present invention relates to preventing or reducing adverse reactions associated with T cell engagers, such as cytokine release syndrome. In particular, the present invention relates to preventing or reducing such side effects using inhibitors 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 (CAR-T cells) expressing chimeric antigen receptors (CARs), have a broad prospect as cancer immunotherapeutics. T cell bispecific antibodies (TCBs) activate T cells by engaging the CD3ε chain of T cell receptors, and simultaneously bind to tumor-associated antigens on target cells. These T cell bispecific antibodies can form immune synapses, where the release of pore-forming cytolytic proteins (perforins) and cytotoxic granules (granzyme B) induces target cell killing. Several TCBs have been described, including CEA-TCB (cibisatamab; Bacac et al. (2016) Clin Cancer Res 22, 3286-97), CD20-TCB (gefitomab; 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 them, gefutomab (CD20-TCB) has shown potent 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 activity, on-target activation of T cells is associated with an intrinsic risk of cytokine release syndrome (CRS), one of the most common safety liabilities associated with treatment with T cell engagement therapies (see, e.g., Shimabukuro-Vornhagen et al. (2018) J Immunother Cancer 6, 56). CRS is characterized by a cytokine storm caused by overactivation of T cells and innate immune cells, leading to symptoms including fever, hypotension, and respiratory defects, and in the worst cases, multiple organ failure. The ASTCT consensus classifies CRS into different grades based on clinical symptoms (fever, hypotension, and hypoxia). For the management of grade 2 and higher CRS, the consensus recommends hospitalization of patients and the use of pressors and oxygen flow. High-dose glucocorticoids and / or IL-6R blockade can also be used to relieve symptoms (Lee et al. (2019) Biol Blood Marrow Transplant 25, 625-38). In the specific case of CD3 bispecific antibodies, ascending or fractionated dosing regimens are widely used clinically to reduce the risk of first-infusion cytokine storm that may be observed after flat-dose administration. Target cell pre-depletion approaches can also be applied to reduce the amount of circulating and lymphoid tissue-resident CD20-expressing B cells and thereby inhibit systemic on-target cytokine release by T-cell engagement therapy against B-cell malignancies: a clinically relevant example is the use of obinutuzumab This example reduced the incidence and severity of CRS in combination with escalating doses of Gefituzumab (CD20-TCB) for pretreatment. Anti-IL-6 therapy (e.g., using tocilizumab) and glucocorticoids are also used in the management of CRS. However, some patients are refractory to these methods, thus reinforcing the need to develop novel methods for alleviating CRS.

[0004] Although CRS symptoms are managed in the clinic, they remain a common dose-limiting safety liability associated with on-target activity of T cell engagers, placing patient safety at risk. Methods to mitigate these life-threatening toxicities are extremely needed, particularly mitigation strategies that reduce cytokine release while preserving therapeutic efficacy. DETAILED DESCRIPTION

[0005] The inventors have found that NLRP3 inhibitors can be used to alleviate CRS through T cell engagement therapy. NLRP3 inhibitors (such as MCC950) were found to reduce TCB-induced cytokine release while retaining TCB-mediated T cell activation and target cell killing. The results show that NLRP3 inhibitors are attractive, potentially superior alternatives or supplements to currently used strategies (such as steroids or IL-6 / IL-6R blockade) for alleviating CRS associated with T cell engagement therapy.

[0006] Thus, in a first aspect, the invention provides a T cell engager for use in treating a disease in an individual, wherein the treatment comprises

[0007] (a) administering a T cell engager to the individual, and

[0008] (b) administering a NLRP3 inhibitor to the individual.

[0009] The invention further provides the use of a T cell engager in the manufacture of a medicament for treating a disease in an individual, wherein the treatment comprises

[0010] (a) administering a T cell engager to the individual, and

[0011] (b) administering a NLRP3 inhibitor to the individual.

[0012] The present invention also provides a method of treating a disease in an individual, wherein the method comprises

[0013] (a) administering a T cell engager to the individual, and

[0014] (b) administering a NLRP3 inhibitor to the individual.

[0015] According to any of the above aspects, administration of a NLRP3 inhibitor can be used to prevent or alleviate adverse effects associated with administration of a T cell engager.

[0016] In another aspect, the invention provides a NLRP3 inhibitor for use in preventing or reducing an adverse reaction associated with administering a T cell engager to a subject.

[0017] The present invention further provides the use of a NLRP3 inhibitor in the manufacture of a medicament for preventing or alleviating adverse reactions associated with the administration of a T cell engager.

[0018] The present invention also provides a method of preventing or reducing adverse reactions associated with administering a T cell engager to a subject, the method comprising administering a NLRP3 inhibitor to the subject.

[0019] The T cell engagers for use, the NLRP3 inhibitors for use, the uses or methods described above and herein may incorporate any of the features described below, alone or in combination (unless the context indicates otherwise).

[0020] Unless otherwise defined herein, the terms used herein are those commonly used in the technical field.

[0021] "NLRP3" stands for protein 3 containing NACHT, LRR, and PYD domains (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 tripartite protein that contains an amino-terminal pyrin domain (PYD), a central NACHT domain (a domain present in NAIP, CIITA, HET-E, and TP1), and a carboxy-terminal leucine-rich repeat domain (LRR domain). The NACHT domain has an adenosine triphosphatase activity that is essential for NLRP3 self-association and function, while the LRR domain is thought to induce autoinhibition by folding back onto the NACHT domain. NLRP3 is a component of the innate immune system and is primarily expressed in macrophages. It acts as an intracellular sensor (pattern recognition receptor (PRR)) that detects a wide range of 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 transducer component, an adaptor protein (ASC, also known as PYCARD), and an effector proinflammatory caspase (caspase 1). The assembly of the NLRP3 inflammasome causes the release of proinflammatory cytokines and cell pyroptosis. For a review, see, for example, Swanson et al. (2019) Nat Rev Immunol 19, 477-489. Human NLRP3, including its sequence, is also described in UniProt Accession No. Q96P20 (Release 213).

[0022] NLRP3 inhibitors are compounds that inhibit the NLRP3 inflammasome, particularly small molecule pharmacological inhibitors. For example, NLRP3 inhibitors may disrupt NLRP3-NLRP3 interactions or NLRP3-ASC interactions, or may target the ATP binding domain of NLRP3, thereby blocking its adenosine triphosphatase activity. For a review, see, for example, Zahid et al. (2019) Frontiers Immunol 10, Art 2538.

[0023] An exemplary NLRP3 inhibitor is MCC950 (also known as CP-456,773), a direct inhibitor of NLRP3 that reportedly targets the NLRP3 ATP hydrolysis motif (Coll et al. (2019) Nat Chem Biol 15:556–9).

[0024] The chemical structure and CAS number of MCC950 are provided below:

[0025]

[0026] CAS 210826-40-7

[0027] In some aspects, the NLRP3 inhibitor is a small molecule NLRP3 inhibitor. In some aspects, the NLRP3 inhibitor is a direct inhibitor of NLRP3. In some aspects, the NLRP3 inhibitor blocks the adenosine triphosphatase domain of NLRP3. In some aspects, the NLRP3 inhibitor is MCC950, or a derivative thereof (retaining NLRP3 inhibitory activity). In some aspects, the NLRP3 inhibitor is MCC950, or a salt or ester thereof (e.g., a sodium salt of MCC950).

[0028] In some aspects, the NLRP3 inhibitor is selnoflast (Suggested INN: List 87; WHO Drug Information, Vol. 36, No. 1, 2022, p. 203), or a salt or ester thereof (eg, the sodium salt of selnoflast).

[0029] The chemical structure and CAS number of Cenolast is provided below:

[0030]

[0031] CAS 2260969-36-4

[0032] In some aspects, the NLRP3 inhibitor is emlenoflast (recommended INN: List 87; WHO Drug Information, Vol. 36, No. 1, 2022, p. 104), or a salt or ester thereof (e.g., the sodium salt of emlenoflast).

[0033] The chemical structure and CAS number of emlenoflast are provided below:

[0034]

[0035] CAS 1995067-59-8

[0036] In some aspects, the NLRP3 inhibitor is CY-09, or a salt or ester thereof (eg, the sodium salt of CY-09).

[0037] The chemical structure and CAS number of CY-09 are provided below:

[0038]

[0039] CAS 1073612-91-5

[0040] Other exemplary NLRP3 inhibitors that can be used in the present invention include, but are not limited to, IZD334 (also known as Somalix; Inflazome), IZD174 (also known as Inzomelid; Inflazome), DFV890 (also known as IFM-2427; IFM Therapeutics), IFM-632 (IFM Therpeutics), 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). 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).

[0041] In some aspects, the NLRP3 inhibitor is selected from the group consisting of MCC950, senolast, emlenoflast, 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 aspects, the NLRP3 inhibitor is selected from the group consisting of MCC950, senolast, emlenoflast, CY-09, IZD334, and IZD174. In some aspects, the NLRP3 inhibitor is selected from the group consisting of MCC950, cenolast, emlenoflast.

[0042] In some aspects, (administration of) a NLRP3 inhibitor causes inhibition of an activity of a T cell engager. In some aspects, (administration of) a NLRP3 inhibitor does not cause inhibition of another activity of a T cell engager. In some aspects, (administration of) a NLRP3 inhibitor causes inhibition of a first activity of a T cell engager, but does not cause inhibition of a second activity of a T cell engager. In some of these aspects, the inhibition is complete inhibition. In some of these aspects, the inhibition is partial inhibition. In some aspects, (administration of) a NLRP3 inhibitor causes partial inhibition of a first activity of a T cell engager, but does not cause inhibition of a second activity of a T cell engager.

[0043] In some aspects, (administration of) an NLRP3 inhibitor causes inhibition of a first activity of a T cell engager and inhibition of a second activity of the T cell engager, wherein the inhibition of the first activity is stronger than the inhibition of the second activity. In some aspects, (administration of) an NLRP3 inhibitor causes inhibition of a first activity of a T cell engager and inhibition of a second activity of the T cell engager, wherein the inhibition of the first activity is complete inhibition and the inhibition of the second activity is partial inhibition.

[0044] The "activity" of a T cell engager refers to a response elicited by the T cell engager in the body of an individual. Such activity may include a cellular response of T cells, particularly CD4+ 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 an effect on a target cell, particularly a target cell (e.g., a tumor cell) expressing a target cell antigen of the T cell engager, such as lysis of the target cell.

[0045] In some aspects, (administering) NLRP3 inhibitors cause inhibition of cytokine secretion (induced by T cell engagers) of immune cells, particularly T cells. In some aspects, the cytokine is one or more cytokines selected from the group consisting of IL-1β, IL-6 and IL-8. Immune cells may include various immune cell types, such as T cells, macrophages, monocytes, NK cells, etc. In some aspects, the T cells are CD8+T cells or CD4+ cells. In some aspects, the inhibition is complete inhibition. In some aspects, the inhibition is partial inhibition.

[0046] In some aspects, (administration of) the NLRP3 inhibitor does not cause inhibition of activation of T cells (induced by the T cell engager). In some aspects, the inhibition is complete inhibition. In some aspects, (administration of) the NLRP3 inhibitor causes inhibition of activation of T cells (induced by the T cell engager), wherein the inhibition is partial inhibition.

[0047] As used herein, "activation of T cells" or "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly CD4+ or CD8+ T cells, selected from: proliferation, differentiation, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein. In particular aspects, T cell activation is the expression of activation markers, particularly the expression of CD25 and / or CD69 (optionally measured by flow cytometry). In particular aspects, T cell activation is determined by measuring the expression of CD25 and / or CD69 on T cells, for example by flow cytometry.

[0048] In some aspects, (administration of) the NLRP3 inhibitor does not cause inhibition of the cytotoxic activity of T cells (induced by the T cell engaging agent). In some aspects, the inhibition is complete inhibition. In some aspects, (administration of) the NLRP3 inhibitor causes inhibition of the cytotoxic activity of T cells (induced by the T cell engaging agent), wherein the inhibition is partial inhibition.

[0049] The "cytotoxic activity" of T cells refers to the induction of lysis (i.e., killing) of target cells by T lymphocytes, particularly CD4+ or CD8+ T cells. Cytotoxic activity generally involves degranulation of T lymphocytes, which is associated with the release of cytotoxic effector molecules such as granzyme B and / or perforin from T lymphocytes.

[0050] In some aspects, (administration of) NLRP3 inhibitors causes inhibition of cytokine secretion (induced by T cell engagers) of T cells, but does not cause inhibition of activation and / or cytotoxic activity (induced by T cell engagers) of T cells. In some of these aspects, the inhibition is complete inhibition. In some aspects, the inhibition is partial inhibition. In some aspects, (administration of) NLRP3 inhibitors causes partial inhibition of cytokine secretion (induced by T cell engagers) of T cells, but does not cause inhibition of activation and / or cytotoxic activity (induced by T cell engagers) of T cells.

[0051] In some aspects, (administration) NLRP3 inhibitors cause inhibition of cytokine secretion (induced by T cell engagers) of T cells, and inhibition of activation and / or cytotoxic activity (induced by T cell engagers) of T cells, wherein the inhibition of cytokine secretion is stronger than the inhibition of activation and / or cytotoxic activity. In some aspects, (administration) NLRP3 inhibitors cause inhibition of cytokine secretion (induced by T cell engagers) of T cells, and inhibition of activation and / or cytotoxic activity (induced by T cell engagers) of T cells, wherein the inhibition of cytokine secretion is complete inhibition, and the inhibition of activation and / or cytotoxic activity is partial inhibition.

[0052] The inhibition herein may be partial inhibition or complete inhibition. Complete inhibition is stronger inhibition than partial inhibition. In some aspects, partial inhibition is no more than 30%, no more than 40%, no more than 50%, no more than 60% or no more than 70% inhibition. In some aspects, partial inhibition is no more than 30% inhibition. In some aspects, partial inhibition is no more than 40% inhibition. In some aspects, partial inhibition is no more than 50% inhibition. In some aspects, partial inhibition is no more than 60% inhibition. In some aspects, partial inhibition is no more than 70% inhibition. In some aspects, complete inhibition is at least 80%, at least 90% or 100% inhibition. In some aspects, complete inhibition is at least 80% inhibition. In some aspects, complete inhibition is at least 90% inhibition. In some aspects, complete inhibition is 100% inhibition. In some aspects, partial inhibition is no more than 50% inhibition, and complete inhibition is at least 80% inhibition. In some aspects, complete inhibition is clinically meaningful and / or statistically significant, and / or partial inhibition is not clinically meaningful and / or statistically significant.

[0053] In some aspects, (administration) NLRP3 inhibitors cause a decrease in serum levels of one or more cytokines in an individual. In some aspects, (administration) NLRP3 inhibitors cause a decrease in secretion of one or more cytokines of immune cells, particularly T cells, in an individual. In some aspects, the one or more cytokines are selected from the group consisting of IL-1β, IL-6, and IL-8. Immune cells may include various immune cell types, such as T cells, macrophages, monocytes, NK cells, etc.

[0054] In some aspects, after the NLRP3 inhibitor has been applied (to an individual) for a given amount of time, the reduction continues. In some aspects, 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 aspects, the reduction continues after the subsequent application of the T cell engager. In particular, even after the NLRP3 inhibitor is stopped / no further application of the NLRP3 inhibitor, the reduction continues. The reduction in serum levels / cytokine secretion is particularly compared to the serum levels / cytokine secretion in individuals (including the same individual) without the application of the NLRP3 inhibitor (that is, in this case, compared to the serum levels / cytokine secretion before the NLRP3 inhibitor is not applied / the NLRP3 inhibitor is applied, the level in serum / cytokine secretion is reduced). The reduction in serum levels / cytokine secretion is particularly compared to serum levels / cytokine secretion in an individual (including the same individual) to whom the T cell engager is administered (particularly for the first time) but the NLRP3 inhibitor is not administered (i.e., in this case, serum levels / cytokine secretion are reduced compared to serum levels / cytokine secretion after administration of the T cell engager / administration of the T cell engager but not administration of the NLRP3 inhibitor / administration of the NLRP3 inhibitor). In the absence of the reduction, serum levels and / or cytokine secretion may be particularly increased / increased relative to (administration of) the T cell engager. In some aspects, the reduction is clinically meaningful and / or statistically significant. In some aspects, the reduction is at least 30%, at least 40%, at least 50%, at least 60% or at least 70%. In some aspects, the reduction is at least 30%. In some aspects, the reduction is at least 40%. In some aspects, the reduction is at least 50%. In some aspects, the reduction is at least 60%. In some aspects, the reduction is at least 70%.

[0055] In some aspects, (administration) of an NLRP3 inhibitor causes inhibition of adverse reactions associated with the administration of a T cell engager. In some aspects, (administration) of an NLRP3 inhibitor does not cause inhibition of a desired reaction associated with the administration of a T cell engager. In some aspects, (administration) of an NLRP3 inhibitor causes inhibition of adverse reactions associated with the administration of a T cell engager, but does not cause inhibition of a desired reaction associated with the administration of a T cell engager. In some of these aspects, the inhibition is complete inhibition. In some of these aspects, the inhibition is partial inhibition. In some aspects, (administration) of an NLRP3 inhibitor causes partial inhibition of adverse reactions associated with the administration of a T cell engager, but does not cause inhibition of a desired reaction associated with the administration of a T cell engager. In some of these aspects, the inhibition is clinically meaningful and / or statistically significant.

[0056] In some aspects, (administration) NLRP3 inhibitors cause inhibition of adverse reactions associated with the administration of T cell engagers and inhibition of desired reactions associated with the administration of T cell engagers, wherein the inhibition of adverse reactions is stronger than the inhibition of desired reactions. In some aspects, (administration) NLRP3 inhibitors cause inhibition of adverse reactions associated with the administration of T cell engagers and inhibition of desired reactions associated with the administration of T cell engagers, wherein the inhibition of adverse reactions is complete inhibition, and the inhibition of beneficial effects is partial inhibition. In some aspects, (administration) NLRP3 inhibitors cause inhibition of adverse reactions associated with the administration of T cell engagers and inhibition of desired reactions associated with the administration of T cell engagers, wherein the inhibition of adverse reactions is clinically significant and / or statistically significant inhibition, and the inhibition of beneficial effects is not clinically significant and / or statistically significant inhibition.

[0057] A "desirable response" is a beneficial and desired response produced by a drug (particularly using a T cell engager in this context) in the treatment of an individual, i.e., a therapeutic or prophylactic effect, such as, for example, killing of tumor cells, reduction or delay of tumor growth, reduction of tumor volume, reduction or prevention of tumor metastasis, increase in progression-free or overall survival, alleviation of disease symptoms, etc.

[0058] An "adverse reaction," sometimes also called a "side effect" or "adverse event," particularly in clinical studies, is an unwanted, undesirable reaction caused by a drug (particularly in this context, with a T cell engager) in the treatment of an individual.

[0059] According to the present invention, adverse reactions are associated with the administration of T cell engagers. In some aspects, adverse reactions are associated with the first administration of T cell engagers. In some aspects, adverse reactions occur when T cell engagers are administered for the first time. In some aspects, adverse reactions occur mainly or only when T cell engagers are administered for the first time. In some aspects, adverse reactions occur within 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours or 96 hours of administration, particularly the first administration of T cell engagers. In some aspects, particularly when only a single administration of T cell engagers is performed (in the process of using T cell engagers for treatment), adverse reactions occur within 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days or 21 days of administering T cell engagers.

[0060] In some aspects, the adverse reaction is cytokine release syndrome (CRS).

[0061] "Cytokine release syndrome" (abbreviated as "CRS") refers to an increase in the levels of cytokines, such as tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin -1β (IL-1β), interleukin -8 (IL-8) and other cytokines in the subject's blood during or shortly after the administration of a therapeutic agent (e.g., a T cell engager) (e.g., within 1 day), leading to adverse symptoms. CRS is an adverse reaction to the therapeutic agent and is timely related to the administration of the therapeutic agent. It usually occurs during or shortly after the administration of the therapeutic agent, i.e., usually within 24 hours after administration (usually infusion), mainly at the first administration. In some cases, such as after the administration of CAR-T cells, CRS may also occur later, such as a few days after the administration of CAR-T cell expansion. The incidence and severity generally decrease with subsequent administration. Symptoms may range from symptomatic discomfort to fatal events, and may include fever, chills, dizziness, hypertension, hypotension, hypoxia, dyspnea, irritability, sweating, flushing, rash, tachycardia, shortness of breath, headache, tumor pain, nausea, vomiting and / or organ failure.CRS can be graded according to 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) established modified cytokine release syndrome grading system. 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).

[0062] In some aspects, the adverse reaction is fever, hypotension and / or hypoxia.

[0063] In some aspects, the adverse reaction is an increase in serum levels of one or more cytokines. The increased serum levels are particularly compared to serum levels in healthy individuals and / or serum levels in individuals (including the same individual) to which the T cell engager is not administered (i.e., in this case, serum levels are increased compared to serum levels to which the T cell engager is not administered). In some aspects, the one or more cytokines are selected from the group consisting of IL-1β, IL-6, and IL-8.

[0064] In some aspects, the NLRP3 inhibitor is administered when the adverse reaction (clinically) appears in (individual). For example, the administration can be performed 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 adverse reaction appears (i.e., clinical symptoms of side effects, such as fever). In some aspects, the NLRP3 inhibitor is administered in response to the adverse reaction (clinically) appearing in (individual).

[0065] Administration of the NLRP3 inhibitor may be before, at the same time and / or after administration of the T cell engager. In some aspects, administration of the NLRP3 inhibitor is before administration of the T cell engager. In some aspects, administration of the NLRP3 inhibitor is performed simultaneously with administration of the T cell engager. In some aspects, administration of the NLRP3 inhibitor is after administration of the T cell engager. When administration of the NLRP3 inhibitor is before or after administration of the T cell engager, such administration of the NLRP3 inhibitor may 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 before or after administration of the T cell engager, respectively. Administration of the NLRP3 inhibitor may be intermittent or continuous. In some aspects, administration of the NLRP3 inhibitor is oral. In some aspects, administration of the NLRP3 inhibitor is parenteral, particularly intravenous.

[0066] In some aspects, the NLRP3 inhibitor is administered at a dose sufficient to cause inhibition of an activity of the T cell engager. In some aspects, the NLRP3 inhibitor is administered at a dose insufficient to cause inhibition of another activity of the T cell engager. In some aspects, the NLRP3 inhibitor is administered at a dose sufficient to cause inhibition of a first activity of the T cell engager but insufficient to cause inhibition of a second activity of the T cell engager. In some of these aspects, the inhibition is complete inhibition. In some of these aspects, the inhibition is partial inhibition. In some aspects, the NLRP3 inhibitor is administered at a dose sufficient to cause partial inhibition of a first activity of the T cell engager but insufficient to cause inhibition of a second activity of the T cell engager.

[0067] In some aspects, the NLRP3 inhibitor is administered at a dose sufficient to cause inhibition of cytokine secretion (induced by T cell engagers) of immune cells (particularly T cells). In some aspects, the cytokine is one or more cytokines selected from the group consisting of IL-1β, IL-6 and IL-8. Immune cells may include various immune cell types, such as T cells, macrophages, monocytes, NK cells, etc. In some aspects, the T cells are CD8+T cells or CD4+ cells. In some aspects, the inhibition is complete inhibition. In some aspects, the inhibition is partial inhibition.

[0068] In some aspects, the NLRP3 inhibitor is administered at a dose that is insufficient to cause inhibition of activation of T cells (induced by the T cell engager). In some aspects, the inhibition is complete inhibition. In some aspects, the inhibition is partial inhibition.

[0069] In some aspects, the NLRP3 inhibitor is administered at a dose that is insufficient to cause inhibition of the cytotoxic activity of T cells (induced by the T cell engager). In some aspects, the inhibition is complete inhibition. In some aspects, the inhibition is partial inhibition.

[0070] In some aspects, the NLRP3 inhibitor is administered at a dose sufficient to cause inhibition of cytokine secretion (induced by the T cell engager) of the T cells but insufficient to cause inhibition of activation and / or cytotoxic activity (induced by the T cell engager) of the T cells. In some of these aspects, the inhibition is complete inhibition. In some aspects, the inhibition is partial inhibition. In some aspects, the NLRP3 inhibitor is administered at a dose sufficient to cause partial inhibition of cytokine secretion (induced by the T cell engager) of the T cells but insufficient to cause inhibition of activation and / or cytotoxic activity (induced by the T cell engager) of the T cells.

[0071] In some aspects, the NLRP3 inhibitor is administered at a dose sufficient to cause a reduction in the serum level of one or more cytokines in an individual. In some aspects, the NLRP3 inhibitor is administered at a dose sufficient to cause a reduction in the secretion of one or more cytokines of immune cells (particularly T cells) in an individual. In some aspects, the one or more cytokines are selected from the group consisting of IL-1β, IL-6 and IL-8. Immune cells may include various immune cell types, such as T cells, macrophages, monocytes, NK cells, etc.

[0072] In some aspects, the NLRP3 inhibitor is administered at a dose sufficient to cause inhibition of adverse reactions associated with the administration of the T cell engager. In some aspects, the NLRP3 inhibitor is administered at a dose insufficient to cause inhibition of the desired response associated with the administration of the T cell engager. In some aspects, the NLRP3 inhibitor is administered at a dose sufficient to cause inhibition of adverse reactions associated with the administration of the T cell engager but insufficient to cause inhibition of the desired response associated with the administration of the T cell engager. In some of these aspects, the inhibition is complete inhibition. In some of these aspects, the inhibition is partial inhibition. In some aspects, the NLRP3 inhibitor is administered at a dose sufficient to cause partial inhibition of adverse reactions associated with the administration of the T cell engager but insufficient to cause inhibition of the desired response associated with the administration of the T cell engager. In some of these aspects, the inhibition is clinically meaningful and / or statistically significant.

[0073] In some aspects, the NLRP3 inhibitor is administered at an effective dose.

[0074] An "effective amount" or "effective dose" of an agent, such as a NLRP3 inhibitor or a T cell engager, refers to an effective amount required, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive effect.

[0075] In some aspects, the NLRP3 inhibitor is administered at a dosage strength equivalent to that available for the NLRP3 inhibitor. Typically, for a given NLRP3 inhibitor, multiple dosage strengths (i.e., dosage forms, such as tablets or capsules, with a specific amount of active ingredient) are available. It would be most convenient to administer the NLRP3 inhibitor at such (commercially) available dosage strengths.

[0076] In some aspects, the NLRP3 inhibitor is administered daily. In some aspects, the NLRP3 inhibitor is administered once a day. In some aspects, the NLRP3 inhibitor is administered once a day at a dosage as mentioned above. In some aspects, the NLRP3 inhibitor is administered during the time period during which the adverse reaction persists (i.e., the NLRP3 inhibitor is administered from the adverse reaction until the adverse reaction is reduced or disappears). In some aspects, the NLRP3 inhibitor is stopped after preventing or alleviating the adverse reaction. In some aspects, the NLRP3 inhibitor is stopped after the adverse reaction is reduced or disappears. The reduction is particularly clinically significant and / or statistically significant. In some aspects, the NLRP3 inhibitor is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times, particularly in the process of using a T cell engager to treat an individual, once, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times. In some aspects, the administration of the NLRP3 inhibitor lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. In some aspects, the administration of the NLRP3 inhibitor is once a day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. The administration of the NLRP3 inhibitor is usually associated with the administration of the T cell engager. In some aspects, the administration of the NLRP3 inhibitor is associated with the first administration of the T cell engager. The first administration is particularly the first administration of the T cell engager during the treatment of an individual with the T cell engager. The administration of the NLRP3 inhibitor may be before, at the same time and / or after the first administration of the T cell engager. In some aspects, the administration of the NLRP3 inhibitor is performed simultaneously with the first administration of the T cell engager. In some aspects, the administration of the NLRP3 inhibitor is before the first administration of the T cell engager. In some aspects, the administration of the NLRP3 inhibitor is after the first administration of the T cell engager. In some aspects, 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. When the NLRP3 inhibitor is administered 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.

[0077] In some aspects, the administration of the T cell engager lasts for a longer period of time than the administration of the NLRP3 inhibitor. In some aspects, the T cell engager is continued to be administered after the administration of the NLRP3 inhibitor is stopped. In some aspects, the administration of the T cell engager is a single administration or repeated administration. In the process of treating an individual with the T cell engager, the T cell engager can be administered once or multiple times. For example, treating an individual with the T cell engager can include multiple treatment cycles, each of which includes one or more administrations of the T cell engager. In some aspects, the administration of the T cell engager includes a first and a second administration.

[0078] For use in the present invention, the T cell engagers will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific 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 timing of administration, and other factors known to practitioners.

[0079] In some aspects, the T cell engager is administered at an effective dose. For systemic administration, the effective dose can be initially estimated based on in vitro assays (such as cell culture assays). The dose can then be formulated in animal models to achieve an IC as determined in cell culture. 50 Circulating concentration range. Such information can be used to more accurately determine useful doses for humans. Initial doses can also be estimated based on in vivo data (e.g., animal models) using techniques known in the art. Doses and intervals can be adjusted individually to provide plasma levels of T cell engagers sufficient to maintain a therapeutic effect. Common patient doses administered by injection range from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. Plasma levels can be measured, for example, by HPLC.

[0080] An effective amount of a T cell engaging agent may be administered to prevent or treat a disease. The appropriate route of administration and dosage of the T cell engaging agent may be determined based on the type of disease to be treated, the type of T cell engaging agent, the severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to treatment, and the discretion of the attending physician. Administration may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic. Various administration schedules are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusions.

[0081] The T cell engager and the NLRP3 inhibitor can be administered by any suitable route, and can be administered by the same route of administration or by different routes of administration. In some aspects, administration of the T cell engager is parenteral, particularly intravenous.

[0082] In some aspects, administering 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 during a course of treatment of the individual with the T cell engager.

[0083] In some aspects, (administering) T cell engagement agents induce (i.e., cause or increase) activation of T cells. In some aspects, (administering) T cell engagement agents induce the cytotoxic activity of T cells. In some aspects, (administering) T cell engagement agents induce the cytokine secretion of T cells. In some aspects, cytokines are one or more cytokines selected from the group consisting of the following items: IL-2, IL-6, IL-1β, IL-8, IFN-γ, IP-10, TNF-α, IL-1Ra, MCP-1 and MIP-1α. In some aspects, cytokines are one or more cytokines selected from the group consisting of IL-1β, IL-6 and IL-8. In some aspects, the T cells are CD8+T cells or CD4+ cells.

[0084] In some aspects, the use of T cell engagement agents results in the activation of T cells, particularly the activation of cytotoxic T cells, particularly at the cancer site (e.g., within a solid tumor cancer). The activation may include the proliferation of T cells, the differentiation of T cells, the cytokine secretion of T cells, the release of cytotoxic effector molecules from T cells, the cytotoxic activity of T cells, and the expression of activation markers by T cells. In some aspects, the use of T cell engagement agents results in an increase in the number of T cells at the cancer site (e.g., within a solid tumor cancer), particularly an increase in the number of cytotoxic T cells.

[0085] "T cell engager" means an immunotherapeutic agent that exerts its effect through the activity of T cells, particularly the activity of cytotoxic T cells. This activity of T cells may include cellular responses of T cells, particularly cellular responses of 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 as considered herein generally include an antigen binding portion that enables it to bind to a target cell antigen on a target cell (such as a tumor cell). Such T cell engagers have an effect on their target cells through the activity of T cells, such as the lysis of target cells. Exemplary T cell engagers include T cell bispecific antibodies, T cells expressing chimeric antigen receptors (CARs) (CAR-T cells), and T cell receptor (TCR)-based approaches, such as ImmTAC ("anti-cancer immunity-driven monoclonal T cell receptor"; a bispecific fusion protein of an engineered TCR and an antibody fragment that is capable of binding to both T cells and target cells) or TCR-modified T cells (TCR-T cells), which are characterized by an engineered T cell receptor that is capable of binding to specific antigenic determinants on target cells.

[0086] In a specific aspect of the invention, the T cell engager is a T cell bispecific antibody.

[0087] In other aspects, the T cell engager is a CAR-T cell. In some aspects, the T cell engager is an ImmTAC. In some aspects, the T cell engager is a TCR-T cell.

[0088] T cell bispecific antibodies useful in the present invention are described below.

[0089] "T cell bispecific antibody" means an antibody capable of binding, including simultaneously, to a T cell (usually via an antigenic determinant expressed on the T cell, such as CD3) and a target cell (usually via an antigenic determinant expressed on the target cell, such as CD20).

[0090] In a preferred aspect according to the present invention, T cell bispecific antibodies can be simultaneously bound to an antigenic determinant (i.e., the first antigen, such as CD3) on a T cell and an antigenic determinant (i.e., the second antigen, such as CD20) on a target cell. In some aspects, T cell bispecific antibodies can crosslink T cells and target cells by being simultaneously bound to CD3 and a target cell antigen. In even more preferred aspects, this simultaneous combination causes target cells, particularly tumor cells expressing target cell antigens (e.g., CD20) to lyse. In some aspects, this simultaneous combination causes the activation of T cells. In some aspects, this simultaneous combination causes the cell response of T cells, which is selected from the group of the following items: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In some aspects, T cell bispecific antibodies are bound to CD3 without being simultaneously bound to a target cell antigen, and do not cause T cell activation. In some aspects, T cell bispecific antibodies can redirect the cytotoxic activity of T cells to target cells. In a preferred aspect, the redirection is independent of the peptide antigen presentation mediated by the target cell MHC and / or the specificity of the T cells.

[0091] The term "bispecific" means that the antibody is able to bind to at least two different antigenic determinants. Typically, a bispecific antibody includes two antigen binding sites, each of which is specific for a different antigenic determinant. In certain aspects, the bispecific antibody is able to simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two unique cells.

[0092] 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 stretch of continuous amino acids or a conformational configuration consisting of different regions of non-continuous amino acids) to which an antigen binding moiety binds, thereby forming an antigen binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, in free matter in serum and / or in the extracellular matrix (ECM).

[0093] As used herein, the term "antigen binding portion" refers to binding, including polypeptide molecules that specifically bind to antigenic determinants. In some aspects, the antigen binding portion can guide the entity to which it is attached (e.g., the second antigen binding portion) to the target site, for example, to a specific type of tumor cell carrying an antigenic determinant. In other aspects, the antigen binding portion can be activated by its target antigen (e.g., T cell receptor complex antigen) signal transduction. The antigen binding portion includes antibodies and fragments thereof as further defined herein. Specific antigen binding portions include the antigen binding domains of antibodies, which include antibody heavy chain variable regions and antibody light chain variable regions. In some aspects, the antigen binding portion may include antibody constant regions as further defined herein and known in the art. Available heavy chain constant regions include any of the following five isotypes: α, δ, ε, γ or μ. Available light chain constant regions include any of the following two isotypes: κ and λ.

[0094] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The term "binding" (bind or binding) herein generally refers to "specific binding". The ability of an antigen binding portion to bind to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance (SPR) technology (analyzed on, for example, 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 aspects, the extent to which the antigen binding portion binds to an unrelated protein is less than about 10%, for example, by SPR, compared to the extent to which the antigen binding portion binds to the antigen. In certain aspects, the antigen binding portion that binds to the antigen, or an antibody comprising the antigen binding portion, has the following dissociation constant (K D ): ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g. 10 -8 M or smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M).

[0095] "Affinity" refers to the strength of the sum 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, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (K D ) indicates that the dissociation constant is the sum of the dissociation rate constant and the association rate constant (k off and k on ). Thus, 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).

[0096] Unless otherwise indicated, "CD3" refers to any natural 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). The term encompasses "full-length" unprocessed CD3, as well as any form of CD3 produced by processing in cells. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In some aspects, CD3 is human CD3, in particular the epsilon subunit (CD3 epsilon) of human CD3. The amino acid sequence of human CD3 epsilon 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] CD3 epsilon is shown in NCBI GenBank No. BAB71849.1. See also SEQ ID NO:2.

[0097] As used herein, "target cell antigen" refers to an antigenic determinant present on the surface of a target cell, such as a cell in a tumor (such as a cancer cell or a cell of a tumor stroma) (in this case, a "tumor cell antigen"). Preferably, the target cell antigen is not CD3, and / or is expressed on a different cell than CD3. In some aspects, the target cell antigen is CD20, particularly human CD20.

[0098] As used herein, the terms "first", "second" or "third" with respect to antigen binding moieties, etc., are used for convenience to distinguish when there are more than one of each type of moiety. Unless explicitly stated, the use of these terms is not intended to confer a particular order or orientation to the bispecific antibodies.

[0099] As used herein, the term "valent" indicates that there is a specified number of antigen binding sites in an antibody. Thus, the term "monovalently binds to an antigen" indicates that there is one (and no more than one) antigen binding site in the antibody that is specific for the antigen.

[0100] The term "antibody" 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.

[0101] The terms "full length antibody," "intact antibody," and "complete antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure.

[0102] "Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269 to 315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments having two antigen binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single domain antibodies are antibody fragments comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain aspects, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B1). Antibody fragments can be prepared by various techniques, including but not limited to proteolytic digestion of intact antibodies, and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0103] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. As used herein, "Kabat numbering" in relation to variable region sequences refers to the numbering system proposed by Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0104] As used herein, the amino acid positions of all constant regions and constant 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 edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "numbered according to Kabat" or "Kabat numbering." Specifically, the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) pages 647 to 660) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661 to 723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3), which is further clarified herein by being referred to in this case as "numbered according to the Kabat EU index."

[0105] As used herein, the term "hypervariable region" or "HVR" refers to the various regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, such as "complementarity determining regions" ("CDRs"). Generally speaking, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include:

[0106] (a) hypervariable loops occurring at the following amino acid residues: 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2), and 96 to 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0107] (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

[0108] (c) Antigenic contact points occur at the following amino acid residues: 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2), and 93 to 101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)).

[0109] Unless otherwise indicated, CDRs are determined according to the method described in Kabat et al., supra. One skilled in the art will appreciate that CDR names may also be determined according to the method described in Chothia, supra, the method described in McCallum, supra, or any other scientifically accepted naming system.

[0110] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain is usually composed of the following four FR domains: FR1, FR2, FR3 and FR4. Therefore, the HVR sequence and FR sequence usually appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0111] The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0112] "Fab molecule" refers to a protein composed of the VH and CH1 domains of the heavy chain ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain") of an immunoglobulin.

[0113] The so-called "cross" Fab molecule (also referred to as "Crossfab") refers to the following Fab molecules: wherein the variable domains or constant domains of the Fab heavy chain and light chain are exchanged (i.e., replaced with each other), i.e., the cross Fab molecule comprises a peptide chain (VL-CH1, in the N-terminal to C-terminal direction) consisting of the light chain variable domain VL and the heavy chain constant domain 1CH1, and a peptide chain (VH-CL, in the N-terminal to C-terminal direction) consisting of the heavy chain variable domain VH and the light chain constant domain CL. For clarity, in the cross Fab molecule in which the variable domains of the Fab light chain and the variable domains of the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the (cross) Fab molecule. Conversely, in the cross Fab molecule in which the constant domains of the Fab light chain and the constant domains of the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (cross) Fab molecule.

[0114] In contrast, the so-called "conventional" Fab molecule refers to a Fab molecule in its natural form, i.e., comprising a heavy chain consisting of a heavy chain variable domain and a constant domain (VH-CH1, in the N-terminal to C-terminal direction), and a light chain consisting of a light chain variable domain and a constant domain (VL-CL, in the N-terminal to C-terminal direction).

[0115] The term "immunoglobulin molecule" refers to a protein with the structure of a naturally occurring antibody. For example, the IgG class immunoglobulin is a heterotetrameric glycoprotein of about 150,000 daltons, which is composed of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH) (also referred to as a variable heavy chain domain or a heavy chain variable region), followed by three constant domains (CH1, CH2, and CH3) (also referred to as a heavy chain constant region). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL) (also referred to as a variable light chain domain or a light chain variable region), followed by a constant light chain (CL) domain (also referred to as a 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 can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types based on the amino acid sequence of their constant domains: called kappa (κ) and lambda (λ). Immunoglobulins are essentially composed of two Fab molecules and an Fc domain connected by the immunoglobulin hinge region.

[0116] The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes a native sequence Fc region and a variant Fc region. Although the boundaries of the IgG heavy chain Fc region may be slightly different, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the antibody produced by the 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. Therefore, the antibody produced by the host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or the antibody may include a cleaved variant of the full-length heavy chain. This may be a case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) in the Fc region may be present or absent. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is numbered according to the EU numbering system (also referred to as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also 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 comprising the C-terminal constant region of an immunoglobulin heavy chain, which polypeptide is capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0117] A "modification that promotes the association of the first and second subunits of the Fc domain" is a manipulation of the peptide backbone or a post-translational modification of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising an Fc domain subunit with the same polypeptide to form a homodimer. As used herein, modifications that promote association particularly include separate modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, wherein the modifications complement each other to promote the association of the two Fc domain subunits. For example, modifications that promote association may change the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (heterologous) dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may be different in the sense that the additional components (e.g., antigen binding moieties) fused to each subunit are not the same. In some aspects, modifications that promote association include amino acid mutations, particularly amino acid substitutions, in the Fc domain. In a particular aspect, the modifications promoting association comprise individual amino acid mutations, in particular amino acid substitutions, in each of the two subunits of the Fc domain.

[0118] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0119] "Percentage (%) of amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignments for determining percentages of amino acid sequence identity can be achieved in various ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA package. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for purposes herein, the BLOSUM50 comparison matrix is ​​used to generate values ​​for % amino acid sequence identity using the ggsearch program of the FASTA package version 36.3.8c or higher. The FASTA package was created 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, sequences can be compared using a public server accessible at http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure that a global rather than a local alignment is performed. The amino acid identity percentages are given in the output alignment header.

[0120] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc domain of an antibody, initiates a signaling event that stimulates cells bearing the receptor to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0121] "Decreased binding" (e.g., reduced binding to an Fc receptor) refers to a decrease in affinity for the corresponding interaction, as measured, for example, by SPR. For clarity, the term also includes a decrease in affinity to zero (or below the detection limit of the analytical method), i.e., complete elimination of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the corresponding interaction.

[0122] By "fused", it is meant that the components (eg, Fab molecule and Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0123] In certain aspects, the T cell bispecific antibody binds to CD3 and a target cell antigen. Thus, in some aspects, 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.

[0124] In some aspects, the first antigen binding moiety and / or the second antigen binding moiety are Fab molecules. In some aspects, the first antigen binding moiety is a cross Fab molecule, wherein the variable region or constant region of the Fab light chain and the Fab heavy chain is exchanged. In such aspects, the second antigen binding moiety is preferably a conventional Fab molecule.

[0125] In some aspects, wherein both the first antigen-binding moiety and the second antigen-binding moiety of the T cell bispecific antibody are Fab molecules, and in one of the antigen-binding moieties (particularly the first antigen-binding moiety), the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other,

[0126] i) in the constant domain CL of the first antigen binding moiety, the amino acid at position 124 is substituted by a positively charged amino acid (according to Kabat numbering), and wherein in the constant domain CH1 of the first antigen binding moiety, the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (according to the Kabat EU index numbering); or

[0127] ii) in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is substituted by a positively charged amino acid (according to Kabat numbering), and wherein in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (according to the Kabat EU index numbering).

[0128] The T cell bispecific antibody does not comprise the modifications mentioned under both i) and ii).The constant domains CL and CH1 of the antigen binding part with VH / VL exchange are not mutually replaced (ie remain unexchanged).

[0129] In more detail,

[0130] i) in the constant domain CL of the first antigen binding portion, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first antigen binding portion, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index numbering); or

[0131] ii) 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) (according to Kabat numbering), and in the constant domain CH1 of the second antigen-binding portion, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0132] In some aspects, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0133] On the other hand, 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) (according to Kabat numbering), 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) (according to the Kabat EU index numbering).

[0134] In a preferred aspect, 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) (according to Kabat numbering), and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), 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) (according to the Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0135] In some aspects, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is substituted by lysine (K) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 is substituted by glutamic acid (E) (according to the Kabat EU index numbering), and the amino acid at position 213 is substituted by glutamic acid (E) (according to the Kabat EU index numbering).

[0136] In some aspects, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is substituted by arginine (R) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 is substituted by glutamic acid (E) (according to the Kabat EU index numbering), and the amino acid at position 213 is substituted by glutamic acid (E) (according to the Kabat EU index numbering).

[0137] In a specific aspect, if the amino acid substitutions according to the above aspects are performed in the constant domain CL and the constant domain CH1 of the second antigen binding moiety, the constant domain CL of the second antigen binding moiety is of the kappa isotype.

[0138] In some aspects, the first antigen binding moiety and the second antigen binding moiety are fused to each other, optionally via a peptide linker.

[0139] In some aspects, the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule and (i) the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, or (ii) the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety.

[0140] In some aspects, the T cell bispecific antibody provides monovalent binding to CD3.

[0141] In certain aspects, T cell bispecific antibodies include a single antigen binding moiety that is bound to CD3 and two antigen binding moieties that are bound to a target cell antigen. Therefore, in some aspects, T cell bispecific antibodies include a third antigen binding moiety that is bound to a target antigen, particularly a Fab molecule, more particularly a conventional Fab molecule. The third antigen binding moiety may be incorporated into all features (such as amino acid substitutions in CDR sequences, variable region sequences and / or constant regions) associated with the second antigen binding moiety described herein, either alone or in combination. In some aspects, the third antigen moiety is identical to the first antigen binding moiety (e.g., also a conventional Fab molecule and comprising the same amino acid sequence).

[0142] In a specific aspect, the T cell bispecific antibody further comprises an Fc domain, which is composed of a first subunit and a second subunit. In some aspects, the Fc domain is an IgG Fc domain. In a specific aspect, the Fc domain is an IgG1 Fc domain. In other aspects, the Fc domain is an IgG4 Fc domain. In a more specific aspect, the Fc domain is an IgG4 Fc domain, which is included in an amino acid substitution at position S228 (numbered according to the Kabat EU index), particularly an amino acid substitution S228P. The amino acid substitution reduces the in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In another specific aspect, the Fc domain is a human Fc domain. In a specific preferred aspect, the Fc domain is a human IgG1 Fc domain. The exemplary sequence of human IgG1 Fc region is given with SEQ ID NO: 3.

[0143] In some aspects wherein the first antigen binding moiety, the second antigen binding moiety, and (where present) the third antigen binding moiety are each a Fab molecule, (a) (i) the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; and (b) the third antigen binding moiety (where present) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0144] In some aspects, the T cell bispecific antibody essentially consists of the first, second and third antigen binding moieties (particularly Fab molecules), the Fc domain composed of the first and second subunits, and optionally one or more peptide linkers.

[0145] The components of the T cell bispecific antibody may be fused directly to each other or preferably via one or more suitable peptide linkers. When the Fab molecule is fused to the N-terminus of a subunit of the Fc domain, the fusion is typically via the immunoglobulin hinge region.

[0146] The antigen binding portion can be fused directly to the Fc domain or to each other, or fused to the Fc or to each other through a peptide linker comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n 、(SG4) n 、(G4S) n 、G4(SG4) n or (G4S) n G5 peptide linker. "n" is typically an integer from 1 to 10, typically from 2 to 4. In some aspects, the peptide linker has a length of at least 5 amino acids, in some aspects 5 to 100 amino acids, and in other aspects 10 to 50 amino acids. In some aspects, the peptide linker is (GxS) n or (GxS) n G m , wherein 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), in some aspects, x = 4 and n = 2 or 3, in other aspects, x = 4 and n = 2, in yet other aspects, x = 4, n = 1 and m = 5. In some aspects, the peptide linker is (G4S)2. In other aspects, the peptide linker is G4SG5. In addition, the linker may comprise (a portion of) an immunoglobulin hinge region. In particular, in the case of fusion of the Fab molecule to the N-terminus of the Fc domain subunit, the fusion may be via an immunoglobulin hinge region or a portion thereof with or without an additional peptide linker.

[0147] In certain aspects, the Fc domain comprises a modification that promotes the association of the first subunit and the second subunit of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain. Therefore, in some aspects, the modification is in the CH3 domain of the Fc domain.

[0148] In a specific aspect, 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 in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. Knob-into-hole technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide, so that the protrusion can be positioned in the cavity to promote the formation of heterodimers and hinder the formation of homodimers. The protrusion is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size as the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0149] Therefore, in some aspects, the amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced by an amino acid residue with a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion can be positioned in the cavity in the CH3 domain of the second subunit, and the amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced by an amino acid residue with a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit can be positioned in the cavity. Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Preferably, the amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V). Protrusions and cavities can be prepared by changing the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis.

[0150] In specific aspects of this type, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbered according to the Kabat EU index). On the other hand, in the first subunit of the Fc domain, the serine residue at position 354 is replaced by a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (particularly the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index). In a preferred aspect, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0151] In some aspects, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0152] In certain aspects, the Fc receptor is an Fcγ receptor. In some aspects, the Fc receptor is a human Fc receptor. In some aspects, the Fc receptor is an activating Fc receptor. In specific aspects, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In some aspects, the effector function is selected from one or more of the groups of complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and cytokine secretion. In certain aspects, the effector function is ADCC.

[0153] Typically, the same one or more amino acid substitutions are present in each of the two subunits of the Fc domain. In some aspects, the one or more amino acid substitutions reduce the binding affinity of the Fc domain to an Fc receptor. In some aspects, the one or more amino acid substitutions reduce the binding affinity of the Fc domain to an Fc receptor to at least half, at least one-fifth, or at least one-tenth of the original.

[0154] In some aspects, the Fc domain is included in the amino acid replacement at the position of the group selected from E233, L234, L235, N297, P331 and P329 (according to the KabatEU index numbering). In more specific aspects, the Fc domain is included in the amino acid replacement at the position of the group selected from L234, L235 and P329 (according to the KabatEU index numbering). In some aspects, the Fc domain comprises amino acid replacement L234A and L235A (according to the KabatEU index numbering). In some such aspects, the Fc domain is an IgG1 Fc domain, specifically human IgG1 Fc domain. In some aspects, the Fc domain is included in the amino acid replacement at position P329. In more specific aspects, amino acid replacement is P329A or P329G, particularly P329G (according to the KabatEU index numbering). In some aspects, the Fc domains are included in the amino acid replacement at position P329, and another amino acid replacement at a position selected from E233, L234, L235, N297 and P331 (according to Kabat EU index numbering). In more specific aspects, another amino acid replacement is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular aspects, the Fc domains are included in position P329, L234 and L235 (according to Kabat EU index numbering) and amino acid replacement. In more specific aspects, the Fc domains include amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). In particular, in preferred aspects, each subunit of the Fc domain comprises 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) (according to the Kabat EU index numbering). In some such aspects, the Fc domain is an IgG1 Fc domain, in particular a human IgG1 Fc domain.

[0155] In some aspects, the target cell antigen of the T cell bispecific antibody is CD20.

[0156] Unless otherwise indicated, "CD20", also referred to as "B lymphocyte antigen B1", refers to any native CD20 from any vertebrate source, including mammals, such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses "full-length", unprocessed CD20, as well as any form of CD20 produced by processing in cells. The term also encompasses naturally occurring variants of CD20, such as splice variants or allelic variants. In some aspects, CD20 is human CD20. Human CD20 is described in UniProt (www.uniprot.org) Accession No. P11836 (entry version 200), and the amino acid sequence of human CD20 is also shown in SEQ ID NO: 24.

[0157] Useful T cell bispecific antibodies that bind to CD20 for use in the present invention are described, for example, in PCT Publication No. WO2016 / 020309 (incorporated herein by reference in its entirety).

[0158] In some aspects, the T cell bispecific antibody comprises a first antigen binding moiety that binds to CD3 and a second antigen binding moiety that binds to CD20.

[0159] In some aspects, the first antigen binding portion comprises: a heavy chain variable region comprising a 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 a light chain CDR (LCDR) 1 of SEQ ID NO:7, LCDR2 of SEQ ID NO:8, and LCDR3 of SEQ ID NO:9.

[0160] In some aspects, the second antigen binding portion comprises: a heavy chain variable region comprising a 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 a light chain CDR (LCDR) 1 of SEQ ID NO: 15, LCDR2 of SEQ ID NO: 16, and LCDR3 of SEQ ID NO: 17.

[0161] In some aspects, the T cell bispecific antibody comprises

[0162] (i) a first antigen binding portion that binds to CD3 and comprises: a heavy chain variable region comprising a heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, a HCDR2 of SEQ ID NO: 5, and a HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR (LCDR) 1 of SEQ ID NO: 7, a LCDR2 of SEQ ID NO: 8, and a LCDR3 of SEQ ID NO: 9; and

[0163] (ii) a second antigen binding portion that binds to CD20 and comprises: a heavy chain variable region comprising a 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 a light chain CDR (LCDR) 1 of SEQ ID NO: 15, LCDR2 of SEQ ID NO: 16, and LCDR3 of SEQ ID NO: 17.

[0164] In some aspects, 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. In some aspects, the first antigen binding portion comprises a heavy chain variable region sequence of SEQ ID NO: 10 and a light chain variable region sequence of SEQ ID NO: 11.

[0165] In some aspects, 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 aspects, the second antigen binding portion comprises a heavy chain variable region sequence of SEQ ID NO: 18 and a light chain variable region sequence of SEQ ID NO: 19.

[0166] In some aspects, the T cell bispecific antibody comprises a third antigen binding moiety that binds to CD20 and / or an Fc domain composed of a first and a second subunit, as described herein.

[0167] In a preferred aspect, the T cell bispecific antibody comprises

[0168] (i) a first antigen binding moiety that binds to CD3, comprising: a heavy chain variable region comprising a heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, a HCDR2 of SEQ ID NO: 5, and a HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR (LCDR) 1 of SEQ ID NO: 7, a LCDR2 of SEQ ID NO: 8, and a LCDR3 of SEQ ID NO: 9, wherein the first antigen binding moiety is a crossover Fab molecule, wherein the Fab light chain is exchanged with the variable region or constant region, in particular the variable region, of the Fab heavy chain;

[0169] (ii) a second antigen-binding moiety and a third antigen-binding moiety that bind to CD20, comprising: a heavy chain variable region comprising a heavy chain CDR (HCDR) 1 of SEQ ID NO: 12, a HCDR2 of SEQ ID NO: 13, and a HCDR3 of SEQ ID NO: 14; and a light chain variable region comprising a light chain CDR (LCDR) 1 of SEQ ID NO: 15, a LCDR2 of SEQ ID NO: 16, and a LCDR3 of SEQ ID NO: 17, wherein the second antigen-binding moiety and the third antigen-binding moiety are each a Fab molecule, in particular a conventional Fab molecule;

[0170] (iii) an Fc domain, which is composed of a first subunit and a second subunit,

[0171] wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0172] In some aspects, the first antigen binding moiety of the T cell bispecific antibody (binding to CD20 and CD3) is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and wherein the second antigen binding moiety and (when present) the third antigen binding moiety of the T cell bispecific antibody are conventional Fab molecules in which, in the constant domain CL, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0173] In particular, in the above aspect, in the constant domain CL of the second Fab molecule and the third Fab molecule under (ii), the amino acid at position 124 may be substituted by lysine (K) (according to Kabat numbering), and the amino acid at position 123 may be lysine (K) or arginine (R), particularly substituted by arginine (R) (according to Kabat numbering), and in the constant domain CH1 of the second Fab molecule and the third Fab molecule under (ii), the amino acid at position 147 may be substituted by glutamic acid (E) (according to Kabat EU index numbering), and the amino acid at position 213 may be substituted by glutamic acid (E) (according to Kabat EU index numbering).

[0174] In some aspects, the first antigen binding portion of the T cell bispecific antibody (binding to 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 aspects, the first antigen binding portion comprises a heavy chain variable region sequence of SEQ ID NO: 10 and a light chain variable region sequence of SEQ ID NO: 11.

[0175] In some aspects, the second antigen binding portion and (when present) the third antigen binding portion of the T cell bispecific antibody (binding to 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: 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 aspects, the second antigen binding portion and (when present) the third antigen binding portion comprises the heavy chain variable region of SEQ ID NO: 18 and the light chain variable region of SEQ ID NO: 19.

[0176] The Fc domain according to the above aspects may incorporate all the features described above for the Fc domain, alone or in combination.

[0177] In some aspects, the Fc domain of the T cell bispecific antibody (binding to CD20 and CD3) comprises a modification that promotes 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 an Fc receptor and / or effector function.

[0178] In some aspects, the antigen binding portion and the Fc region are fused to each other via a peptide linker, particularly via a peptide linker in SEQ ID NO:21 and SEQ ID NO:23.

[0179] In some aspects, the T cell bispecific antibody (binding to CD20 and CD3) comprises: a polypeptide (particularly two polypeptides) comprising a sequence 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 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 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 at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:23. In some aspects, the T cell bispecific antibody (binding to CD20 and CD3) comprises: a polypeptide (particularly 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.

[0180] In a preferred aspect, the T cell bispecific antibody is gefuzumab (WHO Drug Information (International Nonproprietary Names of Drugs), Suggested INN: List 83, 2020, Vol. 34, No. 1, p. 39).

[0181] In some aspects, the disease (to be treated by the T cell engager) is cancer.

[0182] As used herein, "treatment" (and grammatical variants thereof such as treat or treat) refers to an attempt to alter the natural course of the disease in the individual being treated, and may be performed for prevention or may be performed during clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis.

[0183] The term "cancer" refers to a physiological condition in mammals that is usually characterized by unregulated cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More non-limiting examples of cancer include hematological 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, gastric cancer, prostate cancer, skin cancer, squamous cell carcinoma, sarcoma, bone cancer, and renal cancer. Other cell proliferation disorders include, but are not limited to, tumors located in the following parts: abdomen, bones, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary glands, testicles, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral nervous system), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital system. Precancerous conditions or lesions and cancer metastasis are also included.

[0184] In some aspects, the cancer is a cancer that expresses a target cell antigen of the T cell engager (eg, a T cell bispecific antibody).

[0185] In some aspects, the cancer is a cancer that expresses CD20 (particularly in aspects where the target cell antigen of the T cell engager (e.g., T cell bispecific antibody) is CD20). "CD20-positive cancer" or "cancer expressing CD20" means a cancer characterized by 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 content), flow cytometry, immunohistochemistry (IHC), or Western blot assays. In some aspects, the cancer expresses CD20. In some aspects, the cancer expresses CD20 in at least 20%, preferably at least 50%, or at least 80% of the tumor cells, as determined by immunohistochemistry (IHC) using an antibody specific for CD20.

[0186] In some aspects, the cancer is a B cell cancer, particularly a CD20-positive B cell cancer (particularly in aspects where the target cell antigen of the T cell engager (e.g., T cell bispecific antibody) is CD20). In some aspects, the cancer is selected from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphocytic 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 aspects, the cancer is selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), 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 aspects, the cancer is NHL. In some aspects, NHL is relapsed or refractory (r / r) NHL. In some aspects, NHL is inert NHL (iNHL) or aggressive NHL (aNHL). In some aspects, the cancer is DLBCL. In some aspects, DLBCL is Richter's transformation. In some aspects, the cancer is high-grade B-cell lymphoma (HGBCL). In some aspects, the cancer is primary mediastinal large B-cell lymphoma (PMBCL). In some aspects, the cancer is FL. In some aspects, FL is relapsed or refractory (r / r) FL. In some aspects, FL is transformed FL. In some aspects, the cancer is MCL. In some aspects, MCL is relapsed or refractory (r / r) MCL. In some aspects, the cancer is MZL.

[0187] In some aspects, cancer can be treated by a T cell engager. In some aspects, a T cell engager is useful for treating cancer.

[0188] In some aspects, a T cell engager (particularly a T cell bispecific antibody that binds to CD20 and CD3, as described above) is administered to an individual with a dosing regimen that includes at least a first dosing cycle and a second dosing cycle, wherein: (a) the first dosing cycle includes a first dose (C1D1) and a second dose (C1D2) of the T cell engager, wherein C1D1 is 2.5 mg and C1D2 is 10 mg; and (b) the second dosing cycle includes a single dose (C2D1) of 16 mg or 30 mg of the T cell engager. In some aspects, a single dose of the second dosing cycle includes 30 mg of the T cell engager. In some aspects, 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 aspects, a single dose (C2D1) of the second dosing cycle is administered on day 1 of the second dosing cycle. In some aspects, the dosing regimen includes 1 to 10 (C3 to C12) additional dosing cycles. In some such aspects, 1 to 10 additional dosing cycles (C3 to C12) include a single dose (C3D1 to C12D1) of 16mg or 30mg of the T cell engager. In some aspects, a single dose (C3D1 to C12D1) of an additional dosing cycle includes 30mg of the T cell engager. In some aspects, a single dose (C3D1 to C12D1) of an additional dosing cycle is applied on the 1st day of each additional dosing cycle. In some aspects, the dosing regimen includes a total of 12 dosing cycles. In some aspects, a treatment cycle includes 14 days or 21 days. In some aspects, a treatment cycle includes 21 days. In some aspects, treatment using the T cell engager stops after 12 dosing cycles.

[0189] "Individual" or "subject" herein is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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 some aspects, an individual or subject is a human. In some aspects, an individual suffers from a disease, particularly a disease that can be treated by a T cell engager or to be treated by a T cell engager. In some aspects, an individual suffers from cancer, particularly a cancer that can be treated by a T cell engager or to be treated by a T cell engager. In particular, an individual herein is any single human subject who is eligible for treatment who is experiencing or has experienced one or more signs, symptoms, or other indicators of cancer. In some aspects, an individual suffers from cancer or has been diagnosed with cancer, particularly any cancer described above. In some aspects, an individual suffers from locally advanced or metastatic cancer or has been diagnosed with locally advanced or metastatic cancer. An individual may have previously been treated with a T cell engager (e.g., a T cell bispecific antibody) or another drug, or has not received such treatment. In certain aspects, the patient has not been previously treated with a T cell engager (e.g., a T cell bispecific antibody). Prior to starting T cell engager therapy, the patient may have been treated with a therapy that includes one or more drugs other than a T cell engager (e.g., other than a T cell bispecific antibody).

[0190] In some aspects, the serum level of one or more cytokines of an individual increases. In some aspects, the serum level of the increase is related to the application of a T cell engagement agent to an individual. The serum level of the increase is particularly compared to the serum level in a healthy individual and / or an individual (including the same individual) to which a T cell engagement agent is not applied (i.e., in this case, compared to the serum level without the application of a T cell engagement agent, the serum level increases). In some aspects, the one or more cytokines are selected from the group consisting of IL-1 β, IL-6 and IL-8.

[0191] The cytokine according to any of the aspects of the present invention may 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 (IFNγ-induced protein 10; also known as CXC 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.

[0192] Preferably, the T cells according to any aspect of the invention are cytotoxic T cells. In some aspects, the T cells are CD4 + T cells or CD8 + In some aspects, the T cell is a CD8 + In some aspects, the T cell is a CD4 + T cells.

[0193] In some aspects, treatment with a T cell engager or administration of a T cell engager may result in a reaction in an individual. In some aspects, the reaction may be a complete reaction. In some aspects, the reaction may be a sustained reaction after treatment has stopped. In some aspects, the reaction may be a complete reaction that continues after treatment has stopped. In other aspects, the reaction may be a partial reaction. In some aspects, the reaction may be a partial reaction that continues after treatment has stopped. In some aspects, treatment with a T cell engager and an NLRP3 inhibitor or administration of a T cell engager and an NLRP3 inhibitor may improve the reaction compared to treatment with a T cell engager alone or administration of a T cell engager alone (i.e., without an NLRP3 inhibitor). In some aspects, treatment or administration of a T cell engager and an NLRP3 inhibitor may increase the response rate in a patient population compared to a corresponding patient population treated with a T cell engager alone (i.e., without an NLRP3 inhibitor).

[0194] T cell engaging agents can be used alone or together with other agents in therapy. For example, T cell engaging agents can be co-administered with at least one additional therapeutic agent. In certain aspects, the additional therapeutic agent is an anticancer agent, such as a chemotherapeutic agent, a tumor cell proliferation inhibitor, or a tumor cell apoptosis activator.

[0195] NLRP3 inhibitors can be used alone or in combination with one or more other agents to prevent or reduce adverse reactions associated with the administration of T cell engagers, particularly CRS. NLRP3 inhibitors can be used, for example, with IL-6R antagonists (e.g., tocilizumab), steroids (e.g., corticosteroids such as methylprednisolone, prednisolone and / or dexamethasone) or TNF-α antagonists (e.g., etanercept).

[0196] In some aspects, NLRP3 inhibitors can be used with B cell depleting agents such as obinutuzumab (proposed INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453). As used herein, obinutuzumab is synonymous with GA101. The trade name is or In some aspects, obinutuzumab is used as a pretreatment agent (“ The "priming agent" is administered prior to administration of a T cell engager, particularly prior to the first administration of a T cell engager (eg, a T cell bispecific antibody that binds to CD20 and CD3, as described above).

[0197] In some aspects, obinutuzumab is administered 1 to 15 days, in particular 1 to 10 days, prior to the (first) administration of the T cell engager. In a specific aspect, obinutuzumab is administered 7 days prior to the (first) administration of the T cell engager.

[0198] In some aspects, Obinutuzumab is administered at a dosage of 1000mg to 2000mg. In some aspects, Obinutuzumab is administered at a single dose of 1000mg. In some aspects, Obinutuzumab is administered at a single dose of 2000mg. In some aspects, Obinutuzumab is administered at a first dose and a second dose of 1000mg each. In some aspects, the first dose and the second dose of Obinutuzumab are administered on the same day, particularly 7 days before (the first time) the T cell engager is administered. In particular aspects, 2000mg Obinutuzumab is administered 7 days before (the first time) the T cell engager is administered with a single dose of 2000mg or the first dose and the second dose of 1000mg each. In some aspects, the first dose and the second dose of Obinutuzumab are administered on different days. In particular aspects, the first dose of Obinutuzumab is administered 7 days before (the first time) the T cell engager is administered, and the second dose of Obinutuzumab is administered 1 day before the T cell engager is administered.

[0199] During the course of treatment with a T cell engager (particularly a T cell bispecific antibody that is bound to CD20 and CD3, as described above), Obinutuzumab can continue to be administered. In some aspects, Obinutuzumab and the T cell engager are administered on the first day of the second dosing cycle (C2) and any subsequent dosing cycle (e.g., the third (C3) to the twelfth (C12) dosing cycle). In some such aspects, Obinutuzumab is administered at a dosage of 1000 mg. In some aspects, treatment is stopped after a total of 12 dosing cycles using the T cell engager.

[0200] Amino acid sequence

[0201]

[0202]

[0203]

[0204] BRIEF DESCRIPTION OF THE DRAWINGS

[0205] Figure 1 .Effect of NLRP3 inhibitors on CD20-TCB-mediated T cell activation, B cell depletion, and cytokine release. Fresh whole blood from a healthy donor (donor 1) was incubated with increasing doses of CD20-TCB and 10 μM NLRP3 inhibitor. At 24 hr, blood from technical replicates was pooled and lysed. A. Killing of CD19+ B cells was measured by flow cytometry. B and C. CD25 expression on CD4+ (B) and CD8+ (C) T cells was measured by flow cytometry. At 24 hr, serum from technical replicates was collected and pooled. DN. The levels of IFN-γ (D), IL-2 (E), IL-1β (F), IL-1Ra (G), TNF-α (H), IP-10 (I), IL-6 (J), IL-8 (K), MCP-1 (L), MIP-1α (M), and MIP-1β (N) were measured by Luminex.

[0206] Figure 2.Effect of NLRP3 inhibitors on CD20-TCB-mediated T cell activation, B cell depletion, and cytokine release. Fresh whole blood from a healthy donor (donor 2) was incubated with increasing doses of CD20-TCB and 10 μM NLRP3 inhibitor. At 24 hr, blood from technical replicates was pooled and lysed. A. Killing of CD19+ B cells was measured by flow cytometry. B and C. CD25 expression on CD4+ (B) and CD8+ (C) T cells was measured by flow cytometry. At 24 hr, serum from technical replicates was collected and pooled. DN. Levels of IFN-γ (D), IL-2 (E), IL-1β (F), IL-1Ra (G), TNF-α (H), IP-10 (I), IL-6 (J), IL-8 (K), MCP-1 (L), MIP-1α (M), and MIP-1β (N) were measured by Luminex.

[0207] Figure 3 .Effect of NLRP3 inhibitors on CD20-TCB-mediated T cell activation, B cell depletion, and cytokine release. Fresh whole blood from a healthy donor (donor 3) was incubated with increasing doses of CD20-TCB and 10 μM NLRP3 inhibitor. At 24 hr, blood from technical replicates was pooled and lysed. A. Killing of CD19+ B cells was measured by flow cytometry. B and C. CD25 expression on CD4+ (B) and CD8+ (C) T cells was measured by flow cytometry. At 24 hr, serum from technical replicates was collected and pooled. DN. Levels of IFN-γ (D), IL-2 (E), IL-1β (F), IL-1Ra (G), TNF-α (H), IP-10 (I), IL-6 (J), IL-8 (K), MCP-1 (L), MIP-1α (M), and MIP-1β (N) were measured by Luminex.

[0208] Figure 4 Effect of NLRP3 inhibitors on CD20-TCB-mediated antitumor efficacy. Humanized NSG mice carrying OCI-Ly18 were treated with 30 mg / kg obinutuzumab ( The mice were pretreated with 20 mg / kg NLRP3 inhibitor (black arrow, 0.5 mg / kg at the first treatment, 1 mg / kg at the second treatment, and 2 mg / kg at the third treatment) by intravenous (iv) injection. 20 mg / kg NLRP3 inhibitor (grey arrow) was given by intraperitoneal injection 1 hr before, 4 hr and 24 hr after the first CD20-TCB therapy, and 1 hr before and 4 hr after the second CD20-TCB therapy. A. Tumor growth inhibition curves, mean - / + SD. Tumor volume was calculated based on caliper measurements performed 2 or 3 times per week. B. CD8+ T cell counts in tumors 24 hr after the third CD20-TCB therapy, box plots represent the minimum to maximum values ​​of n=5 mice. Tumors were digested and CD8+ T cell counts were measured by flow cytometry.

[0209] Figure 5 Effects of NLRP3 inhibitors on CD20-TCB-induced IL-2, IFN-γ, TNF-α, IL-1β, and IL-6. Figure 4 Treatment was performed as described in. Blood was collected by tail vein bleeding 3 hr after the first CD20-TCB therapy. Serum levels of A. IL-2, B. IFN-γ, C. TNF-α, D. IL-1β, and E. IL-6 were measured by Luminex. n=mean + / - SEM of 5 mice.

[0210] Figure 6 .Heatmap showing the effect of NLRP3 inhibitors on CD20-TCB-mediated cytokine release. Figure 4 The treatment was performed as described in. Blood was collected by tail vein bleeding 3 hr after the first CD20-TCB therapy. For n=5 mice per group, 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 in serum were measured by Luminex.

[0211] Figure 7 Effect of NLRP3 inhibitors on weight loss induced by the first CD20-TCB therapy. Figure 4 Treatments were performed as described in. The body weight of each animal was measured before and after the first treatment with CD20-TCB to calculate the body weight change [%]. Mean + / - SEM.

[0212] Examples

[0213] The following are examples of methods and compositions of the invention.It is understood that various other aspects may be practiced, given the general description provided above.

[0214] Example 1. NLRP3 inhibitors prevent TCB-mediated cytokine release with minimal effects on T cell activation and T cell cytotoxicity

[0215] To assess the effect of NLRP3 inhibitors on TCB-mediated target cell killing and T cell activation, we performed a whole blood assay using fresh whole blood from three healthy donors incubated with increasing concentrations of CD20-TCB (SEQ ID NOs 4-23) combined with 10 μM NLRP3 inhibitor (MCC950, Sigma-Aldrich). At 24 hr, killing of CD19+ B cells in whole blood and expression of CD25 on CD4+ and CD8+ T cells were measured by flow cytometry.

[0216] NLRP3 inhibitors minimally interfered with CD20-TCB-induced B cell depletion, as indicated by the percentage of CD19+ B cells among CD45+ cells ( Figure 1 A. Figure 2 A and Figure 3 A). Consistent with this, NLRP3 inhibitors did not block the expression of the activation marker CD25 on CD4+ and CD8+ T cells, suggesting that the inhibitors did not interfere with CD20-TCB-induced T cell activation. ( Figure 1 B and Figure 1 C. Figure 2 B and Figure 2 C and Figure 3 B and Figure 3 C).

[0217] The cytokine levels in whole blood serum treated with CD20-TCB were measured by Luminex in the presence and absence of NLRP3 inhibitors to determine the effect of NLRP3 inhibitors on cytokine release. The results showed that NLRP3 inhibitors reduced the levels of IL-1β and IL-8 in all three donors and the levels of IL-6 in two of the three donors. However, the inhibitor did not affect the levels of IFN-γ, IL-2, TNF-α, IP-10, IL-1Ra, MCP-1, MIP-1α, and MIP-1β ( Figure 1 DN, Figure 2 DN and Figure 3 DN).

[0218] In conclusion, NLRP3 inhibitors preserved T cell cytotoxicity and T cell activation after stimulation with CD20-TCBs, while reducing the release of IL-6, IL-1β, and IL-8. Given the role of these cytokines in the pathophysiology of CRS, this data suggests that NLRP3 inhibitors may be attractive compounds for alleviating CRS in the context of T cell engagement therapy.

[0219] Example 2. NLRP3 inhibitors retain in vivo efficacy while preventing TCB-mediated cytokine release and body weight loss.

[0220] The diffuse large B-cell lymphoma (DLBCL) tumor model in humanized NSG mice was used to evaluate the effects of NLRP3 inhibitors on the antitumor efficacy and cytokine release mediated by CD20-TCB in vivo. Briefly, humanized NSG mice were implanted with OCI-LY18 tumor cells. When the tumor volume reached 200mm 3 At 4 d, mice were randomly divided into 14 groups based on tumor size. They were then treated with 30 mg / kg obinutuzumab ( The mice were pretreated with CD20-TCB for 7 days to deplete peripheral B cells. The mice were injected intravenously with 0.5 mg / kg, 1 mg / kg and 2 mg / kg CD20-TCB weekly. Figure 4 A. NLRP3 inhibitors were administered 1 hr before, 4 hr and 24 hr after the first CD20-TCB treatment and 1 hr before and 4 hr after the second CD20-TCB treatment.

[0221] To assess the effect of NLRP3 inhibitors on tumor growth, tumor volumes were calculated based on caliper measurements taken twice or three times per week. In addition, CD8+ T cell counts were measured by flow cytometry in tumors collected 24 hr after the third treatment with CD20-TCB. Therefore, NLRP3 inhibitors did not interfere with the antitumor efficacy mediated by CD20-TCB ( Figure 4 A), nor does it interfere with CD8+ T cell infiltration in tumors ( Figure 4 B).

[0222] Cytokine levels in serum collected by tail vein bleeding were measured 3 hr after the first CD20-TCB treatment to determine the effect of NLRP3 inhibitors on CD20-TCB-mediated cytokine release in vivo. Consistent with previous in vitro findings, NLRP3 inhibitors preserved IL-2, IFN-γ, TNF-α, while reducing CD20-TCB-induced IL-6, IL-8, and IL-1β levels ( Figure 5 A to Figure 5 E. Figure 6). In addition, NLRP3 inhibitors altered the cytokine profile induced by CD20-TCB treatment, as indicated by the heat map showing 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, which were measured 3 hr after the first treatment with CD20-TCB ( Figure 6 ). Finally, we measured the body weight of each animal before and after the first treatment with CD20-TCB to calculate weight change. We found that NLRP3 inhibitors prevented CD20-TCB-induced weight loss, suggesting that the inhibitors could prevent clinical signs of CRS. ( Figure 7 ).

[0223] In summary, NLRP3 inhibitors preserved CD20-TCB antitumor activity while reducing IL-1β and IL-6 levels. This prevented weight loss induced by the first treatment with CD20-TCB. In summary, the data support the use of NLRP3 inhibitors to alleviate CRS symptoms that may be induced by treatment with TCB.

[0224] ***

[0225] Although the present invention has been previously described in considerable detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. A T cell engager for use in treating a disease in an individual, wherein the treatment comprises (a) administering the T cell engager to the individual, and (b) administering a NACHT, LRR and PYD domain-containing protein 3 (NLRP3) inhibitor to the individual.

2. Use of a T cell engager in the manufacture of a medicament for treating a disease in an individual, wherein the treatment comprises (a) administering the T cell engager to the individual, and (b) administering a NLRP3 inhibitor to the individual.

3. A method for treating a disease in an individual, wherein the method comprises (a) administering a T cell engager to the individual, and (b) administering a NLRP3 inhibitor to the individual.

4. The T cell engager, use or method according to any one of claims 1 to 3, wherein the administration of the NLRP3 inhibitor is used to prevent or alleviate adverse reactions associated with the administration of the T cell engager.

5. A NLRP3 inhibitor for use in preventing or alleviating adverse reactions associated with administering a T cell engager to an individual.

6. Use of a NLRP3 inhibitor in the manufacture of a medicament for preventing or alleviating adverse reactions associated with the administration of a T cell engager.

7. A method for preventing or reducing an adverse reaction associated with administering a T cell engager to an individual, the method comprising administering a NLRP3 inhibitor to the individual.

8. The T cell engager, NLRP3 inhibitor, use or method of any one of the preceding claims, wherein the NLRP3 inhibitor is MCC950 or a derivative thereof.

9. The T cell engager, NLRP3 inhibitor, use or method of any of the preceding claims, wherein the NLRP3 inhibitor is selected from the group consisting of MCC950, cenosat, emlenoflast, 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.

10. The T cell engager, NLRP3 inhibitor, use or method of any one of the preceding claims, wherein (administration of) the NLRP3 inhibitor results in inhibition of an adverse effect associated with administration of the T cell engager.

11. The T cell engager, NLRP3 inhibitor, use or method of any one of the preceding claims, wherein (administration of) the NLRP3 inhibitor does not result in inhibition of a desired response associated with administration of the T cell engager.

12. The T cell engager, NLRP3 inhibitor, use or method of claim 10 or 11, wherein the inhibition is complete inhibition, or clinically meaningful and / or statistically significant inhibition.

13. The T cell engager, NLRP3 inhibitor, use or method according to any one of claims 4 to 12, wherein the adverse reaction is (i) Cytokine release syndrome (CRS); (ii) fever, hypotension and / or hypoxia; and / or (iii) elevated 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.

14. The T cell engager, NLRP3 inhibitor, use or method according to any one of claims 4 to 13, wherein the NLRP3 inhibitor is administered based on the (clinically) manifestation of the adverse effect (in the individual).

15. The T cell engager, NLRP3 inhibitor, use or method of any one of the preceding claims, wherein administration of the NLRP3 inhibitor is (i) before, simultaneously with and / or after administration of said T cell engager; (ii) intermittently or continuously; and / or (iii) oral or parenteral administration, especially intravenous administration.

16. The T cell engager, NLRP3 inhibitor, use or method of any of the preceding claims, wherein administration of the NLRP3 inhibitor is associated with a first administration of the T cell engager, and optionally is before, simultaneously with and / or after the first administration of the T cell engager.

17. The T cell engager, NLRP3 inhibitor, use or method of any one of the preceding claims, wherein administering the T cell engager is (i) administering at an effective dose; (ii) parenteral administration, in particular intravenous administration; and / or (iii) administering the T cell engager to the individual for the first time.

18. The T cell engager, NLRP3 inhibitor, use or method according to any one of the preceding claims, wherein the T cell engager is a T cell bispecific antibody or a CAR-T cell.

19. The T cell engager, NLRP3 inhibitor, use or method of claim 18, wherein the T cell bispecific antibody binds to CD3 and a target cell antigen.

20. The T cell engager, NLRP3 inhibitor, use or method of claim 18 or 19, wherein 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.

21. The T cell engager, NLRP3 inhibitor, use or method of claim 19 or 20, wherein the target cell antigen is CD20.

22. The T cell engager, NLRP3 inhibitor, use or method of claim 21, wherein the T cell bispecific antibody comprises (i) a first antigen binding portion that binds to CD3 and comprises: a polypeptide comprising SEQ ID NO: 4, HCDR2 of SEQ ID NO:5, and HCDR3 of SEQ ID NO:6; and a light chain variable region comprising a light chain CDR (LCDR) 1 of SEQ ID NO:7, LCDR2 of SEQ ID NO:8, and LCDR3 of SEQ ID NO:9; and (ii) a second antigen binding moiety that binds to CD20 and comprises: a moiety comprising SEQ ID NO: The heavy chain variable region comprises a 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 comprises a light chain CDR (LCDR) 1 of SEQ ID NO: 15, LCDR2 of SEQ ID NO: 16 and LCDR3 of SEQ ID NO:

17.

23. The T cell engager, NLRP3 inhibitor, use or method according to claim 21 or 22, wherein 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.

24. The T cell engager, NLRP3 inhibitor, use or method according to any one of claims 21 to 23, wherein the T cell bispecific antibody comprises (i) a first antigen binding moiety that binds to CD3, comprising: a heavy chain variable region comprising a heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, a HCDR2 of SEQ ID NO: 5, and a HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR (LCDR) 1 of SEQ ID NO: 7, a LCDR2 of SEQ ID NO: 8, and a LCDR3 of SEQ ID NO: 9, wherein the first antigen binding moiety is a crossover Fab molecule in which the variable regions or constant regions of the Fab light chain and the Fab heavy chain are exchanged; (ii) a second antigen-binding portion that binds to CD20 and a third antigen-binding portion comprising: a heavy chain CDR (HCDR) 1 comprising SEQ ID NO: 12, a heavy chain CDR (HCDR) 2 comprising SEQ ID NO: 13 and HCDR2 of SEQ ID NO: 14 and HCDR3 of SEQ ID NO: 15; and a light chain variable region comprising a light chain CDR (LCDR) 1 of SEQ ID NO: 15, LCDR2 of SEQ ID NO: 16 and LCDR3 of SEQ ID NO: 17, wherein the second antigen-binding moiety and the third antigen-binding moiety are each Fab molecules, in particular conventional Fab molecules; (iii) an Fc domain, which is composed of a first subunit and a second subunit, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

25. The T cell engager, NLRP3 inhibitor, use or method of any one of claims 21 to 24, wherein the first antigen binding portion of the T cell bispecific antibody comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 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, and / or the second antigen binding portion and (when present) the third antigen binding portion of the T cell bispecific antibody comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 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.

26. A T cell engager, NLRP3 inhibitor, use or method according to any one of claims 21 to 25, wherein the first antigen binding portion of the T cell bispecific antibody is a crossover Fab molecule, wherein the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and wherein the second antigen binding portion and (when present) the third antigen binding portion of the T cell bispecific antibody are conventional Fab molecules, wherein in the constant domain CL, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

27. The T cell engager, NLRP3 inhibitor, use or method according to any one of claims 23 to 26, wherein the Fc domain of the T cell bispecific antibody 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 an Fc receptor and / or effector function.

28. The T cell engager, NLRP3 inhibitor, use or method of any one of claims 18 to 27, wherein the T cell bispecific antibody is Gefituzumab.

29. The T cell engager, NLRP3 inhibitor, use or method according to any one of the preceding claims, wherein the disease (to be treated by the T cell engager) is cancer, in particular a cancer expressing the target cell antigen of the T cell engager.

30. The T cell engager, NLRP3 inhibitor, use or method of claim 29, wherein the cancer is (i) cancers expressing CD20, (ii) B cell cancer, and / or (ii) selected from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphocytic 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).

31. The invention as hereinbefore described.

Images