Type II anti-CD20 antibody for reducing formation of anti-drug antibodies

Abstract

23620964_1 (GHMatters) P108213.AU.1 5 Abstract The present invention relates to methods of treating a disease, and methods for reduction of the formation of anti-drug antibodies (ADAs) in response to the administration of a therapeutic agent. The invention further relates to methods of treating a disease, particularly a B-cell proliferative disorder, and methods for reduction of adverse effects in response to the administration of a therapeutic agent, particularly a T-cell activating therapeutic agent. 20 24 20 06 23 17 J ul 2 02 6 1 7 J u l 2 0 2 6 A b s t r a c t 5 2 0 2 4 2 0 0 6 2 3

Description

The following statement is a full description of this invention, including the best method of performing known to me: F. Hoffmann-La Roche AG -i- 2024200623   01 Feb 2024 Type II anti-CD20 antibody for reducing formation of anti-drug antibodies Cross-reference to related application The present application is a divisional application of Australian Patent Application No. 2016368469, the entire disclosure of which is incorporated into the present specification by this cross-reference. 5                                   Field of the Invention The present invention relates to methods of treating a disease, and methods for reduction of the formation of anti-drug antibodies (ADAs) in response to the administration of a therapeutic agent. The invention further relates to methods of treating a disease, particularly a B-cell proliferative disorder, and methods for reduction of adverse effects in response to the 10 administration of a therapeutic agent, particularly a T-cell activating therapeutic agent. Background The number of biotechnology-derived therapeutic agents available for use in clinical settings has dramatically increased in recent years, and includes recombinant human cytokines (e.g. a 15 and P interferon, interleukin-2), cellular growth factors (e.g. GM-CSF), hormones (e.g. glucagon), neuromuscular antagonists (e.g. botulinum toxin), blood products (e.g. clotting factor VIII), recombinant receptors (e.g. etanercept) and monoclonal antibodies. Although therapeutic proteins are generally considered safe and non-toxic, antibodies against these therapeutic agents, known as anti-drug antibodies (ADAs), can develop during treatment. 20 ADAs have been observed in connection with various therapeutic agents, such as erythropoietin, factor VIII , insulin, immunotoxins and monoclonal antibodies (Schellekens and Casadevall, J Neurol (2004), 251 [Suppl 2]:II / 4-II / 9; Mossoba et al., Clin Cancer Res (2011) 17(11): 3697-3705; Hsu et al., British Journal of Dermatology (2014) 170, 261-273). ADA formation is frequent for example in autoimmune patients treated with TNF blockers 25 and impacts clinical outcome (Schaeverbecke et al., Rheumatology (2015) doi: 10.1093 / rheumatology / kev277). The development of ADAs may influence serum concentrations and function of therapeutic agents. The presence of ADAs may increase clearance of the therapeutic agent through formation of immune complexes between therapeutic agent and antibody (neutralizing, non-30 neutralizing or both), thus reducing the therapeutic agent’s half-life. Furthermore, the activity and effectiveness of the therapeutic agent may be decreased through binding of antibody to 2024200623   01 Feb 2024 the therapeutic agent. AD As can also be associated with allergic or hypersensitivity reactions and other adverse events. Since these adverse events associated with immune responses can influence the safety and efficacy profile of therapeutics, identification and development of strategies to overcome or 5   inhibit AD As is of great interest. Several protein engineering approaches have been investigated to reduce the immunogenicity of protein therapeutics, including for example masking or alteration of protein B cell epitopes or modification of protein T cell epitopes. However, clinical safety and success of these approaches has not been tested and will require a significant degree of time to evaluate. 10 Therefore, there exists an immediate need to develop new interventions using FDA-approved reagents to prevent ADA responses. Chemotherapy-based approaches aimed at host immune suppression have been reported (Mossoba et al., Clin Cancer Res (2011) 17(11): 3697-3705). The anti-CD20 antibody rituximab has been used in combination with methotrexate and 15 intravenous immune globulin to achieve tolerance to enzyme replacement therapy in a Morbus Pompe patient (Mendelsohn et al., NEJM (2009) 360:2, 194-195). However, in a clinical trial, host pretreatment with rituximab did not inhibit the human immune response against the immunotoxin LMB-1 (Hassan et al., Clin Cancer Res (2004) 10, 16-18). B-cell proliferative disorders describe a heterogeneous group of malignancies that includes 20 both leukemias and lymphomas. Lymphomas develop from lymphatic cells and include two main categories: Hodgkin lymphomas (HL) and the non-Hodgkin lymphomas (NHL). In the United States, lymphomas of B cell origin constitute approximately 80-85% of all nonHodgkin lymphoma cases, and there is considerable heterogeneity within the B-cell subset, based upon genotypic and phenotypic expression patterns in the B-cell of origin. For 25 example, B cell lymphoma subsets include the slow-growing indolent and incurable diseases, such as Follicular lymphoma (FL) or chronic lymphocytic leukemia (CLL), as well as the more aggressive subtypes, mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). Despite the availability of various agents for the treatment of B-cell proliferative disorders, 30 there is an ongoing need for development of safe and effective therapies to prolong remission and improve cure rates in patients. 2024200623   01 Feb 2024 A strategy currently being investigated is the engagement of T cells against malignant B cells. In order to effectively engage T cells against malignant B cells, two recent approaches have been developed. These two approaches are: 1) the administration of T cells engineered ex vivo to recognize tumour cells (also known as chimeric antigen receptor-modified T cell 5 therapy [CAR-T cells]) (Maude et al., N Engl J Med (2014) 371,1507-1517); and, 2) the administration of agents that activate endogenous T cells, such as bispecific antibodies (Oak and Bartlett, Expert Opin Investig Drugs (2015) 24, 715-724). An example of the first approach is reported in the study by Maude et al., in which 30 adult and pediatric patients were treated with autologous T cells transduced with a CD19-directed 10 chimeric antigen receptor lentiviral vector (CTL019 CAR-T cells). The result was a sustained remission based upon a 6-month event-free survival rate of 67% and an overall survival rate of 78%. However, all patients had cytokine release syndrome (CRS) (associated with tumour burden), with 27% of patients having severe CRS. Central nervous system toxicities of unknown cause were also noted at high frequencies. 15 In contrast, the second approach, which involves activating endogenous T cells to recognize tumour targets, bypasses this hurdle of scalability, and can also provide competitive efficacy, safety data and potentially long term durations of response. In different CD20+ hematologic malignancies, this approach is best exemplified by blinatumomab, a CD 19 CD3 targeting T cell bispecific molecule (Bargou et al., Science (2008) 321, 974-977) that was recently 20 approved for patients with minimal residual disease-positive acute lymphocytic leukemia (ALL). This compound, which is composed of two single chain Fv fragments (the so called BiTE® format), directs the lysis of CD19+ cells by cytolytic T cells. The primary constraint of blinatumomab is its short half-life (approximately 2 hours), which necessitates continuous infusion via a pump over 4-8 weeks. Nonetheless, it has potent efficacy in patients with both 25 relapsed / refractory Non-Hodgkin Lymphoma (r / r NHL) and ALL, with step-up dosing (SUD) required to mitigate severe cytokine release syndrome and CNS toxicities (Nagorsen and Baeuerle, Exp Cell Res (2011) 317, 1255-1260). The CD20 CD3 targeting T cell bispecific molecule, CD20XCD3 bsAB, is another example of a next generation of B cell targeting antibody. CD20XCD3 bsAB is a T cell bispecific 30 (TCB) antibody targeting CD20 expressed on B cells and CD3 epsilon chain (CD3e) present on T cells. The mechanism of action of CD20XCD3 bsAB comprises simultaneous binding to CD20+ B cells and CD3+ T cells, leading to T-cell activation and T-cell mediated killing of B cells. In 2024200623   01 Feb 2024 the presence of CD20+ B cells, whether circulating or tissue resident, pharmacologically active doses will trigger T-cell activation and associated cytokine release. CD20XCD3 bsAB has shown enhanced potency in nonclinical models over competitive T cell engaging agents and, having an IgG-based format, has a greatly improved half-life over blinatumomab. 5 Cytokine release is the result of activation of T cells. In a phase 1 study conducted by TeGenero (Suntharalingam et al., N Engl J Med (2006) 355,1018-1028), all 6 healthy volunteers experienced near fatal, severe cytokine release syndrome (CRS) rapidly postinfusion of an inappropriately-dosed, T-cell stimulating super-agonist anti-CD28 monoclonal antibody. More recently, in the above-mentioned study by Maude et al. of CD19-targeting, 10 chimeric antigen receptor T cell (CAR-T cell) treatment of patients with relapsed ALL, all 30 patients had cytokine release, which was categorized as severe in 27% of the patients. CRS is a common but severe complication of CAR-T cell therapy (reviewed in Xu and Tang, Cancer Letters (2014) 343, 172-178). Severe CRS and CNS toxicity have also been frequently observed with the CD19-CD3 T cell 15 bispecific agent, blinatumomab (Klinger et al., Blood. 2012;l 19(26):6226-6233). In patients receiving blinatumomab in all clinical trials, neurological toxicities have occurred in approximately 50% of patients, and the types of toxicities observed are well-defined in the package insert. It is not well understood if or how CNS toxicity is related to earlier cytokine release or T cell 20 activation. Similar to blinatumomab, CNS AEs (ranging from delirium to global encephalopathy) were reported for 43% (13 / 30) of the patients with r / r ALL treated with CD19-targeting CAR-T cells (Maude et al., N Engl J Med (2014) 371,1507-1517; Ghorashian et al., Br J Haematol (2015) 169, 463-478). Neurologic toxic effects typically occurred after symptoms of CRS had peaked and started to resolve; however no direct, 25 unequivocal association with severe CRS was found. The authors proposed that the mechanism of neurotoxicity could involve direct CAR-T-cell-mediated toxicity or it could be cytokine-mediated. In contrast, an association between severe CRS and neurotoxicity (e.g., encephalopathy) has been suggested in another study of CD19-targeting CAR-T cell therapy (Davila et al., Sci Transl Med (2014) 6, 224ra25) and speculated to be due to general T cell 30 activation, versus direct CAR-T-induced damage. Cytokine release and / or CNS-related toxicities are particularly pronounced in T cell bispecific antibodies that link CD3+ cells to B cells, as compared to other T cell bispecific antibodies that link CD3+ cells to tissue-restricted (i.e., non-circulating) target cells. 2024200623   01 Feb 2024 There is thus a need for methods to reduce or prevent such adverse effects of these promising agents which have the potential to significantly contribute to the treatment of patients with B-cell proliferative disorders such as NHL and CLL. It is to be understood that if any prior art publication is referred to herein, such reference does 5 not constitute an admission that the publication forms a part of the common general knowledge in the art in Australia or any other country. Summary of the Invention The present invention is based on the surprising finding that (i) the formation of ADAs in 0 response to administration of an immunogenic therapeutic agent to a subject can effectively and sustainably be prevented, and (ii) the cytokine release associated with administration of a therapeutic agent, particularly a T-cell activating therapeutic agent such as CD20XCD3 bsAB, to a subject can be significantly reduced, by pre-treatment of said subject with a Type II anti-CD20 antibody, such as obinutuzumab. 15 Obinutuzumab is a humanized glyco-engineered type II anti-CD20 mAb that binds with high-affinity to the CD20 antigen, inducing antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), low complement-dependent cytotoxicity (CDC) activity, and high direct cell death induction. To date, the safety profile of obinutuzumab (including cytokine release) has been assessed and managed in hundreds of 20 patients in ongoing obinutuzumab clinical trials. Without wishing to be bound by theory, the use of obinutuzumab (GAZYVA®) pretreatment (GPT) should aid in the rapid depletion of B cells, both in the peripheral blood and in secondary lymphoid organs, such that the risk of highly relevant adverse events (AEs) from strong systemic T cell activation by (T-cell activating) therapeutic agents (e.g. CRS) is 25 reduced, while supporting exposure levels of therapeutic agents that are high enough from the start of dosing to mediate tumour cell elimination. In addition to supporting the safety profile of (T-cell activating) therapeutic agents such as CD20XCD3 bsAB, GPT should also help prevent the formation of anti-drug antibodies (ADAs) to therapeutic molecules. For patients, GPT should translate into better drug exposure with an enhanced safety profile. 2024200623   01 Feb 2024 GPT should be more effective in accomplishing the above goals compared to other methods used, such as step up dosing (SUD). For example, a single dose of obinutuzumab should allow relapsed / refractory patients to receive the full therapeutic dose of T-cell activating therapeutic agent such as CD20XCD3 bsAB, once determined, without a time delay from 5 step up dosing. In contrast thereto, it was recently reported that the blinatumomab dosing regimen for patients with r / r DLBCL in an ongoing Phase 2 trial incorporates a double step up approach (i.e., 9 —>28—>112 pg / m2 / day), thus, requiring 14 days to reach the maximum dose of 112 pg / m2 / day (Viardot el at., Hematol Oncol (2015) 33, 242(Abstract 285)). As shown in the Examples, following pretreatment with obinutuzumab, administration of 10 CD20XCD3 bsAB to cynomolgus monkeys was tolerated up to a level that was ten times higher than that tolerated without GPT. Efficient peripheral blood B-cell depletion and antitumour activity along with strongly reduced cytokine release in the peripheral blood associated with the first CD20XCD3 bsAB injection was observed upon GPT. Accordingly, in a first aspect the present invention provides a method for (i) reducing the 15 formation of anti-drug antibodies (ADAs) against a therapeutic agent in a subject and / or (ii) reducing cytokine release associated with administration of a therapeutic agent, particularly a T-cell activating therapeutic agent, in a subject, comprising administration of a Type II anti-CD20 antibody to the subject prior to administration of the therapeutic agent. In one embodiment the period of time between the administration of the Type II anti-CD20 antibody 20 and administration of the therapeutic agent is sufficient for reduction of the number of B-cells in the subject in response to the administration of the Type II anti-CD20 antibody. In a further aspect, the invention provides a method of treating a disease in a subject, the method comprising a treatment regimen comprising (i) administration to the subject of a Type II anti-CD20 antibody, 25 and consecutively after a period of time (ii) administration to the subject of a therapeutic agent, wherein the period of time between the administration of the Type II anti-CD20 antibody and the administration of the therapeutic agent is sufficient for reduction of the number of B-cells in the subject in response to the administration of the Type II anti-CD20 antibody. 30 In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADAs) in the subject in response to the administration of the therapeutic agent as 2024200623   01 Feb 2024 compared to a corresponding treatment regimen without the administration of the Type II anti-CD20 antibody. In another embodiment, the treatment regimen effectively reduces cytokine release associated with the administration of the therapeutic agent in the subject as compared to a corresponding 5 treatment regimen without the administration of the Type II anti-CD20 antibody. In such embodiment, the therapeutic agent preferably is a T cell activating therapeutic agent. In a further aspect, the invention provides a Type II anti-CD20 antibody for use in a method for (i) reducing the formation of anti-drug antibodies (ADAs) against a therapeutic agent in a subject and / or (ii) reducing cytokine release associated with the administration a therapeutic 10 agent, particularly a T-cell activating therapeutic agent, in a subject, comprising administration of the Type II anti-CD20 antibody to the subject prior to administration of the therapeutic agent. In one embodiment, the period of time between the administration of the Type II anti-CD20 antibody and administration of the therapeutic agent is sufficient for reduction of the number 15   of B-cells in the subject in response to the administration of the CD20 antibody. In a further aspect, the invention provides a Type II anti-CD20 antibody for use in a method of treating a disease in a subject, the method comprising a treatment regimen comprising (i) administration to the subject of the Type II anti-CD20 antibody, and consecutively after a period of time 20 (ii) administration to the subject of a therapeutic agent, wherein the period of time between the administration of the Type II anti-CD20 antibody and the administration of the therapeutic agent is sufficient for reduction of the number of B-cells in the subject in response to the administration of the Type II anti-CD20 antibody. In one embodiment, the treatment regimen effectively reduces the formation of anti-drug 25 antibodies (ADAs) against the therapeutic agent in the subject (in response to the administration of the therapeutic agent) as compared to a corresponding treatment regimen without the administration of the anti-CD20 antibody. In another embodiment, the treatment regimen effectively reduces cytokine release associated with the administration of the therapeutic agent in the subject as compared to a corresponding 30 treatment regimen without the administration of the Type II anti-CD20 antibody. In such embodiment, the therapeutic agent preferably is a T cell activating therapeutic agent. 2024200623   01 Feb 2024 In a further aspect, the invention provides the use of a Type II anti-CD20 antibody in the manufacture of a medicament for (i) reduction of the formation of anti-drug antibodies (ADAs) against a therapeutic agent in a subject and / or (ii) the reduction of cytokine release associated with administration of a therapeutic agent, particularly a T-cell activating 5 therapeutic agent, in a subject, wherein the medicament is to be used in a treatment regimen comprising (i) administration to the subject of the Type II anti-CD20 antibody, and consecutively after a period of time (ii) administration to the subject of a therapeutic agent, 10 wherein the period of time between the administration of the Type II anti-CD20 antibody and the administration of the therapeutic agent is sufficient for reduction of the number of B-cells in the subject in response to the administration of the Type II anti-CD20 antibody. In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADAs) against the therapeutic agent in the subject as compared to a 15 corresponding treatment regimen without the administration of the anti-CD20 antibody. In another embodiment, the treatment regimen effectively reduces cytokine release associated with administration of the therapeutic agent in the subject as compared to a corresponding treatment regimen without the administration of the Type II anti-CD20 antibody. In such embodiment, the therapeutic agent preferably is a T cell activating therapeutic agent. 20 In still a further aspect, the invention provides a kit for (i) the reduction of the formation of anti-drug antibodies (ADAs) against a therapeutic agent in a subject and / or (ii) the reduction of cytokine release associated with administration of a therapeutic agent, particularly a T-cell activating therapeutic agent, in a subject, comprising a package comprising a Type II anti-CD20 antibody composition and instructions for using the Type II anti-CD20 antibody 25 composition in a treatment regimen comprising (i) administration to the subject of the Type II anti-CD20 antibody composition, and consecutively after a period of time (ii) administration to the subject of a therapeutic agent, wherein the period of time between the administration of the Type II anti-CD20 antibody 30 composition and the administration of the therapeutic agent is sufficient for reduction of the number of B-cells in the subject in response to the administration of the Type II CD20 antibody. 2024200623   01 Feb 2024 In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADAs) against the therapeutic agent in the subject as compared to a corresponding treatment regimen without the administration of the Type II anti-CD20 antibody composition. 5 In another embodiment, the treatment regimen effectively reduces cytokine release associated with administration of the therapeutic agent in the subject as compared to a corresponding treatment regimen without the administration of the Type II anti-CD20 antibody composition. In such embodiment, the therapeutic agent preferably is a T cell activating therapeutic agent. In one embodiment, the kit further comprises a therapeutic agent composition. 10 The invention in a further aspect as provides a therapeutic agent for use in a method of treating a disease in a subject, the method comprising a treatment regimen comprising (i) administration to the subject of a Type II anti-CD20 antibody, and consecutively after a period of time (ii) administration to the subject of the therapeutic agent, 15 wherein the period of time between the administration of the Type II anti-CD20 antibody and the administration of the therapeutic agent is sufficient for reduction of the number of B-cells in the subject in response to the administration of the CD20 antibody. In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADAs) in the subject in response to the administration of the therapeutic agent as 20 compared to a corresponding treatment regimen without the administration of the Type II anti-CD20 antibody. In another embodiment, the treatment regimen effectively reduces cytokine release associated with administration of the therapeutic agent in the subject as compared to a corresponding treatment regimen without the administration of the Type II anti-CD20 antibody. In such 25 embodiment, the therapeutic agent preferably is a T cell activating therapeutic agent. The invention in still a further aspect provides the use of a therapeutic agent in the manufacture of a medicament for treatment of a disease in a subject, wherein the treatment comprises a treatment regimen comprising (i) administration to the subject of a Type II anti-CD20 antibody, 30 and consecutively after a period of time (ii) administration to the subject of the therapeutic agent, 2024200623   01 Feb 2024 wherein the period of time between the administration of the Type II anti-CD20 antibody and the administration of the therapeutic agent is sufficient for reduction of the number of B-cells in the subject in response to the administration of the Type II anti-CD20 antibody. In one embodiment, the treatment regimen effectively reduces the formation of anti-drug 5 antibodies (ADAs) in the subject in response to the administration of the therapeutic agent as compared to a corresponding treatment regimen without the administration of the Type II anti-CD20 antibody. In another embodiment, the treatment regimen effectively reduces cytokine release associated with administration of the therapeutic agent in the subject as compared to a corresponding 10 treatment regimen without the administration of the Type II anti-CD20 antibody. In such embodiment, the therapeutic agent preferably is a T cell activating therapeutic agent. The invention in a further aspect provides a kit for the treatment of a disease in a subject, comprising a package comprising a therapeutic agent composition and instructions for using the therapeutic agent composition in a treatment regimen comprising 15   (i) administration to the subject of a Type II anti-CD20 antibody, and consecutively after a period of time (ii) administration to the subject of the therapeutic agent composition, wherein the period of time between the administration of the Type II anti-CD20 antibody and the administration of the therapeutic agent composition is sufficient for reduction of the 20 number of B-cells in the subject in response to the administration of the Type II anti-CD20 antibody. In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADAs) against the therapeutic agent in the subject as compared to a corresponding treatment regimen without the administration of the Type II anti-CD20 25 antibody composition. In another embodiment, the treatment regimen effectively reduces cytokine release associated with administration of the therapeutic agent in the subject as compared to a corresponding treatment regimen without the administration of the Type II anti-CD20 antibody composition. In such embodiment, the therapeutic agent preferably is a T cell activating therapeutic agent. 30 In one embodiment, the kit further comprises a Type II anti-CD20 antibody composition. 2024200623   01 Feb 2024 -n- The methods, uses, Type II anti-CD20 antibodies, therapeutic agents and kits of the invention may incorporate, singly or in combination, any of the features described hereinbelow. In one embodiment, the Type II anti-CD20 antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5   5, and the HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9. In a more specific embodiment, the Type II anti-CD20 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of 10 SEQ ID NO: 11. In one embodiment, the Type II anti-CD20 antibody is an IgG antibody, particularly an IgGi antibody. In one embodiment, the Type II anti-CD20 antibody is engineered to have an increased proportion of non-fucosylated oligosaccharides in the Fc region as compared to a non- 15 engineered antibody. In one embodiment, at least about 40% of the N-linked oligosaccharides in the Fc region of the Type II anti-CD20 antibody are non-fucosylated. In a particular embodiment the anti-CD20 antibody is obinutuzumab. In some embodiments, in particular in relation aspects of the invention concerned with the reduction of the formation of anti-drug antibodies (ADAs) against a therapeutic agent in a 20 subject, the therapeutic agent comprises a polypeptide. In some embodiments, in particular in relation aspects of the invention concerned with the reduction of the formation of anti-drug antibodies (ADAs) against a therapeutic agent in a subject, the therapeutic agent comprises an antibody. In one such embodiment, the antibody specifically binds to carcinoembryonic antigen (CEA). 25 In one embodiment, the antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19. In a further embodiment, the antibody comprises the heavy chain variable region 30 sequence of SEQ ID NO: 20 and the light chain variable region sequence of SEQ ID NO: 21. 2024200623   01 Feb 2024 In another such embodiment, the antibody specifically binds to CD3, particularly CD3 epsilon. In one embodiment, the antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising the light 5 chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37. In a further embodiment, the antibody comprises the heavy chain variable region sequence of SEQ ID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39. In some embodiments, in particular in relation aspects of the invention concerned with the 10 reduction of the formation of anti-drug antibodies (ADAs) against a therapeutic agent in a subject, the therapeutic agent comprises a cytokine. In one such embodiment, the cytokine is interleukin-2 (IL-2). In another such embodiment, the cytokine is a mutant human IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 15 sequence SEQ ID NO: 12). In some embodiments, in particular in relation aspects of the invention concerned with the reduction of the formation of anti-drug antibodies (ADAs) against a therapeutic agent in a subject, the therapeutic agent comprises an immunoconjugate. In one such embodiment, the immunoconjugate comprises (a) an antibody that specifically 20 binds to CEA and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19, and (b) a mutant human IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A and 25 L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 12). In a particular such embodiment, the therapeutic agent comprises cergutuzumab amunaleukin (CEA-IL2v). In some embodiments, in particular in relation aspects of the invention concerned with the reduction of the formation of anti-drug antibodies (ADAs) against a therapeutic agent in a 30 subject, the therapeutic agent comprises a bispecific antibody that specifically binds to CEA and to CD3. In one such embodiment the therapeutic agent comprises a bispecific antibody comprising 2024200623   01 Feb 2024 (i) an antigen binding moiety that specifically binds to CD3and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 5   36 and the LCDR3 of SEQ ID NO: 37; and (ii) an antigen binding moiety that specifically bind to CEA and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 10   18 and the LCDR3 of SEQ ID NO: 19. In a particular embodiment, the therapeutic agent comprises CEA TCB. In some embodiments, in particular in relation aspects of the invention concerned with the reduction of cytokine release associated with the administration of a therapeutic agent in a subject, the therapeutic agent is a T cell activating therapeutic agent. 15 In one embodiment, the T-cell activating therapeutic agent comprises an antibody, particularly a multispecific (e.g. a bispecific) antibody. In one embodiment, the antibody specifically binds to an activating T cell antigen. In one embodiment, the antibody specifically binds to an antigen selected from the group of CD3, CD28, CD137 (also known as 4-1BB), CD40, CD226, OX40, GITR, CD27, HVEM, 20 and CD 127. In one embodiment, the antibody specifically binds to CD3, particularly CD3e. In one embodiment, the antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising the light chain CDR 25 (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37. In one embodiment, the antibody comprises the heavy chain variable region sequence of SEQ ID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39. In one embodiment, the antibody specifically binds to a B-cell antigen, particularly a 30 malignant B-cell antigen. In one embodiment, the antibody specifically binds to an antigen selected from the group consisting of CD20, CD19, CD22, ROR-1, CD37 and CD5, particularly to CD20 or CD19. 2024200623   01 Feb 2024 In one embodiment, the antibody specifically binds to CD20. In one embodiment, the antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising the light chain CDR 5 (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9. In one embodiment, the antibody comprises the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO: 11. In one embodiment, the antibody is a multispecific antibody, particularly a bispecific 10 antibody. In one embodiment, the multispecific antibody specifically binds to (i) an activating T cell antigen and (ii) a B cell antigen. In one embodiment, the multispecific antibody specifically binds to (i) CD3 and (ii) an antigen selected from CD20 and CD 19. 15 In one embodiment, the multispecific antibody specifically binds to CD3 and CD20. In some embodiments, in particular in relation aspects of the invention concerned with the reduction of cytokine release associated with the administration of a therapeutic agent in a subject, the therapeutic agent comprises a bispecific antibody comprising (i) an antigen binding moiety that specifically binds to CD3 and comprises a heavy chain 20 variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and (ii) an antigen binding moiety that specifically binds to CD20 and comprises a heavy chain 25 variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9. In a particular embodiment, the therapeutic agent comprises CD20XCD3 bsAB. 30 In some embodiments, in particular in relation aspects of the invention concerned with the reduction of cytokine release associated with the administration of a therapeutic agent in a subject, the therapeutic agent comprises a chimeric antigen receptor (CAR) or a T cell expressing a CAR, particularly a CAR that specifically binds to a B-cell antigen, more 2024200623   01 Feb 2024 particularly a CAR that specifically binds to an antigen selected from the group of CD20, CD 19, CD22, ROR-1, CD37 and CD5. In some embodiments, in particular in relation aspects of the invention concerned with the reduction of cytokine release associated with the administration of a therapeutic agent in a 5 subject, the disease is a B cell proliferative disorder, particularly a CD20-positive B-cell disorder. In one embodiment, the disease is selected from the group consisting of NonHodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL), Multiple myeloma (MM), 10 and Hodgkin lymphoma (HL). Brief Description of the Drawings Figure 1. Prior treatment with obinutuzumab but not rituximab or vehicle results in the attenuation of tetanus toxoid specific de novo IgG antibody responses in cynomolgus 15   monkeys. Rituxan indicates rituximab and GAI01 obinutuzumab, respectively. Figure 2. Memory recall responses by measles specific IgG antibody production in response to immune re-challenge with a measles / rubella booster vaccination in animals with baseline titers to measles is not affected by either obinutuzumab or rituximab in cynomolgus monkeys. Rituxan indicates rituximab and GAI01 obinutuzumab, respectively. 20 Figure 3. B cell counts (CD45+CD19+) in peripheral blood samples before start of obinutuzumab pre-treatment (BL = baseline), before start of treatment with RO6895882 (C1D1 = Cycle 1 Day 1) and during treatment with RO6895882. Lines / symbols represent individual patients. From the C1D1 time points onwards, no B cells were detectable in the peripheral blood samples. 25 Figure 4. Reduction of CD 19+ cells (B cells) detected by flow cytometry in tumor biopsies collected at baseline (BL) and after treatment with obinutuzumab (treated). On-treatment samples were obtained either before or during treatment with RO6895882. The percentage of CD45+ cells (lymphocytes) staining positive for CD 19 (B lymphocytes) was strongly reduced (B). No clear change was observed for the percentage of CD 16+ cells (Natural Killer 30 Cells) (A) or CD3+ cells (T lymphocytes) (C). Lines represent individual patients. 2024200623   01 Feb 2024 Figure 5. Reduction of B cells in tumor biopsies collected at baseline (BL) and after treatment (treated) with obinutuzumab measured by immunohistochemistry. On-treatment samples were obtained either before or during treatment with RO6895882. The density of B lymphocytes was measured by staining with CD20 (A, B) and PAX 5 (C, D). Both methods 5 detected a depletion of B lymphocytes in tumor and surrounding normal tissue. Lines represent individual patients. Figure 6. Exemplary configurations of the T cell activating bispecific antigen binding molecules (TCBs) useful in the invention. (A, D) Illustration of the “1+1 CrossMab” molecule. (B, E) Illustration of the “2+1 IgG Crossfab” molecule with alternative order of 10 Crossfab and Fab components (“inverted”). (C, F) Illustration of the “2+1 IgG Crossfab” molecule. (G, K) Illustration of the “1+1 IgG Crossfab” molecule with alternative order of Crossfab and Fab components (“inverted”). (H, L) Illustration of the “1+1 IgG Crossfab” molecule. (I, M) Illustration of the “2+1 IgG Crossfab” molecule with two CrossFabs. (J, N) Illustration of the “2+1 IgG Crossfab” molecule with two CrossFabs and alternative order of 15 Crossfab and Fab components (“inverted”). (O, S) Illustration of the “Fab-Crossfab” molecule. (P, T) Illustration of the “Crossfab-Fab” molecule. (Q, U) Illustration of the “(Fab)2-Crossfab” molecule. (R, V) Illustration of the “Crossfab-(Fab)2” molecule. (W, Y) Illustration of the “Fab-(Crossfab)2” molecule. (X, Z) Illustration of the “(Crossfab)2-Fab” molecule. Black dot: optional modification in the Fc domain promoting heterodimerization. 20   ++, —: amino acids of opposite charges optionally introduced in the CHI and CL domains. Crossfab molecules are depicted as comprising an exchange of VH and VL regions, but may - in embodiments wherein no charge modifications are introduced in CHI and CL domains -alternatively comprise an exchange of the CHI and CL domains. Figure 7. B cell and T cell counts in the peripheral blood in the different treatment groups. 25 Flow cytometry analysis of CD19+ B cells (A) and CD3+ T cells (B) in the peripheral blood of vehicle and CD20XCD3 bsAB-treated fully humanized NOG mice, 24 hours and 72 hours after first and second CD20XCD3 bsAB administration. Black arrows indicate days of CD20XCD3 bsAB administration. Figure 8. Cytokines released in peripheral blood among the different treatment groups. 30 Multiplex analysis of cytokines in blood of vehicle and treated mice, 24 hours and 72 hours after the first and second administration of CD20XCD3 bsAB. Histogram bars represent the 2024200623   01 Feb 2024 mean of 5 animals with error bars indicating the standard deviation. Representative graphs for IFNy (A), TNFa (B) and IL-6 (C) are shown. Compare the cytokine release of the first injection of CD20XCD3 bsAB with and without obinutuzumab pre-treatment (bars to be compared are indicated by connecting lines). 5 Figure 9. Anti-tumour activity of CD20XCD3 bsAB, obinutuzumab, and obinutuzumab pretreatment (Gpt) + CD20XCD3 bsAB. Anti-tumour activity of CD20XCD3 bsAB and obinutuzumab as monotherapy or Gpt + CD20XCD3 bsAB in fully humanized NOG mice. Black arrow indicates start of therapy. (8<n<10). Tumour model: WSU-DLCL2. Figure 10. Cytokines released in peripheral blood of cynomolgus monkeys following dosing 10 with CD20XCD3 bsAB and Gpt + CD20XCD3 bsAB treatments. (A) IFNy, (B) IL-8, (C) TNFa, (D) IL-2, (E) IL-6. Figure 11. Anti-tumor activity upon step-up dosing of CD20XCD3 bsAB and obinutuzumab pretreatment (Gpt) in fully humanized NOG mice bearing WSU-DLCL2 tumors. Mice received a first therapy (arrow) either as a fractionated dose of CD20XCD3 bsAB (0.15, 0.05, 15   0.015 mg / kg IV) or Gpt (10 mg / kg obinutuzumab), followed by weekly intravenous injections of CD20XCD3 bsAB at 0.5 mg / kg (full dose) for 9 treatment cycles (i.e., 9 weeks). In the vehicle group, one single mouse is shown from day 18. For the other groups, n = 9 or 10. Tumor model: WSU-DLCL2 injected subcutaneously. [CD20XCD3 bsAB 0.05 mg / kg + CD20XCD3 bsAB 0.05 mg / kg] vs [obinutuzumab 10 mg / kg + CD20XCD3 bsAB 20   0.5 mg / kg] *p = 0.018 (One-way ANOVA analysis of sAUC with Dunnet’s method). Figure 12. T-cell staining in lungs from fully humanized NOG mice bearing WSU-DLCL2 tumors, (A-D) 7 days after the first treatment, and (E-H) 24 hours after the second treatment. Treatment groups are as follows (single treatment or first + second treatment): (A) vehicle, (B) obinutuzumab 10 mg / kg, (C) CD20XCD3 bsAB 0.15 mg / kg, (D) 25 CD20XCD3 bsAB 0.5 mg / kg, (E) vehicle + vehicle, (F) obinutuzumab 10 mg / kg + CD20XCD3 bsAB 0.5 mg / kg, (G) CD20XCD3 bsAB 0.15 mg / kg + CD20XCD3 bsAB 0.5 mg / kg, (H) vehicle + CD20XCD3 bsAB 0.5 mg / kg. Lung sections are immunohistochemically-stained with anti-CD3 antibody (dark); nuclei were counterstained with hematoxylin. Magnification 20x. Arrows point to increase in 30 perivascular CD3 positive cells. 2024200623   01 Feb 2024 Figure 13. Lung of humanized NOG mouse sacrificed 24 hours after single treatment with 0.5 mg / kg of CD20XCD3 bsAB. Margination and adhesion of T cells (arrows) to the endothelium in vessels. Few T cells have transmigrated to the perivascular space (asterisks). (A) 20x magnification, (B) 40x magnification. 5 Figure 14. Serum concentration-time curves of CD20XCD3 bsAB following a single intravenous administration with or without obinutuzumab Pretreatment in cynomolgus monkeys. Cynomolgus monkeys were administered a single IV dose of 100-1000 pg / kg CD20XCD3 bsAB with or without obinutuzumab pretreatment (Gpt) (50 mg / kg, 4 days prior to CD20XCD3 bsAB administration). Six animals are represented in each dose group and the 0 data is presented as mean ± SD. Detailed Description of the Invention Definitions Terms are used herein as generally used in the art, unless otherwise defined in the following. 15 In the claims which follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. 20 CD20 (also known as B-lymphocyte antigen CD20, B-lymphocyte surface antigen Bl, Leu-16, Bp35, BM5, and LF5; the human protein is characterized in UniProt database entry Pl 1836) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD expressed on pre-B and mature B lymphocytes (Valentine, M.A. et al., J. Biol. Chern. 25   264 (1989) 11282-11287; Tedder, T.F., et al., Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 208 212; Stamenkovic, 1., et al., J. Exp. Med. 167 (1988) 1975-1980; Einfeld, D.A., et al., EMBO J. 7 (1988) 711-717; Tedder, T.F., et al., J. Immunol. 142 (1989) 2560-2568). The corresponding human gene is Membrane-spanning 4-domains, subfamily A, member 1, also 2024200623   01 Feb 2024 known as MS4A1. This gene encodes a member of the membrane-spanning 4A gene family. Members of this nascent protein family are characterized by common structural features and similar intron / exon splice boundaries and display unique expression patterns among hematopoietic cells and nonlymphoid tissues. This gene encodes the B-lymphocyte surface 5 molecule which plays a role in the development and differentiation of B-cells into plasma cells. This family member is localized to llql2, among a cluster of family members. Alternative splicing of this gene results in two transcript variants which encode the same protein. The term “CD20” as used herein, refers to any native CD20 from any vertebrate source, 10 including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CD20 as well as any form of CD20 that results from processing in the cell. The term also encompasses naturally occurring variants of CD20, e.g., splice variants or allelic variants. In one embodiment, CD20 is human CD20. The amino acid sequence of an exemplary human CD20 is shown in SEQ ID 15 NO: 1. The terms “anti-CD20 antibody” and “an antibody that binds to CD20” refer to an antibody that is capable of binding CD20 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD20. In one embodiment, the extent of binding of an anti-CD20 antibody to an unrelated, non-CD20 protein is less than about 10% 20 of the binding of the antibody to CD20 as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD20 has a dissociation constant (Kd) of < IpM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. IO'8 M or less, e.g. from 10'8 M to 10'13 M, e.g., from 10'9 M to 10'13 M). In certain embodiments, an anti-CD20 antibody binds to an epitope of CD20 that is conserved among CD20 from 25 different species. By “Type II anti-CD20 antibody” is meant an anti-CD20 antibody having binding properties and biological activities of Type II anti-CD20 antibodies as described in Cragg et al., Blood 103 (2004) 2738-2743; Cragg et al., Blood 101 (2003) 1045-1052, Klein et al., mAbs 5 (2013), 22-33, and summarized in Table 1 below. 2024200623   01 Feb 2024 Table 1. Properties of type I and type II anti-CD20 antibodies type I anti-CD20 antibodies type II anti-CD20 antibodies Bind class I CD20 epitope Bind class II CD20 epitope Localize CD20 to lipid rafts Do not localize CD20 to lipid rafts High CDC * Low CDC * ADCC activity * ADCC activity * Full binding capacity to B cells Approx, half binding capacity to B cells Weak homotypic aggregation Homotypic aggregation Low cell death induction Strong cell death induction * if IgGi isotype Examples of type II anti-CD20 antibodies include e.g. obinutuzumab (GAI 01), tositumumab (Bl), humanized B-Lyl antibody IgGl (a chimeric humanized IgGl antibody as disclosed in 5 WO 2005 / 044859), 11B8 IgGl (as disclosed in WO 2004 / 035607) and AT80 IgGl. Examples of type I anti-CD20 antibodies include e.g. rituximab, ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab, PRO131921, ublituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgGl (as disclosed in WO 2005 / 103081), 2F2 IgGl (as disclosed in WO 2004 / 035607 and WO 2005 / 103081) and 2H7 IgGl (as disclosed in WO 2004 / 056312). 10 The term “humanized B-Lyl antibody” refers to humanized B-Lyl antibody as disclosed in WO 2005 / 044859 and WO 2007 / 031875, which were obtained from the murine monoclonal anti-CD20 antibody B-Lyl (variable region of the murine heavy chain (VH): SEQ ID NO: 2; variable region of the murine light chain (VL): SEQ ID NO: 3 (see Poppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131-139) by chimerization with a human constant domain from 15 IgGl and following humanization (see WO 2005 / 044859 and WO 2007 / 031875). These “humanized B-Lyl antibodies” are disclosed in detail in WO 2005 / 044859 and WO 2007 / 031875. As used herein, the term "cytokine" refers to a molecule that mediates and / or regulates a biological or cellular function or process (e.g. immunity, inflammation, and hematopoiesis). 20 The term "cytokine" as used herein includes "lymphokines," "chemokines," "monokines," and "interleukins". Examples of useful cytokines include, but are not limited to, GM-CSF, 2024200623   01 Feb 2024 IL-la, IL-10, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN-a, IFN-0, IFN-y, MIP-la, MIP-10, TGF-0, TNF-a, and TNF-0. A particular cytokines is IL-2. The term “cytokine” as used herein is meant to also include cytokine variants comprising one or more amino acid mutations in the amino acid sequences of the corresponding wild-type cytokine, 5 such as for example the IL-2 variants described in Sauve et al., Proc Natl Acad Sci USA 88, 4636-40 (1991); Hu et al., Blood 101, 4853-4861 (2003) and US Pat. Publ. No. 2003 / 0124678; Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000); Heaton et al., Cancer Res 53, 2597-602 (1993) and US Pat. No. 5,229,109; US Pat. Publ. No. 2007 / 0036752; WO 2008 / 0034473; WO 2009 / 061853; or in WO 2012 / 107417. 10 The term “interleukin-2” or “IL-2” as used herein, refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2, e.g. splice variants or allelic variants. The amino acid sequence of 15 an exemplary human IL-2 is shown in SEQ ID NO: 12. Unprocessed human IL-2 additionally comprises an N-terminal 20 amino acid signal peptide having the sequence of SEQ ID NO: 31, which is absent in the mature IL-2 molecule. The term “interleukin-2” as used herein is meant to also include IL-2 variants comprising one or more amino acid mutations in the amino acid sequences of the corresponding wild-type cytokine, such as for example the IL-2 20 variants described in Sauve et al., Proc Natl Acad Sci USA 88, 4636-40 (1991); Hu et al., Blood 101, 4853-4861 (2003) and US Pat. Publ. No. 2003 / 0124678; Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000); Heaton et al., Cancer Res 53, 2597-602 (1993) and US Pat. No. 5,229,109; US Pat. Publ. No. 2007 / 0036752; WO 2008 / 0034473; WO 2009 / 061853; or in WO 2012 / 107417. 25 The term "IL-2 mutant" or "mutant IL-2 polypeptide" as used herein is intended to encompass any mutant forms of various forms of the IL-2 molecule including full-length IL-2, truncated forms of IL-2 and forms where IL-2 is linked to another molecule such as by fusion or chemical conjugation. "Full-length" when used in reference to IL-2 is intended to mean the mature, natural length IL-2 molecule. For example, full-length human IL-2 refers to 30 a molecule that has 133 amino acids (see e.g. SEQ ID NO: 12). The various forms of IL-2 mutants are characterized in having a at least one amino acid mutation affecting the 2024200623   01 Feb 2024 interaction of IL-2 with CD25. This mutation may involve substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at that position. Mutants obtained by amino acid substitution are preferred. Unless otherwise indicated, an IL-2 mutant may be referred to herein as an IL-2 mutant peptide sequence, an IL-2 mutant polypeptide, 5 IL-2 mutant protein or IL-2 mutant analog. Designation of various forms of IL-2 is herein made with respect to the sequence shown in SEQ ID NO: 12. Various designations may be used herein to indicate the same mutation. For example a mutation from phenylalanine at position 42 to alanine can be indicated as 42A, A42, A42, F42A, or Phe42Ala. As used herein, the term “release of cytokines” or "cytokine release" is synonymous with 10 “cytokine storm” or “cytokine release syndrome” (abbreviated as “CRS”), and refers to an increase in the levels of cytokines, particularly tumor necrosis factor alpha (TNF-a), interferon gamma (IFN-y), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-2 (IL-2) and / or interleukin-8 (IL-8), in the blood of a subject during or shortly after (e.g. within 1 day of) administration of a therapeutic agent, resulting in adverse symptoms. Cytokine release is a 15 type of infusion-related reaction (IRR), which are common adverse drug reactions to therapeutic agent and timely related to administration of the therapeutic agent. IRRs typically occur during or shortly after an administration of the therapeutic agent, i.e. typically within 24 hours after infusion, predominantly at the first infusion. In some instances, e.g. after the administration of CAR-T cells, CRS can also occur only later, e.g. several days after 20 administration upon expansion of the CAR-T cells. The incidence and severity typically decrease with subsequent infusions. Symptoms may range from symptomatic discomfort to fatal events, and may include fever, chills, dizziness, hypertension, hypotension, dyspnea, restlessness, sweating, flushing, skin rash, tachycardia, tachypnoea, headache, tumour pain, nausea, vomiting and / or organ failure. 25 The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to CD25 or to an Fc receptor. Amino acid sequence deletions and insertions include amino- and / or 30 carboxy-terminal deletions and insertions of amino acids. Particular amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an IL- 2024200623   01 Feb 2024 2 polypeptide or an Fc region, non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard 5 amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may 10 also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc region to glycine can be indicated as 329G, G329, G329, P329G, or Pro329Gly. The term “CD25” or “a-subunit of the IL-2 receptor” as used herein, refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g. humans) and 15 rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed CD25 as well as any form of CD25 that results from processing in the cell. The term also encompasses naturally occurring variants of CD25, e.g. splice variants or allelic variants. In certain embodiments CD25 is human CD25. The amino acid sequence of human CD25 is shown in UniProt (www.uniprot.org) accession no. P01589, or NCBI 20 (www.ncbi.nlm.nih.gov / ) RefSeq NP 000408. The term “high-affinity IL-2 receptor” as used herein refers to the heterotrimeric form of the IL-2 receptor, consisting of the receptor y-subunit (also known as common cytokine receptor y-subunit, yc, or CD132), the receptor P-subunit (also known as CD122 or p70) and the receptor a-subunit (also known as CD25 or p55). The term “intermediate-affinity IL-2 25 receptor” by contrast refers to the IL-2 receptor including only the y-subunit and the P-subunit, without the a-subunit (for a review see e.g. Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)). “Affinity” refers to the strength of the sum total 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 30 indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., receptor and a 2024200623   01 Feb 2024 ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by well 5 established methods known in the art. A particular method for measuring affinity is Surface Plasmon Resonance (SPR). “Reduction” (and grammatical variations thereof such as “reduce” or “reducing”), for example reduction of the number of B cells or the formation of AD As or cytokine release, refers to a decrease in the respective quantity, as measured by appropriate methods known in 10 the art. For clarity the term includes also reduction to zero (or below the detection limit of the analytical method), i.e. complete abolishment or elimination. Conversely, “increased” refers to an increase in the respective quantity. By “regulatory T cell” or “Treg cell” is meant a specialized type of CD4+ T cell that can suppress the responses of other T cells. Treg cells are characterized by expression of the a-15 subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and play a critical role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors. Treg cells require IL-2 for their function and development and induction of their suppressive characteristics. 20 As used herein, the term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen binding moiety is able to direct the entity to which it is attached (e.g. a cytokine or a second antigen binding moiety) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant. Antigen binding moieties include antibodies and fragments thereof 25 as further defined herein. Preferred antigen binding moieties include an antigen binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding moieties may include antibody constant regions as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: a, 5, a, y, or p. Useful light chain constant 30 regions include any of the two isotypes: k and X. 2024200623   01 Feb 2024 By "specifically binds" is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, 5 e.g. surface plasmon resonance technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217229 (2002)). As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope," and refers to a site (e.g. a contiguous stretch of amino acids or a conformational 10 configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, free in blood serum, and / or in the extracellular matrix (ECM). 15 As used herein, term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids, are included within the 20 definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring 25 amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 30   2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three- 2024200623   01 Feb 2024 dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded. By an "isolated" polypeptide or a variant, or derivative thereof is intended a polypeptide that 5 is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. 10 “Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. 15 Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the 20 sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. 25 TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino 30 acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that 2024200623   01 Feb 2024 has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence 5 alignment program ALIGN-2 in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein 10 are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. As used herein, the term "effector moiety" refers to a polypeptide, e.g., a protein or glycoprotein, that influences cellular activity, for example, through signal transduction or other cellular pathways. Accordingly, the effector moiety can be associated with receptor-15 mediated signaling that transmits a signal from outside the cell membrane to modulate a response in a cell bearing one or more receptors for the effector moiety. In one embodiment, an effector moiety can elicit a cytotoxic response in cells bearing one or more receptors for the effector moiety. In another embodiment, an effector moiety can elicit a proliferative response in cells bearing one or more receptors for the effector moiety. In another 20 embodiment, an effector moiety can elicit differentiation in cells bearing receptors for the effector moiety. In another embodiment, an effector moiety can alter expression (i.e. upregulate or downregulate) of an endogenous cellular protein in cells bearing receptors for the effector moiety. Non-limiting examples of effector moieties include cytokines, growth factors, hormones, enzymes, substrates, and cofactors. An effector moiety can be associated 25 with an antigen binding moiety such as an antibody in a variety of configurations to form an immunoconjugate. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents a cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, 30   Pb212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, 2024200623   01 Feb 2024 adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal 5 origin, including fragments and / or variants thereof; and the various antitumor or anticancer agents disclosed below. 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 10 exhibit the desired antigen binding activity. The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a 15 portion of an intact antibody that binds 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 multispecific antibodies formed from antibody fragments. The term “antibody fragment” as used herein also encompasses single-domain antibodies. 20 The term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by 25 three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five classes, called a (IgA), 5 (IgD), a (IgE), y (IgG), or p (IgM), some 30 of which may be further divided into subclasses, e.g. yi (IgGi), y2 (IgG2), 73 (IgGs), 74 (IgG4), 2024200623   01 Feb 2024 ai (IgAi) and a2 (IgA2). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (k) and lambda (X), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region. 5 The term "antigen binding domain" refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). 10 The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. 15 Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. 20 This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable 25 domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. 2024200623   01 Feb 2024 The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and / or form structurally defined loops (“hypervariable loops”) and / or contain the antigen-contacting residues (“antigen contacts”). Generally, 5 antibodies comprise six HVRs: three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 9196 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917(1987)); 10          (b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31 35b (Hl), 50-65 (H2), and 95-102 (H3) (Kabat et al.. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 73215   745 (1996)); and (d) combinations of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (Hl), 26-35b (Hl), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR 20 residues) are numbered herein according to Kabat et al., supra. "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. 25 The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgGs, IgG4, IgAi, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively. 2024200623   01 Feb 2024 The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is 5 usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy 10 chain, or it may include a cleaved variant of the full-length heavy chain (also referred to herein as a “cleaved variant heavy chain”). This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447), of the Fc region may or may not be present. Unless 15 otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). A “subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, 20 i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain. A “modification promoting heterodimerization” is a manipulation of the peptide backbone or the post-translational modifications of a polypeptide, e.g. an immunoglobulin heavy chain, 25 that reduces or prevents the association of the polypeptide with an identical polypeptide to form a homodimer. A modification promoting heterodimerization as used herein particularly includes separate modifications made to each of two polypeptides desired to form a dimer, wherein the modifications are complementary to each other so as to promote association of the two polypeptides. For example, a modification promoting heterodimerization may alter 30 the structure or charge of one or both of the polypeptides desired to form a dimer so as to make their association sterically or electrostatically favorable, respectively. Heterodimerization occurs between two non-identical polypeptides, such as two 2024200623   01 Feb 2024 immunoglobulin heavy chains wherein further immunoconjugate components fused to each of the heavy chains (e.g. IL-2 polypeptide) are not the same. In the immunoconjugates useful in the present invention, the modification promoting heterodimerization is in the heavy chain(s), specifically in the Fc domain, of an immunoglobulin molecule. In some 5 embodiments the modification promoting heterodimerziation comprises an amino acid mutation, specifically an amino acid substitution. In a particular embodiment, the modification promoting heterodimerization comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two immunoglobulin heavy chains. Similarly, a “modification promoting the association of the first and the second subunit of the 10 Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit 15 of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, a modification promoting association may alter 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, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit 20 and a polypeptide comprising the second Fc domain subunit, which might be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding moieties) are not the same. In some embodiments the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a particular embodiment, the modification promoting association comprises a separate amino acid 25 mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain. An “activating Fc receptor” is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Activating Fc receptors include FcyRIIIa (CD16a), Fey RI (CD64), FcyRIIa 30   (CD32), and FcaRI (CD89). 2024200623   01 Feb 2024 The term “effector functions” when used in reference to antibodies refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity 5 (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation. As used herein, the term “effector cells” refers to a population of lymphocytes that display effector moiety receptors, e.g. cytokine receptors, and / or Fc receptors on their surface 10 through which they bind an effector moiety, e.g. a cytokine, and / or an Fc region of an antibody and contribute to the destruction of target cells, e.g. tumor cells. Effector cells may for example mediate cytotoxic or phagocytic effects. Effector cells include, but are not limited to, effector T cells such as CD8+cytotoxic T cells, CD4+ helper T cells, y5 T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages / monocytes. 15 As used herein, the terms “engineer, engineered, engineering,” are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches. “Engineering”, 20 particularly with the prefix “glyco-”, as well as the term “glycosylation engineering” includes metabolic engineering of the glycosylation machinery of a cell, including genetic manipulations of the oligosaccharide synthesis pathways to achieve altered glycosylation of glycoproteins expressed in cells. Furthermore, glycosylation engineering includes the effects of mutations and cell environment on glycosylation. In one embodiment, the glycosylation 25 engineering is an alteration in glycosyltransferase activity. In a particular embodiment, the engineering results in altered glucosaminyltransferase activity and / or fucosyltransferase activity. Glycosylation engineering can be used to obtain a “host cell having increased GnTIII activity” (e.g. a host cell that has been manipulated to express increased levels of one or more polypeptides having P(l,4)-N-acetylglucosaminyltransferase III (GnTIII) activity), a “host 30 cell having increased Manll activity” (e.g. a host cell that has been manipulated to express increased levels of one or more polypeptides having a-mannosidase II (Manll) activity), or a 2024200623   01 Feb 2024 “host cell having decreased a(l,6) fiicosyltransferase activity” (e.g. a host cell that has been manipulated to express decreased levels of a(l,6) fiicosyltransferase). The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny 5 of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A host cell 10 is any type of cellular system that can be used to generate proteins used for the present invention. In one embodiment, the host cell is engineered to allow the production of an antibody with modified oligosaccharides. In certain embodiments, the host cells have been manipulated to express increased levels of one or more polypeptides having P(l,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In certain embodiments the host cells 15 have been further manipulated to express increased levels of one or more polypeptides having a-mannosidase II (Manll) activity. Host cells include cultured cells, e.g. mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, 20 transgenic plant or cultured plant or animal tissue. As used herein, the term "polypeptide having GnTIII activity" refers to polypeptides that are able to catalyze the addition of a N-acetylglucosamine (GlcNAc) residue in P-1,4 linkage to the P-linked mannoside of the trimannosyl core of N-linked oligosaccharides. This includes fusion polypeptides exhibiting enzymatic activity similar to, but not necessarily identical to, 25 an activity of P(l,4)-N-acetylglucosaminyltransferase III, also known as P-l,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl-transferase (EC 2.4.1.144), according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of GnTIII, but 30 rather substantially similar to the dose-dependency in a given activity as compared to the GnTIII (i.e. the candidate polypeptide will exhibit greater activity or not more than about 25- 2024200623   01 Feb 2024 fold less and, preferably, not more than about ten-fold less activity, and most preferably, not more than about three-fold less activity relative to the GnTIII). In certain embodiments the polypeptide having GnTIII activity is a fusion polypeptide comprising the catalytic domain of GnTIII and the Golgi localization domain of a heterologous Golgi resident polypeptide. 5 Particularly, the Golgi localization domain is the localization domain of mannosidase II or GnTI, most particularly the localization domain of mannosidase II. Alternatively, the Golgi localization domain is selected from the group consisting of: the localization domain of mannosidase I, the localization domain of GnTII, and the localization domain of a 1,6 core fucosyltransferase. Methods for generating such fusion polypeptides and using them to 10 produce antibodies with increased effector functions are disclosed in WO2004 / 065540, U.S. Provisional Pat. Appl. No. 60 / 495,142 and U.S. Pat. Appl. Publ. No. 2004 / 0241817, the entire contents of which are expressly incorporated herein by reference. As used herein, the term “Golgi localization domain” refers to the amino acid sequence of a Golgi resident polypeptide which is responsible for anchoring the polypeptide to a location 15 within the Golgi complex. Generally, localization domains comprise amino terminal "tails" of an enzyme. As used herein, the term "polypeptide having Manll activity" refers to polypeptides that are able to catalyze the hydrolysis of the terminal 1,3- and 1,6-linked a-D-mannose residues in the branched GlcNAcMansGlcNAc2 mannose intermediate of N-linked oligosaccharides. 20 This includes polypeptides exhibiting enzymatic activity similar to, but not necessarily identical to, an activity of Golgi a-mannosidase II, also known as mannosyl oligosaccharide 1,3-1,6-a-mannosidase II (EC 3.2.1.114), according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to 25 the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or fragments thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. As used herein, the term “increased / reduced ADCC” is defined as either an increase / reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium 30 surrounding the target cells, by the mechanism of ADCC defined above, and / or a reduction / increase in the concentration of antibody, in the medium surrounding the target 2024200623   01 Feb 2024 cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The increase / reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled 5 in the art), but that has not been engineered. For example the increase in ADCC mediated by an antibody produced by host cells engineered to have an altered pattern of glycosylation (e.g. to express the glycosyltransferase, GnTIII, or other glycosyltransferases) by the methods described herein, is relative to the ADCC mediated by the same antibody produced by the same type of non-engineered host cells. 10 By “antibody having increased / reduced antibody dependent cell-mediated cytotoxicity (ADCC)” is meant an antibody having increased / reducedADCC as determined by any suitable method known to those of ordinary skill in the art. One accepted in vitro ADCC assay is as follows: 1) the assay uses target cells that are known to express the target antigen 15 recognized by the antigen-binding region of the antibody; 2) the assay uses human peripheral blood mononuclear cells (PBMCs), isolated from blood of a randomly chosen healthy donor, as effector cells; 3) the assay is carried out according to following protocol: i) the PBMCs are isolated using standard density centrifugation 20 procedures and are suspended at 5 x 106 cells / ml in RPMI cell culture medium; ii) the target cells are grown by standard tissue culture methods, harvested from the exponential growth phase with a viability higher than 90%, washed in RPMI cell culture medium, labeled with 100 micro-Curies of 51Cr, washed twice with cell culture medium, and resuspended in cell culture medium at a density of 105 cells / ml; 25                  iii) 100 microliters of the final target cell suspension above are transferred to each well of a 96-well microtiter plate; iv) the antibody is serially-diluted from 4000 ng / ml to 0.04 ng / ml in cell culture medium and 50 microliters of the resulting antibody solutions are added to the target cells in the 96-well microtiter plate, testing in triplicate various antibody concentrations 30 covering the whole concentration range above; 2024200623   01 Feb 2024 v) for the maximum release (MR) controls, 3 additional wells in the plate containing the labeled target cells, receive 50 microliters of a 2% (V7V) aqueous solution of non-ionic detergent (Nonidet, Sigma, St. Louis), instead of the antibody solution (point iv above); 5                  vi) for the spontaneous release (SR) controls, 3 additional wells in the plate containing the labeled target cells, receive 50 microliters of RPMI cell culture medium instead of the antibody solution (point iv above); vii) the 96-well micro titer plate is then centrifuged at 50 x g for 1 minute and incubated for 1 hour at 4°C; 10                 viii) 50 microliters of the PBMC suspension (point i above) are added to each well to yield an effector:target cell ratio of 25:1 and the plates are placed in an incubator under 5% CO2 atmosphere at 37°C for 4 hours; ix) the cell-free supernatant from each well is harvested and the experimentally released radioactivity (ER) is quantified using a gamma counter; 15                  x) the percentage of specific lysis is calculated for each antibody concentration according to the formula (ER-MR) / (MR-SR) x 100, where ER is the average radioactivity quantified (see point ix above) for that antibody concentration, MR is the average radioactivity quantified (see point ix above) for the MR controls (see point v above), and SR is the average radioactivity quantified (see point ix above) for the SR controls (see 20 point vi above); 4) “increased / reduced ADCC” is defined as either an increase / reduction in the maximum percentage of specific lysis observed within the antibody concentration range tested above, and / or a reduction / increase in the concentration of antibody required to achieve one half of the maximum percentage of specific lysis observed within the antibody 25 concentration range tested above. The increase / reduction in ADCC is relative to the ADCC, measured with the above assay, mediated by the same antibody, produced by the same type of host cells, using the same standard production, purification, formulation and storage methods, which are known to those skilled in the art, but that has not been engineered. As used herein, the term "immunoconjugate" refers to a polypeptide molecule that includes at 30 least one effector moiety, such as a cytokine, and an antigen binding moiety, such as an antibody. In certain embodiments, the immunoconjugate comprises not more than one effector moiety. Particular immunoconjugates useful in the invention essentially consist of 2024200623   01 Feb 2024 one effector moiety and an antibody joined by one or more peptide linkers. Particular immunoconjugates according to the invention are fusion proteins, i.e. the components of the immunconjugate are joined by peptide bonds. The term "monoclonal antibody" as used herein refers to an antibody obtained from a 5 population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different 10 antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal 15 antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. 20 As used herein, the terms "first", "second", “third” etc. with respect to antigen binding moieties etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation unless explicitly so stated. The terms “multispecific” and “bispecific” mean that the antigen binding molecule is able to 25 specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments a bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells. 2024200623   01 Feb 2024 The term “valent” as used herein denotes the presence of a specified number of antigen binding sites in an antigen binding molecule. As such, the term “monovalent binding to an antigen” denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the antigen binding molecule. 5 An “antigen binding site” refers to the site, i.e. one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site. 10 A “T cell activating therapeutic agent” as used herein refers to a therapeutic agent capable of inducing T cell activation in a subject, particularly a therapeutic agent designed for inducing T-cell activation in a subject. Examples of T cell activating therapeutic agents include bispecific antibodies that specifically bind an activating T cell antigen, such as CD3, and a target cell antigen, such as CD20 or CD 19. Further examples include chimeric antigen 15 receptors (CARs) which comprise a T cell activating domain and an antigen binding moiety that specifically binds to a target cell antigen, such as CD20 or CD 19. An “activating T cell antigen” as used herein refers to an antigenic determinant expressed by a T lymphocyte, particularly a cytotoxic T lymphocyte, which is capable of inducing or enhancing T cell activation upon interaction with an antigen binding molecule. Specifically, 20 interaction of an antigen binding molecule with an activating T cell antigen may induce T cell activation by triggering the signaling cascade of the T cell receptor complex. An exemplary activating T cell antigen is CD3. “T cell activation” as used herein refers to one or more cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine 25 secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. The T cell activating bispecific antigen binding molecules and T cell activating therapeutic agents used in the present invention are capable of inducing T cell activation. Suitable assays to measure T cell activation are known in the art described herein. 2024200623   01 Feb 2024 A “target cell antigen” as used herein refers to an antigenic determinant presented on the surface of a target cell, for example a cell in a tumor such as a cancer cell or a cell of the tumor stroma. A “B-cell antigen” as used herein refers to an antigenic determinant presented on the surface 5 of a B lymphocyte, particularly a malignant B lymphocyte (in that case the antigen also being referred to as “malignant B-cell antigen”). A “T-cell antigen” as used herein refers to an antigenic determinant presented on the surface of a T lymphocyte, particularly a cytotoxic T lymphocyte. A “Fab molecule” refers to a protein consisting of the VH and CHI domain of the heavy 10 chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin. By “chimeric antigen receptor” or “CAR” is meant a genetically engineered receptor protein comprising an antigen binding moiety, e.g. a single-chain variable fragment (scFv) of a targeting antibody, a transmembrane domain, an intracellular T-cell activating signaling 15 domain (e.g. the CD3 zeta chain of the T-cell receptor) and optionally one or more intracellular co-stimulatory domains (e.g. of CD28, CD27, CD137 (4-1BB), 0x40). CARs mediate antigen recognition, T cell activation, and — in the case of second-generation CARs — costimulation to augment T cell functionality and persistence. For a review see e.g. Jackson et al., Nat Rev Clin Oncol (2016) 13, 370-383. 20 By “B cell proliferative disorder” is meant a disease wherein the number of B cells in a patient is increased as compared to the number of B cells in a healthy subject, and particularly wherein the increase in the number of B cells is the cause or hallmark of the disease. A “CD20-positive B cell proliferative disorder” is a B cell proliferative disorder wherein B-cells, particularly malignant B-cells (in addition to normal B-cells), express CD20. 25 Exemplary B cell proliferation disorders include Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL), as well as some types of Multiple myeloma (MM) and Hodgkin lymphoma (HL). 2024200623   01 Feb 2024 By “fused” is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers. An “anti-drug antibody” or “ADA” refers to an antibody that binds to a therapeutic agent and may influence serum concentrations and function of the therapeutic agent in a subject. The 5 presence of ADAs may increase clearance of the therapeutic agent through formation of immune complexes between therapeutic agent and antibody (neutralizing, non-neutralizing or both), thus reducing the therapeutic agent’s half-life. Furthermore, the activity and effectiveness of the therapeutic agent may be decreased through binding of antibody to the therapeutic agent (particularly in the case of neutralizing ADAs). ADAs can also be 10 associated with allergic or hypersensitivity reactions and other adverse events. An "effective amount" of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered. A "therapeutically effective amount" of an agent, e.g. a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired 15 therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease. By “therapeutic agent” is meant an active ingredient, e.g. of a pharmaceutical composition, that is administered to a subject in an attempt to alter the natural course of a disease in the subject being treated, and can be performed either for prophylaxis or during the course of 20 clinical pathology. An “immunotherapeutic agent” refers to a therapeutic agent that is administered to a subject in an attempt to restore or enhance the subject’s immune response, e.g. to a tumor. An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and 25 non-human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Preferably, the individual or subject is a human. The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and 2024200623   01 Feb 2024 which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A 5 pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of 10 clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, methods of the invention are used to delay 15 development of a disease or to slow the progression of a disease. The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products. 20   “CD3” refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CD3 as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, e.g., splice variants or allelic variants. In 25 one embodiment, CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3a). The amino acid sequence of human CD3a is shown in UniProt (www.uniprot.org) accession no. P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP 000724.1. See also SEQ ID NO: 115. The amino acid sequence of cynomolgus [Macaca fascicularis] CD3a is shown in NCBI GenBank no. BAB71849.1. See also SEQ ID NO: 116. 2024200623   01 Feb 2024 “CD19” refers to B-lymphocyte antigen CD 19, also known as B-lymphocyte surface antigen B4 or T-cell surface antigen Leu-12 and includes any native CD19 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CD 19 as well 5 as any form of CD 19 that results from processing in the cell. The term also encompasses naturally occurring variants of CD19, e.g., splice variants or allelic variants. In one embodiment, CD 19 is human CD 19. The amino acid sequence of an exemplary human CD 19 is shown in UniProt (www.uniprot.org) accession no. Pl5391 (version 174), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_001770.5, and SEQ ID NO: 117. 10 “Carcinoembryonic antigen” or “CEA” (also known as Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)) refers to any native CEA from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CEA as well as any form of CEA that results from processing in 15 the cell. The term also encompasses naturally occurring variants of CEA, e.g., splice variants or allelic variants. In one embodiment, CEA is human CEA. The amino acid sequence of human CEA is shown in UniProt (www.uniprot.org) accession no. P06731, or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004354.2. “Fibroblast activation protein” or “FAP” (also known as seprase) refers to any native FAP 20 from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed FAP as well as any form of FAP that results from processing in the cell. The term also encompasses naturally occurring variants of FAP, e.g., splice variants or allelic variants. In one embodiment, FAP is human 25 FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession no. Q12884, or NCBI (www.ncbi.nlm.nih.gov / ) RefSeqNP_004451.2. By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain 30   composed of the light chain variable domain VL and the heavy chain constant domain 1 CHI (VL-CH1, in N- to C-terminal direction), and a peptide chain composed of the heavy chain 2024200623   01 Feb 2024 variable domain VH and the light chain constant domain CL (VH-CL, in N- to C-terminal direction). For clarity, in a crossover Fab molecule wherein the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1 CHI is referred to herein as the “heavy chain” of the (crossover) Fab 5 molecule. Conversely, in a crossover Fab molecule wherein the constant domains of the Fab light chain and 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 (crossover) Fab molecule. In contrast thereto, by a “conventional” Fab molecule is meant a Fab molecule in its natural 10 format, i.e. comprising a heavy chain composed of the heavy chain variable and constant domains (VH-CH1, in N- to C-terminal direction), and a light chain composed of the light chain variable and constant domains (VL-CL, in N- to C-terminal direction). Type II anti-CD20 antibodies The CD20 molecule (also called human B-lymphocyte-restricted differentiation antigen or 15   Bp35) is a hydrophobic transmembrane protein expressed on the surface of malignant and non-malignant pre-B and mature B lymphocytes that has been described extensively (Valentine, M.A., et al., J. Biol. Chern. 264 (1989) 11282-11287; and Einfeld, D.A., et al., EMBO J. 7 (1988) 711-717; Tedder, T.F., et al., Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 208212; Stamenkovic, I., et al., J. Exp. Med. 167 (1988) 1975-1980; Tedder, T.F., et al., J. 20 Immunol. 142 (1989) 2560-2568). CD20 is highly expressed by over 90% of B cell non-Hodgkin's lymphomas (NHL) (Anderson, K.C., et al., Blood 63 (1984) 1424-1433) but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissues (Tedder, T.F., et al., J, Immunol. 135 (1985) 973- 979). 25 There exist two different types of anti-CD20 antibodies differing significantly in their mode of CD20 binding and biological activities (Cragg, M.S., et al., Blood 103 (2004) 2738-2743; and Cragg, M.S., et al., Blood 101 (2003) 1045-1052). Type I anti-CD20 antibodies primarily utilize complement to kill target cells, while Type II antibodies primarily operate through direct induction of cell death. 2024200623   01 Feb 2024 Type I and Type II anti-CD20 antibodies and their characteristics are reviewed e.g. in Klein et al., mAbs 5 (2013), 22-33. Type II anti-CD20 antibodies do not localize CD20 to lipid rafts, show low CDC activity, show only about half the binding capacity to B cells as compared to Type I anti-CD20 antibodies, and induce homotypic aggregation and direct cell 5 death. In constrast thereto, Type I antibodies localize CD20 to lipid rafts, show high CDC activity, full binding capacity to B cells, and only weak induction of homotypic aggregation and direct cell death. Obinutuzumab and tositumumab (CAS number 192391-48) are examples of Type II anti-CD20 antibodies, while rituximab, ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab, 10   PRO131921 and ublituximab are examples of Type I anti-CD20 antibodies. According to the invention, the anti-CD20 antibody is a Type II anti-CD20 antibody. In one embodiment according to the present invention, the Type II anti-CD20 antibody is capable of reducing the number of B cells in a subject. In one embodiment the Type II anti-CD20 antibody is an IgG antibody, particularly an IgGl antibody. In one embodiment, the Type II 15 anti-CD20 antibody is a full-length antibody. In one embodiment, the Type II anti-CD20 antibody comprises an Fc region, particularly an IgG Fc region or, more particularly, an IgGl Fc region. In one embodiment the Type II anti-CD20 antibody is a humanized B-Lyl antibody. Particularly, the Type II anti-CD20 antibody is a humanized, IgG-class Type II anti-CD20 antibody, having the binding specificity of the murine B-Lyl antibody (Poppema 20 and Visser, Biotest Bulletin 3, 131-139 (1987); SEQ ID NOs 2 and 3). In one embodiment, the Type II anti-CD20 antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of 25 SEQ ID NO: 9. Particularly, the heavy chain variable region framework regions (FRs) FR1, FR2, and FR3 of said Type II anti-CD20 antibody are human FR sequences encoded by the VH110 human germ-line sequence, the heavy chain variable region FR4 of said anti-CD20 antibody is a human FR sequence encoded by the JH4 human germ-line sequence, the light chain variable region FRs FR1, FR2, and FR3 of said Type II anti-CD20 antibody are human 30 FR sequences encoded by the VK 2 40 human germ-line sequence, and the light chain variable region FR4 of said anti-CD20 antibody is a human FR sequence encoded by the JK4 human germ-line sequence. In one embodiment, the Type II anti-CD20 antibody comprises 2024200623   01 Feb 2024 the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO: 11. In a particular embodiment, the Type II anti-CD20 antibody is obinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453). As used herein, obinutuzumab 5 is synonymous for GA101. The tradename is GAZYVA® or GAZYVARO®. This replaces all previous versions (e.g. Vol. 25, No. 1, 2011, p.75-76), and is formerly known as afutuzumab (recommended INN, WHO Drug Information, Vol. 23, No. 2, 2009, p. 176; Vol. 22, No. 2, 2008, p. 124). In one embodiment, the Type II anti-CD20 antibody is tositumomab. 10 The Type II anti-CD20 antibody useful in the present invention may be engineered to have increased effector function, as compared to a corresponding non-engineered antibody. In one embodiment the antibody engineered to have increased effector function has at least 2-fold, at least 10-fold or even at least 100-fold increased effector function, compared to a corresponding non-engineered antibody. The increased effector function can include, but is 15 not limited to, one or more of the following: increased Fc receptor binding, increased Clq binding and complement dependent cytotoxicity (CDC), increased antibody-dependent cell-mediated cytotoxicity (ADCC), increased antibody-dependent cellular phagocytosis (ADCP), increased cytokine secretion, increased immune complex-mediated antigen uptake by antigen-presenting cells, increased binding to NK cells, increased binding to macrophages, 20 increased binding to monocytes, increased binding to polymorphonuclear cells, increased direct signaling inducing apoptosis, increased crosslinking of target-bound antibodies, increased dendritic cell maturation, or increased T cell priming. In one embodiment the increased effector function one or more selected from the group of increased Fc receptor binding, increased CDC, increased ADCC, increased ADCP, and 25 increased cytokine secretion. In one embodiment the increased effector function is increased binding to an activating Fc receptor. In one such embodiment the binding affinity to the activating Fc receptor is increased at least 2-fold, particularly at least 10-fold, compared to the binding affinity of a corresponding non-engineered antibody. In a specific embodiment the activating Fc receptor is selected from the group of FcyRIIIa, FcyRI, and FcyRIIa. In one 30 embodiment the activating Fc receptor is FcyRIIIa, particularly human FcyRIIIa. In another embodiment the increased effector function is increased ADCC. In one such embodiment the ADCC is increased at least 10-fold, particularly at least 100-fold, compared to the ADCC 2024200623   01 Feb 2024 mediated by a corresponding non-engineered antibody. In yet another embodiment the increased effector function is increased binding to an activating Fc receptor and increased ADCC. Increased effector function can be measured by methods known in the art. A suitable assay 5 for measuring ADCC is described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 13511361 (1987). Alternatively, non-radioactive assays methods may be employed (see, for 10 example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in a animal model such as that disclosed 15 in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998). Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. According to a particular embodiment, binding affinity to an activating Fc receptor is measured by surface plasmon resonance using a 20 BIACORE® T100 machine (GE Healthcare) at 25°C. Alternatively, binding affinity of antibodies for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as NK cells expressing Fcyllla receptor. Clq binding assays may also be carried out to determine whether the antibody is able to bind Clq and hence has CDC activity. See e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. 25 To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 10451052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)). Increased effector function may result e.g. from glycoengineering of the Fc region or the introduction of amino acid mutations in the Fc region of the antibody. In one embodiment the 30 anti-CD20 antibody is engineered by introduction of one or more amino acid mutations in the Fc region. In a specific embodiment the amino acid mutations are amino acid substitutions. In 2024200623   01 Feb 2024 an even more specific embodiment the amino acid substitutions are at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). Further suitable amino acid mutations are described e.g. in Shields et al., J Biol Chern 9(2), 6591-6604 (2001); U.S. Patent No. 6,737,056; WO 2004 / 063351 and WO 2004 / 099249. Mutant Fc regions can be 5 prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing. In another embodiment the Type II anti-CD20 antibody is engineered by modification of the 10 glycosylation in the Fc region. In a specific embodiment the Type II anti-CD20 antibody is engineered to have an increased proportion of non-fucosylated oligosaccharides in the Fc region as compared to a non-engineered antibody. An increased proportion of non-fucosylated oligosaccharides in the Fc region of an antibody results in the antibody having increased effector function, in particular increased ADCC. 15 In a more specific embodiment, at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 40%, of the N-linked oligosaccharides in the Fc region of the Type II anti-CD20 antibody are non-fucosylated. In one embodiment, between about 40% and about 80% of the N-linked 20 oligosaccharides in the Fc region of the Type II anti-CD20 antibody are non-fucosylated. In one embodiment, between about 40% and about 60% of the N-linked oligosaccharides in the Fc region of the Type II anti-CD20 antibody are non-fucosylated. The non-fucosylated oligosaccharides may be of the hybrid or complex type. In another specific embodiment the Type II anti-CD20 antibody is engineered to have an 25 increased proportion of bisected oligosaccharides in the Fc region as compared to a nonengineered antibody. In a more specific embodiment, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 40%, of the N-linked oligosaccharides in the 30 Fc region of the Type II anti-CD20 antibody are bisected. In one embodiment, between about 40% and about 80% of the N-linked oligosaccharides in the Fc region of the anti-CD20 2024200623   01 Feb 2024 antibody are bisected. In one embodiment, between about 40% and about 60% of the N-linked oligosaccharides in the Fc region of the Type II anti-CD20 antibody are bisected. The bisected oligosaccharides may be of the hybrid or complex type. In yet another specific embodiment the anti-CD20 antibody is engineered to have an 5 increased proportion of bisected, non-fucosylated oligosaccharides in the Fc region, as compared to a non-engineered antibody. In a more specific embodiment, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 15%, more preferably at 10 least about 25%, of the N-linked oligosaccharides in the Fc region of the anti-CD20 antibody are bisected, non-fucosylated. The bisected, non-fucosylated oligosaccharides may be of the hybrid or complex type. The oligosaccharide structures in the antibody Fc region can be analysed by methods well known in the art, e.g. by MALDI TOF mass spectrometry as described in Umana et al., Nat 15 Biotechnol 17, 176-180 (1999) or Ferrara et al., Biotechn Bioeng 93, 851-861 (2006). The percentage of non-fucosylated oligosaccharides is the amount of oligosaccharides lacking fucose residues, relative to all oligosaccharides attached to Asn 297 (e. g. complex, hybrid and high mannose structures) and identified in an N-glycosidase F treated sample by MALDI TOF MS. Asn 297 refers to the asparagine residue located at about position 297 in the Fc 20 region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. The percentage of bisected, or bisected non-fucosylated, oligosaccharides is determined analogously. In one embodiment the Type II anti-CD20 antibody is engineered to have modified 25 glycosylation in the Fc region, as compared to a non-engineered antibody, by producing the antibody in a host cell having altered activity of one or more glycosyltransferase. Glycosyltransferases include P(l,4)-N-acetylglucosaminyltransferase III (GnTIII), 3(1,4)-galactosyltransferase (GalT), 3( 1,2)-N-acetylglucosaminyltransferase I (GnTI), 3O,2)-N-acetylglucosaminyltransferase II (GnTII) and a(l,6)-fucosyltransferase. In a specific 30 embodiment the Type II anti-CD20 antibody is engineered to have an increased proportion of non-fucosylated oligosaccharides in the Fc region, as compared to a non-engineered 2024200623   01 Feb 2024 antibody, by producing the antibody in a host cell having increased 0(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In an even more specific embodiment the host cell additionally has increased a-mannosidase II (Manll) activity. The glycoengineering methodology that can be used for engineering antibodies useful for the present invention has 5 been described in greater detail in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342 (U.S. Pat. No. 6,602,684; EP 1071700); WO 2004 / 065540 (U.S. Pat. Appl. Publ. No. 2004 / 0241817; EP 1587921), WO 03 / 011878 (U.S. Pat. Appl. Publ. No. 2003 / 0175884), the entire content of each of which is incorporated herein by reference in its entirety. Antibodies glycoengineered using this 10 methodology are referred to as GlycoMabs herein. Generally, any type of cultured cell line, including the cell lines discussed herein, can be used to generate cell lines for the production of anti-TNC A2 antibodies with altered glycosylation pattern. Particular cell lines include CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, 15 and other mammalian cells. In certain embodiments, the host cells have been manipulated to express increased levels of one or more polypeptides having 0(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In certain embodiments the host cells have been further manipulated to express increased levels of one or more polypeptides having a-mannosidase II (Manll) activity. In a specific embodiment, the polypeptide having GnTIII 20 activity is a fusion polypeptide comprising the catalytic domain of GnTIII and the Golgi localization domain of a heterologous Golgi resident polypeptide. Particularly, said Golgi localization domain is the Golgi localization domain of mannosidase II. Methods for generating such fusion polypeptides and using them to produce antibodies with increased effector functions are disclosed in Ferrara et al., Biotechn Bioeng 93, 851-861 (2006) and 25   WO2004 / 065540, the entire contents of which are expressly incorporated herein by reference. The host cells which contain the coding sequence of an antibody useful for the invention and / or the coding sequence of polypeptides having glycosyltransferase activity, and which express the biologically active gene products may be identified e.g. by DNA-DNA or DNA-RNA hybridization; the presence or absence of "marker" gene functions; assessing the level 30   of transcription as measured by the expression of the respective mRNA transcripts in the host cell; or detection of the gene product as measured by immunoassay or by its biological 2024200623   01 Feb 2024 activity - methods which are well known in the art. GnTIII or Man II activity can be detected e.g. by employing a lectin which binds to biosynthetis products of GnTIII or Manll, respectively. An example for such a lectin is the E4-PHA lectin which binds preferentially to oligosaccharides containing bisecting GlcNAc. Biosynthesis products (i.e. specific 5 oligosaccharide structures) of polypeptides having GnTIII or Manll activity can also be detected by mass spectrometric analysis of oligosaccharides released from glycoproteins produced by cells expressing said polypeptides. Alternatively, a functional assay which measures the increased effector function, e.g. increased Fc receptor binding, mediated by antibodies produced by the cells engineered with the polypeptide having GnTIII or Manll 10 activity may be used. In another embodiment the anti-CD20 antibody is engineered to have an increased proportion of non-fucosylated oligosaccharides in the Fc region, as compared to a non-engineered antibody, by producing the antibody in a host cell having decreased a(l,6)-fucosyltransferase activity. A host cell having decreased a(l,6)-fucosyltransferase activity may be a cell in 15 which the a(l,6)-fucosyltransferase gene has been disrupted or otherwise deactivated, e.g. knocked out (see Yamane-Ohnuki et al., Biotech Bioeng 87, 614 (2004); Kanda et al., Biotechnol Bioeng, 94(4), 680-688 (2006); Niwa et al., J Immunol Methods 306, 151-160 (2006)). Other examples of cell lines capable of producing defucosylated antibodies include Lee 13 20 CHO cells deficient in protein fucosylation (Ripka et al., Arch Biochem Biophys 249, 533 545 (1986); US Pat. Appl. No. US 2003 / 0157108; and WO 2004 / 056312, especially at Example 11). The antibodies useful in the present invention can alternatively be glycoengineered to have reduced fucose residues in the Fc region according to the techniques disclosed inEP 1 176 195 Al, WO 03 / 084570, WO 03 / 085119 and U.S. Pat. Appl. Pub. Nos. 25   2003 / 0115614, 2004 / 093621, 2004 / 110282, 2004 / 110704, 2004 / 132140, US Pat. No. 6,946,292 (Kyowa), e.g. by reducing or abolishing the activity of a GDP-fucose transporter protein in the host cells used for antibody production. Glycoengineered antibodies useful in the invention may also be produced in expression systems that produce modified glycoproteins, such as those taught in WO 03 / 056914 30 (GlycoFi, Inc.) or in WO 2004 / 057002 and WO 2004 / 024927 (Greenovation). 2024200623   01 Feb 2024 Therapeutic agents The present invention is useful in connection with various therapeutic agents, particularly with therapeutic agents that are immunogenic in the subject (i.e. have the ability of inducing an immune response in the subject) and / or that activate T-cells in the subject. Such 5 therapeutic agents include, for example, recombinant proteins. In one embodiment, the therapeutic agent induces the formation of AD As in a subject when administered to the subject in a treatment regimen without the administration of a Type II anti-CD20 antibody. In one embodiment, the therapeutic agent induces cytokine release in a subject when administered to the subject in a treatment regimen without the administration of 10 a Type II anti-CD20 antibody. In one embodiment, the therapeutic agent induces formation of AD As and cytokine release in a subject when administered to the subject in a treatment regimen without the administration of a Type II anti-CD20 antibody. In one embodiment, the therapeutic agent is a biologic agent. In one embodiment, the therapeutic agent comprises a polypeptide, particularly a recombinant polypeptide. In one 15 embodiment, the therapeutic agent comprises a polypeptide that does not naturally occur in the subject and / or is immunogenic in the subject. In one embodiment, the therapeutic agent is to be systemically administered. In one embodiment, the therapeutic agent is to be administered by infusion, particulary intravenous infusion. In one embodiment, the therapeutic agent comprises an antigen binding polypeptide. In one 20 embodiment, the therapeutic agent comprises a polypeptide selected from the group of an antibody, an antibody fragment, an Fc domain, and an immunoconjugate. In one embodiment, the therapeutic agent comprises a polypeptide selected from the group of an antibody, an antibody fragment, an antigen receptor or an antigen-binding fragment thereof, and a receptor ligand or a receptor-binding fragment thereof. In one embodiment, the 25 therapeutic agent comprises an antibody. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a polyclonal antibody. In one embodiment the antibody is a human antibody. In one embodiment, the antibody is humanized antibody. In one embodiment the antibody is a chimeric antibody. In one embodiment the antibody is fulllength antibody. In one embodiment the antibody is an IgG-class antibody, particularly an 30 IgGl subclass antibody. In one embodiment, the antibody is a recombinant antibody. 2024200623   01 Feb 2024 In certain embodiments, the therapeutic agent comprises an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., 5 Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046. In one embodiment, the antibody fragment is a Fab fragment or a scFv 10 fragment. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129134 (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). 15 Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl). Antibody fragments can be made by various techniques, including but not limited to 20 proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein. In certain embodiments, the therapeutic agent comprises a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al.. Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody 25 comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof. 2024200623   01 Feb 2024 In certain embodiments, the therapeutic agent comprises a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) 5 are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody 10 specificity or affinity. Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al.. Nature 332:323-329 (1988); Queen et al., Proc. Nat’I Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., 15 Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling). 20 Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 25   (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chern. 272:10678-10684 (1997) andRosoket al., J. Biol. Chern. 271:22611-22618 (1996)). In certain embodiments, the therapeutic agent comprises a human antibody. Human 30 antibodies can be produced using various techniques known in the art. Human antibodies are 2024200623   01 Feb 2024 described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human 5 variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic 10 animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HuMab® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007 / 0061900, describing VelociMouse® technology). Human variable regions from intact antibodies 15 generated by such animals may be further modified, e.g., by combining with a different human constant region. Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., 20 Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al.. Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM 25 antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). 30 Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may 2024200623   01 Feb 2024 then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below. Antibodies comprised in the therapeutic agent may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of 5 methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et aL, Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 10   222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004). In certain phage display methods, repertoires of VH and VL genes are separately cloned by 15 polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. 20 Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et aL, EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to 25 accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360. 30 Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein. 2024200623   01 Feb 2024 In certain embodiments, the therapeutic agent comprises a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, the binding specificities are for different antigens. In certain embodiments, the binding specificities are for different 5 epitopes on the same antigen. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express an antigen. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different 10 specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., US 15 Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et aL, J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see,e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific 20 antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991). Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein (see, e.g. US 2006 / 0025576A1). The antibody or fragment herein also includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to two different antigens (see, US 2008 / 0069820, for 25 example). “Crossmab” antibodies are also included herein (see e.g. WO2009080251, WO2009080252, WO2009080253, WO2009080254). Another technique for making bispecific antibody fragments is the "bispecific T cell engager" or BiTE® approach (see, e.g., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and 30   WO2008 / 119567). This approach utilizes two antibody variable domains arranged on a single 2024200623   01 Feb 2024 polypeptide. For example, a single polypeptide chain includes two single chain Fv (scFv) fragments, each having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of a length sufficient to allow intramolecular association between the two domains. This single polypeptide further includes a polypeptide spacer 5 sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific for different cell types, such that cells of two different cell types are brought into close proximity or tethered when each scFv is engaged with its cognate epitope. One particular embodiment of this approach includes a scFv recognizing a cellsurface antigen expressed by an immune cell, e.g., a CD3 polypeptide on a T cell, linked to 10 another scFv that recognizes a cell-surface antigen expressed by a target cell, such as a malignant or tumor cell. As it is a single polypeptide, the bispecific T cell engager may be expressed using any prokaryotic or eukaryotic cell expression system known in the art, e.g., a CHO cell line. However, specific purification techniques (see, e.g., EPl691833) may be necessary to 15 separate monomeric bispecific T cell engagers from other multimeric species, which may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing secreted polypeptides is first subjected to a metal affinity chromatography, and polypeptides are eluted with a gradient of imidazole concentrations. This eluate is further purified using anion exchange chromatography, and 20 polypeptides are eluted using with a gradient of sodium chloride concentrations. Finally, this eluate is subjected to size exclusion chromatography to separate monomers from multimeric species. Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60 (1991). 25 In certain embodiments, an antibody comprised in the therapeutic agent may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene 30 glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either 2024200623   01 Feb 2024 homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its 5 stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be 10 improved, whether the antibody derivative will be used in a therapy under defined conditions, etc. The therapeutic agent may also comprise an antibody conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or 15 fragments thereof), or radioactive isotopes. In one embodiment, the therapeutic agent comprises an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 Bl); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and 20 MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 25   (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and 30   CC1065. 2024200623   01 Feb 2024 In another embodiment, the therapeutic agent comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, 5 Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. In another embodiment, the therapeutic agent comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes 10 are available for the production of radioconjugates. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example Tc"m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-15   111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters 20 (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoylj-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). 25 Carbon-14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94 / 11026. The linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 30   52:127-131 (1992); U.S. Patent No. 5,208,020) may be used. 2024200623   01 Feb 2024 In some embodiments, the therapeutic agent may comprise an monoclonal antibody such as, but not limited to, alemtuzumab (LEMTRADA®), bevacizumab (AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), pertuzumab (OMNITARG®, 2C4), trastuzumab (HERCEPTIN®), tositumomab (Bexxar®), abciximab (REOPRO®), 5 adalimumab (HUMIRA®), apolizumab, aselizumab, atlizumab, bapineuzumab, basiliximab (SIMULECT®), bavituximab, belimumab (BENLYSTA®) briankinumab, canakinumab (ILARIS®), cedelizumab, certolizumab pegol (CIMZIA®), cidfusituzumab, cidtuzumab, cixutumumab, clazakizumab, crenezumab, daclizumab (ZENAPAX®), dalotuzumab, denosumab (PROLIA®, XGEVA®), eculizumab (SOLIRIS®), efalizumab, epratuzumab, 10 erlizumab, felvizumab, fontolizumab, golimumab (SIMPONI®), ipilimumab, imgatuzumab, infliximab (REMICADE®), labetuzumab, lebrikizumab, lexatumumab, lintuzumab, lucatumumab, lulizumab pegol, lumretuzumab, mapatumumab, matuzumab, mepolizumab, mogamulizumab, motavizumab, motovizumab, muronomab, natalizumab (TYSABRI®), necitumumab (PORTRAZZA®), nimotuzumab (THERACIM®), nolovizumab, 15 numavizumab, olokizumab, omalizumab (XOLAIR®), onartuzumab (also known as MetMAb), palivizumab (SYNAGIS®), pascolizumab, pecfusituzumab, pectuzumab, pembrolizumab (KEYTRUDA®), pexelizumab, priliximab, ralivizumab, ranibizumab (LUCENTIS®), reslivizumab, reslizumab, resyvizumab, robatumumab, rontalizumab, rovelizumab, ruplizumab, sarilumab, secukinumab, seribantumab, sifalimumab, 20 sibrotuzumab, siltuximab (SYLVANT®) siplizumab, sontuzumab, tadocizumab, talizumab, tefibazumab, tocilizumab (ACTEMRA®), toralizumab, tucusituzumab, umavizumab, urtoxazumab, ustekinumab (STELARA®), vedolizumab (ENTYVIO®), visilizumab, zanolimumab, zalutumumab. In one embodiment, the therapeutic agent comprises an antibody indicated for the treatment 25 of cancer. In one embodiment, the therapeutic agent comprises an antibody indicated for the treatment of an autoimmune disease. In one embodiment, the therapeutic agent is an immunotherapeutic agent. In one embodiment the therapeutic agent is indicated for the treatment of cancer. In some embodiments, in particular in relation aspects of the invention concerned with the reduction of cytokine release associated with the administration of a 30 therapeutic agent in a subject, the cancer is a B-cell proliferative disorder. In one embodiment, the cancer is a CD20-positive B-cell proliferative disorder. In one embodiment, the cancer is selected from the group consisting of Non-Hodgkin lymphoma (NHL), acute 2024200623   01 Feb 2024 lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL), Multiple myeloma (MM), and Hodgkin lymphoma (HL). In one embodiment, the therapeutic agent is an immunotherapeutic agent. 5 In one embodiment, the therapeutic agent is an immunosuppressive agent. In one embodiment, the therapeutic agent is indicated for the treatment of an autoimmune disease. Without wishing to be bound to theory, it is thought that enhancing T cell stimulation, by promoting an activating co-stimulatory molecule or by inhibiting a negative co-stimulatory molecule, may promote tumor cell death thereby treating or delaying progression of cancer. 0 In some embodiments, the therapeutic agent may comprise an agonist directed against an activating co-stimulatory molecule. In some embodiments, an activating co-stimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD 127. In some embodiments, the agonist directed against an activating co-stimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD 137, 15   CD27, HVEM, or CD127. In some embodiments, the therapeutic agent may comprise an antibody targeting GITR. In some embodiments, the antibody targeting GITR is TRX518. In some the therapeutic agent may comprise an antagonist directed against an inhibitory costimulatory molecule. In some embodiments, an inhibitory co-stimulatory molecule may include CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-20   H4, IDO, TIGIT, MICA / B, or arginase. In some embodiments, the antagonist directed against an inhibitory co-stimulatory molecule is an antagonist antibody that binds to CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase. In some embodiments, the therapeutic agent may comprise an anti-PD-1 antibody. In one embodiment the anti-PD-1 antibody is selected from the group consisting of MDX-1106 25 (nivolumab), MK-3475 (pembrolizumab, formerly known as lambrolizumab), CT-011 (pidilizumab). MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in WO2006 / 121168. MK-3475, also known as pembrolizumab or (formerly) lambrolizumab, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT, hBAT-1 or pidilizumab, is an anti-PD-1 3 0   antibody described in WO2009 / 101 611. 2024200623   01 Feb 2024 In some embodiments, the therapeutic agent may comprise an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In one embodiment, the therapeutic agent may comprise AMP-224, also known as B7-DCIg (a PD-5 L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342). In some embodiments, the therapeutic agent may comprise an anti-PD-Ll antibody. In one embodiment the anti-PD-Ll antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. Antibody YW243.55.S70 is an anti-PD-Ll antibody described in WO 2010 / 077634. MDX-1105, also known as BMS-10   936559, is an anti-PD-Ll antibody described in WO2007 / 005874. MEDI4736 is an anti-PD- Ll monoclonal antibody described in WO2011 / 066389 and US2013 / 034559. In one embodiment, the anti-PD-Ll antibody is atezolizumab. In some embodiments, the therapeutic agent may comprise an antagonist directed against CTLA-4 (also known as CD 152), for example, a blocking antibody. In some embodiments, 15 the therapeutic agent may comprise ipilimumab (also known as MDX-010, MDX-101, or YERVOY®). In some embodiments, the therapeutic agent may comprise tremelimumab (also known as ticilimumab or CP-675,206). In some embodiments, the therapeutic agent may comprise an antagonist directed against B7-H3 (also known as CD276), for example, a blocking antibody. In some embodiments, the therapeutic agent may comprise MGA271. In 20 some embodiments, the therapeutic agent may comprise an antagonist directed against a TGF beta, for example, metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299. In some embodiments, the therapeutic agent may comprise an agonist directed against CD 137 (also known as TNFRSF9, 4-IBB, or ILA), for example, an activating antibody. In some 25 embodiments, the therapeutic agent may comprise urelumab (also known as BMS-663513). In some embodiments, the therapeutic agent may comprise ligand of CD 137 (also known as TNFRSF9, 4-1BB, or ILA), such as 4-1BBL. In some embodiments, the therapeutic agent may comprise an agonist directed against CD40, for example, an activating antibody. In some embodiments, the therapeutic agent may comprise CP-870893. In some embodiments, the 30 therapeutic agent may comprise an agonist directed against OX40 (also known as CD 134), for example, an activating antibody. In some embodiments, the therapeutic agent may 2024200623   01 Feb 2024 comprise an anti-OX40 antibody (e.g., AgonOX). In some embodiments, the therapeutic agent may comprise a ligand of OX40, such as OX40L. In some embodiments, the therapeutic agent may comprise an agonist directed against CD27, for example, an activating antibody. In some embodiments, the therapeutic agent may comprise CDX-1127. 5 In some embodiments, the therapeutic agent may comprise a T cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR). In some embodiments, the therapeutic agent may comprise a T cell comprising a dominant-negative TGF beta receptor, e.g, a dominant-negative TGF beta type II receptor. In some embodiments, the therapeutic agent may comprise an antibody-drug conjugate. In 10 some embodiments, the antibody-drug conjugate comprises mertansine or monomethyl auristatin E (MMAE). In some embodiments, the therapeutic agent may comprise an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599). In some embodiments, the therapeutic agent may comprise trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or KADCYLA®). In some embodiments, the therapeutic 15 agent may comprise DMUC5754A. In some embodiments, the therapeutic agent may comprise an antibody-drug conjugate targeting the endothelin B receptor (EDNBR), for example, an antibody directed against EDNBR conjugated with MMAE (also known as DEDN6526A). In some embodiments, the therapeutic agent may comprise gemtuzumab ozogamicin (MYLOTARG®). In some embodiments, the therapeutic agent may comprise 20 inotuzumab ozogamicin. In some embodiments, the therapeutic agent may comprise bivatuzumab mertansine. In some embodiments, the therapeutic agent may comprise cantuzumab mertansine. In some embodiments, the therapeutic agent may comprise cantuzumab ravtansine. In some embodiments, the therapeutic agent may comprise brentuximab vedotin (ADECTRIS®). In some embodiments, the therapeutic agent may 25 comprise pinatuzumab vedotin. In some embodiments, the therapeutic agent may comprise polatuzumab vedotin In some embodiments, the therapeutic agent may comprise glembatumumab vedotin. In some embodiments, the therapeutic agent may comprise lorvotuzumab mertansine. In some embodiments, the therapeutic agent may comprise tacatuzumab tetraxetan. In some embodiments, the therapeutic agent may comprise 30 vandortuzumab vedotin (DSTP3086S). In some embodiments, the therapeutic agent may comprise ibritumomab tiuxetan (ZEVALIN®) 2024200623   01 Feb 2024 In some embodiments, the therapeutic agent may comprise an antibody directed against angiopoietin 2 (also known as Ang2). In some embodiments, the therapeutic agent may comprise MEDI3617. In some embodiments, the therapeutic agent may comprise an antibody targeting CSF-1R 5 (also known as M-CSFR or CD 115). In some embodiments, the therapeutic agent may comprise IMC-CS4 (LY3022855)). In some embodiments, the therapeutic agent may comprise emactuzumab. In some embodiment, the therapeutic agent may comprise a cytokine. In some embodiments, the therapeutic agent may comprise an interferon, for example interferon alpha or interferon 10 gamma. In some embodiments the therapeutic agent may comprise Roferon-A (also known as recombinant Interferon alpha-2a). In some embodiments, the therapeutic agent may comprise GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or LEUKIN E®). In some embodiments, the therapeutic agent may comprise aldesleukin (PROLEUKIN®). In some embodiments, the therapeutic 15   agent may comprise IL-12. In some embodiments, the therapeutic agent may comprise IL-10. In some embodiments, the therapeutic agent may comprise an IL-2 fusion protein. In some embodiments, the therapeutic agent may comprise tucotuzumab celmoleukin. In some embodiments, the therapeutic agent may comprise darleukin. In some embodiments, the therapeutic agent may comprise teleukin. 20 In some embodiments, the therapeutic agent may comprise an IL-10 fusion protein. In some embodiments, the therapeutic agent may comprise dekavil. In some embodiments, the therapeutic agent may comprise a TNF fusion protein. In some embodiments, the therapeutic agent may comprise fibromun. In some embodiments, the therapeutic agent may comprise a bispecific antibody. In some 25 embodiments, the therapeutic agent may comprise a bispecific antibody, such as, but not limited to, duligotuzumab, MM-111, MM141, TF2, ABT-981, ABT-122, LY3164530, SAR156597, GSK2434735, ozoralizumab, ALX-0761, ALX-0061, ALX-0141, ACE910. In some embodiments, the therapeutic agent may comprise a bispecific antibody capable of binding to a T cell and a target cell, e.g. a tumor cell. In some embodiment, the therapeutic 2024200623   01 Feb 2024 agent may comprise a bispecific antibody that specifically binds to CD3 on a T cell and to a target cell antigen. In some embodiment, the therapeutic agent may comprise a bispecific T cell engager (BiTE®). In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and CD 19. In one embodiment, the bispecific antibody is 5 blinatumomab (BLINCYTO®). In one embodiment, the bispecific antibody is AFM11. In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and EpCAM. In one embodiment, the bispecific antibody is catumaxomab (REVOMAB®). In one embodiment, the bispecific antibody is solitomab (AMG 110, MT 110). In some embodiments, the therapeutic agent may comprise a bispecific antibody 10 directed against CD3 and Her2. In one embodiment, the bispecific antibody is ertumaxomab. In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and PSMA. In one embodiment, the bispecific antibody is BAY2010112 (AMG212, MT112). In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and CEA. In one embodiment, the bispecific antibody is 15 MEDI565 (AMG211, MT111). In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and CD33. In one embodiment, the bispecific antibody is AMG330. In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and CD 123. In one embodiment, the bispecific antibody is MGD006. In one embodiment, the bispecific antibody is XmAb®14045. In some 20 embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and CD38. In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and gpA33. In one embodiment, the bispecific antibody is MGD007. In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and CD20. In one embodiment, the bispecific antibody is XmAb® 13676. In one 25 embodiment, the bispecific antibody is REGN1979. In one embodiment, the bispecific antibody is FBTA05 (Lymphomun). In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD30 and CD16A. In one embodiment, the bispecific antibody is AFM13. In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against DR5 30 and FAP. In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against Ang2 and VEGF. In one embodiment, the bispecific antibody is vanucizumab. 2024200623   01 Feb 2024 In some embodiments, the therapeutic agent may comprise an Fc domain. In some embodiments, the therapeutic agent may comprise a fusion protein comprising an Fc domain. In some embodiments, the therapeutic agent may comprise a recombinant receptor or a fragment thereof. In some embodiments, the receptor is a T cell receptor. In some 5 embodiments, the receptor is a TNF receptor. In some embodiments, the therapeutic agent may comprise etanercept (ENBREL®). In some embodiments, the receptor is a VEGF receptor. In some embodiments, the therapeutic agent may comprise ziv-aflibercept (ZALTRAP®). In some embodiments, the therapeutic agent may comprise aflibercept (EYLEA®). In some embodiments, the receptor is an IL-1 receptor. In some embodiments, 0 the therapeutic agent may comprise rilonacept (ARCALYST®). In some embodiments, the therapeutic agent may comprise IMCgplOO. In some embodiments, the therapeutic agent may comprise a chimeric antigen receptor (CAR). In some embodiments, the therapeutic agent may comprise a Factor IX-Fc fusion protein. In some embodiments, the therapeutic agent may comprise a Factor VIII-Fc fusion protein. In some embodiments, the therapeutic agent 15 may comprise a CTLA-4-Fc fusion protein, such as e.g. belatacept, abatacept (ORENCIA®). In one embodiment, the therapeutic agent may comprise romiplostin. In some embodiments, the therapeutic agent may comprise a recombinant receptor ligand, such as a TNF receptor ligand. In some embodiments, the therapeutic agent may comprise a generic, biosimilar or non-20 comparable biologic version of an agent, e.g. an antibody, named herein. In one embodiment, the therapeutic agent does not comprise obinutuzumab. T cell activating therapeutic agents The following describes in further detail T cell activating therapeutic agents for which the 25 invention may be useful, in particular aspects of the invention concerned with the reduction of cytokine release associated with the administration of a therapeutic agent in a subject. In some embodiments, the therapeutic agent comprises an antibody that specifically binds to an activating T cell antigen. In one embodiment, the therapeutic agent may comprise an 2024200623   01 Feb 2024 antibody that specifically binds to an antigen selected from the group of CD3, CD28, CD137 (also known as 4-IBB), CD40, CD226, OX40, GITR, CD27, HVEM, and CD 127. In one embodiment, the therapeutic agent comprises an antibody that specifically binds to CD3, particularly CD3e. 5 In one embodiment, the therapeutic agent comprises an antibody that is or can compete for binding with antibody H2C (PCT publication no. WO2008 / 119567), antibody V9 (Rodrigues et al., Int J Cancer Suppl 7, 45-50 (1992) and US patent no. 6,054,297), antibody FN18 (Nooij et al., Eur J Immunol 19, 981-984 (1986)), antibody SP34 (Pessano et al., EMBO J 4, 337-340 (1985)), antibody OKT3 (Kung et al., Science 206, 347-349 (1979)), antibody 10 WT31 (Spits et al., J Immunol 135, 1922 (1985)), antibody UCHT1 (Burns et al., J Immunol 129, 1451-1457 (1982)), antibody 7D6 (Coulie et al., Eur J Immunol 21, 1703-1709 (1991)) or antibody Leu-4. In some embodiments, the therapeutic agent may also comprise an antibody that specifically binds to CD3 as described in WO 2005 / 040220, WO 2005 / 118635, WO 2007 / 042261, WO 2008 / 119567, WO 2008 / 119565, WO 2012 / 162067, WO 15   2013 / 158856, WO 2013 / 188693, WO 2013 / 186613, WO 2014 / 110601, WO 2014 / 145806, WO 2014 / 191113, WO 2014 / 047231, WO 2015 / 095392, WO 2015 / 181098, WO 2015 / 001085, WO 2015 / 104346, WO 2015 / 172800, WO 2016 / 020444, or WO 2016 / 014974. In one embodiment, the therapeutic agent may comprise an antibody that specifically binds to a B-cell antigen, particularly a malignant B-cell antigen. In one embodiment, the therapeutic 20 agent may comprise an antibody that specifically binds to an antigen selected from the group consisting of CD20, CD19, CD22, ROR-1, CD37 and CD5, particularly to CD20 or CD19. In some embodiments, the therapeutic agent may comprise an antibody selected from rituximab, ocrelizumab, ofatumumab, ocaratuzumab, veltuzumab, and ublituximab. In some embodiments, the therapeutic agent may comprise a multispecific antibody, 25 particularly a bispecific antibody. In some embodiments, the therapeutic agent may comprise a bispecific antibody capable of binding to a T cell and a target cell, e.g. a tumor cell. In some embodiments, the target cell is a B-cell, particularly a malignant B-cell. In some embodiments, the therapeutic agent may comprise a bispecific antibody that specifically binds to (i) an activating T cell antigen and (ii) a B cell antigen. In some embodiments, the 2024200623   01 Feb 2024 therapeutic agent may comprise a bispecific antibody that specifically binds to CD3 on a T cell and to a target cell antigen. In some embodiments, the target cell antigen is a B-cell antigen, particularly a malignant B-cell antigen. In some embodiments, the therapeutic agent may comprise a bispecific T cell engager (BiTE®). 5 In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and CD20. In one embodiment, the bispecific antibody is XmAb® 13676. In one embodiment, the bispecific antibody is REGN1979. In one embodiment, the bispecific antibody is FBTA05 (Lymphomun). In some embodiments, the therapeutic agent may comprise a bispecific antibody directed 10 against CD3 and CD 19. In one embodiment, the bispecific antibody is blinatumomab (BLINCYTO®). In one embodiment, the bispecific antibody is AFM11. In one embodiment, the bispecific antibody is MGD011 (JNJ-64052781). In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and CD38. In one embodiment, the bispecific antibody is XmAb®13551, 15 XmAb® 15426, or XmAb®14702. In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and BCMA. In one embodiment, the bispecific antibody is BI836909. In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and CD33. In one embodiment, the bispecific antibody is AMG330. 20 In some embodiments, the therapeutic agent may comprise a bispecific antibody directed against CD3 and CD123. In one embodiment, the bispecific antibody is MGD006. In one embodiment, the bispecific antibody is XmAb® 14045. In one embodiment, the bispecific antibody is JNJ-63709178. In some embodiments, the therapeutic agent may comprise a recombinant receptor or a 25 fragment thereof. In some embodiments, the receptor is a T cell receptor (TCR). In some embodiments, the therapeutic agent may comprise a chimeric antigen receptor (CAR). 2024200623   01 Feb 2024 In some embodiments, the therapeutic agent may comprise a T cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR). In some embodiments, the therapeutic agent may comprise a T cell expressing a recombinant T cell receptor (TCR). In one embodiment, the therapeutic agent may comprise a CAR that specifically binds to a B-5 cell antigen, particularly a malignant B-cell antigen. In one embodiment, the therapeutic agent may comprise a CAR that specifically binds to an antigen selected from the group consisting of CD20, CD 19, CD22, ROR-1, CD37 and CD5, particularly to CD20 or CD 19. In some embodiments, the therapeutic agent may comprise a CAR directed to CD 19, or a T cell expressing a CAR directed to CD 19. In some embodiments, the therapeutic agent may 10 comprise KTE-C19, CTL019, JCAR-014, JCAR-015, JCAR-017, BPX-401, UCART19, In some embodiments, the therapeutic agent may comprise a CAR directed to CD22, or a T cell expressing a CAR directed to CD22. In some embodiments, the therapeutic agent may comprise JCAR-018 or UCART22. In some embodiments, the therapeutic agent may comprise an agonist directed against an T 15 cell activating co-stimulatory molecule. In some embodiments, a T cell activating costimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD 137, CD27, HVEM, or CD127. In some embodiments, the agonist directed against a T cell activating costimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the therapeutic agent may 20 comprise an antibody targeting GITR. In some embodiments, the antibody targeting GITR is TRX518. In some embodiments, the therapeutic agent may comprise an agonist directed against CD 137 (also known as TNFRSF9, 4-IBB, or ILA), for example, an activating antibody. In some embodiments, the therapeutic agent may comprise urelumab (also known as BMS-663513). 25 In some embodiments, the therapeutic agent may comprise ligand of CD 137 (also known as TNFRSF9, 4-1BB, or ILA), such as 4-1BBL. In some embodiments, the therapeutic agent may comprise an agonist directed against CD40, for example, an activating antibody. In some embodiments, the therapeutic agent may comprise CP-870893. In some embodiments, the therapeutic agent may comprise an agonist directed against OX40 (also known as CD 134), 2024200623   01 Feb 2024 for example, an activating antibody. In some embodiments, the therapeutic agent may comprise an anti-OX40 antibody (e.g., AgonOX). In some embodiments, the therapeutic agent may comprise a ligand of OX40, such as OX40L. In some embodiments, the therapeutic agent may comprise an agonist directed against CD27, for example, an activating 5 antibody. In some embodiments, the therapeutic agent may comprise CDX-1127. Particular therapeutic agents (i)    Reduction of the formation of anti-drug antibodies (ADAs) The therapeutic agents described in the following are particularly useful in the invention, in 10   particular in relation aspects of the invention concerned with the reduction of the formation of anti-drug antibodies (ADAs) against a therapeutic agent in a subject. In some embodiments, the therapeutic agent comprises an antibody that specifically binds to carcinoembryonic antigen (CEA). In one embodiment, the antibody that specifically binds to CEA comprises a heavy chain 15 variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19. In a further embodiment, the antibody that specifically binds CEA comprises a heavy chain variable region sequence that is at least 80%, 20   85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID NO: 20 and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 21. In a further embodiment, the antibody that specifically binds CEA comprises the heavy chain variable region sequence of SEQ ID NO: 20 and the light chain variable region sequence of SEQ ID NO: 21. 25 In one embodiment, the antibody that specifically binds to CEA comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 136, the HCDR2 of SEQ ID NO: 137, and the HCDR3 of SEQ ID NO: 138; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 139, the LCDR2 of SEQ ID NO: 140 and the LCDR3 of SEQ ID NO: 141. In a further embodiment, the antibody that 30 specifically binds CEA comprises a heavy chain variable region sequence that is at least 80%, 2024200623   01 Feb 2024 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID NO: 142 and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 143. In a further embodiment, the antibody that specifically binds CEA comprises the heavy chain variable region sequence of SEQ ID NO: 5   142 and the light chain variable region sequence of SEQ ID NO: 143. In one embodiment, the antibody that specifically binds to CEA is a full-length antibody. In one embodiment, the antibody that specifically binds to CEA is an antibody of the human IgG class, particularly an antibody of the human IgGi class. In one embodiment, the antibody that specifically binds to CEA is an antibody fragment, particularly a Fab molecule or a scFv 10 molecule, more particularly a Fab molecule. In one embodiment, the antibody that specifically binds to CEA is a humanized antibody. In some embodiments, the therapeutic agent comprises an antibody that specifically binds to fibroblast activation protein (FAP). In one embodiment, the antibody that specifically binds FAP comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 15   96%, 97%, 98%, or 99% identical to of SEQ ID NO: 25 and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 26. In a further embodiment, the antibody that specifically binds FAP comprises the heavy chain variable region sequence of SEQ ID NO: 25 and the light chain variable region sequence of SEQ ID NO: 26. 20 In one embodiment, the antibody that specifically binds to FAP is a full-length antibody. In one embodiment, the antibody that specifically binds to FAP is an antibody of the human IgG class, particularly an antibody of the human IgGi class. In one embodiment, the antibody that specifically binds to FAP is an antibody fragment, particularly a Fab molecule or a scFv molecule, more particularly a Fab molecule. In one embodiment, the antibody that 25 specifically binds to FAP is a human antibody. In some embodiments, the therapeutic agent comprises an antibody that specifically binds to CD3, particularly CD3 epsilon. In one embodiment, the antibody that specifically binds to CD3 comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a 30 light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37. In a further embodiment, the 2024200623   01 Feb 2024 antibody that specifically binds CD3 comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID NO: 38 and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39. In a further embodiment, the antibody that 5 specifically binds CD3 comprises the heavy chain variable region sequence of SEQ ID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39. In one embodiment, the antibody that specifically binds to CD3 is a full-length antibody. In one embodiment, the antibody that specifically binds to CD3 is an antibody of the human IgG class, particularly an antibody of the human IgGi class. In one embodiment, the antibody that 10 specifically binds to CD3 is an antibody fragment, particularly a Fab molecule or a scFv molecule, more particularly a Fab molecule. In a particular embodiment, the antibody that specifically binds to CD3 is a crossover Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other). In one embodiment, the antibody that specifically binds to CD3 is a humanized 15 antibody. In some embodiments, the therapeutic agent comprises a cytokine. In one embodiment the cytokine is selected from the group consisting of , GM-CSF, IL-la, IL-1 P, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN-a, IFN-p, IFN-y, MIP-la, MIP-lp, TGF-p, TNF-a, and TNF-p. In one embodiment, the cytokine is IL-2, particularly human IL-2. The 20 sequence of wild-type human IL-2 is shown in SEQ ID NO: 12. In one embodiment, the therapeutic agent comprises a mutant IL-2 polypeptide having reduced binding affinity to the a-subunit of the IL-2 receptor as compared to wild-type IL-2. Together with the P- and y-subunits (also known as CD122 and CD132, respectively), the a-subunit (also known as CD25) forms the heterotrimeric high-affinity IL-2 receptor, while the 25 dimeric receptor consisting only of the P- and y-subunits is termed the intermediate-affinity IL-2 receptor. A mutant IL-2 polypeptide with reduced binding to the a-subunit of the IL-2 receptor has a reduced ability to induce IL-2 signaling in regulatory T (Treg) cells, induces less activation-induced cell death (AICD) in T cells, and has a reduced toxicity profile in vivo, compared to a wild-type IL-2 polypeptide (see e.g. WO 2012 / 107417, incorporated 30 herein by reference in its entirety). 2024200623   01 Feb 2024 In a more specific embodiment, the mutant IL-2 polypeptide comprises three amino acid substitutions at the positions corresponding to residue 42, 45 and 72 of human IL-2. In an even more specific embodiment, the mutant IL-2 polypeptide is a human IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the 5 human IL-2 sequence SEQ ID NO: 12). In one embodiment the mutant IL-2 polypeptide additionally comprises an amino acid mutation at a position corresponding to position 3 of human IL-2, which eliminates the O-glycosylation site of IL-2. In one embodiment said amino acid mutation which eliminates the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2 is an amino acid substitution selected from the 10 group of T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. Particularly, said additional amino acid mutation is an amino acid substitution replacing a threonine residue by an alanine residue. A particular mutant IL-2 polypeptide useful in the invention comprises four amino acid substitutions at positions corresponding to residues 3, 42, 45 and 72 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A and L72G. This mutant IL-2 15 polypeptide exhibits no detectable binding to CD25, reduced ability to induce apoptosis in T cells, reduced ability to induce IL-2 signaling in Treg cells, and a reduced toxicity profile in vivo (see e.g. WO 2012 / 107417, incorporated herein by reference in its entirety). However, it retains ability to activate IL-2 signaling in effector cells, to induce proliferation of effector cells, and to generate IFN-y as a secondary cytokine by NK cells. 20 The IL-2 or mutant IL-2 polypeptide according to any of the above embodiments may comprise additional mutations that provide further advantages such as increased expression or stability. For example, the cysteine at position 125 may be replaced with a neutral amino acid such as serine, alanine, threonine or valine, yielding C125S IL-2, C125A IL-2, C125T IL-2 or C125V IL-2 respectively, as described in U.S. Patent no. 4,518,584. As described 25 therein, one may also delete the N-terminal alanine residue of IL-2 yielding such mutants as des-Al C125S or des-Al C125A. Alternatively or conjunctively, the IL-2 mutant may include a mutation whereby methionine normally occurring at position 104 of wild-type human IL-2 is replaced by a neutral amino acid such as alanine (see U.S. Patent no. 5,206,344). The resulting mutants, e. g., des-Al M104A IL-2, des-Al M104A C125S IL-2, 30   M104A IL-2, M104A Cl25A IL-2, des-Al M104A Cl25A IL-2, or M104A C125S IL-2 (these and other mutants may be found in U.S. Patent No. 5,116,943 and in Weiger et al., Eur J Biochem 180, 295-300 (1989)) may be used in conjunction with the particular IL-2 mutations described herein. 2024200623   01 Feb 2024 Thus, in certain embodiments the IL-2 or mutant IL-2 polypeptide comprises an additional amino acid mutation at a position corresponding to residue 125 of human IL-2. In one embodiment said additional amino acid mutation is the amino acid substitution Cl25A. In certain embodiments the mutant IL-2 polypeptide is essentially a full-length IL-2 5 molecule, particularly a human full-length IL-2 molecule. In one embodiment, the mutant IL- 2 polypeptide comprises a polypeptide sequence that is at least 80%, at least 85%, or at least 90% identical to the sequence of SEQ ID NO: 12. In a specific embodiment the mutant IL-2 polypeptide comprises the polypeptide sequence of SEQ ID NO: 13. 10 In some embodiments, the therapeutic agent comprises an immunoconjugate. Particular immunoconjugates are described in WO 2012 / 107417 and WO 2012 / 146628 (each incorporated herein by reference in its entirety). In one embodiment, the immunoconjugate comprises an antibody that specifically binds to CEA as described herein, and a mutant IL-2 polypeptide as described herein. In one 15 embodiment, the antibody is a full-length antibody. In one embodiment the therapeutic agent comprises an immunoconjugate comprising (i) an antibody of the human IgGi subclass that specifically binds to CEA and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of 20             SEQ ID NO: 16; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19; and (ii) a mutant human IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 25               12). In one embodiment, the immunoconjugate comprises an antibody that specifically binds to FAP as described herein, and a mutant IL-2 polypeptide as described herein. In one embodiment, the antibody is a full-length antibody. In one embodiment the therapeutic agent comprises an immunoconjugate comprising 2024200623   01 Feb 2024 (i) an antibody of the human IgGi subclass that specifically binds to FAP and comprises the heavy chain variable region of SEQ ID NO: 25; and the light chain variable region of SEQ ID NO: 26; and (ii) a mutant human IL-2 polypeptide comprising the amino acid substitutions F42A, 5            Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 12). In one embodiment, the immunoconjugate comprises no more than one mutant IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide is fused to the carboxyterminal amino acid of one of the antibody heavy chains, optionally through a linker peptide. 10 Suitable, non-immunogenic linker peptides include, for example, (G4S)n, (SGQn or G4(SG4)n linker peptides, wherein n is generally a number between 1 and 10, typically between 2 and 4. In one embodiment, the linker peptide is (G4S)3. In one embodiment, the immunoconjugate comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of 15 SEQ ID NO: 22, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 23, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 24. In one embodiment, the immunoconjugate comprises a polypeptide comprising the sequence 20 of SEQ ID NO: 22, a polypeptide comprising the sequence of SEQ ID NO: 23, and a polypeptide comprising the sequence of SEQ ID NO: 24. In one embodiment, the immunoconjugate is cergutuzumab amunaleukin (see WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Recommended INN: List 75, 2016, pre-publication copy” (incorporated herein by reference 25 in its entirety). In one embodiment, the immunoconjugate comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 27, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 28, and a polypeptide 2024200623   01 Feb 2024 comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 29. In one embodiment, the immunoconjugate comprises a polypeptide comprising the sequence of SEQ ID NO: 27, a polypeptide comprising the sequence of SEQ ID NO: 28, and a 5 polypeptide comprising the sequence of SEQ ID NO: 29. In one embodiment, the therapeutic agent comprises a bispecific antibody. Particular bispecific antibodies are described in PCT publication nos. WO 2013 / 026833 and WO 2014 / 131712 and in PCT application no. PCT / EP2016 / 073171 (each incorporated herein by reference in its entirety). 10 In one embodiment, the bispecific antibody comprises an antibody that specifically binds to CEA as described herein, and an antibody that specifically binds to CD3 as described herein. In one embodiment, the bispecific antibody comprises a first antibody that specifically binds to CD3 as described herein, and a second and a third antibody that specifically bind to CEA as described herein. In one embodiment, the first antibody is a crossover Fab molecule as 15 described herein, and the second and the first antibody are each a conventional Fab molecule. In one embodiment, the bispecific antibody further comprises an Fc domain as described herein. The bispecific antibody may have the antibody formats described herein and may comprise the antigen binding moieties described herein. The bispecific antibody may comprise modifications in the Fc region and / or the antigen binding moieties as described 20 herein. In one embodiment the therapeutic agent comprises a bispecific antibody comprising (i) a first antigen binding moiety that specifically binds to CD3, comprising a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region 25 comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged; (ii) a second and a third antigen binding moiety that specifically bind to CEA, comprising a 30 heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a light chain 2024200623   01 Feb 2024 variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19, wherein the second and third antigen binding moiety are each a Fab molecule, particularly a conventional Fab molecule; (iii) an Fc domain composed of a first and a second subunit capable of stable association, 5 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 10 domain. In one embodiment, the first antigen binding moiety that specifically binds to CD3, comprises the heavy chain variable region of SEQ ID NO: 38, and the light chain variable region of SEQ ID NO: 39. In one embodiment, the second and third antigen binding moieties that specifically bind to CEA comprise the heavy chain variable region of SEQ ID NO: 20, 15 and the light chain variable region of SEQ ID NO: 21. In one embodiment, the antigen binding moieties and the Fc region are fused to each other by peptide linkers, particularly by peptide linkers as in SEQ ID NO: 42 and SEQ ID NO: 43.In one embodiment, the bispecific antibody comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ 20 ID NO: 40, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 41, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 42, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 43. 25 In one embodiment, the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 40, a polypeptide comprising the sequence of SEQ ID NO: 41, a polypeptide comprising the sequence of SEQ ID NO: 42, and a polypeptide comprising the sequence of SEQ ID NO: 43. (CEA TCB) In one embodiment the therapeutic antibody comprises a bispecific antibody comprising 30   (i) a first antigen binding moiety that specifically binds to CD3, comprising a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 2024200623   01 Feb 2024 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions, particularly the variable 5 regions, of the Fab light chain and the Fab heavy chain are exchanged; (ii) a second and a third antigen binding moiety that specifically bind to CEA, comprising a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 136, the HCDR2 of SEQ ID NO: 137, and the HCDR3 of SEQ ID NO: 138; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 139, the LCDR2 10 of SEQ ID NO: 140 and the LCDR3 of SEQ ID NO: 141, wherein the second and third antigen binding moiety are each a Fab molecule, particularly a conventional Fab molecule; (iii) an Fc domain composed of a first and a second subunit capable of stable association, 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 15 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. In one embodiment, the first antigen binding moiety that specifically binds to CD3, 20 comprises the heavy chain variable region of SEQ ID NO: 38, and the light chain variable region of SEQ ID NO: 39. In one embodiment, the second and third antigen binding moiety that specifically bind to CEA comprise the heavy chain variable region of SEQ ID NO: 142, and the light chain variable region of SEQ ID NO: 143. In one embodiment, the antigen binding moieties and the Fc region are fused to each other by 25 peptide linkers, particularly by peptide linkers as in SEQ ID NO: 145 and SEQ ID NO: 146. In one embodiment, in the constant domain CL of the second and the third Fab molecule under (ii) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat), and in the constant domain CHI 30 of the second and the third Fab molecule under (ii) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino 2024200623   01 Feb 2024 acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index). In one embodiment, the bispecific antibody comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of 5 SEQ ID NO: 144, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 145, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 146, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ 10 ID NO: 147. In one embodiment, the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 144, a polypeptide comprising the sequence of SEQ ID NO: 145, a polypeptide comprising the sequence of SEQ ID NO: 146, and a polypeptide comprising the sequence of SEQ ID NO: 147. 15 (ii) Reduction of cytokine release The therapeutic agents described in the following are particularly useful in the invention, in particular in relation aspects of the invention concerned with the reduction of cytokine release associated with the administration of a therapeutic agent in a subject. The aspects of the invention concerned with the reduction of cytokine release associated with 20 the administration of a therapeutic agent in a subject are particularly useful in connection with therapeutic agents that are activating T-cells in the subject (T cell activating therapeutic agents), i.e. have the ability of inducing T-cell activation in the subject. Such therapeutic agents include, for example, antibodies directed to T-cell antigens (particularly activating T-cell antigens), or T-cells modified with chimeric antigen receptors (CAR) or recombinant T-25 cell receptors (TCR). The aspects of the invention concerned with the reduction of cytokine release associated with the administration of a therapeutic agent in a subject are particularly useful in connection with B-cell targeted T-cell activating therapeutic agents. In some embodiments, the therapeutic agent comprises an antibody that specifically binds to CD3, particularly CD3 epsilon. 2024200623   01 Feb 2024 In one embodiment, the antibody that specifically binds to CD3 comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 5   36 and the LCDR3 of SEQ ID NO: 37. In a further embodiment, the antibody that specifically binds CD3 comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID NO: 38 and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39. In a further embodiment, the antibody that 10 specifically binds CD3 comprises the heavy chain variable region sequence of SEQ ID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39. In one embodiment, the antibody that specifically binds to CD3 comprises a heavy chain variable region comprising the heavy chain HVR 1 (Hl-HVR) of SEQ ID NO: 120, the H2-HVR of SEQ ID NO: 121, and the H3-HVR of SEQ ID NO: 122; and a light chain variable 15 region comprising the light chain HVR 1 (Ll-HVR) of SEQ ID NO: 123, the L2-HVR of SEQ ID NO: 124 and the L3-HVR of SEQ ID NO: 125. In a further embodiment, the antibody that specifically binds CD3 comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID NO: 126 and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 20 or 99% identical to the sequence of SEQ ID NO: 127. In a further embodiment, the antibody that specifically binds CD3 comprises the heavy chain variable region sequence of SEQ ID NO: 126 and the light chain variable region sequence of SEQ ID NO: 127. In one embodiment, the antibody that specifically binds to CD3 is a full-length antibody. In one embodiment, the antibody that specifically binds to CD3 is an antibody of the human IgG 25 class, particularly an antibody of the human IgGi class. In one embodiment, the antibody that specifically binds to CD3 is an antibody fragment, particularly a Fab molecule or a scFv molecule, more particularly a Fab molecule. In a particular embodiment, the antibody that specifically binds to CD3 is a crossover Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each 30 other). In one embodiment, the antibody that specifically binds to CD3 is a humanized antibody. 2024200623   01 Feb 2024 In one embodiment, the therapeutic agent comprises a multispecific antibody, particularly a bispecific antibody. In one embodiment, the multispecific antibody specifically binds to (i) an activating T cell antigen and (ii) a B cell antigen. Particular bispecific antibodies are described in PCT publication no. WO 2016 / 020309 and PCT application no. 5 PCT / EP2016 / 073041, as well as PCT publication no. WO 2015 / 095392 (each incorporated herein by reference in its entirety). In one embodiment, the bispecific antibody specifically binds to CD3 and CD20. In one embodiment, the bispecific antibody comprises an antigen binding moiety that specifically binds to CD20, and an antigen binding moiety that specifically binds to CD3. In one 10 embodiment, the bispecific antibody comprises a first antigen binding moiety that specifically binds to CD3, and a second and a third antigen binding moiety that specifically bind to CD20. In one embodiment, the first antigen binding moiety is a crossover Fab molecule, and the second and the first antigen binding moiety are each a conventional Fab molecule. In one embodiment, the bispecific antibody further comprises an Fc domain. The bispecific antibody 15 may have the antibody formats described herein and may comprise the antigen binding moieties described herein. The bispecific antibody may comprise modifications in the Fc region and / or the antigen binding moieties as described herein. In one embodiment, the therapeutic agent comprises a bispecific antibody comprising (i) an antigen binding moiety that specifically binds to CD3 and comprises a heavy chain 20 variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and (ii) an antigen binding moiety that specifically binds to CD20 and comprises a heavy chain 25 variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9. In one embodiment, the therapeutic agent comprises a bispecific antibody comprising 30   (i) an antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region of SEQ ID NO: 38; and a light chain variable region of SEQ ID NO: 39; and 2024200623   01 Feb 2024 (ii) an antigen binding moiety that specifically binds to CD20 and comprises a heavy chain variable region of SEQ ID NO: 10; and a light chain variable region of SEQ ID NO: 11. In a particular embodiment, the therapeutic agent comprises a bispecific antibody comprising a) a first Fab molecule which specifically binds to a first antigen; 5 b) a second Fab molecule which specifically binds to a second antigen, and wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other; c) a third Fab molecule which specifically binds to the first antigen; and d) an Fc domain composed of a first and a second subunit capable of stable association; 10 wherein (i) the first antigen is CD20 and the second antigen is CD3, particularly CD3 epsilon; (ii) the first Fab molecule under a) and the third Fab molecule under c) each comprise the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 4, the heavy chain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID NO: 6, the light chain CDR 1 of 15 SEQ ID NO: 7, the light chain CDR 2 of SEQ ID NO: 8 and the light chain CDR 3 of SEQ ID NO: 9, and the second Fab molecule under b) comprises the heavy chain CDR 1 of SEQ ID NO: 32, the heavy chain CDR 2 of SEQ ID NO: 33, the heavy chain CDR 3 of SEQ ID NO: 34, the light chain CDR 1 of SEQ ID NO: 35, the light chain CDR 2 of SEQ ID NO: 36 and the light chain CDR 3 of SEQ ID NO: 37; 20 (iii) in the constant domain CL of the first Fab molecule under a) and the third Fab molecule under c) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat), and wherein in the constant domain CHI of the first Fab molecule under a) and the third Fab molecule under c) the amino 25 acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); and (iv) the first Fab molecule under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule under b), and the second Fab 30 molecule under b) and the third Fab molecule under c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under d). 2024200623   01 Feb 2024 In one embodiment, the first Fab molecule under a) and the third Fab molecule under c) each comprise a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 10, and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 11. 5 In one embodiment, the first Fab molecule under a) and the third Fab molecule under c) each comprise the heavy chain variable region sequence of SEQ ID NO: 10, and the light chain variable region sequence of SEQ ID NO: 11. In one embodiment, the second Fab molecule under b) comprises a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 10   38, and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39. In still a further embodiment, the second Fab molecule under b) comprises the heavy chain variable region sequence of SEQ ID NO: 38, and the light chain variable region sequence of SEQ ID NO: 39. 15 In a particular embodiment, the bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 44, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 45, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 46, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the 20 sequence of SEQ ID NO: 47. In a further particular embodiment, the bispecific antibody comprises a polypeptide sequence of SEQ ID NO: 44, a polypeptide sequence of SEQ ID NO: 45, a polypeptide sequence of SEQ ID NO: 46 and a polypeptide sequence of SEQ ID NO: 47. (CD20XCD3 bsAB) In one embodiment, the therapeutic agent comprises a bispecific antibody comprising 25   (i) an antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region comprising the heavy chain HVR 1 (Hl-HVR) of SEQ ID NO: 120, the H2-HVR of SEQ ID NO: 121, and the H3-HVR of SEQ ID NO: 122; and a light chain variable region comprising the light chain HVR 1 (Ll-HVR) of SEQ ID NO: 123, the L2-HVR of SEQ ID NO: 124 and the L3-HVR of SEQ ID NO: 125; and 2024200623   01 Feb 2024 (ii) an antigen binding moiety that specifically binds to CD20 and comprises a heavy chain variable region comprising the heavy chain HVR 1 (Hl-HVR) of SEQ ID NO: 128, the H2-HVR of SEQ ID NO: 129, and the H3-HVR of SEQ ID NO: 130; and a light chain variable region comprising the light chain HVR 1 (LI-HVR) of SEQ ID NO: 131, the L2-HVR of 5 SEQ ID NO: 132 and the L3-HVR of SEQ ID NO: 133. In one embodiment, the therapeutic agent comprises a bispecific antibody comprising (i) an antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region of SEQ ID NO: 126; and a light chain variable region of SEQ ID NO: 127; and 10 (ii) an antigen binding moiety that specifically binds to CD20 and comprises a heavy chain variable region of SEQ ID NO: 134; and a light chain variable region of SEQ ID NO: 135. In one embodiment, the bispecific antibody comprises an antigen binding moiety that specifically binds to CD 19, and an antigen binding moiety that specifically binds to CD3. In one embodiment, the bispecific antibody comprises a first antigen binding moiety that 15 specifically binds to CD3, and a second and a third antigen binding moiety that specifically bind to CD 19. In one embodiment, the first antigen binding moiety is a crossover Fab molecule, and the second and the first antigen binding moiety are each a conventional Fab molecule. In one embodiment, the bispecific antibody further comprises an Fc domain. The bispecific antibody may comprise modifications in the Fc region and / or the antigen binding 20 moieties as described herein. In one embodiment, the therapeutic agent comprises a bispecific antibody comprising (i) an antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region 25 comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and (ii) an antigen binding moiety that specifically binds to CD 19 and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 48, the HCDR2 of SEQ ID NO: 49, and the HCDR3 of SEQ ID NO: 50; and a light chain variable region 30 comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 51, the LCDR2 of SEQ ID NO: 52 and the LCDR3 of SEQ ID NO: 53. 2024200623   01 Feb 2024 In one embodiment, the therapeutic agent comprises a bispecific antibody comprising (i) an antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region of SEQ ID NO: 38; and a light chain variable region of SEQ ID NO: 39; and (ii) an antigen binding moiety that specifically binds to CD 19 and comprises a heavy chain 5 variable region of SEQ ID NO: 54; and a light chain variable region of SEQ ID NO: 55. In a particular embodiment, the therapeutic agent comprises a bispecific antibody comprising a) a first Fab molecule which specifically binds to a first antigen; b) a second Fab molecule which specifically binds to a second antigen, and wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by 10 each other; c) a third Fab molecule which specifically binds to the first antigen; and d) an Fc domain composed of a first and a second subunit capable of stable association; wherein (i) the first antigen is CD 19 and the second antigen is CD3, particularly CD3 epsilon; 15 (ii) the first Fab molecule under a) and the third Fab molecule under c) each comprise the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 48, the heavy chain CDR 2 of SEQ ID NO: 49, the heavy chain CDR 3 of SEQ ID NO: 50, the light chain CDR 1 of SEQ ID NO: 51, the light chain CDR 2 of SEQ ID NO: 52 and the light chain CDR 3 of SEQ ID NO: 53, and the second Fab molecule under b) comprises the heavy chain CDR 20   1 of SEQ ID NO: 32, the heavy chain CDR 2 of SEQ ID NO: 33, the heavy chain CDR 3 of SEQ ID NO: 34, the light chain CDR 1 of SEQ ID NO: 35, the light chain CDR 2 of SEQ ID NO: 36 and the light chain CDR 3 of SEQ ID NO: 37; (iii) in the constant domain CL of the first Fab molecule under a) and the third Fab molecule under c) the amino acid at position 124 is substituted by lysine (K) (numbering according to 25 Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat), and wherein in the constant domain CHI of the first Fab molecule under a) and the third Fab molecule under c) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering 30 according to Kabat EU index); and (iv) the first Fab molecule under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule under b), and the second Fab 2024200623   01 Feb 2024 molecule under b) and the third Fab molecule under c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under d). In one embodiment, the first Fab molecule under a) and the third Fab molecule under c) each comprise a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical 5 to the sequence of SEQ ID NO: 54, and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 55. In one embodiment, the first Fab molecule under a) and the third Fab molecule under c) each comprise the heavy chain variable region sequence of SEQ ID NO: 54, and the light chain variable region sequence of SEQ ID NO: 55. 10 In one embodiment, the second Fab molecule under b) comprises a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 38, and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39. In still a further embodiment, the second Fab molecule under b) comprises the heavy chain 15 variable region sequence of SEQ ID NO: 38, and the light chain variable region sequence of SEQ ID NO: 39. In a particular embodiment, the bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 47, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 56, a 20 polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 57, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 58. In a further particular embodiment, the bispecific antibody comprises a polypeptide sequence of SEQ ID NO: 47, a polypeptide sequence of SEQ ID NO: 56, a polypeptide sequence of SEQ ID NO: 57 and a polypeptide sequence of SEQ ID 25 NO: 58. In one embodiment, the therapeutic agent comprises a bispecific antibody comprising (i) an antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 2024200623   01 Feb 2024 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and (ii) an antigen binding moiety that specifically binds to CD 19 and comprises a heavy chain 5 variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 59, the HCDR2 of SEQ ID NO: 60, and the HCDR3 of SEQ ID NO: 61; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 62, the LCDR2 of SEQ ID NO: 63 and the LCDR3 of SEQ ID NO: 64. In one embodiment, the therapeutic agent comprises a bispecific antibody comprising 10   (i) an antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region of SEQ ID NO: 38; and a light chain variable region of SEQ ID NO: 39; and (ii) an antigen binding moiety that specifically binds to CD 19 and comprises a heavy chain variable region of SEQ ID NO: 65; and a light chain variable region of SEQ ID NO: 66. In a particular embodiment, the therapeutic agent comprises a bispecific antibody comprising 15 a) a first Fab molecule which specifically binds to a first antigen; b) a second Fab molecule which specifically binds to a second antigen, and wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other; c) a third Fab molecule which specifically binds to the first antigen; and 20 d) an Fc domain composed of a first and a second subunit capable of stable association; wherein (i) the first antigen is CD 19 and the second antigen is CD3, particularly CD3 epsilon; (ii) the first Fab molecule under a) and the third Fab molecule under c) each comprise the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 59, the heavy 25 chain CDR 2 of SEQ ID NO: 60, the heavy chain CDR 3 of SEQ ID NO: 61, the light chain CDR 1 of SEQ ID NO: 62, the light chain CDR 2 of SEQ ID NO: 63 and the light chain CDR 3 of SEQ ID NO: 64, and the second Fab molecule under b) comprises the heavy chain CDR 1 of SEQ ID NO: 32, the heavy chain CDR 2 of SEQ ID NO: 33, the heavy chain CDR 3 of SEQ ID NO: 34, the light chain CDR 1 of SEQ ID NO: 35, the light chain CDR 2 of SEQ ID 30 NO: 36 and the light chain CDR 3 of SEQ ID NO: 37; 2024200623   01 Feb 2024 (iii) in the constant domain CL of the first Fab molecule under a) and the third Fab molecule under c) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat), and wherein in the constant 5 domain CHI of the first Fab molecule under a) and the third Fab molecule under c) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); and (iv) the first Fab molecule under a) is fused at the C-terminus of the Fab heavy chain to the 10 N-terminus of the Fab heavy chain of the second Fab molecule under b), and the second Fab molecule under b) and the third Fab molecule under c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under d). In one embodiment, the first Fab molecule under a) and the third Fab molecule under c) each comprise a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical 15 to the sequence of SEQ ID NO: 65, and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 66. In one embodiment, the first Fab molecule under a) and the third Fab molecule under c) each comprise the heavy chain variable region sequence of SEQ ID NO: 65, and the light chain variable region sequence of SEQ ID NO: 66. 20 In one embodiment, the second Fab molecule under b) comprises a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 38, and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39. In still a further embodiment, the second Fab molecule under b) comprises the heavy chain 25 variable region sequence of SEQ ID NO: 38, and the light chain variable region sequence of SEQ ID NO: 39. In a particular embodiment, the bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 47, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 148, a 30 polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID 2024200623   01 Feb 2024 NO: 149, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 150. In a further particular embodiment, the bispecific antibody comprises a polypeptide sequence of SEQ ID NO: 47, a polypeptide sequence of SEQ ID NO: 148, a polypeptide sequence of SEQ ID NO: 149 and a polypeptide sequence of SEQ ID 5 NO: 150. Antibody formats The components of an antibody comprised in the therapeutic agent, particularly a multispecific antibody, can be fused to each other in a variety of configurations. Exemplary 10 configurations are depicted in Figure 6. In particular embodiments, the antigen binding moieties comprised in the antibody are Fab molecules. In such embodiments, the first, second, third etc. antigen binding moiety may be referred to herein as first, second, third etc. Fab molecule, respectively. Furthermore, in particular embodiments, the antibody comprises an Fc domain composed of a first and a 15 second subunit capable of stable association. In some embodiments, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. In one such embodiment, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In a specific such 20 embodiment, the antibody essentially consists of the first and the second Fab molecule, the Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second 25 subunit of the Fc domain. Such a configuration is schematically depicted in Figures 6G and 6K. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other. In another such embodiment, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In a specific 2024200623   01 Feb 2024 such embodiment, the antibody essentially consists of the first and the second Fab molecule, the Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first and the second Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. Such a 5 configuration is schematically depicted in Figures 6A and 6D. The first and the second Fab molecule may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment the first and the second Fab molecule are each fused to the Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgGi hinge region, particularly where the Fc domain is an IgGi Fc domain. 10 In other embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In one such embodiment, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In a specific such embodiment, the antibody essentially consists of the first and the second Fab molecule, 15 the Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. Such a configuration is schematically depicted in Figures 6H and 20   6L. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other. The Fab molecules may be fused to the Fc domain or to each other directly or through a peptide linker, comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable, non-immunogenic 25 peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n or G4(SG4)n peptide linkers, “n” is generally an integer from 1 to 10, typically from 2 to 4. In one embodiment said peptide linker has a length of at least 5 amino acids, in one embodiment a length of 5 to 100, in a further embodiment of 10 to 50 amino acids. In one embodiment said peptide linker is (GxS)n or (GxS)nGm with G=glycine, S=serine, and (x=3, n= 3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, 30   n=2, 3, 4 or 5 and m= 0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in a further embodiment x=4 and n=2. In one embodiment said peptide linker is (648)2. A particularly 2024200623   01 Feb 2024 suitable peptide linker for fusing the Fab light chains of the first and the second Fab molecule to each other is (648)2. An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and the second Fab fragments comprises the sequence (D)-(G4S)2 (SEQ ID NOs 118 and 119). Another suitable such linker comprises the sequence (648)4. Additionally, 5 linkers may comprise (a portion of) an immunoglobulin hinge region. Particularly where a Fab molecule is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker. An antibody with a single antigen binding moiety (such as a Fab molecule) capable of 10 specific binding to a target cell antigen (for example as shown in Figure 6A, D, 6, H, K, L) is useful, particularly in cases where internalization of the target cell antigen is to be expected following binding of a high affinity antigen binding moiety. In such cases, the presence of more than one antigen binding moiety specific for the target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availablity. 15 In many other cases, however, it will be advantageous to have an antibody comprising two or more antigen binding moieties (such as Fab moelcules) specific for a target cell antigen (see examples shown in Figure 6B, 6C, 6E, 6F, 61, 6J. 6M or 6N), for example to optimize targeting to the target site or to allow crosslinking of target cell antigens. Accordingly, in particular embodiments, the antibody further comprises a third Fab molecule 20 which specifically binds to the first antigen. The first antigen preferably is the target cell antigen. In one embodiment, the third Fab molecule is a conventional Fab molecule. In one embodiment, the third Fab molecule is identical to the first Fab molecule (i.e. the first and the third Fab molecule comprise the same heavy and light chain amino acid sequences and have the same arrangement of domains (i.e. conventional or crossover)). In a particular 25 embodiment, the second Fab molecule specifically binds to an activating T cell antigen, particularly CD3, and the first and third Fab molecule specifically bind to a target cell antigen. In alternative embodiments, the antibody further comprises a third Fab molecule which specifically binds to the second antigen. In these embodiments, the second antigen preferably 30 is the target cell antigen. In one such embodiment, the third Fab molecule is a crossover Fab 2024200623   01 Feb 2024 molecule (a Fab molecule wherein the variable domains VH and VL or the constant domains CL and CHI of the Fab heavy and light chains are exchanged / replaced by each other). In one such embodiment, the third Fab molecule is identical to the second Fab molecule (i.e. the second and the third Fab molecule comprise the same heavy and light chain amino acid 5 sequences and have the same arrangement of domains (i.e. conventional or crossover)). In one such embodiment, the first Fab molecule specifically binds to an activating T cell antigen, particularly CD3, and the second and third Fab molecule specifically bind to a target cell antigen. In one embodiment, the third Fab molecule is fused at the C-terminus of the Fab heavy chain 10 to the N-terminus of the first or second subunit of the Fc domain. In a particular embodiment, the second and the third Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In a specific such embodiment, 15 the antibody essentially consists of the first, the second and the third Fab molecule, the Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc 20 domain, and wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such a configuration is schematically depicted in Figure 6B and 6E (particular embodiments, wherein the third Fab molecule is a conventional Fab molecule and preferably identical to the first Fab molecule), and Figure 61 and 6M (alternative embodiments, wherein the third Fab molecule is a 25 crossover Fab molecule and preferably identical to the second Fab molecule). The second and the third Fab molecule may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment the second and the third Fab molecule are each fused to the Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgGi hinge region, particularly where the Fc 30 domain is an IgGi Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other. 2024200623   01 Feb 2024 In another embodiment, the first and the third Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In a specific such embodiment, the antibody 5 essentially consists of the first, the second and the third Fab molecule, the Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and 10 wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such a configuration is schematically depicted in Figure 6C and 6F (particular embodiments, wherein the third Fab molecule is a conventional Fab molecule and preferably identical to the first Fab molecule) and in Figure 6J and 6N (alternative embodiments, wherein the third Fab molecule is a crossover Fab 15 molecule and preferably identical to the second Fab molecule). The first and the third Fab molecule may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment the first and the third Fab molecule are each fused to the Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgGi hinge region, particularly where the Fc domain is an IgGi Fc domain. 20 Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other. In configurations of the antibody wherein a Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of each of the subunits of the Fc domain through an immunoglobulin hinge regions, the two Fab molecules, the hinge regions and the Fc domain 25 essentially form an immunoglobulin molecule. In a particular embodiment the immunoglobulin molecule is an IgG class immunoglobulin. In an even more particular embodiment the immunoglobulin is an IgGi subclass immunoglobulin. In another embodiment the immunoglobulin is an IgG4 subclass immunoglobulin. In a further particular embodiment the immunoglobulin is a human immunoglobulin. In other embodiments the 30 immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. 2024200623   01 Feb 2024 In some of the antibodies, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are fused to each other, optionally via a peptide Inker. Depending on the configuration of the first and the second Fab molecule, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the 5 second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. Fusion of the Fab light chains of the first and the second Fab molecule further reduces mispairing of unmatched Fab heavy and light chains, and also reduces the number of plasmids needed for expression of some of the antibodies. 10 In certain embodiments the antibody comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with 15 an Fc domain subunit (VL(2)-CH1(2)-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(i)-CHl(i)-CH2-CH3(-CH4)). In some embodiments the antibody further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of 20 the second Fab molecule (VH(2)-CL(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond. In certain embodiments the antibody comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the 25 Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(2)-CL(2)-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc 30 domain subunit (VH(i)-CHl(i)-CH2-CH3(-CH4)). In some embodiments the antibody further comprises a polypeptide wherein the Fab light chain variable region of the second Fab 2024200623   01 Feb 2024 molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL(2)-CH1(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond. 5 In some embodiments, the antibody comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with 10 the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL(2)-CHl(2)-VH(i)-CHl(i)-CH2-CH3(-CH4)). In other embodiments, the antibody comprises a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule which in turn shares a carboxy-terminal peptide bond with 15 the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(i)-CHl(i)-VL(2)-CHl(2)-CH2-CH3(-CH4)). In some of these embodiments the antibody further comprises a crossover Fab light chain 20 polypeptide of the second Fab molecule, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH(2)-CL(2)), and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In others of these embodiments the antibody further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab 25 molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule (VH(2)-CL(2)-VL(i)-CL(i)), or a polypeptide wherein the Fab light chain polypeptide of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule which in 30 turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VL(i)-CL(i)-VH(2)-CL(2)), as appropriate. 2024200623   01 Feb 2024 The antibody according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(3)-CH1(3)-CH2-CH3(-CH4)) and the Fab light chain polypeptide of a third Fab molecule (VL(3)-5   CL(3)). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond. In some embodiments, the antibody comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule 10 comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(2)-CL(2)-VH(i)-CHl(i)-CH2-CH3(-CH4)). In other embodiments, the antibody comprises a polypeptide wherein the Fab heavy chain of the 15 first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with 20 an Fc domain subunit (VH(i)-CHl(i)-VH(2)-CL(2)-CH2-CH3(-CH4)). In some of these embodiments the antibody further comprises a crossover Fab light chain polypeptide of the second Fab molecule, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL(2)-CH 1(2)), and the Fab light chain 25 polypeptide of the first Fab molecule (VL(i)-CL(i)). In others of these embodiments the antibody further comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule (VL(2)-CHl(2)-VL(i)-30   CL(i)), or a polypeptide wherein the Fab light chain polypeptide of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the 2024200623   01 Feb 2024 second Fab molecule which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VL(i)-CL(i)-VH(2)-CL(2)}, as appropriate. The antibody according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of a third 5 Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(3)- CH1(3)-CH2-CH3(-CH4)) and the Fab light chain polypeptide of a third Fab molecule (VL(3)-CL(3)). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond. In some embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy 10 chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In certain such embodiments, the antibody does not comprise an Fc domain. In certain embodiments, the antibody essentially consists of the first and the second Fab molecule, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. Such a 15 configuration is schematically depicted in Figures 60 and 6S. In other embodiments, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In certain such embodiments, the antibody does not comprise an Fc domain. In certain embodiments, the antibody essentially consists of the first and the second Fab molecule, and optionally one or 20 more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. Such a configuration is schematically depicted in Figures 6P and 6T. In some embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the antibody 25 further comprises a third Fab molecule, wherein said third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In particular such embodiments, said third Fab molecule is a conventional Fab molecule. In other such embodiments, said third Fab molecule is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the 30 constant domains CL and CHI of the Fab heavy and light chains are exchanged / replaced by 2024200623   01 Feb 2024 each other. In certain such embodiments, the antibody essentially consists of the first, the second and the third Fab molecule, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the third Fab molecule is fused at the C-5 terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. Such a configuration is schematically depicted in Figure 6Q and 6U (particular embodiments, wherein the third Fab molecule is a conventional Fab molecule and preferably identical to the first Fab molecule). In some embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy 10 chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the antibody further comprises a third Fab molecule, wherein said third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the second Fab molecule. In particular such embodiments, said third Fab molecule is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL 15 or the constant domains CHI and CL of the Fab heavy and light chains are exchanged / replaced by each other. In other such embodiments, said third Fab molecule is a conventional Fab molecule. In certain such embodiments, the antibody essentially consists of the first, the second and the third Fab molecule, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the 20 Fab heavy chain of the second Fab molecule, and the third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the second Fab molecule. Such a configuration is schematically depicted in Figure 6W and 6Y (particular embodiments, wherein the third Fab molecule is a crossover Fab molecule and preferably identical to the second Fab molecule). 25 In some embodiments, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the antibody further comprises a third Fab molecule, wherein said third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the first Fab molecule. In particular such embodiments, said third Fab molecule is a conventional Fab 30 molecule. In other such embodiments, said third Fab molecule is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the 2024200623   01 Feb 2024 constant domains CHI and CL of the Fab heavy and light chains are exchanged / replaced by each other. In certain such embodiments, the antibody essentially consists of the first, the second and the third Fab molecule, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of 5 the Fab heavy chain of the first Fab molecule, and the third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the first Fab molecule. Such a configuration is schematically depicted in Figure 6R and 6V (particular embodiments, wherein the third Fab molecule is a conventional Fab molecule and preferably identical to the first Fab molecule). 10 In some embodiments, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the antibody further comprises a third Fab molecule, wherein said third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In particular such embodiments, said third Fab molecule is a crossover Fab 15 molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CHI and CL of the Fab heavy and light chains are exchanged / replaced by each other. In other such embodiments, said third Fab molecule is a conventional Fab molecule. In certain such embodiments, the antibody essentially consists of the first, the second and the third Fab molecule, and optionally one or more peptide linkers, wherein the 20 second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. Such a configuration is schematically depicted in Figure 6X and 6Z (particular embodiments, wherein the third Fab molecule is a crossover Fab molecule and preferably 25 identical to the first Fab molecule). In certain embodiments the antibody comprises a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second 30 Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region) (VH(i)-CHl(i)-VL(2)-CHl(2)). In some 2024200623   01 Feb 2024 embodiments the antibody further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH(2)-CL(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). 5 In certain embodiments the antibody comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with 10 the Fab heavy chain of the first Fab molecule (VL(2)-CHl(2)-VH(i)-CHl(i)). In some embodiments the antibody further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH(2)-CL(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). 15 In certain embodiments the antibody comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with 20 the Fab heavy chain of the first Fab molecule (VH(2)-CL(2)-VH(i)-CHl(i)). In some embodiments the antibody further comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL(2)-CH1(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). 25 In certain embodiments the antibody comprises a polypeptide wherein the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the 30 second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region) (VH(3)-CHl(3)-VH(i)-CHl(i)- 2024200623   01 Feb 2024 VL(2)-CH1(2)). In some embodiments the antibody further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH(2)-CL(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In some 5 embodiments the antibody further comprises the Fab light chain polypeptide of a third Fab molecule (VL(3)-CL(3)). In certain embodiments the antibody comprises a polypeptide wherein the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy 10 chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region) (VH(3)-CHl(3)-VH(i)-CHl(i)-VH(2)-CL(2)). In some embodiments the antibody further comprises a polypeptide wherein the 15 Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL(2)-CH1(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In some embodiments the antibody further comprises the Fab light chain polypeptide of a third Fab molecule (VL(3)-CL(3)). 20 In certain embodiments the antibody comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with 25 the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule (VL(2)-CHl(2)-VH(i)-CHl(i)-VH(3)-CH1(3)). In some embodiments the antibody further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH(2)- 30   CL(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In some 2024200623   01 Feb 2024 embodiments the antibody further comprises the Fab light chain polypeptide of a third Fab molecule (VL(3)-CL(3)). In certain embodiments the antibody comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the 5 Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule (VH(2)-CL(2)-VH(i)-CHl(i)- 10   VH(3)-CH1(3)). In some embodiments the antibody further comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL(2)-CH1(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In some embodiments the antibody further comprises the Fab light chain polypeptide of a third Fab 15 molecule (VL(3)-CL(3)). In certain embodiments the antibody comprises a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second 20 Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of a third Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a third Fab molecule (i.e. the third Fab molecule comprises a crossover Fab heavy chain, wherein the 25 heavy chain variable region is replaced by a light chain variable region) (VH(i)-CHl(i)-VL(2)-CH1(2)-VL(3)-CH1(3)). In some embodiments the antibody further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxyterminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH(2)-CL(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In 30 some embodiments the antibody further comprises a polypeptide wherein the Fab heavy 2024200623   01 Feb 2024 chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a third Fab molecule (VH(3)-CL(3)). In certain embodiments the antibody comprises a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain 5 variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a third Fab molecule, which in turn shares a 10 carboxy-terminal peptide bond with the Fab light chain constant region of a third Fab molecule (i.e. the third Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region) (VH(i)-CHl(i)-VH(2)-CL(2)-VH(3)-CL(3)). In some embodiments the antibody further comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy - 15 terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL(2)-CH1(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In some embodiments the antibody further comprises a polypeptide wherein the Fab light chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a third Fab molecule (VL(3)-CH1(3)). 20 In certain embodiments the antibody comprises a polypeptide wherein the Fab light chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a third Fab molecule (i.e. the third Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab 25 light chain variable region of the second Fab molecule, which in turn shares a carboxyterminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VL(3)- 30   CHl(3)-VL(2)-CHl(2)-VH(i)-CHl(i)). In some embodiments the antibody further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a 2024200623   01 Feb 2024 carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH(2)-CL(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In some embodiments the antibody further comprises a polypeptide wherein the Fab heavy chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond 5 with the Fab light chain constant region of a third Fab molecule (VH(3)-CL(3)). In certain embodiments the antibody comprises a polypeptide wherein the Fab heavy chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a third Fab molecule (i.e. the third Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light 10 chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxyterminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a 15 carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VH(3)- CL(3)-VH(2)-CL(2)-VH(i)-CHl(i)). In some embodiments the antibody further comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL(2)-CH1(2)) and the Fab light chain polypeptide of the first Fab molecule (VL(i)-20   CL(i)). In some embodiments the antibody further comprises a polypeptide wherein the Fab light chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a third Fab molecule (VL(3)-CH1(3)). According to any of the above embodiments, components of the antibody (e.g. Fab molecules, Fc domain) may be fused directly or through various linkers, particularly peptide 25 linkers comprising one or more amino acids, typically about 2-20 amino acids, that are described herein or are known in the art. Suitable, non-immunogenic peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n or G4(SG4)n peptide linkers, wherein n is generally an integer from 1 to 10, typically from 2 to 4. 30 Fc domain 2024200623   01 Feb 2024 An antibody, e.g. a bispecific antibody or an immunoconjugate, comprised in the therapeutic agent may comprise an Fc domain which consists of a pair of polypeptide chains comprising heavy chain domains of an antibody molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and 5 CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment, the Fc domain is an IgG Fc domain. In a particular embodiment the Fc domain is an IgGi Fc domain. In another embodiment the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino 10 acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment the Fc domain is human. An exemplary sequence of a human IgGi Fc region is given in SEQ ID NO: 30. 15       (i)    Fc domain modifications promoting heterodimerization Antibodies, particularly bispecific antibodies or immunoconjugates, comprised in the therapeutic agent may comprise different components (e.g. antigen binding domains, cytokines) fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. 20 Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of such antibodies in recombinant production, it will thus be advantageous to introduce in the Fc domain of the antibody a modification promoting the association of the desired polypeptides. Accordingly, in particular embodiments the Fc domain comprises a modification promoting 25 the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment said modification is in the CH3 domain of the Fc domain. There exist several approaches for modifications in the CH3 domain of the Fc domain in 30 order to enforce heterodimerization, which are well described e.g. in WO 96 / 27011, 2024200623   01 Feb 2024 WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both 5 engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed). These different approaches for improved heavy chain heterodimerization are 10 contemplated as different alternatives in combination with heavy-light chain modifications (e.g. variable or constant region exchange / replacement in Fab arms, or introduction of substitutions of charged amino acids with opposite charges in the CHI / CL interface) which reduce light chain mispairing and Bence Jones-type side products. In a specific embodiment said modification promoting the association of the first and the 15 second subunit of the Fc domain is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain. The knob-into-hole technology is described e.g. in 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 20 method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or 25 tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). Accordingly, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side 30 chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the 2024200623   01 Feb 2024 CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. 5 Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protuberance and cavity can be made by altering the nucleic acid encoding the 10 polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain (the “knobs” subunit) the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the “hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one 15 embodiment, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index). In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid 20 residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). 25 In a particular embodiment, 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 Kabat EU index). 2024200623   01 Feb 2024 In a particular embodiment the mutant IL-2 polypeptide in the immunoconjugate described herein, or the CD3 antigen binding moiety in the bispecific antibody described herein, is fused to the first subunit of the Fc domain (comprising the “knob” modification). Without wishing to be bound by theory, fusion of the IL-2 polypeptide or CD3 antigen binding moiety 5 to the knob-containing subunit of the Fc domain will (further) minimize the generation of immunoconjugates comprising two IL-2 polypeptides or bispecific antibodies comprising two CD3 antigen binding moieties, respectively (steric clash of two knob-containing polypeptides). Other techniques of CH3-modification for enforcing the heterodimerization are contemplated 10 as alternatives according to the invention and are described e.g. in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291. In one embodiment the heterodimerization approach described in EP 1870459 Al, is used 15 alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. One preferred embodiment are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (numbering according 20 to Kabat EU index). In another embodiment the antibody comprises amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and 25 amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numberings according to Kabat EU index). In another embodiment the antibody comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the antibody 30 comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of 2024200623   01 Feb 2024 the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings 5 according to Kabat EU index). In one embodiment the heterodimerization approach described in WO 2013 / 157953 is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351D (numberings according to Kabat EU index). In a further embodiment the first CH3 domain comprises further amino 10 acid mutation L351K. In a further embodiment the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numberings according to Kabat EU index). In one embodiment the heterodimerization approach described in WO 2012 / 058768 is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutations 15   L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g. selected from a) T411N, T411R, T41 IQ, T41 IK, T41 ID, T41 IE or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, 20   N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numberings according to Kabat EU index). In a further embodiment a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F. In a further embodiment a first CH3 domain comprises amino acid mutation Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a 25 further embodiment the second CH3 domain further comprises amino acid mutations K392E, T41 IE, D399R and S400R (numberings according to Kabat EU index). In one embodiment the heterodimerization approach described in WO 2011 / 143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to Kabat EU index). 2024200623   01 Feb 2024 In one embodiment the heterodimerization approach described in WO 2011 / 090762, which also uses the knobs-into-holes technology described above, is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A. In one embodiment a first CH3 domain 5 comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (numberings according to Kabat EU index). In one embodiment the antibody or its Fc domain is of IgG2 subclass and the heterodimerization approach described in WO 2010 / 129304 is used alternatively. In an alternative embodiment a modification promoting association of the first and the second 10 subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009 / 089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In one such 15 embodiment a first CH3 domain comprises amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K392D or N392D) and a second CH3 domain comprises amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K, D356K, or E357K, and more preferably D399K and E356K). In a 20 further embodiment the first CH3 domain further comprises amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). In a further embodiment the first CH3 domain further or alternatively comprises amino acid substitution of K439 and / or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to 25 Kabat EU index). In yet a further embodiment the heterodimerization approach described in WO 2007 / 147901 is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (numberings according to Kabat EU index). 2024200623   01 Feb 2024 In still another embodiment the heterodimerization approach described in WO 2007 / 110205 can be used alternatively. In one embodiment, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid 5 substitutions D356K and D399K (numbering according to Kabat EU index). (ii) Fc domain modifications reducing Fc receptor binding and / or effector function The Fc domain confers to an antibody, such as a bispecific antibody or immunoconjugate, favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the 10 same time it may, however, lead to undesirable targeting of the antibody to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with other immunostimulatory properties the antibody may have and the long half-life of the antibody, results in excessive activation of cytokine receptors and severe side effects upon 15 systemic administration. Accordingly, in particular embodiments, the Fc domain of the antibody, particularly bispecific antibody or immunoconjugate, comprised in the therapeutic agent exhibits reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgGi Fc domain. In one such embodiment the Fc domain (or the molecule, e.g. antibody, 20 comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGi Fc domain (or a corresponding molecule comprising a native IgGi Fc domain), and / or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native 25 IgGi Fc domain domain (or a corresponding molecule comprising a native IgGi Fc domain). In one embodiment, the Fc domain (or the molecule, e.g. antibody, comprising said Fc domain) does not substantially bind to an Fc receptor and / or induce effector function. In a particular embodiment the Fc receptor is an Fey receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a 30 specific embodiment the Fc receptor is an activating human Fey receptor, more specifically 2024200623   01 Feb 2024 human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. In one embodiment the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment the effector function is ADCC. In one embodiment the Fc domain exhibits substantially similar binding affinity to neonatal Fc 5 receptor (FcRn), as compared to a native IgGi Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the molecule, e.g. antibody, comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGi Fc domain (or the corresponding molecule comprising a native IgGi Fc domain) to FcRn. 10 In certain embodiments the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a non-engineered Fc domain. In particular embodiments, the Fc domain comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of 15 the Fc domain. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid 20 mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10fold, at least 20-fold, or even at least 50-fold. In one embodiment the molecule, e.g. antibody, comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to a corresponding molecule comprising a non-engineered Fc domain. In a particular embodiment 25 the Fc receptor is an Fey receptor. In some embodiments the Fc receptor is a human Fc receptor. In some embodiments the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments binding affinity to a complement 30 component, specifically binding affinity to Clq, is also reduced. In one embodiment binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when 2024200623   01 Feb 2024 the Fc domain (or the molecule, e.g. antibody, comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or a corresponding molecule comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or molecule (e.g. antibody) comprising said Fc domain, may exhibit greater 5 than about 80% and even greater than about 90% of such affinity. In certain embodiments the Fc domain is engineered to have reduced effector function, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibodydependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular 10 phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment the reduced 15 effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a corresponding molecule comprising a non-engineered Fc domain). 20 In one embodiment the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or effector function is an amino acid substitution. In one embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). In a more specific embodiment the Fc domain comprises an amino acid substitution at 25 a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In some embodiments the Fc domain comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. In one embodiment the Fc domain comprises an amino acid substitution at position P329. In a more specific 30 embodiment the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index). In one embodiment the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from 2024200623   01 Feb 2024 E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific embodiment the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). 5 In more particular embodiments the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. The “P329G LALA” combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGi Fc domain, as described in PCT publication no. WO 2012 / 130831, 10 incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions. IgGi antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgGi antibodies. Hence, in some embodiments the Fc domain is an 15 IgGi Fc domain, particularly a human IgGi Fc domain. In one embodiment the IgGi Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P (numberings according to Kabat EU index). To further reduce its binding affinity to an Fc receptor and / or its effector function, in one embodiment the IgGi Fc domain comprises an amino acid substitution at position L235, specifically the amino acid 20 substitution L235E (numberings according to Kabat EU index). In another embodiment, the IgGi Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numberings according to Kabat EU index). In a particular embodiment, the IgGi Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numberings 25 according to Kabat EU index). Such IgGi Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012 / 130831, incorporated herein by reference in its entirety. In a particular embodiment the Fc domain exhibiting reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgGi Fc domain, is a 30 human IgGi Fc domain comprising the amino acid substitutions L234A, L235A and 2024200623   01 Feb 2024 optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numberings according to Kabat EU index). In certain embodiments N-glycosylation of the Fc domain has been eliminated. In one such embodiment the Fc domain comprises an amino acid mutation at position N297, particularly 5 an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) or glycine (N297G) (numberings according to Kabat EU index). In addition to the Fc domains described hereinabove and in PCT publication no. WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 10   327 and 329 (U.S. Patent No. 6,737,056) (numberings according to Kabat EU index). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581). Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or 15 modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing. Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE 20 Healthcare), and Fc receptors such as may be obtained by recombinant expression. Alternatively, binding affinity of Fc domains or molecules comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor. Effector function of an Fc domain, or a molecule (e.g. an antibody) comprising an Fc domain, 25 can be measured by methods known in the art. A suitable assay for measuring ADCC is described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). 30 Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ 2024200623   01 Feb 2024 non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of 5 interest may be assessed in vivo, e.g. in a animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998). In some embodiments, binding of the Fc domain to a complement component, specifically to Clq, is reduced. Accordingly, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. Clq 10 binding assays may be carried out to determine whether the Fc domain, or molecule (e.g. antibody) comprising the Fc domain, is able to bind Clq and hence has CDC activity. See e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and 15 Cragg and Glennie, Blood 103, 2738-2743 (2004)). Antigen Binding Moieties The antibody comprised in the therapeutic agent may be bispecific, i.e. it comprises at least two antigen binding moieties capable of specific binding to two distinct antigenic 20 determinants. According to particular embodiments, the antigen binding moieties are Fab molecules (i.e. antigen binding domains composed of a heavy and a light chain, each comprising a variable and a constant domain). In one embodiment said Fab molecules are human. In another embodiment said Fab molecules are humanized. In yet another embodiment said Fab molecules comprise human heavy and light chain constant domains. 25 In some embodiments, at least one of the antigen binding moieties is a crossover Fab molecule. Such modification reduces mispairing of heavy and light chains from different Fab molecules, thereby improving the yield and purity of the antibody in recombinant production. In a particular crossover Fab molecule useful for the antibody, the variable domains of the Fab light chain and the Fab heavy chain (VL and VH, respectively) are exchanged. Even with 30 this domain exchange, however, the preparation of the antibody may comprise certain side 2024200623   01 Feb 2024 products due to a so-called Bence Jones-type interaction between mispaired heavy and light chains (see Schaefer et al, PNAS, 108 (2011) 11187-11191). To further reduce mispairing of heavy and light chains from different Fab molecules and thus increase the purity and yield of the desired antibody, charged amino acids with opposite charges may be introduced at 5 specific amino acid positions in the CHI and CL domains of either the Fab molecule(s) specifically binding to a target cell antigen, or the Fab molecule specifically binding to an activating T cell antigen. Charge modifications are made either in the conventional Fab molecule(s) comprised in the antibody (such as shown e.g. in Figures 6 A-C, G-J), or in the VH / VL crossover Fab molecule(s) comprised in the antibody (such as shown e.g. in Figure 6 10 D-F, K-N) (but not in both). In particular embodiments, the charge modifications are made in the conventional Fab molecule(s) comprised in the antibody (which in particular embodiments specifically bind(s) to the target cell antigen). In a particular embodiment according to the invention, the antibody is capable of simultaneous binding to a target cell antigen, particularly a tumor cell antigen, and an 15 activating T cell antigen, particularly CD3. In one embodiment, the antibody is capable of crosslinking a T cell and a target cell by simultaneous binding to a target cell antigen and an activating T cell antigen. In an even more particular embodiment, such simultaneous binding results in lysis of the target cell, particularly a tumor cell. In one embodiment, such simultaneous binding results in activation of the T cell. In other embodiments, such 20 simultaneous binding results in a cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from the group of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. In one embodiment, binding of the antibody to the activating T cell antigen, particularly CD3, without simultaneous binding to the target cell antigen does not result in T cell activation. 25 In one embodiment, the antibody is capable of re-directing cytotoxic activity of a T cell to a target cell. In a particular embodiment, said re-direction is independent of MHC-mediated peptide antigen presentation by the target cell and and / or specificity of the T cell. Particularly, a T cell according to any of the embodiments of the invention is a cytotoxic T cell. In some embodiments the T cell is a CD4+ or a CD8+ T cell, particularly a CD8+ T cell. 30       (i) Activating T cell antigen binding moiety 2024200623   01 Feb 2024 In some embodiments, an antibody comprised in the therapeutic agent, particularly a bispecific antibody, comprises at least one antigen bind...

Claims

1. A method of treating a disease in a subject, the method comprising a treatment regimen comprising(iii) administration to the subject of a Type II anti-CD20 antibody,5 and consecutively after a period of time(iv) administration to the subject of a therapeutic agent, particularly a T-cell activating therapeutic agent,wherein the period of time between the administration of the Type II anti-CD20 antibody and the administration of the therapeutic agent is sufficient for reduction of the number of B-cells 10 in the subject in response to the administration of the Type II anti-CD20 antibody.

2. The method of claim 1, wherein the treatment regimen effectively reduces cytokine release associated with the administration of the therapeutic agent in the subject as compared to a corresponding treatment regimen without the administration of the Type II anti-CD20 antibody.15   3. A method for reducing cytokine release associated with the administration of thetherapeutic agent, particularly a T-cell activating therapeutic agent, in a subject, comprising administration of a Type II anti-CD20 antibody to the subject prior to administration of the therapeutic agent.

4. The method of claim 3, wherein the period of time between the administration of the Type 20 II anti-CD20 antibody and administration of the therapeutic agent is sufficient for reductionof the number of B-cells in the subject in response to the administration of the Type II anti-CD20 antibody.

5. The method of any one of claims 1-4, wherein the period of time between the administration of the Type II anti-CD20 antibody and administration of the therapeutic agent 25 is (i) 3 days to 21 days, 5 days to 20 days, 7 days to 21 days, 7 days to 14 days, 5 days to 15 days, 7 days to 15 days, 8 days to 15 days, 10 days to 20 days, 10 days to 15 days, 11 days to 14 days, or 12 days to 13 days; or (ii) 5 days to 10 days, particularly 7 days.

6. The method of any one of claims 1-5, wherein the Type II anti-CD20 antibody comprises (i) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID2024200623   01 Feb 2024NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9; and / or (ii) the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO: 11.5   7. The method of any one of claims 1-6, wherein the Type II anti-CD20 antibody is an IgGantibody, particularly an IgGi antibody; and / or wherein the Type II anti-CD20 antibody comprises an Fc region, particularly an IgG Fc region, more particularly an IgGl Fc region, optionally wherein at least about 40% of the N-linked oligosaccharides in the Fc region of the Type II anti-CD20 antibody are non-fucosylated.10   8. The method of any one of claims 1-7, wherein the Type II anti-CD20 antibody isobinutuzumab.

9. The method of any one of claims 1-8, wherein the administration of the Type II anti-CD20 antibody is (i) a single administration, or (ii) two or more separate administrations.

10. The method of any one of claims 1-9, wherein the administration of the Type II anti-15 CD20 antibody is a dose of about 1000 mg or about 2 g Type II anti-CD20 antibody.

11. The method of any one of claims 1-10, wherein the administration of the Type II anti-CD20 antibody is a single administration of a dose of about 1000 mg Type II anti-CD20 antibody, and the period of time between the administration of the Type II anti-CD20 antibody and administration of the therapeutic agent is 7 days.20    12. The method of any one of claims 1-11, wherein the therapeutic agent is administeredparenterally, particularly intravenously.

13. The method of any one of claims 1-12, wherein the therapeutic agent comprises an antibody, particularly a multispecific antibody.

14. The method of claim 13, wherein the antibody comprised in the therapeutic agent2 5   specifically binds to an activating T cell antigen, particularly an antigen selected from thegroup consisting of CD3, CD28, CD137 (also known as 4-1BB), CD40, CD226, OX40, GITR, CD27, HVEM, and CD127, more particularly CD3, most particularly CD3e, and optionally comprises2024200623   01 Feb 2024(iii) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and / or5 (iv) a heavy chain variable region sequence of SEQ ID NO: 38 and a light chain variable region sequence of SEQ ID NO: 39.

15. The method of claim 13 or 14, wherein the antibody comprised in the therapeutic agent specifically binds to a B-cell antigen, particularly an antigen selected from the group consisting of CD20, CD19, CD22, ROR-1, CD37 and CD5, more particularly CD20 or10 CD 19, most particularly CD20, and optionally comprises(iii) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9; and / or15 (iv) a heavy chain variable region sequence of SEQ ID NO: 10 and a light chain variable region sequence of SEQ ID NO: 11.

16. The method of any one of claims 1-15, wherein the therapeutic agent comprises a bispecific antibody that specifically binds to CD3 and to a target cell antigen, particularly a B cell antigen, more particularly CD20 or CD 19, most particularly CD20.20   17. The method of any one of claims 1-16, wherein the therapeutic agent comprises abispecific antibody comprising(a) an antigen binding moiety that specifically binds to CD3 and comprises (i) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region 25 comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO:36 and the LCDR3 of SEQ ID NO: 37, and / or (ii) a heavy chain variable region of SEQ ID NO: 38; and a light chain variable region of SEQ ID NO: 39; and(b) an antigen binding moiety that specifically binds to CD20 and comprises (i) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR230 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 82024200623   01 Feb 2024and the LCDR3 of SEQ ID NO: 9, and / or (ii) a heavy chain variable region of SEQ ID NO: 10; and a light chain variable region of SEQ ID NO: 11.

18. The method of claim 16 or 17, wherein the bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:5   44, a polypeptide that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to thesequence of SEQ ID NO: 45, a polypeptide that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 46, and a polypeptide that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 47.

19. The method of any one of claims 1-18, wherein the therapeutic agent comprises10 CD20XCD3 bsAB.

20. The method of any one of claims 1-12, wherein the therapeutic agent comprises a T cell expressing a chimeric antigen receptor (CAR), particularly a CAR that specifically binds to a B-cell antigen, more particularly a CAR that specifically binds to an antigen selected from the group of CD20, CD19, CD22, ROR-1, CD37 and CD5.15   21. The method of any one of claims 1-20, wherein the disease is (i) cancer, (ii) a B cellproliferative disorder, particularly a CD20-positive B-cell disorder, and / or (iii) a disease selected from the group consisting of Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zone20 lymphoma (MZL), Multiple myeloma (MM) and Hodgkin lymphoma (HL).

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