Combination therapy with anti-CD19 / anti-CD28 bispecific antibodies

Abstract

The present invention relates to a combination therapy that combines an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, and the use of this combination therapy for the treatment of B-cell cancers, such as diffuse large B-cell lymphoma (DLBCL).

Description

[Technical Field]

[0001] The present invention relates to a combination therapy employing an anti-CD20 / anti-CD3 bispecific antibody in combination with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, and the use of this combination therapy for the treatment of B-cell proliferative disorders such as diffuse large B-cell lymphoma (DLBCL). [Background technology]

[0002] B-cell proliferative disorders refer to a heterogeneous group of malignancies, including both leukemias and lymphomas. Lymphomas arise from lymphocytes and include two major categories: Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). In the United States, lymphomas of B-cell origin account for approximately 80–85% of all non-Hodgkin lymphoma cases, and within B-cell subsets, there is considerable heterogeneity based on the genotype and phenotypic expression patterns of B-cell origin. For example, the B-cell lymphoma subset includes slow-growing, indolent, and incurable diseases such as follicular lymphoma (FL) or chronic lymphocytic leukemia (CLL), as well as more aggressive subtypes such as mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Diffuse large B-cell lymphoma (DLBCL) is the most common type of NHL, accounting for approximately 30%–40% of all NHL diagnoses, followed by follicular lymphoma (FL; 20%–25% of all NHL diagnoses) and mantle cell lymphoma (MCL; 6%–10% of all NHL diagnoses). B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in adults, with approximately 15,000 new cases occurring annually in the United States (American Cancer Society 2015). Despite the availability of various drugs for the treatment of B-cell proliferative disorders, there is a continuing need to develop safe and effective therapies to extend remission duration and improve cure rates in patients.

[0003] Anti-CD20 / anti-CD3 bispecific antibodies target CD20 expressed on B cells and CD3 epsilon chain (CD3ε) present on T cells. Co-binding activates T cells and triggers T cell-mediated killing of B cells. In the presence of CD20-expressing B cells, whether circulating or tissue-resident, pharmacologically active amounts of anti-CD20 / anti-CD3 bispecific antibodies trigger T cell activation and subsequent cytokine release. Glofitamab is a T cell-targeting bispecific antibody (TCB) that targets CD20 expressed on B cells and CD3 epsilon chain (CD3ε) present on T cells. In parallel with B cell depletion in peripheral blood, anti-CD20 / anti-CD3 bispecific antibodies cause a transient reduction in peripheral blood T cells and a peak in cytokine release within 24 hours after the first administration, followed by rapid T cell recovery and a return of cytokine levels to baseline within 72 hours. Therefore, to achieve complete elimination of tumor cells, additional agents are needed to maintain T cell activation and generate a sustained immune response against cancer cells.

[0004] 4-1BB (CD137) is an inducible member of the tissue necrosis factor (TNF) receptor superfamily expressed by activated T cells. Many other immune cells also express 4-1BB, including NK cells, B cells, NKT cells, monocytes, neutrophils, mast cells, dendritic cells (DCs), and non-hematopoietic cells such as endothelial cells and smooth muscle cells. 4-1BB expression in various cell types is mostly inducible and triggered by various stimulatory signals, including T cell receptor (TCR) or B cell receptor stimulation, or signaling induced through costimulatory molecules or inflammatory cytokine receptors. The 4-1BB ligand (4-1BBL or CD137L) was identified in 1993. 4-1BBL expression has been shown to be restricted to professional antigen-presenting cells (APCs), such as B cells, dendritic cells, and macrophages. 4-1BBL expression is characteristic of T cells, including both αβ and γδ T cell subsets, and endothelial cells.

[0005] Costimulation via the 4-1BB receptor (e.g., by 4-1BB ligand binding) stimulates T cells (CD4 + and CD8 + 4-1BB activates multiple signaling cascades within T cells (both T cell subsets) and potently enhances T cell activation. In combination with TCR triggering, agonistic antibodies specific for 4-1BB promote T cell proliferation, stimulate lymphokine secretion, and reduce T lymphocyte susceptibility to activation-induced cell death. This mechanism was further advanced as the first proof-of-concept in cancer immunotherapy. Preclinical models in which agonistic antibodies against 4-1BB were administered to tumor-bearing mice demonstrated potent antitumor effects. Subsequently, accumulating evidence demonstrated that 4-1BB typically exerts its antitumor efficacy only when combined with other immunomodulatory compounds, chemotherapeutic agents, tumor-specific vaccines, or radiation therapy (Bartkowiak and Curran, 2015).

[0006] Signaling of the TNFR superfamily requires cross-linking of trimerized ligands to bind to the receptor. 4-1BB agonistic antibodies are similar, requiring wild-type Fc binding. However, systemic administration of 4-1BB-specific agonistic antibodies with functionally active Fc domains results in CD8+ / CD8+ binding associated with liver toxicity. + Although T cell influx was induced, the liver toxicity was reduced or significantly ameliorated in mice without functional Fc receptors. In clinical trials, an Fc-competent 4-1BB agonist antibody (BMS-663513) (NCT00612664) caused grade 4 hepatitis, leading to study discontinuation. Therefore, more effective and safe 4-1BB agonists are needed. One example is an antigen-binding molecule consisting of a trimeric, biologically active 4-1BB ligand, an antigen-binding domain specific for the tumor antigen CD19, and a silent Fc domain (hereafter referred to as CD19-4-1BBL). This structure, described in International Publication No. WO 2016 / 075278, replaces nonspecific FcγR-mediated crosslinking, which is responsible for Fc-mediated toxicity, with B cell-specific crosslinking targeting CD19.

[0007] CD19 is expressed on the surface of B cells and is almost exclusively B cells, making it an ideal target for immunotherapy of B cell malignancies. CD19 is more widely expressed than CD20 during B cell development; therefore, CD20-positive cells typically also express CD19. During B cell differentiation into plasma cells (antibody-secreting cells), B cells downregulate CD20 expression. CD20 expression is sometimes downregulated in B cell lymphomas, but CD19 remains positive. Therefore, targeting both CD19 and CD20 may broadly cover diseased B cells in lymphomas. It is also possible that selective pressure for CD20 extends to both CD19 and CD20. While it is unclear whether CD19 is directly involved in B cell oncogenesis, its expression is highly conserved in most B cell neoplasms, including acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B cell lymphoma. In acute leukemia, CD19 is stably and persistently expressed in almost all subtypes, whereas CD20 is expressed in a minority of leukemias.

[0008] CD28 is a founding member of a subfamily of costimulatory molecules characterized by a single transmembrane domain containing key signaling motifs and paired V-set immunoglobulin superfamily (IgSF) domains linked to a cytoplasmic domain. Other members of this subfamily include ICOS, CTLA-4, PD1, PD1H, TIGIT, and BTLA. CD28 expression is restricted to T cells and is found on all naive and most antigen-experienced cell subsets, including those expressing PD-1 or CTLA-4. CD28 and CTLA-4 are highly homologous and compete for binding with the B7 molecules CD80 and CD86, which are expressed on dendritic cells, B cells, macrophages, and tumor cells. CTLA-4 has a higher affinity for B7 family ligands, allowing it to bind preferentially to CD28, suppressing effector T cell responses. In contrast, PD-1 has been shown to inhibit CD28 signaling by partially dephosphorylating the CD28 cytoplasmic domain. Recent evidence indicates that the anti-cancer effects of PD-L1 / PD-1 and CTLA-4 checkpoint inhibitors depend on CD28. CD28 is constitutively expressed on the cell surface of both CD4 and CD8 T cells. Upon signal 1 transduction via TCR or CD3 engagement, CD28-mediated costimulation activates multiple signaling cascades within T cells, enhancing T cell-mediated immune responses. CD28-mediated signal 2 is thought to occur via co-clustering at the immune synapse. CD28 agonistic antibodies can enhance immune responses by administering them in combination with signal 1 providers, such as antibodies targeting CD3. Immune stimulation is a complex chain, and an uncontrolled response carries significant risks. In 2006, a life-threatening cytokine storm occurred during a phase I clinical trial of the human CD28 antibody TGN1412. In contrast to TGN1412, the anti-CD19 / anti-CD28 bispecific antibody avoids autonomous T cell activation, as it induces T cell proliferation, cytokine secretion, and tumor cell killing only in the presence of CD19-expressing tumor cells and signal 1 via TCR or CD3 engagement.

[0009] With the recent development of second-generation or later T cells expressing genetically engineered chimeric antigen receptors (CAR-T) for patients with CD19-positive malignancies, CD28 has once again attracted significant attention as an immunotherapy target. Multiple clinical trials of autologous anti-CD19-directed CAR-T therapy containing the CD28 signaling domain in heavily pretreated NHL patients have demonstrated response rates ranging from 64% to 82%, proving the concept's validity. However, CAR-T cell therapy still faces significant limitations, including life-threatening CAR-T cell-associated toxicity and resistance in B-cell malignancies, post-infusion adverse events such as cytokine release syndrome (CRS) and neurotoxicity, and host rejection of non-human CARs. Common barriers include limited cell manufacturing, baseline T cell quality, and time to infusion. Autologous CAR-T cell therapy requires 4–6 weeks to prepare genetically engineered CAR-T cells from the patient's T cells, a delay that can adversely affect treatment outcomes. In contrast, bispecific antibodies are readily available.

[0010] Because current standard therapies are unable to treat all patients suffering from B-cell proliferative disorders, there is a great need to develop new, potent and specific therapies. Summary of the Invention

[0011] The present invention relates to the use of anti-CD20 / anti-CD3 bispecific antibodies and anti-CD19 / anti-CD28 bispecific antibodies in combination with CD19-targeted 4-1BB (CD137) agonists in combination therapies for the treatment of cancer, particularly B-cell proliferative disorders. The combination therapies described herein have been shown to be more effective in inhibiting tumor growth and eliminating tumor cells than treatment with anti-CD20 / anti-CD3 bispecific antibodies and anti-CD19 / anti-CD28 bispecific antibodies alone or with CD19-targeted 4-1BB (CD137) agonists alone.

[0012] In one aspect, the present invention provides a combination of an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist for use in combination therapy for the treatment of a B-cell proliferative disorder.

[0013] In another aspect, the invention provides for the use of an anti-CD20 / anti-CD3 bispecific antibody in combination with an anti-CD19 / anti-CD28 bispecific antibody and an anti-CD19 targeted 4-1BB (CD137) agonist in the manufacture of a medicament for use in combination therapy for the treatment of a B-cell proliferative disorder.

[0014] In yet another aspect, the present invention provides a method of treating B-cell cancer in an individual in need thereof, comprising administering to the individual a combination therapy combining an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and an anti-CD19-targeted 4-1BB (CD137) agonist.

[0015] In a further aspect, the present invention provides a kit for use in combination therapy comprising a first medicament comprising an anti-CD20 / anti-CD3 bispecific antibody, a second medicament comprising an anti-CD19 / anti-CD28 bispecific antibody, and a third medicament comprising a CD19-targeted 4-1BB (CD137) agonist, optionally further comprising a package insert containing instructions for administering the first medicament in combination with the second medicament to treat cancer in an individual.

[0016] In another aspect, a medicament is provided comprising an anti-CD20 / anti-CD3 bispecific antibody for treating a B-cell proliferative disorder, wherein said anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist.

[0017] In certain embodiments, there is provided a use, method of use, kit or medicament as described herein above, combining an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, wherein said combination therapy comprises a first therapeutic regimen of the anti-CD20 / anti-CD3 bispecific antibody in combination with the anti-CD19 / anti-CD28 bispecific antibody, and a second therapeutic regimen of the anti-CD20 / anti-CD3 bispecific antibody in combination with the CD19-targeted 4-1BB (CD137) agonist.

[0018] In one embodiment, the first treatment regimen consists of 1 to 5 treatment cycles, and the second treatment regimen begins with the next treatment cycle. In another embodiment, the first treatment regimen consists of 1 to 5 treatment cycles, and the second treatment regimen begins with the next treatment cycle. In one embodiment, the first treatment regimen consists of 4 treatment cycles, and the second treatment regimen begins with treatment cycle 5. In one embodiment, there is a one-week time interval between the end of the first treatment regimen and the start of the second treatment regimen.

[0019] In all these aspects, there is provided a combination therapy, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, wherein pretreatment with a type II anti-CD20 antibody has taken place, preferably with obinutuzumab, prior to the combination therapy, and wherein the period between pretreatment and combination therapy is sufficient to deplete B cells in the individual in response to the type II anti-CD20 antibody, preferably obinutuzumab.

[0020] In one embodiment of the present invention, the CD19-targeted 4-1BB agonist comprises three ectodomains of 4-1BBL, each ectodomain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. More particularly, the ectodomain of 4-1BBL comprises the amino acid sequence of SEQ ID NO:5. In one embodiment, the CD19-targeted 4-1BB agonist comprises an Fc domain, particularly an IgG1 or IgG4 Fc domain, comprising one or more amino acid substitutions that reduce or eliminate Fc receptor binding and / or effector function. More particularly, the CD19-targeted 4-1BB agonist comprises an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A, and P329G (EU numbering according to Kabat).

[0021] In a further aspect, there is provided a use, method of use, kit or medicament as described herein, combining an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, wherein the CD19-targeted 4-1BB agonist comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12. H and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15. L In particular, the CD19-targeted 4-1BB agonist comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 16. H CD19) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 17 L It contains an antigen-binding domain capable of specifically binding to CD19, including CD19.

[0022] In another aspect, there is provided an anti-CD20 / anti-CD3 bispecific antibody that combines an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist for use, wherein the CD19-targeted 4-1BB agonist is (a) a first polypeptide comprising: (a1) a first ectodomain of 4-1BBL or a fragment thereof, the C-terminus of which is fused to the N-terminus of a second ectodomain of 4-1BBL or a fragment thereof; (a2) a second ectodomain of 4-1BBL or a fragment thereof, the C-terminus of which is fused to the N-terminus of a CL domain; (a3) ​​the CL domain, the C-terminus of which is fused to the N-terminus of one of the subunits of an Fc domain (e.g., a first subunit); and (a4) one of the subunits of the Fc domain (e.g., the first subunit); (b) a second polypeptide comprising: (b1) a third ectodomain of 4-1BBL or a fragment thereof, the C-terminus of which is fused to the N-terminus of a CH1 domain; and (b2) the CH1 domain; (c) a third polypeptide comprising (c1) a heavy chain of a Fab molecule that binds to CD19, the C-terminus of which is fused to the N-terminus of another one of the subunits of the Fc domain (e.g., a second subunit); and (c2) another one of the subunits of the Fc domain (e.g., a second subunit); (d) a fourth polypeptide comprising the light chain of the Fab molecule that binds to CD19; and There is provided a use, method, kit or medicament according to any one of the preceding paragraphs comprising:

[0023] In one aspect there is provided a use, method of use, kit or medicament as described herein above, combining an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, wherein the CD19-targeted 4-1BB agonist comprises a first polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18, and a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19. a second polypeptide consisting of an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:20; a third polypeptide consisting of an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:20; and a fourth polypeptide consisting of an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:21.

[0024] More specifically, the CD19-targeted 4-1BB agonist comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 18, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 19, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 20, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 21.

[0025] In a further aspect, there is provided a use, method of use, kit or medicament as herein before disclosed, combining an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V H CD3) and the light chain variable region (V L a first antigen-binding domain comprising a heavy chain variable region (V H CD20) and the light chain variable region (V L In one embodiment, the first antigen-binding domain comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 22, the CDR-H2 sequence of SEQ ID NO: 23, and the CDR-H3 sequence of SEQ ID NO: 24.H and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 25, the CDR-L2 sequence of SEQ ID NO: 26, and the CDR-L3 sequence of SEQ ID NO: 27. L In one embodiment, the first antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 28. H CD3) and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 29 L In one embodiment, the second antigen-binding domain comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 30, the CDR-H2 sequence of SEQ ID NO: 31, and the CDR-H3 sequence of SEQ ID NO: 32. H CD20), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 33, the CDR-L2 sequence of SEQ ID NO: 34, and the CDR-L3 sequence of SEQ ID NO: 35. L In one embodiment, the second antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 36. H CD20) and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 37 L CD20).

[0026] In a further aspect there is provided the use of an anti-CD20 / anti-CD3 bispecific antibody in combination with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, a use, method, kit or medicament as herein before disclosed, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises a third antigen-binding domain that binds to CD20. In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody is a first polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 38, a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39, a third polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40, and fourth and fifth polypeptides comprising amino acid sequences at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41. More particularly, the anti-CD20 / anti-CD3 bispecific antibody comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 38, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 39, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 40, and fourth and fifth polypeptides comprising the amino acid sequence of SEQ ID NO: 41. More particularly, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

[0027] In one aspect there is provided a use, method of use, kit or medicament as herein before disclosed which combines an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, wherein the anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (V H CD28) and the light chain variable region (V L The first antigen-binding domain consists of a heavy chain variable region (V H CD19) and the light chain variable region (V LIn one embodiment, the anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 42, the CDR-H2 sequence of SEQ ID NO: 43, and the CDR-H3 sequence of SEQ ID NO: 44. H CD28), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 45, the CDR-L2 sequence of SEQ ID NO: 46, and the CDR-L3 sequence of SEQ ID NO: 47 L In one embodiment, the anti-CD19 / anti-CD28 bispecific antibody comprises a first antigen-binding domain comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 48. H CD28) and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 49 L In one embodiment, the anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 10, the CDR-H2 sequence of SEQ ID NO: 11, and the CDR-H3 sequence of SEQ ID NO: 12. H CD19); and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 13, the CDR-L2 sequence of SEQ ID NO: 14, and the CDR-L3 sequence of SEQ ID NO: 15 L In one embodiment, the anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 16. H CD19) and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 17 L and a second antigen-binding domain comprising a nucleotide sequence encoding the nucleotide sequence of interest (e.g., nucleotide sequence 10 ...

[0028] In one embodiment, there is provided a use, method of use, kit, or medicament as described herein, combining an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, wherein the anti-CD19 / anti-CD28 bispecific antibody comprises an Fc domain, particularly an IgG1 or IgG4 Fc domain, which Fc domain comprises one or more amino acid substitutions that reduce or eliminate binding to an Fc receptor and / or effector function. More particularly, the anti-CD19 / anti-CD28 bispecific antibody comprises an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A, and P329G (EU numbering according to Kabat).

[0029] In certain embodiments, the anti-CD19 / anti-CD28 bispecific antibody is a first polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:50; a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:51; a third polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:52; and a fourth polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:53. More particularly, the anti-CD19 / anti-CD28 bispecific antibody comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:50, a second polypeptide comprising the amino acid sequence of SEQ ID NO:51, a third polypeptide comprising the amino acid sequence of SEQ ID NO:52, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:53.

[0030] In a further aspect, the present invention provides the use of an anti-CD20 / anti-CD3 bispecific antibody in combination with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, a use, method, kit or medicament as hereinbefore described, wherein the combination therapy is administered at intervals of about 1 to 3 weeks.

[0031] In one embodiment, there is provided a use, method of use, kit, or medicament as disclosed herein above, which combines an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, wherein the B-cell proliferative disorder is selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin's lymphoma (HL). In a particular embodiment, the B-cell proliferative disorder is diffuse large B-cell lymphoma (DLBCL).

[0032] In a further aspect, the invention provides an anti-CD20 / anti-CD3 bispecific antibody, use, method, or medicament combining an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a CD19-targeted 4-1BB (CD137) agonist for use, wherein the anti-CD20 / anti-CD3 bispecific antibody, anti-CD19 / anti-CD28 bispecific antibody, and CD19-targeted 4-1BB (CD137) agonist are administered intravenously. In another aspect, the anti-CD20 / anti-CD3 bispecific antibody, anti-CD19 / anti-CD28 bispecific antibody, and CD19-targeted 4-1BB (CD137) agonist are administered subcutaneously. [Brief explanation of the drawings]

[0033] [Figure 1]Figures 1A to 1C are schematic diagrams of the specific CD19-4-1BBL antigen-binding molecule, specific anti-CD20 / anti-CD3 bispecific antibody, and specific anti-CD19 / anti-CD28 bispecific antibody used in the Examples. These molecules are described in detail in Examples 1, 2, and 3, respectively. Solid black dots represent knob-into-hole modifications. * denotes amino acid modifications in the CH1 and CL domains (so-called charge variants). Figure 1A shows an antigen-binding molecule comprising a monovalent CD19 4-1BBL trimer with modifications in the CH1 and CL domains adjacent to the 4-1BBL dimer and 4-1BBL monomer. This molecule is referred to herein as CD19-4-1BBL. Figure 1B shows an exemplary bispecific anti-CD20 / anti-CD3 antibody in a 2+1 format (referred to as CD20-TCB or glofitamab). Figure 1C shows an exemplary bispecific anti-CD19 / anti-CD28 antibody in a 1+1 CrossFab format, in which the VH and VL domains of the CD19 antigen-binding domain are swapped such that the VH domain is part of the light chain and the VL domain is part of the heavy chain. [Figure 2] Figures 2A-2D show that the combination of CD20-TCB with CD19-4-1BBL or CD19-CD28 enhances T cell activation, as measured by the release of selected cytokines, compared with CD20-TCB alone in patients with stage IVB splenectomy B-cell lymphoma. The figures show the release of the cytokines granzyme B (GzB, Figure 2A), IFNγ (Figure 2B), IL-8 (Figure 2C), and IL-2 (Figure 2D). [Figure 3] Figure 3 shows the design of an efficacy study to evaluate the effect of the triple combination of CD20-TCB with CD19-4-1BBL and CD19-CD28 in human OCI-Ly18 xenografts using humanized NSG mice. The design of different treatment groups A to G (10 mice each) shows various injections at different time points. [Figure 4]Figures 4A through 4G show the results of an efficacy study of OCI-Ly18 xenografts in humanized NSG mice. The figures plot tumor growth for individual mice across seven treatment groups on the y-axis. Figure 4A shows tumor growth for individual mice in the vehicle group; Figure 4B shows tumor growth for individual mice treated with CD20-TCB; Figure 4C shows tumor growth for mice treated with CD20-TCB and CD19-CD28; and Figure 4D shows tumor growth for mice treated with CD20-TCB and CD19-4-1BBL. Figure 4E shows tumor growth for a mouse initially treated with CD20-TCB and CD19-4-1BBL combination therapy and then switched to CD20-TCB and CD19-CD28 combination therapy on day 66. Figure 4F shows tumor growth in mice initially treated with the combination of CD20-TCB and CD19-CD28, followed (day 66) by the combination of CD20-TCB and CD19-4-1BBL. The group receiving the alternating treatment regimen, which began with CD19-CD28 for the first four cycles and continued with the CD19-4-1BBL combination until the end of the study, achieved complete tumor control for 120 days in all animals, whereas the group receiving the alternating treatment regimen, which began with CD19-4-1BBL for the first four cycles and continued with the CD19-CD28 combination, failed to completely inhibit tumor growth. Figure 4G shows tumor growth in mice receiving the triple combination of CD20-TCB, CD19-4-1BBL, and CD19-CD28. Interestingly, co-administration of CD20-TCB, CD19-CD28, and CD19-4-1BBL did not improve tumor growth suppression compared with treatment with CD20-TCB or CD19-4-1BBL alone. [Figure 5] Figure 5 shows a survival analysis using a tumor volume cutoff of 1500 m3 for the triple-drug efficacy study. The probability of survival (%) is plotted against days. Mice treated first with the combination of CD20-TCB and CD19-CD28 and second with the combination of CD20-TCB and CD19-4-1BBL had the highest survival rates (100%). [Figure 6]Figure 6 shows the study design for an efficacy study to evaluate the effect of the triple combination of CD20-TCB with CD19-4-1BBL and CD19-CD28 in human OCI-Ly18 xenografts using humanized BRGS-CD47 mice. The design of the different treatment groups, A to G (10 mice each), i.e., various injections at different time points, is shown. The combination treatment was initiated one week earlier than the first study using humanized NSG mice. [Figure 7]Figures 7A through 7G show the results of an efficacy study of OCI-Ly18 xenografts in humanized BRGS-CD47 mice. The figures plot tumor growth for individual mice across seven treatment groups on the y-axis. Figure 7A shows tumor growth for individual mice in the vehicle group; Figure 7B shows tumor growth for mice treated with CD20-TCB alone; Figure 7C shows tumor growth for mice treated with CD20-TCB and CD19-CD28; and Figure 7D shows tumor growth for mice treated with CD20-TCB and CD19-4-1BBL. Combination treatment began on day 27. Figure 7E shows tumor growth for mice initially treated with CD20-TCB and CD19-4-1BBL combination therapy and then switched to CD20-TCB and CD19-CD28 combination therapy on day 55. Figure 7F shows tumor growth in mice initially treated with the CD20-TCB and CD19-CD28 combination, followed (day 55) by the CD20-TCB and CD19-4-1BBL combination. Most animals receiving the alternating treatment regimen, which began with CD19-CD28 for the first four cycles and continued with the CD19-4-1BBL combination until the end of the study, achieved good tumor control over the 94-day period. However, the group receiving the alternating treatment regimen, which began with CD19-4-1BBL for the first four cycles and continued with the CD19-CD28 combination, failed to completely inhibit tumor growth. Figure 7G shows tumor growth in mice receiving the triple combination of CD20-TCB, CD19-4-1BBL, and CD19-CD28. The tumor control in this combination group was stronger than that in the CD20-TCB and CD19-4-1BBL combination group in our study, likely due to the earlier initiation of the combination treatment. The group that received an alternating treatment regimen starting with CD19-CD28 for the first four cycles followed by CD19-4-1BBL combination treatment until the end of the study (Figure 7F) achieved tumor control similar to that achieved with the 94-day combination treatment. [Figure 8]Figures 8A-8C show a comparison of the corresponding treatment regimens from both studies. The figures show the difference in tumor growth between mice treated first with the combination of CD20-TCB and CD19-4-1BBL and then, on day 55, with the combination of CD20-TCB and CD19-CD28 (Figure 8A), the difference in tumor growth between mice treated first with the combination of CD20-TCB and CD19-CD28 and then, on day 55, with the combination of CD20-TCB and CD19-4-1BBL (Figure 8B), and the difference in tumor growth between mice simultaneously treated with the triple combination of CD20-TCB, CD19-4-1BBL, and CD19-CD28 (Figure 8C), demonstrating that the combination treatment initiated one week earlier resulted in better tumor control than the combination treatment initiated later. DETAILED DESCRIPTION OF THE INVENTION

[0034] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. In interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa.

[0035] As used herein, the term "antigen-binding molecule" broadly refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules include antibodies, antibody fragments, and antigen-binding protein scaffolds. As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope. The exceptions, however, include variant antibodies, including, for example, naturally occurring variants or those that may arise during the manufacture of monoclonal antibody preparations, which are generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule consists of two antigen-binding sites, each specific for a different antigenic determinant. However, a bispecific antigen-binding molecule may contain an additional antigen-binding site that binds to an additional antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can simultaneously bind two antigenic determinants, particularly two antigenic determinants expressed on two different cells or the same cell. Therefore, the term "bispecific" according to the present disclosure may also include trispecific molecules, such as a bispecific molecule consisting of a CD28 antibody and two antigen-binding domains directed to two different target cell antigens.

[0036] As used herein, the term "antigen-binding domain that binds to a B cell surface antigen" or "site capable of specifically binding to a B cell surface antigen" refers to a polypeptide molecule that specifically binds to an antigenic determinant on the B cell surface. In one embodiment, the antigen-binding domain is capable of activating signaling through a target cell antigen. In particular embodiments, the antigen-binding domain is capable of directing an entity to which it is bound (e.g., a CD28 agonist) to a target site, for example, on a B cell. Antigen-binding domains capable of specifically binding to a B cell surface antigen include antibodies and fragments thereof as further defined herein. Furthermore, antigen-binding domains capable of specifically binding to a B cell surface antigen include binding domains based on scaffold antigen-binding proteins, as further defined herein, such as designed repeat proteins or designed repeat domains (see, e.g., WO 2002 / 020565).

[0037] As used herein, the term "valent" refers to the presence of a specific number of binding sites specific for one distinct antigenic determinant in an antigen-binding molecule specific for one distinct antigenic determinant. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, or six binding sites specific for a given antigenic determinant in an antigen-binding molecule, respectively. In certain embodiments of the present invention, a bispecific antigen-binding molecule according to the present invention can be monovalent with respect to a given antigenic determinant, i.e., have only one binding site for said antigenic determinant, or can be bivalent or tetravalent with respect to a given antigenic determinant, i.e., have two or four binding sites, respectively, for said antigenic determinant.

[0038] As used herein, the terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably to refer to antibodies having a structure substantially similar to that of a native antibody. "Native antibodies" refer to naturally occurring immunoglobulin molecules with a variety of structures. For example, native IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also known as heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a light chain constant domain (CL), also known as a light chain constant region. Antibody heavy chains are classified into one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM), some of which may be further classified into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Antibody light chains are classified into one of two types, called κ (kappa) and λ (lambda), based on the amino acid sequence of their constant domains.

[0039] An "antibody fragment" refers to a molecule other than a complete antibody that constitutes a portion of the complete antibody and binds to the same antigen as the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, crossFab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and single-domain antibodies. For a review of certain types of antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having increased in vivo half-lives, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two bivalent or bispecific antigen-binding sites. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 B1).Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies, as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0040] Papain digestion of a complete antibody produces two identical antigen-binding fragments called "Fab" fragments, each containing the variable domains of the heavy and light chains, the constant domain of the light chain, and the first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term "Fab fragment" refers to an antibody fragment containing the variable light (VL) domain and the constant domain of the light chain (CL), and a variable heavy (VH) domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment that contains a free thiol group at the cysteine ​​residue in the constant domain. Pepsin treatment produces an F(ab')2 fragment containing two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0041] A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which the variable or constant domains of the Fab heavy and light chains have been swapped (i.e., replaced with each other). That is, a crossover Fab molecule is composed of a peptide chain comprising a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1, N- to C-terminal), and a peptide chain comprising a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, N- to C-terminal). For clarity, in crossover Fab molecules in which the variable domains of the Fab light chain and the Fab heavy chain are swapped, the peptide chain comprising the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in crossover Fab molecules in which the constant domains of the Fab light chain and the Fab heavy chain are swapped, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0042] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its natural form, i.e., a Fab molecule comprising a heavy chain composed of a heavy chain variable and constant domain (VH-CH1, N- to C-terminal), and a light chain composed of a light chain variable and constant domain (VL-CL, N- to C-terminal).

[0043] A single-chain variable fragment (scFv) is a fusion protein in which the variable regions of an antibody's heavy chain (VH) and light chain (VL) are linked by a short linker peptide of about 10 to 25 amino acids. The linker usually contains a large amount of glycine to improve flexibility and serine or threonine to improve solubility. H N-terminus and V L The C-terminus of the fragment can be linked to the C-terminus of the fragment, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. Furthermore, antibody fragments consist of a single polypeptide chain characterized by the characteristics of a VH domain, i.e., the ability to assemble with a VL domain, or the characteristics of a VL domain, i.e., the ability to assemble with a VH domain and bind to a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0044] An "antigen-binding molecule that binds to the same epitope" as a reference molecule refers to an antigen-binding molecule that inhibits the binding of the reference molecule to its antigen by 50% or more in a competitive assay; conversely, the reference molecule inhibits the binding of the antigen-binding molecule to its antigen by 50% or more in a competitive assay.

[0045] The term "antigen-binding domain" refers to a portion of an antigen-binding molecule that comprises a region that specifically binds to and is complementary to a part or all of an antigen. When an antigen is large, an antigen-binding molecule may bind only to a specific portion of the antigen, which is called an epitope. An antigen-binding domain can be provided, for example, by one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0046] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide polymer (e.g., a stretch of consecutive amino acids or a configuration consisting of different regions of non-contiguous amino acids) to which an antigen-binding site binds, forming an antigen-binding site-antigen complex. Useful antigenic determinants can be found, for example, on tumor cell surfaces, virus-infected cell surfaces, other diseased cell surfaces, immune cell surfaces, serum free bodies, and / or the extracellular matrix (ECM). Proteins useful as antigens herein can be any naturally occurring protein from vertebrates, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. In certain embodiments, the antigen is a human protein. When referring to a particular protein herein, the term encompasses not only the "full-length," unprocessed protein, but also all forms of the protein that result from processing within a cell. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants.

[0047] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed with a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the binding of the antigen-binding molecule to the antigen, as measured, for example, by SPR. In certain embodiments, molecules that bind to an antigen have a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0048] "Affinity" or "binding affinity" refers to the strength of the sum of noncovalent interactions between a single binding site of a molecule, such as an antibody, and a binding partner, such as an antigen. Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed as a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities can involve different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0049] As used herein, "B cell surface antigen" refers to an antigenic determinant displayed on the surface of B lymphocytes, particularly malignant B lymphocytes (in this case, the antigen is also called "malignant B cell surface antigen"). Some B cell surface antigens are interesting from the perspective of immunotherapy of hematological malignancies. In one embodiment, the B cell surface antigen is selected from the group consisting of CD19, CD79b, CD20, CD22, and CD37.

[0050] The term "CD19" refers to the B lymphocyte antigen CD19, also known as B lymphocyte surface antigen B4 or T cell surface antigen Leu-12, and includes native CD19 from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human CD19 is set forth in Uniprot Accession No. P15391 (version 160, SEQ ID NO: 54). This term encompasses not only "full-length," unprocessed human CD19, but also any form of human CD19 resulting from intracellular processing, so long as the antibodies reported herein bind to it. CD19 is a structurally distinct cell surface receptor expressed on the surface of human B cells, including, but not limited to, pre-B cells, early developmental B cells (i.e., immature B cells), mature B cells that have undergone terminal differentiation into plasma cells, and malignant B cells. CD19 is expressed on most pre-B acute lymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B-cell chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, hairy cell leukemia, common acute lymphocytic leukemia, and some non-acute lymphoblastic leukemias. CD19 expression on plasma cells suggests that CD19 may also be expressed in differentiated B-cell neoplasms such as multiple myeloma. Therefore, the CD19 antigen is a target for immunotherapy in the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia.

[0051] "CD20" refers to the B lymphocyte antigen CD20, also known as B lymphocyte surface antigen B1 or leukocyte surface antigen Leu-16, and includes native forms of CD20 from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human CD20 is set forth in Uniprot Accession No. P11836 (version 149, SEQ ID NO: 55). CD20 is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kDa that is expressed in pre-B lymphocytes and mature B lymphocytes. The corresponding human gene is transmembrane 4 domain, subfamily A, member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this emerging protein family are characterized by common structural features and similar intron / exon splice boundaries and show unique expression patterns between hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that plays a role in B cell development and differentiation into plasma cells. The family is located at 11q12 and is part of a cluster of family members. Alternative splicing of this gene results in two transcript variants that encode the same protein. The term "CD20" encompasses not only "full-length," unprocessed CD20, but also the intracellularly processed version. The term also encompasses naturally occurring variants of CD20, such as splice variants or allelic variants.

[0052] The terms "anti-CD20 antibody" and "antibody that binds to CD20" refer to an antibody that can bind to CD20 with sufficient affinity so as to be useful as a CD20-targeting diagnostic and / or therapeutic agent. In one embodiment, the extent of binding of an anti-CD20 antibody to an unrelated, non-CD20 protein is less than about 10% of the binding of the antibody to CD20 as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD20 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10-8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 In certain embodiments, the anti-CD20 antibody binds to an epitope of CD20 that is conserved among CD20 of different species.

[0053] By "type II anti-CD20 antibody" is meant an anti-CD20 antibody having the binding characteristics and biological activity 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 A below. TIFF2025541593000001.tif72170

[0054] Examples of type II anti-CD20 antibodies include obinutuzumab (GA101), tositumumab (B1), humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody disclosed in WO 2005 / 044859), 11B8 IgG1 (disclosed in WO 2004 / 035607), AT80 IgG1, and the like.

[0055] In one embodiment, the type II anti-CD20 antibody has the heavy chain variable region sequence of SEQ ID NO: 36 (V H CD20) and the light chain variable region sequence of SEQ ID NO: 37 (V LIn another embodiment, the type II anti-CD20 antibody is modified to increase the proportion of nonfucosylated oligosaccharides in the Fc region compared to the unmodified 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 nonfucosylated. In a specific embodiment, the type II anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 56 and a light chain comprising the amino acid sequence of SEQ ID NO: 57. This antibody has been named GA101 or obinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453). The trade name is GAZYVA® or GAZYVARO®.

[0056] Examples of Type I anti-CD20 antibodies include, for example, rituximab, ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab, PRO131921, ublituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (disclosed in WO 2005 / 103081), 2F2 IgG1 (disclosed in WO 2004 / 035607 and WO 2005 / 103081), and 2H7 IgG1 (disclosed in WO 2004 / 056312).

[0057] The term "reduction" (and grammatical variations thereof, such as "reduce" or "reducing") refers to a decrease in the respective amount, as measured by an appropriate method known in the art, e.g., a decrease in the number of B cells or a decrease in cytokine release. For clarity, the term also includes a reduction to zero (or below the detection limit of an analytical method), i.e., complete abolition or elimination. Conversely, "increased" means that the respective quantity has increased.

[0058] As used herein, "T cell antigen" refers to an antigenic determinant displayed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes.

[0059] As used herein, "T cell activating therapeutic agent" refers to a therapeutic agent capable of inducing T cell activation in a subject, particularly a therapeutic agent designed to induce T cell activation in a subject. Examples of T cell activating therapeutic agents include bispecific antibodies that specifically bind to an activating T cell antigen, such as CD3, and a target cell antigen, such as CD20 or CD19. Further examples include chimeric antigen receptors (CARs) that include a T cell activation domain and an antigen-binding portion that specifically binds to a target cell antigen, such as CD20 or CD19.

[0060] As used herein, "activating T cell antigen" refers to an antigenic determinant expressed by T lymphocytes, particularly cytotoxic T lymphocytes, which can induce or enhance the activation of T cells through interaction with antigen-binding molecules. Specifically, the interaction between antigen-binding molecules and activating T cell antigens can induce the activation of T cells by triggering the signal transduction cascade of the T cell receptor complex. An exemplary activating T cell antigen is CD3.

[0061] The term "CD3," unless otherwise specified, refers to native CD3 from vertebrates, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD3 as well as CD3 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is set forth in UniProt (www.uniprot.org) Accession No. P07766 (version 144) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeqNP_000724.1. See also SEQ ID NO: 58. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank no. BAB71849.1. See also SEQ ID NO: 59.

[0062] The term "CD28" (cluster of differentiation 28, Tp44) refers to the CD28 protein from vertebrates, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. CD28 is expressed on T cells and provides costimulatory signals necessary for T cell activation and survival. T cell stimulation via CD28 in addition to the T cell receptor (TCR) can provide a potent signal for the production of various interleukins. CD28 is the receptor for the CD80 (B7.1) and CD86 (B7.2) proteins and is the only B7 receptor constitutively expressed on naive T cells. The amino acid sequence of human CD28 is set forth in UniProt (www.uniprot.org) accession number P10747 (SEQ ID NO: 60).

[0063] An "agonistic antibody" is an antibody that exhibits agonism toward a specific receptor. Generally, when an agonistic ligand (factor) binds to a receptor, the tertiary structure of the receptor protein changes, activating the receptor (if the receptor is a membrane protein, it usually transmits cell growth signals, etc.). If the receptor is a dimer-forming type, the agonistic antibody dimerizes the receptor at the appropriate distance and angle, exhibiting the same effect as the ligand. An appropriate anti-receptor antibody can mimic the dimerization of the receptor by the ligand and therefore can be an agonistic antibody.

[0064] "CD28 agonistic antibodies" or "CD28 conventional agonistic antibodies" are antibodies that mimic the CD28 natural ligands (CD80 or CD86) and enhance T cell activation in the presence of a T cell receptor signal ("signal 2"). Two signals are required for full T cell activation. Under physiological conditions, "signal 1" results from the interaction of the T cell receptor (TCR) molecule with a peptide / major histocompatibility complex (MHC) complex on an antigen-presenting cell (APC), and "signal 2" results from engagement with a costimulatory receptor, such as CD28. CD28 agonistic antigen-binding molecules can costimulate T cells (signal 2). Furthermore, when combined with molecules specific for the TCR complex, they can induce T cell proliferation and cytokine secretion. However, CD28 agonistic antigen-binding molecules cannot fully activate T cells without further TCR stimulation. However, there is a subclass of CD28-specific antigen-binding molecules known as CD28 superagonistic antigen-binding molecules. A "CD28 superagonistic antibody" is a CD28 antibody that can fully activate T cells without additional TCR stimulation. CD28 superagonistic antibodies can induce T cell proliferation and cytokine secretion without prior T cell activation (Signal 1). An example of a CD28 superagonistic antibody is TGN1412 (disclosed in WO 2006 / 050949).

[0065] The terms "anti-CD28 antibody," "anti-CD28," "CD28 antibody," and "antibody that specifically binds to CD28" refer to an antibody that can bind to CD28 with sufficient affinity so as to be useful as a diagnostic and / or therapeutic agent that targets CD28. In one embodiment, the extent of binding of an anti-CD28 antibody to an unrelated, non-CD28 protein is less than about 10% of the binding of the antibody to CD28 as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS). In certain embodiments, an antibody that binds to CD28 has a dissociation constant (K D ) is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -6M or less, e.g., 10 -6 M to 10 -13 M, e.g., 10 -8 M to 10 -10 M).

[0066] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain involved in binding of an antigen-binding molecule to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). One VH or VL domain may be sufficient to confer antigen-binding specificity. When used herein in connection with a variable region sequence, "Kabat numbering" refers to the numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0067] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), and is referred to herein as "Kabat numbering" or "Kabat numbering." Specifically, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) pp. 647-660) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pp. 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3), which is further clarified herein by referring to "numbering according to the Kabat EU index" in this case.

[0068] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and determines antigen-binding specificity, e.g., a "complementarity-determining region" ("CDR"). Generally, antibodies have six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include the following: (a) Hypervariable loops occurring at amino acid residues 26–32 (L1), 50–52 (L2), 91–96 (L3), 26–32 (H1), 53–55 (H2), and 96–101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901–917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0069] Unless otherwise specified, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that CDR designations can also be determined according to Chothia, McCallum, or other scientifically accepted nomenclature.

[0070] As used herein, the term "affinity maturation" in the context of an antigen-binding molecule (e.g., an antibody) refers to an antigen-binding molecule that is derived from a reference antigen-binding molecule, for example, by mutation, binds to the same antigen as the reference antibody, preferably binds to the same epitope, and has a higher affinity for the antigen than the affinity of the reference antigen-binding molecule for the antigen. Affinity maturation generally involves modifying one or more amino acid residues in one or more CDRs of the antigen-binding molecule. Typically, the affinity-matured antigen-binding molecule binds to the same epitope as the original reference antigen-binding molecule.

[0071] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0072] As used herein, an "acceptor human framework" refers to a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework that is "derived" from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0073] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0074] 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 some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0075] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the HVRs (e.g., CDRs) corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. A humanized antibody may optionally 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. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been additionally modified or altered from that of the original antibody to generate properties according to the invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0076] A "human" antibody is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody of non-human origin that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0077] The term "CH1 domain" refers to the portion of an antibody heavy chain polypeptide extending from approximately EU position 118 to EU position 215 (EU numbering system according to Kabat). In one embodiment, the CH1 domain has the amino acid sequence ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 61). Typically, a segment having the amino acid sequence EPKSC (SEQ ID NO: 62) connects the CH1 domain to the hinge region.

[0078] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 domains in a wild-type antibody heavy chain, e.g., from about position 216 to about position 230 according to the EU numbering system of Kabat, or from about position 226 to about position 230 according to the EU numbering system of Kabat. Hinge regions of other IgG subclasses can be determined by aligning the hinge region cysteine ​​residues with the IgG1 subclass sequence. Hinge regions are usually dimeric molecules composed of two polypeptides with identical amino acid sequences. The hinge region generally contains up to 25 amino acid residues and is flexible, allowing the associated target binding sites to move independently. The hinge region can be subdivided into three domains: upper, middle, and lower (see, e.g., Roux, et al., J. Immunol. 161 (1998) 4083).

[0079] As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. An IgG Fc region contains the IgG CH2 domain and the IgG CH3 domain.

[0080] The "CH2 domain" of a human IgG Fc region typically extends from amino acid residue EU231 to amino acid residue EU340 (EU numbering system according to Kabat). In one embodiment, the CH2 domain has the amino acid sequence APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 63). The CH2 domain is unique in that it is not tightly paired with other domains. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native Fc region. It has been speculated that the carbohydrate chains may act as a domain-to-domain pairing and help stabilize the CH2 domains. Burton, Mol. Immunol. 22 (1985) 161-206. In one embodiment, carbohydrate chains are attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain.

[0081] "CH3 domain" refers to the portion of an antibody heavy chain polypeptide that comprises the stretch of C-terminal residues of the CH2 domain in the Fc region and extends from approximately EU position 341 to EU position 446 (EU numbering system according to Kabat). In one embodiment, the CH3 domain has the amino acid sequence GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO: 64). The CH3 region herein may be a native-sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "protuberance" ("knob") in one chain and a corresponding introduced "cavity" ("hole") in the other chain (see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains, as described herein. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may be present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0082] The "knob-into-hole" technique has been described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a "protuberance" ("knob") at the interface of a first polypeptide and a corresponding "cavity" ("hole") at the interface of a second polypeptide, positioning the protuberance in the cavity to promote heterodimer formation and prevent homodimer formation. The protuberance is constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A complementary depression of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). The protrusion and depression can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In a more specific embodiment, the Fc domain subunit that constitutes the knob modification further comprises the amino acid substitution S354C, and the Fc domain subunit that constitutes the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues forms a disulfide bridge between the two subunits of the Fc region, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0083] The term "region equivalent to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as variants with modifications that result in substitutions, additions, or deletions but do not substantially reduce the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantially losing biological function. Such variants can be selected according to general rules known in the art to minimize the effect on activity (see, for example, Bowie, JU et al., Science 247:1306-10 (1990)).

[0084] The term "wild-type Fc domain" refers to an amino acid sequence identical to that of a naturally occurring Fc domain. Wild-type human Fc domains include native human IgG1 Fc regions (non-A and A allotypes), native human IgG2 Fc regions, native human IgG3 Fc regions, and native human IgG4 Fc regions, as well as naturally occurring variants thereof. The human IgG1 Fc region is set forth in SEQ ID NO: 65.

[0085] The term "variant (human) Fc domain" refers to an amino acid sequence that differs from a "wild-type" (human) Fc domain amino acid sequence by at least one "amino acid mutation." In one embodiment, the variant Fc region has at least one amino acid mutation compared to a native Fc region, e.g., about 1 to about 10 amino acid mutations, and in one embodiment, about 1 to about 5 amino acid mutations in a native Fc region. In one embodiment, the (variant) Fc region has at least about 95% homology to the wild-type Fc region.

[0086] The term "effector function" refers to the biological activities attributable to the Fc region of an antibody and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0087] Effector functions dependent on Fc receptor binding are mediated by the interaction of the Fc region of antibodies with specialized cell surface receptors on hematopoietic cells, known as Fc receptors (FcRs). Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the elimination of antibody-coated pathogens through phagocytosis of immune complexes and the lysis of corresponding antibody-coated red blood cells and various other cellular targets (e.g., tumor cells) via antibody-dependent cell-mediated cytotoxicity (ADCC) (see Van de Winkel, J. G. and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes, and the Fc receptors for IgG antibodies are called FcγRs. Fc receptor binding is described, for example, in Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ, et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE, et al., Ann. Hematol. 76 (1998) 231-248.

[0088] Cross-linking of receptors (FcγRs) to the Fc region of IgG antibodies not only regulates immune complex clearance and antibody production, but also elicits a wide variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and the release of inflammatory mediators. In humans, FcγRs are divided into three classes: FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils. Modification of the Fc region of IgG at one or more of the amino acid residues E233-G236, P238, D265, N297, A327, and P329 (numbering according to the EU index of Kabat) reduces FcγRI binding. Substitution of IgG2 residues 233-236 with IgG1 and IgG4 reduced FcγRI binding by 103-fold and abolished the human monocyte response to antibody-sensitized erythrocytes (Armour, KL, et al., Eur. J. Immunol. 29 (1999) 2613-2624). FcγRII (CD32) binds complexed IgG with intermediate to low affinity and is widely expressed. This receptor is divided into two subtypes, FcγRIIA and FcγRIIB. FcγRIIA is present on many cells involved in killing (e.g., macrophages, monocytes, and neutrophils) and is thought to activate the killing process. FcγRIIB appears to be involved in inhibitory processes and is present on B cells, macrophages, mast cells, and eosinophils. In B cells, it appears to function to suppress further immunoglobulin production and isotype switching, for example, to the IgE class. In macrophages, FcγRIIB inhibits phagocytosis via FcγRIIA. In eosinophils and mast cells, the B form is thought to help suppress activation of these cells by binding IgE to a different receptor. Decreased binding to FcγRIIA is observed, for example, in antibodies comprising an IgG Fc region having a mutation in at least one of amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (Kabat EU index numbering). FcγRIII (CD16) binds IgG with intermediate to low affinity and exists in two forms: FcγRIIIA, which is present on NK cells, macrophages, eosinophils, some monocytes, and T cells, and mediates ADCC; FcγRIIIB, which is highly expressed on neutrophils. Reduced binding to FcγRIIIA is observed, for example, in antibodies containing an IgG Fc region with mutations at one or more of the following amino acid residues: E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (Kabat EU index numbering).

[0089] Mapping of the binding site in human IgG1 for Fc receptors, the mutation sites described above, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al. J. Biol. Chem. 276 (2001) 6591-6604.

[0090] The term "ADCC" or "antibody-dependent cellular cytotoxicity" refers to an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which an antibody or its derivative containing an Fc region specifically binds, typically via a protein portion at the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as either a decrease in the number of target cells lysed in a given time period at a given concentration of antibody in the medium surrounding the target cells by the ADCC mechanism defined above, and / or an increase in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time period by the ADCC mechanism. The reduced ADCC is compared to the ADCC mediated by the same antibody produced by the same type of host cell and using the same standard production, purification, formulation, and storage methods (known to those skilled in the art), but without genetic engineering. For example, the reduced ADCC mediated by an antibody with an ADCC-reducing amino acid substitution in the Fc domain is compared to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT Publication No. WO 2006 / 082515 or PCT Publication No. WO 2012 / 130831). For example, the ability of an antibody to induce the early steps of mediating ADCC is assessed by measuring binding to Fcγ receptor-expressing cells, such as cells recombinantly expressing FcγRI and / or FcγRIIA or NK cells (which inherently express FcγRIIIA). In particular, binding to FcγR on NK cells is measured.

[0091] An "activating Fc receptor" is an Fc receptor that, upon binding to the Fc region of an antibody, initiates a signaling event that stimulates the receptor-bearing cell to exert effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt Accession No. P08637, version 141).

[0092] The term "effector function" refers to the biological activities attributable to the Fc region of an antibody and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0093] 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, through which they bind effector moieties, e.g., cytokines, and / or the Fc region of antibodies, and contribute to the destruction of target cells, e.g., tumor cells. Effector cells mediate, for example, cytotoxicity and phagocytosis. Effector cells include CD8 + Cytotoxic T cells, CD4 + These include, but are not limited to, effector T cells such as helper T cells, γδ T cells, NK cells, lymphocyte-activated killer (LAK) cells, and macrophages / monocytes.

[0094] "Ectodomain" refers to the domain of a membrane protein that extends into the extracellular space (i.e., the space outside the target cell). The ectodomain is typically the portion that initiates contact with the surface of the protein and leads to signal transduction. Thus, the ectodomain of 4-1BBL, as defined herein, refers to the portion of 4-1BBL that extends outside the cell (the extracellular domain), but also includes shorter portions or fragments thereof that are responsible for trimerization and binding to the corresponding receptor, 4-1BB. Thus, the term "ectodomain of 4-1BBL or a fragment thereof" refers to the extracellular domain of 4-1BBL that forms the extracellular domain, or the portion capable of binding to the receptor (the receptor-binding domain).

[0095] "4-1BBL," or "4-1BB ligand," or "CD137L," is a member of the costimulatory TNF ligand family and can costimulate T cell proliferation and cytokine production. Costimulatory TNF family ligands costimulate TCR signaling by interacting with their corresponding TNF receptors, which recruit TNFR-associated factors (TRAFs) and initiate a signaling cascade leading to T cell activation. 4-1BBL is a type II transmembrane protein. Full-length 4-1BBL, having the amino acid sequence of SEQ ID NO: 66, has been described to form trimers on the cell surface. Trimerization is enabled by a specific motif in the ectodomain of 4-1BBL. This motif is referred to herein as the "trimerization region." Amino acids 50 to 254 of the human 4-1BBL sequence (SEQ ID NO: 9) form the extracellular domain of 4-1BBL, but fragments of this domain can also form trimers. In specific embodiments of the present invention, the term "4-1BBL ectodomain or a fragment thereof" refers to a polypeptide having an amino acid sequence selected from SEQ ID NO: 4 (amino acids 52 to 254 of human 4-1BBL), SEQ ID NO: 1 (amino acids 71 ​​to 254 of human 4-1BBL), SEQ ID NO: 3 (amino acids 80 to 254 of human 4-1BBL), SEQ ID NO: 2 (amino acids 85 to 254 of human 4-1BBL), SEQ ID NO: 5 (amino acids 71 ​​to 248 of human 4-1BBL), SEQ ID NO: 6 (amino acids 85 to 248 of human 4-1BBL), SEQ ID NO: 7 (amino acids 80 to 248 of human 4-1BBL), SEQ ID NO: 8 (amino acids 52 to 248 of human 4-1BBL), and SEQ ID NO: 9 (amino acids 50 to 254 of human 4-1BBL). However, other fragments of the ectodomain capable of trimerization are also encompassed herein.

[0096] As used herein, the term "4-1BB" or "CD137" refers to naturally occurring 4-1BB from vertebrates, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," unprocessed 4-1BB and 4-1BB that results from intracellular processing. The term also encompasses naturally occurring variants of 4-1BB, such as splice variants or allelic variants. The amino acid sequence of an exemplary human 4-1BB is set forth in SEQ ID NO: 67 (Uniprot Accession No. Q07011).

[0097] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. A suitable non-immunogenic linker peptide is, for example, (G4S)2 (SEQ ID NO: 68).

[0098] As used within this application, the term "amino acid" refers to the group of naturally occurring carboxy α-amino acids, including alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0099] By "fused" or "linked" is meant that the components (eg, a polypeptide and a 4-1BBL ectodomain) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0100] "Percent (%) amino acid sequence identity" to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of those in the art, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, SAWI, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including the algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes of this specification, percent amino acid sequence identity values ​​are calculated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, along with user documentation, has been submitted to the U.S. Copyright Office (Washington, DC 20559) and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program must be compiled to be used on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and should not be altered.In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or to a given amino acid sequence B (which can alternatively be stated as "given amino acid sequence A has a certain % amino acid sequence identity to, with, or to a given amino acid sequence B, or is composed of a given amino acid sequence A") is calculated as follows: X / Y×100

[0101] where X is the number of amino acid residues that the sequence alignment program ALIGN-2 scores as identical matches in the alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if 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 and B will not be equal to the % amino acid sequence identity of B and A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0102] In certain embodiments, amino acid sequence variants of the antigen-binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antigen-binding molecule. Amino acid sequence variants of antigen-binding molecules can be produced by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletion, insertion, and / or substitution of residues within the antibody amino acid sequence. Any combination of deletion, insertion, and substitution can be used to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding. Sites of interest for substitutional mutagenesis include HVRs and framework regions (FRs). Conservative substitutions are listed in Table C under the heading "Preferred Substitutions" and are further described below with reference to amino acid side-chain classes (1) to (6). Amino acid substitutions can be introduced into a molecule of interest, and the products can be screened for desired activities, such as retained or improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0103] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism by which immune effector cells lyse antibody-coated target cells. Target cells are cells to which an antibody or fragment thereof containing an Fc region specifically binds, typically via the protein portion at the N-terminus of the Fc region. As used herein, the term "increase / decrease in ADCC" is defined as either an increase / decrease in the number of target cells lysed in a given time period by the ADCC mechanism defined above at a given concentration of antibody in the medium surrounding the target cells, and / or an increase / decrease in the antibody concentration in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time period by the ADCC mechanism. The increase / decrease in ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell and using the same standard production, purification, formulation, and storage methods (known to those skilled in the art), but without genetic engineering. For example, the increased ADCC mediated by an antibody produced by a host cell engineered by the methods described herein to have an altered glycosylation pattern (e.g., to express glycosyltransferase, GnTIII, or other glycosyltransferase) is compared to the ADCC mediated by the same antibody produced by a non-engineered host cell of the same type.

[0104] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581). Certain antibody variants with improved or reduced binding to FcRs have been described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).) In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0105] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcγR binding, e.g., substitutions at positions 234 and 235 (EU numbering of residues) in the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in the Fc region derived from a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A, and P329G (LALA-PG) in the Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012 / 130831). In another embodiment, the substitutions are L234A, L235A, and D265A (LALA-DA) in the Fc region derived from a human IgG1 Fc region.

[0106] In some embodiments, modifications to the Fc region are made that alter (i.e., either improve or decrease) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0107] Antibodies with extended half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (e.g., U.S. Pat. No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0108] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253, and / or 310, and / or 435 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A in the Fc region derived from a human IgG1 Fc region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0109] In another embodiment, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 310, and / or 433, and / or 436 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 310, 433, and 436. In one embodiment, the substitutions are H310A, H433A, and Y436A in the Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2014 / 177460).

[0110] An "effective amount" of a drug refers to the amount needed to produce a physiological change in the cells or tissue to which it is administered.

[0111] A "therapeutically effective amount" of a drug, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of a drug, for example, eliminates, reduces, delays, minimizes, or prevents the side effects of a disease.

[0112] 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 non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0113] The term "pharmaceutical composition" refers to a formulation that is in a form that is effective for the biological activity of the active ingredient contained therein and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0114] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, preservatives, etc.

[0115] "Package insert" means the instructions customarily accompanying the packaging of a marketed therapeutic drug product that contain information regarding the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings concerning the use of such therapeutic drug product.

[0116] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention that seeks to alter the natural course of the treated individual and can be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, improving or palliating the disease state, achieving remission or improving prognosis, etc. In some embodiments, the molecules of the invention are used to delay the onset of disease or to slow the progression of disease.

[0117] As used herein, the term "cancer" refers to proliferative diseases such as lymphoma, lymphocytic leukemia, and melanoma.

[0118] "B cell proliferative disorder" refers to a disease in which a patient's B cell count is increased compared to the B cell count of a healthy individual, particularly a disease in which an increased B cell count is the cause or characteristic of the disease. "CD20-positive B cell proliferative disorder" refers to a B cell proliferative disorder in which B cells, particularly malignant B cells (in addition to normal B cells), express CD20. Exemplary B cell proliferative disorders include non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and certain types of multiple myeloma (MM) and Hodgkin's lymphoma (HL). In particular, the B-cell proliferative disorder is non-Hodgkin's lymphoma (NHL or diffuse large B-cell lymphoma (DLBCL). In particular embodiments, the B-cell proliferative disorder is diffuse large B-cell lymphoma (DLBCL).

[0119] The present invention relates to the use of an anti-CD20 / anti-CD3 bispecific antibody in combination with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeting 4-1BB (CD137) agonist in combination therapy for the treatment of B-cell proliferative disorders. A scheduling study of glofitamab and CD19-CD28 in humanized NSG mice suggested that a safe and potent treatment regimen was possible by pre-treating Gazyva with glofitamab and the anti-CD19 / anti-CD28 bispecific antibody CD19-CD28, administered 3 days apart in the first treatment cycle. In huNSG mice harboring the difficult-to-treat disseminated WSU-DLCL2 DLBCL tumor model, combination treatment with glofitamab and CD19-CD28 resulted in tumor-free animals compared with either monotherapy group. In vivo studies of the mechanism of action revealed that glofitamab-mediated T cell infiltration in tumor tissue was strongly enhanced in both CD4+ and CD8+ T cell subsets without demonstrating an increase in regulatory T cell activity. In a subcutaneous OCI-Ly18 DLBCL model, second-line treatment with CD19-CD28 extended the duration of glofitamab response and delayed tumor recurrence in vivo. Interestingly, alternating CD19-CD28 with a CD19-targeting 4-1BB (CD137) agonist (CD19-4-1BBL) completely prevented tumor recurrence during glofitamab treatment for over 120 days when CD19-CD28 was administered in the first treatment cycle followed by CD19-4-1BBL in subsequent cycles. Finally, CD19-CD28 enhanced glofitamab-mediated cytokine secretion and T cell activation in DLBCL patient samples ex vivo, demonstrating activity against T cells from healthy donors as well as patient-derived T cells. Collectively, preclinical data provide strong evidence that combining CD19-CD28 with CD20TCB in patients with relapsed / relapsed non-Hodgkin's lymphoma can further deepen and prolong therapeutic response, although the optimal schedule is alternating treatment with a CD20TCB (glofitamab) and a CD19-targeting 4-1BB (CD137) agonist (CD19-4-1BBL).

[0120] Exemplary anti-CD20 / anti-CD3 bispecific antibodies for use in the present invention As used herein, an anti-CD20 / anti-CD3 bispecific antibody is a bispecific antibody that comprises a first antigen-binding domain that binds to CD3 and a second antigen-binding domain that binds to CD20.

[0121] Thus, the anti-CD20 / anti-CD3 bispecific antibodies used herein comprise a heavy chain variable region (V H CD3) and the light chain variable region (V L a first antigen-binding domain comprising a heavy chain variable region (V H CD20) and the light chain variable region (V L It contains a second antigen-binding domain comprising a nucleotide sequence encoding the nucleotide sequence of interest (e.g., nucleotide sequence of interest, e ...

[0122] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody for use in combination comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 22, the CDR-H2 sequence of SEQ ID NO: 23, and the CDR-H3 sequence of SEQ ID NO: 24. H and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 25, the CDR-L2 sequence of SEQ ID NO: 26, and the CDR-L3 sequence of SEQ ID NO: 27. L More particularly, the anti-CD20 / anti-CD3 bispecific comprises a first antigen-binding domain comprising a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28. H CD3) and / or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 29 L In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a first antigen-binding domain consisting of a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 28. H CD3) and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 29 L CD3).

[0123] 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 a human IgG class antibody, particularly a human IgG1 class antibody. In one embodiment, the antibody that specifically binds to CD3 is an antibody fragment, particularly a Fab molecule or scFv molecule, more particularly a Fab molecule. In a particular embodiment, the antibody that specifically binds to CD3 is a crossover Fab molecule in which the variable or constant domains of the Fab heavy and light chains have been exchanged (i.e., replaced with each other). In one embodiment, the antibody that specifically binds to CD3 is a humanized antibody.

[0124] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 30, the CDR-H2 sequence of SEQ ID NO: 31, and the CDR-H3 sequence of SEQ ID NO: 32. H CD20), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 33, the CDR-L2 sequence of SEQ ID NO: 34, and the CDR-L3 sequence of SEQ ID NO: 35 L More particularly, the anti-CD20 / anti-CD3 bispecific comprises a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 36. H CD20), and / or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 37. L In a further embodiment, the anti-CD20 / anti-CD3 bispecific comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 36. H CD20) and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 37 L and a second antigen-binding domain comprising a nucleotide sequence identical to that of the nucleotide sequence of the target antigen (e.g., nucleotide sequence 1000-10 ...

[0125] In another particular embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a third antigen-binding domain that binds to CD20. In particular, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 30, the CDR-H2 sequence of SEQ ID NO: 31, and the CDR-H3 sequence of SEQ ID NO: 32. H CD20); and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 33, the CDR-L2 sequence of SEQ ID NO: 34, and the CDR-L3 sequence of SEQ ID NO: 35 L More particularly, the anti-CD20 / anti-CD3 bispecific comprises a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 36. H CD20), and / or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 37. L In a further embodiment, the anti-CD20 / anti-CD3 bispecific comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 36. H CD20) and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 37 L and a third antigen-binding domain comprising a nucleotide sequence identical to that of the nucleotide sequence of the target antigen (e.g., nucleotide sequence 1000-10 ...

[0126] In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a bispecific antibody, wherein the first antigen-binding domain is a Cross-Fab molecule in which the variable or constant domains of the Fab heavy and light chains have been exchanged, and the second and third antigen-binding domains (if present) are conventional Fab molecules.

[0127] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a bispecific antibody, wherein (i) the second antigen-binding domain 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 domain, the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain, the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0128] Fab molecules can be fused to the Fc domain or to each other directly or via a peptide linker comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, the (G4S)2 peptide linker (SEQ ID NO: 68). Furthermore, the linker can include (a portion of) an immunoglobulin hinge region. In particular, when a Fab molecule is fused to the N-terminus of an Fc domain subunit, it may be fused via the immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

[0129] In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises an Fc domain comprising one or more amino acid substitutions that reduce Fc receptor binding and / or effector function. In particular, the anti-CD20 / anti-CD3 bispecific antibody comprises an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0130] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody comprises a first polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 38, a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39, a third polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40, and fourth and fifth polypeptides comprising amino acid sequences at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41. In further specific embodiments, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 38, the polypeptide sequence of SEQ ID NO: 39, the polypeptide sequence of SEQ ID NO: 40, and the polypeptide sequence of SEQ ID NO: 41 (CD20 TCB) twice. In a particular embodiment, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

[0131] Glofitamab (WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Recommended INN: List 83, 2020, vol. 34, no. 1, p. 39; Proposed INN: List 121 WHO Drug Information, Vol. 33, No. 2, 2019, page 276; also known as CD20-TCB, RO7082859, or RG6026; CAS#: 2229047-91-8) is a full-length T cell-binding bispecific antibody with a 2:1 molecular configuration that bivalently binds to CD20 on B cells and monovalently binds to CD3, specifically the CD3 epsilon chain (CD3e), on T cells. The CD3-binding domain is fused head-to-tail to one of the CD20-binding domains via a flexible linker. This structure allows glofitamab to exhibit superior in vitro potency compared to other CD20-CD3 bispecific antibodies in a 1:1 configuration and to produce profound antitumor effects in preclinical DLBCL models. The bivalency of CD20 maintains this potency even in the presence of competing anti-CD20 antibodies, providing the opportunity for pretreatment or cotreatment with these agents. Glofitamab contains an engineered heterodimeric Fc region that completely abolishes FcgR and C1q binding. By simultaneously binding to CD3e in the T cell receptor (TCR) complex on human CD20-expressing tumor cells and T cells, glofitamab induces tumor cell lysis, as well as T cell activation, proliferation, and cytokine release. Glofitamab-induced B cell lysis is CD20-specific and does not occur when CD20 is not expressed or when T cells do not simultaneously bind (crosslink) CD20-expressing cells. In addition to killing, T cells undergo activation by CD3 crosslinking, as detected by an increase in T cell activation markers (CD25, CD69), cytokine release (IFNγ, TNFα, IL-2, IL-6, IL-10), cytotoxic granule release (Granzyme B), and T cell proliferation. A schematic diagram of the molecular structure of glofitamab is shown in Figure 1B.

[0132] Other bispecific antibodies, among others, are described in PCT Publication Nos. WO 2016 / 020309 or WO 2015 / 095392. In a further embodiment, the antibody is mosunetuzumab.

[0133] In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody may also comprise a bispecific T cell engager (BiTE®). In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody is XmAb® 13676. In another embodiment, the bispecific antibody is REGN1979. In another embodiment, the bispecific antibody is FBTA05 (Lymphomun).

[0134] Exemplary 4-1BB Agonists for Use in the Present Invention In particular, the CD19-targeted 4-1BB (CD137) agonist used in combination with an anti-CD20 / anti-CD3 bispecific antibody is a molecule containing 4-1BBL. In particular, the 4-1BB agonist used in the present invention comprises the three ectodomains of 4-1BBL or a fragment thereof.

[0135] In a particular embodiment, the CD19-targeted 4-1BB (CD137) agonist is a molecule comprising the three ectodomains of 4-1BBL or a fragment thereof, wherein the ectodomain of 4-1BBL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, in particular the amino acid sequence of SEQ ID NO:5.

[0136] It has been shown that 4-1BB agonists having at least one antigen-binding domain capable of specifically binding to CD19 are not internalized by B cells via CD19 and therefore do not lose their ability to interact with the tumor microenvironment. In one embodiment, a 4-1BB agonist is provided that is not internalized by B cells and retains its activity.

[0137] In another embodiment, the CD19-targeted 4-1BB (CD137) agonist is an antigen-binding molecule consisting of the three ectodomains of 4-1BBL or a fragment thereof and at least one portion capable of specifically binding to CD19, wherein the antigen-binding domain capable of specifically binding to CD19 is cynomolgus cross-reactive, i.e., the antigen-binding domain capable of specifically binding to CD19 specifically binds to human and cynomolgus CD19.

[0138] In a further embodiment, the CD19-targeted 4-1BB (CD137) agonist is an antigen-binding molecule comprising the three ectodomains of 4-1BBL or a fragment thereof and at least one portion capable of specifically binding to CD19, wherein the antigen-binding domain capable of specifically binding to CD19 comprises a heavy chain variable region (VH1) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12. H (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15. L CD19).

[0139] In a further embodiment, the CD19-targeted 4-1BB (CD137) agonist is an antigen-binding molecule comprising the three ectodomains of 4-1BBL or a fragment thereof and at least one antigen-binding domain capable of specifically binding to CD19, wherein the antigen-binding domain capable of specifically binding to CD19 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 16. H CD19) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 17 L CD19).

[0140] In another embodiment, the CD19-targeted 4-1BB (CD137) agonist is an antigen-binding molecule further comprising an Fc domain comprising a first subunit and a second subunit capable of stable association. In one embodiment, the CD19-targeted 4-1BB (CD137) agonist is an antigen-binding molecule comprising an IgG Fc domain, specifically an IgG1 Fc domain or an IgG4 Fc domain. In particular, the CD19-targeted 4-1BB (CD137) agonist is an antigen-binding molecule comprising an Fc domain containing one or more amino acid substitutions that reduce Fc receptor binding and / or effector function. In a specific embodiment, the CD19-targeted 4-1BB (CD137) agonist is an antigen-binding molecule comprising an IgG1 Fc domain containing the amino acid substitutions L234A, L235A, and P329G.

[0141] In one embodiment, the CD19-targeted 4-1BB (CD137) agonist is an antigen-binding molecule comprising: (a) at least one antigen-binding domain capable of specifically binding to CD19; (b) a first and a second polypeptide linked to each other by a disulfide bond, Here, the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof linked to each other by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL or fragments thereof.

[0142] In another embodiment, the CD19-targeted 4-1BB agonist is (a) a first polypeptide comprising: (a1) a first ectodomain of 4-1BBL or a fragment thereof, the C-terminus of which is fused to the N-terminus of a second ectodomain of 4-1BBL or a fragment thereof; (a2) a second ectodomain of 4-1BBL or a fragment thereof, the C-terminus of which is fused to the N-terminus of a CL domain; (a3) ​​the CL domain, the C-terminus of which is fused to the N-terminus of one of the subunits of an Fc domain (e.g., a first subunit); and (a4) one of the subunits of the Fc domain (e.g., the first subunit); (b) a second polypeptide comprising: (b1) a third ectodomain of 4-1BBL or a fragment thereof, the C-terminus of which is fused to the N-terminus of a CH1 domain; and (b2) the CH1 domain; (c) a third polypeptide comprising (c1) a heavy chain of a Fab molecule that binds to CD19, the C-terminus of which is fused to the N-terminus of another one of the subunits of the Fc domain (e.g., a second subunit); and (c2) another one of the subunits of the Fc domain (e.g., a second subunit); (d) a fourth polypeptide comprising the light chain of the Fab molecule that binds to CD19; and Includes:

[0143] In certain embodiments, the CD19-targeted 4-1BB agonist is a first polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18; a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19; a third polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20; or a fourth polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21. More particularly, the CD19-targeted 4-1BB agonist comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 18, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 19, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 20, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the CD19-targeted 4-1BB agonist is CD19-targeted 4-1BB ligand (CD19-4-1BBL) or englumafusp alfa.

[0144] Englumafusp alfa (WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Recommended INN:List89, 2023, vol. 37, no. 1, p. 97, also known as CD19-4-1BBL, RO7227166, RG6076, CAS#: 2417199-08-5) is a 4-1BB ligand (CD19-4-1BBL) targeting CD19. The basic mechanism of action of this molecule is to crosslink 4-1BB-positive activated effector cells with CD19-positive tumor targets, thereby enhancing the effector function of tumor-infiltrating T cells or NK cells upon activation by tumor-targeting T cell bispecific antibodies (TCBs) or antibody-dependent cellular cytotoxicity (ADCC), respectively. Crosslinking of 4-1BB results in immune cell costimulation, i.e., enhanced efficacy or function (e.g., proliferation, interferon-γ and IL2 production, protecting cells from death (e.g., upregulation of anti-apoptotic pathway genes), promoting the development of immunological memory, and generating durable immune responses. By selectively promoting immune responses in the CD19-expressing tumor microenvironment, the risk of off-target immune responses is limited. The safety of englumafusp alfa is further enhanced by abolishing the interaction of the IgG1 antibody moiety with Fcγ receptors (FcγR) and C1q complexes, thereby suppressing FcγR-mediated coactivation of innate immune effector cells such as NK cells and macrophages / monocytes, thereby preventing the induction of ADCC and antibody-dependent cellular phagocytosis (ADCP). A schematic diagram of the molecular structure of englumafusp alfa is shown in Figure 1A.

[0145] In a further embodiment, the 4-1BB agonist is an anti-CD19 / anti-4-1BB bispecific antibody.

[0146] Exemplary Anti-CD28 Bispecific Antibodies for Use in the Invention As used herein, a bispecific anti-CD28 antibody is a bispecific agonistic CD28 antibody comprising an antigen-binding domain capable of specifically binding to CD28, an antigen-binding domain capable of specifically binding to a B cell surface antigen, and an Fc domain consisting of first and second subunits capable of stable association and containing one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to Fc receptors and / or effector functions. In one embodiment, the bispecific CD28 agonistic antibody described herein is characterized by monovalent binding to CD28. In a further embodiment, the bispecific CD28 agonistic antibody described herein is characterized by monovalent binding to a B cell surface antigen. In particular, the B cell surface antigen is CD19.

[0147] In one embodiment, a bispecific CD28 agonist antibody as defined herein above is provided, wherein the Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain. In certain embodiments, the Fc domain comprising a first subunit and a second subunit capable of stable association is an IgG1 Fc domain. The Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or reduce or eliminate effector function. In one embodiment, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one embodiment, the Fc domain is an Fc domain of the human IgG1 subclass and comprises amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0148] In one embodiment, the anti-CD19 / anti-CD28 bispecific antibody used herein comprises a heavy chain variable region (V H CD28) and the light chain variable region (V L a first antigen-binding domain comprising a heavy chain variable region (V H CD19) and the light chain variable region (V L It contains a second antigen-binding domain comprising a nucleotide sequence similar to that of the nucleotide sequence of interest (e.g., nucleotide sequence 10 ...

[0149] In a further embodiment, the anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 42, the CDR-H2 sequence of SEQ ID NO: 43 and the CDR-H3 sequence of SEQ ID NO: 44. H CD28), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 45, the CDR-L2 sequence of SEQ ID NO: 46, and the CDR-L3 sequence of SEQ ID NO: 47 L In a further embodiment, the anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (V) consisting of an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 48. H CD28), and / or a light chain variable region (V) consisting of an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 49. L In particular, the anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (V) consisting of the amino acid sequence of SEQ ID NO: 48. H CD28) and / or a light chain variable region (V) consisting of the amino acid sequence of SEQ ID NO: 49 L It contains a first antigen-binding domain consisting of the nucleotide sequence of the target antigen (e.g., CD28).

[0150] In one embodiment, the anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 10, the CDR-H2 sequence of SEQ ID NO: 11, and the CDR-H3 sequence of SEQ ID NO: 12. H CD19); and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 13, the CDR-L2 sequence of SEQ ID NO: 14, and the CDR-L3 sequence of SEQ ID NO: 15 L In one embodiment, the anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16. HCD19) and / or a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17. L In one embodiment, the anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 16. H CD19) and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 17 L and a second antigen-binding domain comprising a nucleotide sequence encoding the nucleotide sequence of interest (e.g., nucleotide sequence 10 ...

[0151] In certain embodiments, the anti-CD19 / anti-CD28 bispecific antibody is a first polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:50; a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:51; a third polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:52; and a fourth polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:53. More specifically, the anti-CD19 / anti-CD28 bispecific antibody comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 50, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 51, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 52, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 53. A schematic diagram of the molecular structure of the anti-CD19 / anti-CD28 bispecific antibody is shown in Figure 1C.

[0152] Preparation of bispecific antibodies for use in the present invention In certain embodiments, the therapeutic agent used in combination comprises a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificities at at least two different sites. In certain embodiments, the binding specificities are for different antigens. In certain embodiments, the binding specificities are for different epitopes on the same antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0153] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and the "knob-in-hole" technique (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies have also been developed using electrostatic steering techniques to create antibody-Fc heterodimeric molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to create bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create 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., 148(5):1547-1553 (1992)). al., J. Immunol., 152:5368 (1994); and trispecific antibodies can also be prepared as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0154] Also included herein are modified antibodies with three or more functional antigen binding sites, including "octopus antibodies" (see, eg, US Patent Application Publication No. 2006 / 0025576).

[0155] The antibodies or fragments herein also include "dual-acting FAbs" or "DABs" that consist of antigen-binding sites that bind to two different antigens (see, e.g., U.S. Patent No. 2008 / 0069820). "Crossmab" antibodies are also included herein (see, e.g., WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, or WO 2009 / 080254).

[0156] Another technique for generating bispecific antibody fragments is the "bispecific T cell engager" or BiTE® approach (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain contains two single-chain Fv (scFv) fragments, each with a variable heavy (VH) domain and a variable light (VL) domain, separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. The single polypeptide further contains a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, which may be specific for a different cell type, and upon association of each scFv with its cognate epitope, cells of two different cell types are brought into close proximity or tethered. One particular embodiment of this approach involves linking an scFv that recognizes a cell surface antigen expressed by an immune cell, e.g., the CD3 polypeptide on a T cell, to another scFv that recognizes a cell surface antigen expressed by a target cell, such as a malignant or tumor cell.

[0157] As a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic expression system known in the art, such as a CHO cell line. However, specific purification techniques (see, e.g., EP 1 691 833) may be required to separate the monomeric bispecific T cell engager from other multimeric species that may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted with an imidazole gradient. The eluate is further purified by anion exchange chromatography, and the polypeptide is eluted with a sodium chloride gradient. Finally, the eluate is subjected to molecular sieve chromatography to separate the monomers from the multimers. In one embodiment, the bispecific biantibody used in the present invention is composed of a single polypeptide chain comprising two single-chain FV fragments (scFVs) fused to each other by a peptide linker.

[0158] Fc domain modifications that reduce Fc receptor binding and / or effector function The Fc domain of the antigen-binding molecule of the present invention is composed of a pair of polypeptide chains that constitute the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, and each subunit contains the IgG heavy chain constant domains CH2 and CH3. The two subunits of the Fc domain can stably associate with each other.

[0159] The Fc domain confers advantageous pharmacokinetic properties to the antigen-binding molecules of the present invention, including a long serum half-life and a favorable tissue-to-blood distribution ratio, which contribute to favorable accumulation in target tissues. However, at the same time, it may lead to the undesirable situation that the bispecific antibodies of the present invention are targeted to cells expressing Fc receptors rather than to preferred antigen-bearing cells. Thus, in certain embodiments, the Fc domain of the antigen-binding molecules of the present invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain. In one embodiment, the Fc does not substantially bind to Fc receptors and / or induce effector function. In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically, human FcγRIIIa, FcγRI, or FcγRIIa, most specifically, human FcγRIIIa. In one embodiment, the Fc domain does not induce effector function. Decreased effector function includes, but is not limited to, one or more of the following: decreased complement-dependent cytotoxicity (CDC), decreased antibody-dependent cell-mediated cytotoxicity (ADCC), decreased antibody-dependent cellular phagocytosis (ADCP), decreased cytokine secretion, decreased immune complex-mediated antigen uptake by antigen-presenting cells, decreased binding to NK cells, decreased binding to macrophages, decreased binding to monocytes, decreased binding to polymorphonuclear cells, decreased direct signaling to induce apoptosis, decreased dendritic cell maturation, or decreased T cell priming.

[0160] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0161] In certain embodiments, the invention provides antibodies, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors.

[0162] In one embodiment, the Fc domain of an antibody of the present invention comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor and / or its effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In particular, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331, and P329 (EU numbering). In particular, the Fc domain comprises amino acid substitutions at positions 234 and 235 (EU numbering) and / or 329 (EU numbering) of the IgG heavy chain. More specifically, an antibody according to the present invention is provided that comprises an Fc domain with amino acid substitutions L234A, L235A, and P329G ("P329G LALA"; EU numbering) of the IgG heavy chain. The amino acid substitutions at L234A and L235A are referred to as the so-called LALA mutation. The amino acid substitution combination "P329G LALA", which almost completely abolishes Fcγ receptor binding in human IgG1 Fc domains, is described in WO 2012 / 130831, which also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function.

[0163] Fc domains with reduced Fc receptor binding and / or effector function also include those with substitutions at one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0164] In another embodiment, the Fc domain is an IgG4 Fc domain. Compared to IgG1 antibodies, IgG4 antibodies have reduced binding affinity to Fc receptors and reduced effector function. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly an amino acid substitution of S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising amino acid substitutions L235E, S228P, and P329G (EU numbering). Such IgG4 Fc domain mutants and their binding properties to Fcγ receptors are also described in WO 2012 / 130831.

[0165] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The exact nucleotide changes can be confirmed, for example, by sequencing.

[0166] Binding to Fc receptors can be readily measured, for example, by ELISA or surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare), which can be obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain or a cell-activating antibody containing an Fc domain to an Fc receptor can be assessed using a cell line known to express the specific Fc receptor, such as human NK cells expressing the FcγIIIa receptor.

[0167] The effector function of an Fc domain, or an antibody of the present invention containing an Fc domain, can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing 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). Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0168] In some aspects, the binding of the Fc domain to complement components, particularly C1q, is reduced. Thus, in some embodiments in which the Fc domain is modified to reduce effector function, the reduction in effector function includes a reduction in CDC. C1q binding assays can be performed to determine whether the bispecific antigen-binding molecules of the present invention can bind to C1q and therefore have CDC activity (see, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402). CDC assays can also be performed to assess complement activation (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0169] Fc domain modifications that promote heterodimerization The bispecific antigen-binding molecules of the present invention contain different antigen-binding sites fused to one or the other of the two subunits of the Fc domain, and therefore the two subunits of the Fc domain may contain two non-identical polypeptide chains. Recombinant coexpression of these polypeptides followed by dimerization allows for several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antibodies of the present invention in recombinant production, it may be advantageous to introduce modifications to the Fc domain of the bispecific antigen-binding molecules of the present invention that promote the association of the desired polypeptides.

[0170] In certain embodiments, the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is the CH3 domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0171] In a specific embodiment, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which comprises a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain. Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a "protuberance" ("knob") at the interface of a first polypeptide and a corresponding "cavity" ("hole") at the interface of a second polypeptide, where the protuberance can be positioned in the cavity to promote heterodimer formation and prevent homodimer formation. The protuberance is constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protuberance is created at the interface of the second polypeptide by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine).

[0172] Thus, in some embodiments, an amino acid residue in the CH3 domain of a first subunit of an Fc domain is substituted with an amino acid residue having a larger side chain volume, thereby forming a protrusion in the CH3 domain of the first subunit that can be positioned in a cavity in the CH3 domain of a second subunit. Then, an amino acid residue in the CH3 domain of a second subunit of an Fc domain is substituted with an amino acid residue having a smaller side chain volume, thereby forming a cavity in the CH3 domain of the second subunit that can be positioned in the protrusion in the CH3 domain of the first subunit. Preferably, the 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, the 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 protrusion and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0173] In a specific such embodiment, the first subunit of the Fc domain has a substitution of a tryptophan residue (T366W) for the threonine residue at position 366, and a valine residue (Y407V) for the tyrosine residue at position 407, and optionally a tryptophan residue (T366S) for the threonine residue at position 366, and an alanine residue (L368A) for the leucine residue at position 368 (numbering according to the Kabat EU index). In a further embodiment, the first subunit of the Fc domain further comprises a substitution of the serine residue at position 354 with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is substituted with a cysteine ​​residue), and the second subunit of the Fc domain further comprises a substitution of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). In a preferred embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0174] The C-terminus of the heavy chain of a bispecific antibody as reported herein can be a complete C-terminus ending in amino acid residue PGK. The C-terminus of the heavy chain can also be a truncated C-terminus in which one or two of the C-terminal amino acid residues are removed. In a preferred embodiment, the C-terminus of the heavy chain is a truncated C-terminus ending in PG. In one aspect of all aspects reported herein, a bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as defined herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the Kabat EU index). In one embodiment of all aspects reported herein, a bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as defined herein comprises a C-terminal glycine residue (G446, numbering according to the Kabat EU index).

[0175] Fab domain modifications In one embodiment, the molecule used herein is a bispecific antibody, in which one of the Fab fragments has either the variable domains VH and VL exchanged, or the constant domains CH1 and CL exchanged. Bispecific antibodies are prepared according to the crossmab technology.

[0176] Multispecific antibodies in which one binding arm has undergone domain substitution / swapping (CrossMabVH-VL or CrossMabCH-CL) are described in detail in WO 2009 / 080252 and Schaefer, W. et al., PNAS, 108 (2011) 11187-1191. These multispecific antibodies can significantly reduce by-products caused by mismatches between a light chain for a first antigen and an incorrect heavy chain for a second antigen (compared to approaches without such domain swapping). In certain embodiments, the additional Fab fragment is a Fab fragment, in which the variable domains VL and VH are substituted with each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

[0177] In one aspect, the present invention relates to a bispecific agonist CD28 antigen-binding molecule characterized by monovalent binding to CD28, comprising: (a) one antigen-binding domain capable of specifically binding to CD28; (b) at least one antigen-binding domain capable of specifically binding to a tumor-associated antigen; and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, the first subunit and the second subunit comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor. In the Fab fragment capable of specifically binding to the tumor-associated antigen, the constant domains CL and CH1 are replaced with each other such that the CH1 domain is part of the light chain and the CL domain is part of the heavy chain.

[0178] In another embodiment, to further improve correct pairing, a bispecific antibody used herein, for example, a bispecific agonistic CD28 antibody characterized by monovalent binding to CD28, comprises (a) one Fab fragment capable of specifically binding to CD28, (b) one Fab domain capable of specifically binding to CD19, and (c) one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to the Fc receptor and / or effector function, comprising first and second subunits capable of stable association, wherein the Fc domains can contain differently charged amino acid substitutions (so-called "charged residues"). These modifications can be introduced into the crossover or non-crossover CH1 and CL domains. In a particular embodiment, the present invention relates to a bispecific CD28 antigen-binding molecule, in which in one of the CL domains, the amino acid at position 123 (EU numbering) is substituted with arginine (R) and the amino acid at position 124 (EU numbering) is substituted with lysine (K), and in one of the CH1 domains, the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are substituted with glutamic acid (E). In a particular embodiment, in the CL domain of a Fab fragment capable of specifically binding to CD28, the amino acid at position 123 (EU numbering) is substituted with arginine (R) and the amino acid at position 124 (EU numbering) is substituted with lysine (K), and in the CH1 domain of a Fab fragment capable of specifically binding to CD28, the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are substituted with glutamic acid (E).

[0179] More particularly, a bispecific as used herein can comprise a Fab in which the amino acid at position 123 (EU numbering) in the CL domain is substituted with arginine (R), the amino acid at position 124 (EU numbering) is substituted with lysine (K), and the amino acids at positions 147 (EU numbering) and 213 (EU numbering) in the CH1 domain adjacent to the TNF ligand family member are substituted with glutamic acid (E).

[0180] Pharmaceutical Compositions, Medicaments, and Routes of Administration In a further embodiment, a pharmaceutical composition or medicament is provided comprising an anti-CD20 / anti-CD3 antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a CD19-targeted 4-1BB (CD137) agonist. In one embodiment, the pharmaceutical composition comprises an antibody provided herein and at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises an antibody provided herein and at least one additional therapeutic agent, e.g., as described below.

[0181] The pharmaceutical compositions disclosed herein comprise a therapeutically effective amount of one or more bispecific antibodies dissolved or dispersed in a pharmaceutically acceptable excipient. The term "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not cause adverse, allergic, or other untoward reactions when administered to animals, such as humans. The preparation of pharmaceutical compositions comprising at least one antibody and, optionally, additional active ingredients will be known to those skilled in the art in light of this disclosure, as exemplified in Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, incorporated herein by reference. In particular, the compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable excipients" include any solvents, buffers, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, salts, stabilizers, and combinations thereof, as known to those skilled in the art.

[0182] Pharmaceutical compositions comprising the bispecific antigen-binding molecules disclosed herein can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate the processing of proteins into pharmaceutically usable preparations. The appropriate formulation depends on the selected route of administration.

[0183] Bispecific antibodies may also be formulated in compositions in free acid or base, neutral, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or free base. These include acid addition salts, such as those formed with free amino groups of proteinaceous compositions, or with inorganic acids such as hydrochloric acid and phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, and ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, and procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0184] The compositions herein may also contain multiple active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other, and such active ingredients are preferably present in combination in amounts that are effective for the purpose intended.

[0185] Formulations to be used for in vivo administration are generally sterile. Sterilization is readily accomplished, for example, by filtration through sterile filtration membranes.

[0186] Administration of bispecific antibodies The anti-CD20 / anti-CD3 bispecific antibodies, anti-CD19 / anti-CD28 bispecific antibodies, and CD19-targeted 4-1BB (CD137) agonists (all referred to herein as agents) can be administered by any suitable means, including parenteral, pulmonary, intranasal, and, if desired for localized treatment, topical administration. However, the methods disclosed herein are particularly useful in connection with therapeutic agents administered parenterally, particularly by intravenous infusion.

[0187] Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration. Administration can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending on whether the administration is short-term or long-term. Various administration schedules are contemplated herein, including, but not limited to, single or multiple doses over various time periods, bolus administration, and pulse infusion. In one embodiment, the therapeutic agent is administered parenterally, particularly intravenously. In a particular embodiment, the agent is administered by intravenous infusion. In another embodiment, the agent is administered subcutaneously.

[0188] Anti-CD20 / anti-CD3 bispecific antibodies, anti-CD19 / anti-CD28 bispecific antibodies, and CD19-targeted 4-1BB (CD137) agonists will be formulated, dosed, and administered in a manner consistent with good manufacturing practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site, method, and schedule of administration of the agent, and other factors known to medical professionals. Anti-CD20 / anti-CD3 bispecific antibodies, anti-CD19 / anti-CD28 bispecific antibodies, and CD19-targeted 4-1BB (CD137) agonists are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of therapeutic agent present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and by any route of administration as described herein, or at dosages that are about 1-99% of the dosages described herein, or at any dosage and by any route of administration that is empirically / clinically determined to be appropriate.

[0189] Anti-CD20 / anti-CD3 bispecific antibodies can be administered by any suitable means, including parenteral, pulmonary, intranasal, and, if necessary for localized treatment, topical administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration. Administration can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending on whether the administration is short-term or long-term. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is administered parenterally, particularly intravenously, for example, by infusion. In one embodiment, the infusion rate of the anti-CD20 / anti-CD3 bispecific antibody, particularly glofitamab, is at least 4 hours. In one embodiment, the infusion time of the anti-CD20 / anti-CD3 bispecific antibody may be shortened or extended. In one embodiment, if there are no infusion-related adverse events, the infusion time of glofitamab in subsequent cycles is shortened to 2 hours ± 15 minutes. In one embodiment, the infusion time is extended to up to 8 hours for subjects at high risk for cytokine release syndrome (CRS). In one embodiment, the infusion time of glofitamab is extended to up to 8 hours for patients who may be at increased risk of CRS, who have experienced IRR or CRS with a previous glofitamab administration, or who are at increased risk of recurrent IRR / CRS with subsequent administration.

[0190] In certain embodiments, disclosed herein are uses, methods, kits, or medicaments that combine an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, where the combination therapy comprises simultaneous administration of the anti-CD20 / anti-CD3 bispecific antibody, the anti-CD19 / anti-CD28 bispecific antibody, and the CD19-targeted 4-1BB (CD137) agonist. In certain embodiments, the combination administration is for one or more treatment cycles, particularly 3 to 8 treatment cycles. The length of the treatment cycle corresponds to 7, 14, or 21 days, particularly 7 or 14 days.

[0191] In particular, the present disclosure also relates to the uses, methods of use, kits or medicaments described herein that combine an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, wherein the combination therapy comprises a first treatment regimen of the anti-CD20 / anti-CD3 bispecific antibody in combination with the anti-CD19 / anti-CD28 bispecific antibody and a second treatment regimen of the anti-CD20 / anti-CD3 bispecific antibody in combination with the CD19-targeted 4-1BB (CD137) agonist.

[0192] In one embodiment, the first therapeutic regimen using the combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-CD19 / anti-CD28 bispecific antibody is administered in a single dose (one treatment cycle). In certain embodiments, the administration of the first therapeutic regimen using the combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-CD19 / anti-CD28 bispecific antibody is administered in two or more treatment cycles. In one embodiment, the first therapeutic regimen consists of one to five treatment cycles, and the second therapeutic regimen begins with the next treatment cycle. In another embodiment, the first therapeutic regimen consists of three to five treatment cycles, and the second therapeutic regimen begins with the next treatment cycle. In one embodiment, the first therapeutic regimen consists of four treatment cycles, and the second therapeutic regimen begins with treatment cycle five. In one embodiment, the anti-CD19 / anti-CD28 bispecific antibody is administered one hour later than the anti-CD20 / anti-CD3 bispecific antibody, but within the same treatment cycle (e.g., seven days). In another embodiment, the anti-CD19 / anti-CD28 bispecific antibody is administered two days after the anti-CD20 / anti-CD3 bispecific antibody, but in the same treatment cycle (e.g., 7 days, 14 days, 21 days).

[0193] In one embodiment, the second therapeutic regimen using the anti-CD20 / anti-CD3 bispecific antibody in combination with the CD19-targeted 4-1BB (CD137) agonist is administered in a single dose (one treatment cycle). In certain embodiments, the administration of the second therapeutic regimen using the anti-CD20 / anti-CD3 bispecific antibody in combination with the CD19-targeted 4-1BB (CD137) agonist is for two or more cycles. In one embodiment, the second therapeutic regimen includes one to five treatment cycles. In another embodiment, the second therapeutic regimen includes three to five treatment cycles. In one embodiment, the second therapeutic regimen includes two or more treatment cycles, after which the first therapeutic regimen is repeated. In one embodiment, the first repeated therapeutic regimen begins with the next treatment cycle. In one embodiment, the first repeated therapeutic regimen is followed by the second repeated therapeutic regimen (alternating administration).

[0194] In one such embodiment, the substance is administered weekly, every two weeks, or every three weeks, particularly every two weeks. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is administered in a therapeutically effective amount. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is administered at a dose of about 50 μg / kg, about 100 μg / kg, about 200 μg / kg, about 300 μg / kg, about 400 μg / kg, about 500 μg / kg, about 600 μg / kg, about 700 μg / kg, about 800 μg / kg, about 900 μg / kg, or about 1000 μg / kg. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is administered at a lower dose than the dose of the anti-CD20 / anti-CD3 bispecific antibody in a corresponding treatment regimen that does not administer an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist. In one embodiment, the administration of the anti-CD20 / anti-CD3 bispecific antibody comprises an initial administration of a first dose of the anti-CD20 / anti-CD3 bispecific antibody and one or more subsequent administrations of a second dose of the anti-CD20 / anti-CD3 bispecific antibody, wherein the second dose is higher than the first dose. In one embodiment, the administration of the anti-CD20 / anti-CD3 bispecific antibody comprises an initial administration of a first dose of the anti-CD20 / anti-CD3 bispecific antibody and one or more subsequent administrations of a second dose of the anti-CD20 / anti-CD3 bispecific antibody, wherein the first dose is not lower than the second dose.

[0195] In one embodiment, the initial treatment regimen with the anti-CD20 / anti-CD3 bispecific antibody in combination with the anti-CD19 / anti-CD28 bispecific antibody is initiated after one to three treatment cycles with a single administration of the anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is administered weekly. The initial treatment regimen with the anti-CD20 / anti-CD3 bispecific antibody in combination with the anti-CD19 / anti-CD28 bispecific antibody is initiated one week after the last single administration of the anti-CD20 / anti-CD3 bispecific antibody alone.

[0196] In another embodiment, administration of the anti-CD20 / anti-CD3 bispecific antibody in a treatment regimen according to the invention is the first administration of the anti-CD20 / anti-CD3 bispecific antibody to the subject (at least within the same course of treatment). In one embodiment, the anti-CD19 / anti-CD28 bispecific antibody is not administered before the administration of the anti-CD20 / anti-CD3 bispecific antibody. In another embodiment, the anti-CD19 / anti-CD28 bispecific antibody is administered before the administration of the anti-CD20 / anti-CD3 bispecific antibody.

[0197] In all of these embodiments, the anti-CD19 / anti-CD28 bispecific antibody and the anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist are for use in combination therapy, and the treatment regimen begins with administration of one or more treatment cycles, particularly 3 to 5 treatment cycles, of the anti-CD20 / anti-CD3 bispecific antibody alone before initiating administration of the combination therapy. In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody is administered at escalating doses in each single-administration treatment cycle (step-up dosing).

[0198] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is for use in combination with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, wherein prior treatment with a Type II anti-CD20 antibody, preferably obinutuzumab, precedes the combination treatment, and the period between prior treatment and combination treatment is sufficient to deplete B cells in the individual in response to the Type II anti-CD20 antibody, preferably obinutuzumab. In certain embodiments, obinutuzumab is administered 7 days prior to the first treatment with the anti-CD20 / anti-CD3 bispecific antibody. In certain embodiments, obinutuzumab is administered 7 days before the first treatment with the anti-CD20 / anti-CD3 bispecific antibody, the anti-CD20 / anti-CD3 bispecific antibody is administered on day 1 of the first treatment cycle, and then the anti-CD20 / anti-CD3 bispecific antibody is administered twice at progressively higher doses on days 3 and 8 of the first treatment cycle before initiating combination treatment in the second treatment cycle.

[0199] T cell activation can lead to severe cytokine release syndrome (CRS). In a phase 1 study conducted by TeGenero (Suntharalingam et al., N Engl J Med (2006) 355, 1018-1028), all six healthy volunteers experienced rapid, near-lethal CRS after infusion of an inappropriate dose of a T cell stimulatory superagonist anti-CD28 monoclonal antibody. Cytokine release associated with administration of T cell-activating therapeutics, such as anti-CD20 / anti-CD3 bispecific antibodies, can be significantly reduced by pretreatment with a type II anti-CD20 antibody, such as obinutuzumab. The use of GAZYVA® pretreatment (Gpt) aids in the rapid depletion of B cells in peripheral blood and secondary lymphoid organs, reducing the risk of adverse events (AEs) associated with strong systemic T cell activation by T cell-activating therapeutics (such as CRS) and simultaneously supporting sufficient exposure levels of the T cell-activating therapeutic to mediate tumor cell elimination from the start of administration. To date, the safety profile of obinutuzumab (including cytokine release) has been evaluated and managed in hundreds of patients in ongoing obinutuzumab clinical trials. Finally, in addition to supporting the safety profile of T cell-activating therapeutics such as anti-CD20 / anti-CD3 bispecific antibodies, Gpt should also help prevent the formation of anti-drug antibodies (ADAs) against these unique molecules.

[0200] The combination therapy described above encompasses coadministration (two or more therapeutic agents in the same or separate formulations) and separate administration, where administration of a therapeutic agent can occur before, simultaneously with, and / or after administration of the additional therapeutic agent or agent. In one embodiment, administration of a therapeutic agent and administration of the additional therapeutic agent occur within about one month, or within about one, two, three weeks, or within about one, two, three, four, five, or six days of each other.

[0201] Therapeutic methods and compositions CD20 and CD19 are expressed on most B cells (pan-B cell markers) except for stem cells and plasma cells, and are frequently expressed on most human B cell malignancies, such as lymphomas and leukemias (e.g., non-Hodgkin's lymphoma and acute lymphoblastic leukemia). Bispecific antibodies that recognize two cell surface proteins on different cell populations hold promise for redirecting cytotoxic immune cells to destroy pathogenic target cells.

[0202] In one aspect, provided is a method of treating B-cell cancer in an individual in need thereof, comprising administering to the individual a combination therapy combining an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and an anti-CD19-targeted 4-1BB (CD137) agonist.

[0203] In one such aspect, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In a further embodiment, provided herein is a method of depleting B cells comprising administering to the subject effective amounts of an anti-CD20 / anti-CD3 antibody and an anti-CD19 / anti-CD28 bispecific antibody and / or a CD19-targeted 4-1BB (CD137) agonist. An "individual" or "subject" according to any of the above aspects is preferably a human.

[0204] In a further embodiment, a composition is provided for use in cancer immunotherapy, comprising an anti-CD20 / anti-CD3 antibody and an anti-CD19 / anti-CD28 bispecific antibody and / or a CD19-targeted 4-1BB (CD137) agonist. In certain embodiments, a composition is provided for use in a method of cancer immunotherapy, comprising an anti-CD20 / anti-CD3 antibody and an anti-CD19 / anti-CD28 bispecific antibody and / or a CD19-targeted 4-1BB (CD137) agonist.

[0205] In a further aspect, provided herein is the use of a composition comprising an anti-CD20 / anti-CD3 antibody and an anti-CD19 / anti-CD28 bispecific antibody and / or a CD19-targeted 4-1BB (CD137) agonist in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a B cell proliferative disorder. In a further embodiment, the medicament of the invention is for use in a method for treating a B cell proliferative disorder, comprising administering an effective amount of the medicament to an individual having a B cell proliferative disorder. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In a further embodiment, the medicament is for depleting B cells. The B-cell proliferative disorder is selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin's lymphoma (HL). In certain embodiments, the B-cell cancer is non-Hodgkin's lymphoma or diffuse large B-cell lymphoma (DLBCL).

[0206] In a further aspect, provided herein are methods for treating B-cell cancer. In one embodiment, the method comprises administering to an individual having such B-cell cancer an effective amount of an anti-CD20 / anti-CD3 bispecific antibody in combination with an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a CD19-targeted 4-1BB (CD137) agonist. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the above embodiments may be a human. The B-cell cancer, in one embodiment, is a B-cell lymphoma or B-cell leukemia. In one embodiment, the B-cell cancer is non-Hodgkin's lymphoma or acute lymphoblastic leukemia.

[0207] The combination therapies described above encompass combined administration (where two or more therapeutic agents are in the same formulation or separate formulations) and separate administration, where administration of an antibody as reported herein can occur before, simultaneously with, and / or after administration of the additional therapeutic agent or agents. In one embodiment, the combined administration of an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist and the administration of the additional therapeutic agent occur within about 1 month, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0208] The combination of the anti-CD20 / anti-CD3 bispecific antibody with the anti-CD19 / anti-CD28 bispecific antibody and the CD19-targeted 4-1BB (CD137) agonist reported herein (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, pulmonary, intranasal, and, if desired for localized treatment, topical administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration. Administration can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending on whether administration is short-term or long-term. Various administration schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time points, bolus administration, and pulse infusion.

[0209] The anti-CD20 / anti-CD3 bispecific antibody in combination with the anti-CD19 / anti-CD28 bispecific antibody and CD19-targeted 4-1BB (CD137) agonist reported herein is formulated, dosed, and administered in a manner consistent with good manufacturing practice. Factors to consider in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site, method, and schedule of drug administration, and other factors known to medical professionals. The antibody is optionally, but not necessarily, combined with one or more drugs currently used to prevent or treat the disorder in question. The effective amount of such other drugs depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and via the routes of administration described herein, or at doses ranging from about 1 to 99% of the dosages described herein, or at any dosage and via any route of administration determined empirically / clinically to be appropriate.

[0210] In another embodiment, the combination of the anti-CD20 / anti-CD3 bispecific antibody with the anti-CD19 / anti-CD28 bispecific antibody and the CD19-targeted 4-1BB (CD137) is for use in combination therapy, wherein pretreatment with a Type II anti-CD20 antibody, preferably obinutuzumab, precedes the combination therapy, and the period of time between the pretreatment and the combination therapy is sufficient to deplete B cells in the individual in response to the Type II anti-CD20 antibody, preferably obinutuzumab.

[0211] T cell activation can lead to severe cytokine release syndrome (CRS). In a phase 1 study conducted by TeGenero (Suntharalingam et al., N Engl J Med (2006) 355, 1018-1028), all six healthy volunteers experienced rapid, near-lethal CRS after infusion of an inappropriate dose of a T cell stimulatory superagonist anti-CD28 monoclonal antibody. Cytokine release associated with administration of T cell-activating therapeutics, such as anti-CD20 / anti-CD3 bispecific antibodies, can be significantly reduced by pretreatment with a type II anti-CD20 antibody, such as obinutuzumab. The use of GAZYVA® pretreatment (Gpt) aids in the rapid depletion of B cells in peripheral blood and secondary lymphoid organs, reducing the risk of adverse events (AEs) associated with strong systemic T cell activation by T cell-activating therapeutics (such as CRS) and simultaneously supporting sufficient exposure levels of the T cell-activating therapeutic to mediate tumor cell elimination from the start of administration. To date, the safety profile of obinutuzumab (including cytokine release) has been evaluated and managed in hundreds of patients in ongoing obinutuzumab clinical trials. Finally, in addition to supporting the safety profile of T cell-activating therapeutics such as anti-CD20 / anti-CD3 bispecific antibodies, Gpt should also help prevent the formation of anti-drug antibodies (ADAs) against these unique molecules.

[0212] Other drugs and treatments The antigen-binding molecules of the present invention can be administered in combination with one or more other drugs in therapy. For example, the fusion proteins of the present invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any drug that can be administered to treat symptoms or diseases in individuals who require such treatment. Such additional therapeutic agents can include active ingredients suitable for the specific indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is another anti-cancer drug.

[0213] Such other agents are preferably present in combination in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of fusion protein used, the type of disorder or treatment, and other factors described above. The antigen-binding molecule is generally used in the same dosages and by the same administration route as described herein, or in a dosage that is about 1 to 99% of the dosage described herein, or in any dosage and by any administration route that is empirically / clinically determined to be appropriate.

[0214] Such combination therapy includes combined administration (two or more therapeutic agents contained in the same or separate compositions) and separate administration, in which case administration of the antigen-binding molecule of the present invention may occur before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant.

[0215] Manufactured products (kits) In another embodiment, a kit containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders is provided. The kit comprises at least one container and a label or package insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, infusion bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for the treatment, prevention, and / or diagnosis of a condition, alone or in combination with another composition, and may have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). The kit contains at least three active agents: an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a CD19-targeted 4-1BB (CD137) agonist.

[0216] In certain embodiments, a kit is provided for treating or delaying the progression of cancer in a subject, comprising: (A) a package containing a first composition comprising an anti-CD20 / anti-CD3 bispecific antibody as an active ingredient and a pharmaceutically acceptable carrier; (B) a second composition comprising an anti-CD19 / anti-CD28 bispecific antibody and a pharmaceutically acceptable carrier as active ingredients; (C) a third composition comprising a CD19-targeted 4-1BB (CD137) agonist and a pharmaceutically acceptable carrier as active ingredients; and (D) instructions for using the compositions in combination therapy.

[0217] The label or package insert indicates how the composition is used to treat the selected condition and describes how to use the composition in combination therapy. Furthermore, the kit may include: (a) a first container containing a composition, the first container containing an anti-CD20 / anti-CD3 bispecific antibody of the present invention; (b) a second container containing a composition, the second container containing an anti-CD19 / anti-CD28 bispecific antibody; and (c) a third container containing a composition, the third container containing a CD19-targeted 4-1BB (CD137) agonist. Furthermore, the kit may include one or more additional containers containing additional active ingredients that can be used in combination. The article of manufacture in this embodiment of the present invention may further include a package insert indicating that the composition can be used to treat a specific condition.

[0218] Alternatively, or additionally, the kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, dextrose solution, etc. It may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filtering agents, needles, syringes, etc.

[0219] Pharmaceuticals In another aspect, a medicament is provided comprising an anti-CD20 / anti-CD3 bispecific antibody for treating a B-cell proliferative disorder, wherein said anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist.

[0220] In one embodiment, a medicament comprising an anti-CD20 / anti-CD3 bispecific antibody is provided for treating a B cell proliferative disorder in combination with an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a CD19-targeted 4-1BB (CD137) agonist. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody, the anti-CD19 / anti-CD28 bispecific antibody, and the CD19-targeted 4-1BB (CD137) agonist are administered together in a single composition or separately in two or more different compositions. In a specific embodiment, the anti-CD20 / anti-CD3 bispecific antibody, the anti-CD19 / anti-CD28 bispecific antibody, and the CD19-targeted 4-1BB (CD137) agonist are administered separately in three different compositions. TIFF2025541593000002.tif251170TIFF2025541593000003.tif252170TIFF2025541593000004.tif252170TIFF2025541593000005.tif252170 TIFF2025541593000006.tif252170TIFF2025541593000007.tif254170TIFF2025541593000008.tif254170TIFF2025541593000009.tif217170

[0221] General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains can be found in the following reference: Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of antibody chains, as defined above, are numbered and referenced according to the numbering system according to Kabat (Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). [Example]

[0222] The following are examples of methods and compositions of the present invention: It will be understood from the above general description that various other embodiments are possible.

[0223] Example 1 Preparation, purification and characterization of CD19-41BBL antigen-binding molecules The CD19-targeted 4-1BB ligand trimer-containing Fc-fusion antigen binding molecule was prepared as described in International Patent Application Publication No. WO 2016 / 075278, particularly Example 7.2.7 (construct 4.5).

[0224] To prepare the construct, a polypeptide containing the dimeric 4-1BB ligand fused to the human CL domain was subcloned in frame with the human IgG1 heavy chain CH2 and CH3 domains on the knob. A polypeptide containing one ectodomain of the 4-1BB ligand was fused to the human IgG1-CH1 domain. To improve correct pairing, crossover CH-CL (charge variant) amino acid mutations were introduced: E123R and Q124K in the CL domain, and K147E and K213E in the CH1 domain (EU numbering according to Kabat).

[0225] The variable regions of the heavy and light chain DNA sequences encoding the CD19 antibody clone 8B8-2B11 were subcloned in frame with either the recess constant heavy chain or the human IgG1 constant light chain. Following the methods described in the WO 2012 / 130831 pamphlet, Pro329Gly, Leu234Ala, and Leu235Ala mutations were introduced into the knob and hole heavy chain constant regions to inhibit Fcγ receptor binding. Combining a dimeric ligand-Fc knob chain containing the S354C / T366W mutation, a monomeric CH1 fusion, a targeted anti-CD19-Fc hole chain containing the Y349C / T366S / L368A / Y407V mutation, and an anti-CD19 light chain allowed the generation of a heterodimer containing an associated trimeric 4-1BB ligand and CD19-binding Fab. This molecule is referred to herein as CD19-4-1BBL.

[0226] CD19-4-1BBL comprises the amino acid sequences of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60. A schematic diagram of an Fc-fusion antigen binding molecule containing a 4-1BB ligand trimer targeting CD19 is shown in Figure 1A.

[0227] The production and properties of CD19-targeting and non-targeting 4-1BB ligand trimer-containing Fc-fusion antigen binding molecules are described in detail in Example 7.4 and Examples 8 to 11 of WO 2016 / 075278, respectively.

[0228] Example 2 Preparation, purification, and characterization of T cell bispecific (TCB) antibodies The TCB molecules were prepared according to the method described in WO 2016 / 020309.

[0229] The anti-CD20 / anti-CD3 bispecific antibody (CD20 CD3 TCB or CD20 TCB or glofitamab) used in the experiments corresponds to molecule B described in Example 1 of WO 2016 / 020309. Molecule B is a "2+1 IgG CrossFab" antibody, consisting of two distinct heavy chains and two distinct light chains. Point mutations in the CH3 domain ("knob-into-hole") were introduced to promote assembly of the two distinct heavy chains. Following the method described in the WO 2012 / 130831 pamphlet, Pro329Gly, Leu234Ala, and Leu235Ala mutations were introduced into the constant regions of the knob and hole heavy chains to inhibit Fcγ receptor binding. The VH and VL domains of the CD3-binding Fab were swapped, and point mutations in the CH and CL domains of the CD20-binding Fab were also performed to promote correct assembly of the two distinct light chains. 2+1 means that the molecule has two antigen-binding domains specific for CD20 and one antigen-binding domain specific for CD3.

[0230] CD20 TCB comprises the amino acid sequences of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60. A schematic diagram of a bispecific antibody in a 2+1 format is shown in Figure 1B.

[0231] This molecule is further characterized in Example 1 of WO 2016 / 020309.

[0232] Example 3 Preparation, purification and characterization of CD19-CD28 bispecific antibodies The CD19-CD28 bispecific antibody was prepared as described in International Patent Application Publication No. WO 2020 / 127618.

[0233] More detailed generation and production are described in Example 18. To generate the respective expression plasmids, the variable domain sequences of the CD19 antibody clone 8B8-2B11 and the CD28 antibody clone SA_v8 were subcloned in frame with the respective constant regions previously inserted into the respective recipient mammalian expression vectors. A schematic diagram of the resulting molecule is shown in Figure 1C. This is a "1+1 IgG1 CrossFab" antibody, consisting of two distinct heavy chains and two distinct light chains. To promote the association of the two distinct heavy chains, point mutations ("knob-into-hole") in the CH3 domain were introduced. Following the method described in the WO 2012 / 130831 pamphlet, Pro329Gly, Leu234Ala, and Leu235Ala mutations were introduced into the knob and hole heavy chain constant regions to inhibit binding to Fcγ receptors. Swapping of the VH and VL domains of the CD19-binding Fab and point mutations in the CH and CL domains of the CD28-binding Fab were performed to facilitate the correct assembly of the two different light chains.

[0234] The CD20 TCB comprises the amino acid sequences of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60.

[0235] Example 4 Ex vivo combination therapy with CD19-CD28, CD19-4-1BBL, and CD20TCB Our hypothesis is that not only CD19-CD28 but also CD19-4-1BBL synergize with CD20-TCB to activate T cells. To test this, we digested malignant spleens from patients with stage IVB B-cell lymphoma, and incubated the cells with either CD20-TCB (25 pM) or two costimulatory factors, CD19-CD28 or CD19-4-1BBL (1 nM), alone or in combination with TCB. After 3 days, cytokine release in the supernatant was measured using a CBA kit (Cytometric Bead Array, BD Biosciences). Figures 2A-D show that CD20-TCB induces cytokine release (GzB, IFNg, IL-2, and IL-8) and that CD19-CD28, as well as CD19-4-1BBL, can further enhance CD20-TCB-induced cytokine release (especially IFNg and IL-2; see Figures 2B-D, respectively). Triple combinations of CD20-TCB and both costimulators (at 0.5 nM or 1 nM of each costimulator) further increased cytokine release (especially IFNg and IL-2) compared with dual combinations of CD20-TCB and CD19-CD28 or CD19-4-1BBL.

[0236] Example 5 Efficacy evaluation study of a triple combination of CD20-TCB, CD19-4-1BBL, and CD19-CD28 using humanized NSG mice The efficacy study described here aimed to evaluate the potential efficacy of the triple combination of CD20-TCB with CD19-CD28 and CD19-41BBL in a CD19 / CD20-positive human lymphoma model using fully humanized NSG mice.

[0237] Human OCI-Ly18 (diffuse large B-cell lymphoma; DLBCL) cells were originally obtained from ATCC and then expanded and deposited in the Roche Glycart in-house cell bank. Cells were cultured in RPMI containing 10% FCS and 1x Glutamax. Cells were cultured at 37°C in a saturated water vapor atmosphere with 5% CO2. Fifty microliters of cell suspension (5x10) was added.6 NALM6 cells) mixed with 50 microliters of Matrigel was injected subcutaneously into the flanks of anesthetized mice using a 22G to 30G needle.

[0238] Female NSG mice (housed at the Jackson Laboratory) aged 4–5 weeks at the start of the experiment were housed under specific pathogen-free conditions with a 12-hour light / 12-hour dark cycle in accordance with official guidelines (GV-Solas; Felasa; TierschG). The experimental research protocol was reviewed and approved by the local government (ZH183 / 2020). After arrival, the mice were housed for one week to allow for acclimatization to the new environment and for observation. Continuous health monitoring was performed at regular intervals. Mice were injected intravenously with 15 mg / kg busulfan, followed one day by 1 x 10 IgG isolated from umbilical cord blood. 5 Human hematopoietic stem cells were injected intravenously. Blood was collected from the mice sublingually 14–16 weeks after stem cell injection and analyzed by flow cytometry for successful humanization. Mice that were efficiently engrafted were randomly assigned to treatment groups according to their human T-cell frequency. At that time, mice were injected sc with tumor cells as described above (Figure 3), and tumors were observed to grow to approximately 250 mm. 3 At the time of reaching 12 days (day 12), mice were treated with either compound or histidine buffer (vehicle; group A). ​​All mice were intravenously injected with 200 μl of the appropriate solution. To obtain the appropriate compound volume per 200 μl, stock solutions (Table 1) were diluted with histidine buffer as needed. For combination treatments (groups C–G), antibodies were mixed and injected simultaneously. All combination treatments began on day 38 with three cycles of Gazyva and CD20-TCB followed by tumor cell injection. As shown in Figure 3, group C received CD19-CD28 and group D received CD19-4-1BBL. Group G received the triple combination injection from day 38 onward. Groups E and F received an alternating treatment schedule, beginning with CD19-CD28 (group F) or CD19-4-1BBL (group E) for four cycles, followed by injection of the other costimulatory molecule for all remaining treatment cycles. TIFF2025541593000010.tif207170

[0239] Tumor growth was measured three times a week with a vernier caliper, and tumor volume was calculated as follows: T v :(W 2 / 2)×L (W: width, L: length)

[0240] The study ended on day 120 after a total of 15 cycles of treatment.

[0241] Figures 4A through 4G show tumor growth as individual tumor growth rates per group and per mouse. As described here, CD20-TCB monotherapy (Figure 4B) initially caused strong tumor growth suppression, followed by tumor recurrence in all treated animals. CD19-CD28 combination treatment (Figure 4C) slightly delayed tumor growth in some mice. The CD19-41BBL combination (Figure 4D) delayed tumor recurrence more uniformly, although most animals met the termination criterion (tumor volume > 2000 mm) before the end of the study. 3 (or more). Treatment groups receiving triple-drug combination injections after day 38 (Figure 4G) showed no further improvement in extending the duration of therapeutic activity compared to the CD19-4-1BBL-only combination group. Interestingly, the group receiving an alternating treatment regimen, starting with CD19-CD28 for the first four cycles and then continuing with CD19-4-1BBL combination treatment until the end of the study (Figure 4F), achieved complete tumor control over 120 days in all treated animals. In contrast, no such tumor control was observed when CD19-4-1BBL was started and then alternated with CD19-CD28 (Figure 4E).

[0242] Tumor volume 1500m 3 Survival analysis using a cutoff of (Figure 5) demonstrated a strong synergistic effect of glofitamab in combination with CD19-CD28 and CD19-4-1BBL when CD19-CD28 was administered in the first four treatment cycles and CD19-4-1BBL in the following treatment cycles.

[0243] Example 6 A study to evaluate the triple combination effect of CD20-TCB, CD19-4-1BBL, and CD19-CD28 using humanized BRGS-CD47 mice The second efficacy study was conducted in humanized BRGS-CD47 mice and aimed to evaluate the effect of the triple combination of CD19-CD28 and CD19-41BBL with CD20-TCB in a CD19 / CD20-positive human lymphoma model, with the combination treatment initiated one week earlier.

[0244] Human OCI-Ly18 (diffuse large B-cell lymphoma; DLBCL) cells were originally obtained from ATCC and then expanded and deposited in the Roche Glycart in-house cell bank. Cells were cultured in RPMI containing 10% FCS and 1x Glutamax. Cells were cultured at 37°C in a saturated water vapor atmosphere with 5% CO2. Fifty microliters of cell suspension (5x10) was added. 6 NALM6 cells) mixed with 50 microliters of Matrigel was injected subcutaneously into the flanks of anesthetized mice using a 22G to 30G needle.

[0245] Female humanized BRGS-CD47 mice were generated at the Jackson Laboratory by injection of human hematopoietic stem cells at 4–5 weeks of age. Once engraftment was confirmed, the humanized mice were transported to Roche at 15–16 weeks of age. Upon arrival, the mice were housed for one week to acclimate to their new environment and for observation. Continuous health monitoring was performed regularly, and the mice were housed under specific pathogen-free conditions with a 12-hour light / 12-hour dark cycle daily, in accordance with the guidelines of the FDA (GV-Solas; Felasa; TierschG). The experimental research protocol was reviewed and approved by the local government (ZH181 / 2020). As shown in Figure 6, mice were injected with tumor cells sc, and tumors grew to approximately 250 mm in size. 3At day 10, mice were treated with either compound or histidine buffer (vehicle; Group A). ​​All mice were intravenously injected with 200 μl of the appropriate solution. To obtain the appropriate compound volume per 200 μl, stock solutions (Table 1) were diluted with histidine buffer as needed. For combination treatments (Groups C–G), antibodies were mixed and injected simultaneously. All combination treatments began on day 27 with two cycles of Gazyva and CD20-TCB followed by tumor cell injection. As shown in Figure 6, Group C received CD19-CD28 and Group D received CD19-4-1BBL. Group G received the triple combination injection from day 27 onward. Groups E and F received an alternating treatment schedule, beginning with CD19-CD28 (Group F) or CD19-4-1BBL (Group E) for four cycles, followed by injection of the other costimulatory molecule for all remaining treatment cycles. TIFF2025541593000011.tif207170

[0246] Tumor growth was measured with calipers three times a week, and tumor volume was calculated as follows: T v :(W 2 / 2)×L (W: width, L: length)

[0247] The study ended on day 94 after a total of 13 cycles of treatment.

[0248] Figures 7A–7G show tumor growth as individual tumor growth rates per group and per mouse. As described here, CD20-TCB monotherapy (Figure 7B) initially caused strong tumor growth suppression, followed by tumor recurrence in all treated animals. CD19-CD28 combination treatment (Figure 7C) delayed tumor growth in some mice and completely suppressed tumors in one mouse. CD19-41BBL combination treatment (Figure 7D) similarly delayed tumor recurrence. Compared with the CD19-4-1BBL and CD19-CD28 combination alone, the triple combination treatment group injected after day 27 (Figure 7G) demonstrated superior tumor control with a prolonged duration of therapeutic activity. This combination group likely achieved stronger tumor control due to the earlier initiation of combination treatment. The group receiving an alternating treatment regimen, starting with CD19-CD28 for the first four cycles followed by CD19-4-1BBL combination therapy until the end of the study (Figure 7F), achieved tumor control similar to that achieved with the 94-day combination regimen. In contrast, alternating CD19-4-1BBL and CD19-CD28 did not achieve such significant tumor control (Figure 7E).

Claims

1. An anti-CD20 / anti-CD3 bispecific antibody in combination with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist for use in combination therapy for the treatment of B-cell proliferative disorders.

2. 1. Use of an anti-CD20 / anti-CD3 bispecific antibody in combination with an anti-CD19 / anti-CD28 bispecific antibody and an anti-CD19-targeted 4-1BB (CD137) agonist in the manufacture of a medicament for use in combination therapy for the treatment of a B-cell proliferative disorder.

3. 1. A method of treating B-cell cancer in an individual in need thereof, comprising administering to said individual a combination therapy comprising an anti-CD20 / anti-CD3 bispecific antibody in combination with an anti-CD19 / anti-CD28 bispecific antibody and an anti-CD19-targeted 4-1BB (CD137) agonist.

4. 1. A kit for use in combination therapy comprising a first medicament comprising an anti-CD20 / anti-CD3 bispecific antibody, a second medicament comprising an anti-CD19 / anti-CD28 bispecific antibody, and a third medicament comprising a CD19-targeted 4-1BB (CD137) agonist, optionally further comprising a package insert containing instructions for administering said first medicament in combination with said second medicament to treat cancer in an individual.

5. A medicament comprising an anti-CD20 / anti-CD3 bispecific antibody for the treatment of a B-cell proliferative disorder, wherein the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist.

6. 6. The anti-CD19 / anti-CD28 bispecific antibody for use and the anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeting 4-1BB (CD137) agonist, use, method, kit or medicament according to any one of claims 1 to 5, wherein the combination therapy comprises a first therapeutic regimen of the anti-CD20 / anti-CD3 bispecific antibody in combination with an anti-CD19 / anti-CD28 bispecific antibody, and a second therapeutic regimen of the anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeting 4-1BB (CD137) agonist.

7. 6. The anti-CD19 / anti-CD28 bispecific antibody for use and anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament of claim 5, wherein the first treatment regimen comprises 1 to 5 treatment cycles and the second treatment regimen begins with a subsequent treatment cycle.

8. 9. The anti-CD19 / anti-CD28 bispecific antibody for use, use, method, kit or medicament in combination with a CD19-targeted 4-1BB (CD137) agonist of claim 7 or claim 8, wherein the first treatment regimen comprises four treatment cycles and the second treatment regimen starts at treatment cycle 5.

9. 7. The anti-CD19 / anti-CD28 bispecific antibody for use and anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament of claim 6, wherein there is a time interval of one week between the end of the first therapeutic regimen and the start of the second therapeutic regimen.

10. 10. An anti-CD20 / anti-CD3 bispecific antibody in combination with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist for use, use, method, kit or medicament according to any one of claims 1 to 9, wherein prior treatment with a type II anti-CD20 antibody, preferably obinutuzumab, occurs before the combination therapy with an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist, and the period between said prior treatment and said combination therapy is sufficient to deplete B cells in the individual in response to said type II anti-CD20 antibody, preferably obinutuzumab.

11. 11. An anti-CD19 / anti-CD28 bispecific antibody for use and an anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament according to any one of claims 1 to 10, wherein said CD19-targeted 4-1BB agonist comprises the three ectodomains of 4-1BBL, each ectodomain comprising an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, in particular SEQ ID NO:

5.

12. 12. An anti-CD19 / anti-CD28 bispecific antibody for use, use, method, kit or medicament in combination with a CD19-targeted 4-1BB (CD137) agonist according to any one of claims 1 to 11, wherein said CD19-targeted 4-1BB agonist comprises an Fc domain, in particular an IgG1 or IgG4 Fc domain, which Fc domain comprises one or more amino acid substitutions that reduce or eliminate binding to an Fc receptor and / or effector function.

13. The CD19-targeted 4-1BB agonist comprises a heavy chain variable region (VH) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:

12. Ｈ (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

15. Ｌ 13. The anti-CD19 / anti-CD28 bispecific antibody for use, use, method, kit or medicament in combination with a CD19-targeted 4-1BB (CD137) agonist according to any one of claims 1 to 12, comprising an antigen-binding domain capable of specifically binding to CD19, the antigen-binding domain comprising a 4-1BB (CD137) agonist.

14. The CD19-targeted 4-1BB agonist comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:

16. Ｈ CD19) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 17 Ｌ 14. An anti-CD19 / anti-CD28 bispecific antibody for use, use, method, kit or medicament in combination with a CD19-targeted 4-1BB (CD137) agonist according to any one of claims 1 to 13, comprising an antigen-binding domain capable of specifically binding to CD19, comprising a 4-1BB (CD137) agonist.

15. the CD19-targeted 4-1BB agonist is (a) a first polypeptide comprising: (a1) a first ectodomain or a fragment thereof of 4-1BBL, the C-terminus of which is fused to the N-terminus of a second ectodomain or a fragment thereof of 4-1BBL; (a2) a second ectodomain or a fragment thereof of 4-1BBL, the C-terminus of which is fused to the N-terminus of a CL domain; (a3) ​​the CL domain, the C-terminus of which is fused to the N-terminus of one of the subunits of an Fc domain (e.g., a first subunit); and (a4) one of the subunits of the Fc domain (e.g., the first subunit); (b) a second polypeptide comprising: (b1) a third ectodomain of 4-1BBL or a fragment thereof, the C-terminus of which is fused to the N-terminus of a CH1 domain; and (b2) the CH1 domain; (c) a third polypeptide comprising: (c1) a heavy chain of a Fab molecule that binds to CD19, the heavy chain of the Fab molecule that binds to CD19, the C-terminus of which is fused to the N-terminus of another one of the subunits of the Fc domain (e.g., a second subunit); and (c2) another one of the subunits of the Fc domain (e.g., the second subunit); (d) a fourth polypeptide comprising a light chain of said Fab molecule that binds to CD19; and 15. The anti-CD19 / anti-CD28 bispecific antibody for use and the anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament of any one of claims 1 to 14, comprising:

16. The CD19-targeted 4-1BB agonist comprises a first polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18; a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19; and a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

20.

16. The anti-CD19 / anti-CD28 bispecific antibody for use and anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament of any one of claims 1 to 15, comprising: a third polypeptide comprising an amino acid sequence that is 100% identical; and a fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

21.

17. 17. The anti-CD19 / anti-CD28 bispecific antibody for use and anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament according to any one of claims 1 to 16, wherein the CD19-targeted 4-1BB agonist is englumafusp alfa.

18. The anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V Ｈ CD3) and light chain variable region (V Ｌ a first antigen-binding domain comprising a heavy chain variable region (V Ｈ CD20) and light chain variable region (V Ｌ and a second antigen-binding domain comprising a CD19-targeted 4-1BB (CD137) agonist.

19. The first antigen-binding domain comprises a heavy chain variable region (V) comprising a CDR-H1 sequence of SEQ ID NO: 22, a CDR-H2 sequence of SEQ ID NO: 23, and a CDR-H3 sequence of SEQ ID NO:

24. Ｈ and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 25, the CDR-L2 sequence of SEQ ID NO: 26, and the CDR-L3 sequence of SEQ ID NO:

27. Ｌ 20. The anti-CD19 / anti-CD28 bispecific antibody for use, use, method, kit or medicament of any one of claims 1 to 18, comprising a CD19-targeted 4-1BB (CD137) agonist.

20. The first antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:

28. Ｈ CD3) and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 29 Ｌ 20. The anti-CD19 / anti-CD28 bispecific antibody for use, use, method, kit or medicament according to any one of claims 1 to 19, comprising a CD19-targeted 4-1BB (CD137) agonist.

21. The second antigen-binding domain comprises a heavy chain variable region (V) comprising a CDR-H1 sequence of SEQ ID NO: 30, a CDR-H2 sequence of SEQ ID NO: 31, and a CDR-H3 sequence of SEQ ID NO:

32. Ｈ CD20), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 33, the CDR-L2 sequence of SEQ ID NO: 34, and the CDR-L3 sequence of SEQ ID NO:

35. Ｌ 21. The anti-CD19 / anti-CD28 bispecific antibody for use, use, method, kit or medicament according to any one of claims 1 to 20, comprising a CD19-targeted 4-1BB (CD137) agonist.

22. the second antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 36 Ｈ CD20), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 37 Ｌ 22. The anti-CD19 / anti-CD28 bispecific antibody for use, use, method, kit or medicament of any one of claims 1 to 21, comprising a CD19-targeted 4-1BB (CD137) agonist.

23. 23. The anti-CD19 / anti-CD28 bispecific antibody for use and anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament according to any one of claims 1 to 22, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises a third antigen-binding domain that binds to CD20.

24. The anti-CD20 / anti-CD3 bispecific antibody comprises: a first polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 38; a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39; and a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

40.

24. The anti-CD19 / anti-CD28 bispecific antibody for use and anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament of any one of claims 1 to 23, comprising: a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41; and fourth and fifth polypeptides that both comprise amino acid sequences that are at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

41.

25. 25. The anti-CD19 / anti-CD28 bispecific antibody for use and the anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament according to any one of claims 1 to 24, wherein said anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

26. The anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (V Ｈ CD28) and the light chain variable region (V Ｌ a first antigen-binding domain comprising a heavy chain variable region (V Ｈ CD19) and the light chain variable region (V Ｌ 26. An anti-CD19 / anti-CD28 bispecific antibody for use and an anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament according to any one of claims 1 to 25, comprising a second antigen-binding domain comprising a CD19-targeted 4-1BB (CD137) agonist.

27. The anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (VH) comprising the CDR-H1 sequence of SEQ ID NO: 42, the CDR-H2 sequence of SEQ ID NO: 43, and the CDR-H3 sequence of SEQ ID NO:

44. Ｈ CD28), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 45, the CDR-L2 sequence of SEQ ID NO: 46, and the CDR-L3 sequence of SEQ ID NO:

47. Ｌ 27. An anti-CD19 / anti-CD28 bispecific antibody for use according to any one of claims 1 to 26, comprising a first antigen-binding domain comprising a CD19-targeted 4-1BB (CD137) agonist, and an anti-CD20 / anti-CD3 bispecific antibody, use, method, kit or medicament in combination with a CD19-targeted 4-1BB (CD137) agonist.

28. The anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:

48. Ｈ CD28), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 49 Ｌ 28. An anti-CD19 / anti-CD28 bispecific antibody for use according to any one of claims 1 to 27, comprising a first antigen-binding domain comprising a CD19-targeted 4-1BB (CD137) agonist, and an anti-CD20 / anti-CD3 bispecific antibody, use, method, kit or medicament in combination with a CD19-targeted 4-1BB (CD137) agonist.

29. The anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (VH) comprising the CDR-H1 sequence of SEQ ID NO: 10, the CDR-H2 sequence of SEQ ID NO: 11, and the CDR-H3 sequence of SEQ ID NO:

12. Ｈ CD19), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 13, the CDR-L2 sequence of SEQ ID NO: 14, and the CDR-L3 sequence of SEQ ID NO:

15. Ｌ 29. An anti-CD19 / anti-CD28 bispecific antibody for use according to any one of claims 1 to 28, comprising a second antigen-binding domain comprising a CD19 targeting 4-1BB (CD137) and an anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeting 4-1BB (CD137) agonist, use, method, kit or medicament.

30. The anti-CD19 / anti-CD28 bispecific antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:

16. Ｈ CD19), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 17 Ｌ 30. An anti-CD19 / anti-CD28 bispecific antibody for use, use, method, kit or medicament in combination with a CD19-targeted 4-1BB (CD137) agonist according to any one of claims 1 to 29, comprising a second antigen-binding domain comprising a CD19 targeting 4-1BB (CD137).

31. 31. An anti-CD19 / anti-CD28 bispecific antibody for use and an anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament according to any one of claims 1 to 30, wherein said anti-CD19 / anti-CD28 bispecific antibody comprises an Fc domain, in particular an IgG1 or IgG4 Fc domain, and comprises one or more amino acid substitutions that reduce or eliminate binding to an Fc receptor and / or effector function.

32. the anti-CD19 / anti-CD28 bispecific antibody comprises a first polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 50; a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 51; and a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

52.

32. The anti-CD19 / anti-CD28 bispecific antibody for use and anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament of any one of claims 1 to 31, comprising a third polypeptide comprising an amino acid sequence that is 100% identical, and a fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

53.

33. 33. The anti-CD19 / anti-CD28 bispecific antibody for use and anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament of any one of claims 1 to 32, wherein the combination therapy is administered at intervals of about 1 to 3 weeks.

34. The B-cell proliferative disorder is selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL), and marginal zone lymphoma (MR).

34. The anti-CD19 / anti-CD28 bispecific antibody for use in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament according to any one of claims 1 to 33, wherein the anti-CD19 / anti-CD28 bispecific antibody is selected from the group consisting of multiple myeloma (MM) and Hodgkin's lymphoma (HL).

35. 35. The anti-CD19 / anti-CD28 bispecific antibody for use and anti-CD20 / anti-CD3 bispecific antibody in combination with a CD19-targeted 4-1BB (CD137) agonist, use, method, kit or medicament according to any one of claims 1 to 34, wherein the B-cell proliferative disorder is non-Hodgkin's lymphoma (NHL) or diffuse large B-cell lymphoma (DLBCL).

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