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

Abstract

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

Description

Technical Field

[0001] The present invention relates to combination therapies employing combinations of anti-CD20 / anti-CD3 bispecific antibodies with anti-CD19 / anti-CD28 bispecific antibodies and 4-1BB (CD137) agonists targeting CD19, and to the use of such combination therapies for the treatment of B-cell proliferative disorders such as diffuse large B-cell lymphoma (DLBCL). Background Art

[0002] B-cell proliferative disorders describe a heterogeneous group of malignancies including leukemias and lymphomas. Lymphomas develop 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 there is considerable heterogeneity within B-cell subsets based on genotype and phenotypic expression patterns in B-cell origin. For example, subsets of B-cell lymphomas include slow-growing indolent diseases and incurable diseases such as follicular lymphoma (FL) or chronic lymphocytic leukemia (CLL), as well as more aggressive subtypes, 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% to 40% of all NHL diagnoses, followed by follicular lymphoma (FL; 20% to 25% of all NHL diagnoses) and mantle cell lymphoma (MCL; 6% to 10% of all NHL diagnoses). B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in adults, with approximately 15,000 new cases per year in the United States (American Cancer Society, 2015). Despite the availability of multiple drugs for the treatment of B-cell proliferative disorders, there remains a need to develop safe and effective therapies to prolong patient remission and improve cure rates.

[0003] An anti-CD20 / anti-CD3 bispecific antibody is a molecule that targets CD20 expressed on B cells and the CD3ε chain (CD3ε) present on T cells. Simultaneous binding leads to T cell activation and T cell-mediated B cell killing. In the presence of B cells expressing CD20, whether circulating or tissue-resident, a pharmacological active dose of an anti-CD20 / anti-CD3 bispecific antibody triggers T cell activation and the release of associated cytokines. Glofitamab is a T cell bispecific (TCB) antibody that targets CD20 expressed on B cells and the CD3ε chain (CD3ε) present on T cells. In parallel with B cell depletion in peripheral blood, an anti-CD20 / anti-CD3 bispecific antibody causes a transient reduction of T cells in peripheral blood within 24 hours after the first administration, and cytokine release peaks, followed by a rapid T cell recovery and cytokine levels returning to baseline within 72 hours. Therefore, in order to achieve complete elimination of tumor cells, additional reagents are needed to preserve T cell activation and deliver a persistent immune response to cancer cells.

[0004] 4-1BB (CD137) is an inducible member of the tumor 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 cells of non-hematopoietic origin, such as endothelial cells and smooth muscle cells. The expression of 4-1BB in different cell types is mostly inducible and is driven by various stimulatory signals, such as T cell receptor (TCR) or B cell receptor triggering and signal transduction induced through co-stimulatory molecules or receptors for pro-inflammatory cytokines. The 4-1BB ligand (4-1BBL or CD137L) was identified in 1993. It has been shown that the expression of 4-1BBL is restricted to professional antigen-presenting cells (APCs) such as B cells, DCs, and macrophages. The inducible expression of 4-1BBL is characteristic of T cells (including both αβ and γδ T cell subsets) and endothelial cells.

[0005] Co-stimulation through the 4-1BB receptor (e.g., ligation by 4-1BBL) activates T cells (CD4 + and CD8 +Multiple signaling cascades within two subsets) potently enhance T cell activation. In combination with TCR triggering, agonistic 4-1BB-specific antibodies enhance T cell proliferation, stimulate lymphokine secretion and reduce the sensitivity of T lymphocytes to activation-induced cell death. This mechanism was further advanced as the first proof of concept for cancer immunotherapy. In preclinical models of tumor-bearing mice treated with agonistic antibodies against 4-1BB, potent anti-tumor effects were generated. Subsequently, increasing evidence indicates that 4-1BB generally exhibits its potency as an anti-tumor agent only when administered in combination with other immunomodulatory compounds, chemotherapeutic agents, tumor-specific vaccination or radiotherapy (Bartkowiak and Curran, 2015).

[0006] Signaling of the TNFR superfamily requires crosslinking of trimeric ligands to engage the receptor, and the same is true for 4-1BB agonistic antibodies that require wild-type Fc binding. However, systemic administration of 4-1BB-specific agonistic antibodies with a functionally active Fc domain results in the influx of CD8 + T cells, and this hepatotoxicity is attenuated or significantly improved in the absence of functional Fc receptors in mice. In clinical practice, the Fc-competent 4-1BB agonist Ab (BMS-663513) (NCT00612664) caused grade 4 hepatitis, leading to termination of the trial. Therefore, there is a need for effective and safer 4-1BB agonists. An example thereof is an antigen-binding molecule consisting of a trimeric and thus bioactive 4-1BB ligand, an antigen-binding domain specific for the tumor antigen CD19, and a silent Fc domain (referred to herein as CD19-4-1BBL). This construct has been described in WO 2016 / 075278 and replaces non-specific FcγR-mediated crosslinking that causes Fc-mediated toxicity by B cell-specific crosslinking targeting CD19.

[0007] CD19 is an ideal target for immunotherapy of B cell malignancies because it is expressed on the surface of B cells and is almost specific to these cells. During B cell development, CD19 is more widely expressed on B cells than CD20, so usually CD20-positive cells will also express CD19. During the differentiation of B cells towards plasma cells (antibody-secreting cells), B cells downregulate CD20 expression. Sometimes, B cell lymphomas also downregulate the expression of CD20 but remain positive for CD19. Therefore, targeting both CD19 and CD20 will widely cover the diseased B cells in lymphoma, which may also shift the selection pressure from CD20 to both CD19 and CD20. Although it is not clear whether CD19 directly contributes to B cell carcinogenesis, its expression is highly conserved in most B cell tumors such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B cell lymphoma. In acute leukemia, CD19 is stable and continuously expressed in almost all subtypes, while only a few leukemias express CD20.

[0008] CD28 is a founding member of the co-stimulatory molecule subfamily, characterized by paired V-set immunoglobulin superfamily (IgSF) domains attached to a single transmembrane domain and a cytoplasmic domain containing key signaling motifs. Other members of this subfamily include ICOS, CTLA-4, PD1, PD1H, TIGIT, and BTLA. CD28 expression is restricted to T cells and is ubiquitous in all naive subsets and most subsets that have experienced antigen stimulation, including those expressing PD-1 or CTLA-4. CD28 and CTLA-4 are highly homologous and compete for binding to the same B7 molecules, CD80 and CD86, which are expressed on dendritic cells, B cells, macrophages, and tumor cells. The higher affinity of CTLA-4 for the B7 ligand family enables CTLA-4 to outcompete CD28 in ligand binding and inhibit effector T cell responses. In contrast, PD-1 has been shown to inhibit CD28 signaling by partially dephosphorylating the cytoplasmic domain of CD28. Recent evidence has demonstrated 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 CD4 and CD8 positive T cells. After providing the so-called signal 1 via TCR or CD3 engagement, co-stimulation via CD28 activates multiple signaling cascades within T cells to enhance T cell-mediated immune responses. CD28-mediated signal 2 is thought to occur via co-clustering at the immunological synapse. CD28 agonist antibodies can be administered together with signal 1 providers, such as antibodies targeting CD3, to enhance immune responses. Immune stimulation is a complex cascade, and uncontrolled responses pose significant risks. The phase I study of the human CD28 antibody TGN1412 triggered a life-threatening cytokine storm in 2006. In contrast to TGN1412, anti-CD19 / anti-CD28 bispecific antibodies avoid autonomous T cell activation because T cell proliferation, cytokine secretion, and tumor cell killing are only induced in the presence of tumor cells expressing CD19 and signal 1 via TCR or CD3 engagement.

[0009] With the recent development of second-generation and later generations of T cells engineered to express chimeric antigen receptors (CAR-T) for patients with CD19-positive malignancies, CD28 has regained significant attention as an immunotherapy target. Proof-of-concept has been demonstrated by several trials showing response rates of 64% to 82% in heavily pretreated NHL patients using autologous anti-CD19-directed CAR-T therapy, including the CD28 signaling domain. However, major limitations of CAR-T cell therapy must still be addressed, including life-threatening CAR-T cell-related toxicity inhibition and resistance in B cell malignancies, adverse events after infusion such as cytokine release syndrome (CRS) and neurotoxicity, and host rejection of non-human CARs. General barriers include cell manufacturing limitations, baseline quality of T cells, and infusion time. For autologous CAR-T cell therapy, it takes 4 to 6 weeks to prepare engineered CAR T cells from a patient's T cells, and the delay may compromise the outcome of treatment. In contrast, bispecific antibodies are off-the-shelf available.

[0010] Given that current standard-of-care treatments do not cure all patients with B cell proliferative disorders, there is clearly a need to develop new therapies that are effective and specific. SUMMARY OF THE INVENTION

[0011] The present invention relates to anti-CD20 / anti-CD3 bispecific antibodies and their use in combination with anti-CD19 / anti-CD28 bispecific antibodies and a 4-1BB (CD137) agonist targeting CD19 for use in combination therapy for treating cancer, particularly for treating B cell proliferative disorders. It has been found that the combination therapy described herein is more effective in inhibiting tumor growth and eliminating tumor cells than treatment with a combination of anti-CD20 / anti-CD3 bispecific antibodies with anti-CD19 / anti-CD28 bispecific antibodies alone or with a 4-1BB (CD137) agonist targeting CD19 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 4-1BB (CD137) agonist targeting CD19 for use in combination therapy for treating B cell proliferative disorders.

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

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

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

[0016] In another aspect, there is provided a medicament comprising an anti-CD20 / anti-CD3 bispecific antibody for treating 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 4-1BB (CD137) agonist targeting CD19.

[0017] In a particular aspect, there is provided a combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a 4-1BB (CD137) agonist targeting CD19 for use as described above, wherein the combination therapy comprises a first treatment regimen with a combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-CD19 / anti-CD28 bispecific antibody and a second treatment regimen with a combination of an anti-CD20 / anti-CD3 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19.

[0018] In one aspect, the first treatment regimen comprises 1 to 5 treatment cycles, and the second treatment regimen starts from the next treatment cycle. In another aspect, the first treatment regimen comprises 1 to 5 treatment cycles, and the second treatment regimen starts from the next treatment cycle. In one aspect, the first treatment regimen comprises 4 treatment cycles, and the second treatment regimen starts from treatment cycle 5. In one aspect, 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, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use, wherein prior to the combination therapy, pre-treatment is carried out with a type II anti-CD20 antibody, preferably obinutuzumab, and wherein the time period between the pre-treatment and the combination therapy is sufficient to reduce B cells in an individual responsive to the type II anti-CD20 antibody, preferably obinutuzumab.

[0020] In one aspect of the invention, the 4-1BB agonist targeting CD19 comprises three extracellular domains of 4-1BBL, each extracellular domain 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 extracellular domain of 4-1BBL comprises the amino acid sequence of SEQ ID NO:5. In one aspect, the 4-1BB agonist targeting CD19 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 Fc receptors and / or effector functions. More particularly, the 4-1BB agonist targeting CD19 comprises an IgG1 Fc domain which comprises the amino acid substitutions L234A, L235A and P329G (EU numbering according to Kabat).

[0021] In a further aspect, there is provided a combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use as described above, wherein the 4-1BB agonist targeting CD19 comprises an antigen-binding domain capable of specifically binding to CD19, the antigen-binding domain comprising a heavy chain variable region (V H CD19) and a light chain variable region (V LCD19), wherein the heavy chain variable region comprises: (i) CDR-H1, which comprises the amino acid sequence of SEQ ID NO:10, (ii) CDR-H2, which comprises the amino acid sequence of SEQ ID NO:11, and (iii) CDR-H3, which comprises the amino acid sequence of SEQ ID NO:12, and the light chain variable region comprises: (iv) CDR-L1, which comprises the amino acid sequence of SEQ ID NO:13, (v) CDR-L2, which comprises the amino acid sequence of SEQ ID NO:14, and (vi) CDR-L3, which comprises the amino acid sequence of SEQ ID NO:15. In particular, the anti-CD19 4-1BB agonist comprises an antigen-binding domain capable of specifically binding to CD19, and the antigen-binding domain comprises a heavy chain variable region (V H CD19) and a light chain variable region (V L CD19), the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:16, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:17.

[0022] In another aspect, there is provided a combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody and an anti-CD19 / anti-CD28 bispecific antibody and an anti-CD19 4-1BB (CD137) agonist for use according to any one of the preceding paragraphs, wherein the anti-CD19 4-1BB agonist comprises

[0023] (a) a first polypeptide, which comprises: (a1) the first extracellular domain of 4-1BBL or a fragment thereof, which is fused at its C-terminus to the N-terminus of the second extracellular domain of 4-1BBL or a fragment thereof, (a2) the second extracellular domain of 4-1BBL or a fragment thereof, which is fused at its C-terminus to the N-terminus of the CL domain, (a3) the CL domain, which is fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain (e.g., the first subunit), and (a4) one of the subunits of the Fc domain (e.g., the first subunit);

[0024] (b) a second polypeptide, which comprises: (b1) the third extracellular domain of 4-1BBL or a fragment thereof, which is fused at its C-terminus to the N-terminus of the CH1 domain, and (b2) the CH1 domain;

[0025] (c) a third polypeptide, which comprises: (c1) the heavy chain of a Fab molecule that binds to CD19, which is fused at its C-terminus to the N-terminus of the other subunit of the Fc domain (e.g., the second subunit), and (c2) the other subunit of the Fc domain (e.g., the second subunit); and

[0026] (d) a fourth polypeptide, which comprises the light chain of a Fab molecule that binds to CD19.

[0027] In one aspect, there is provided a combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use as described above, wherein the 4-1BB agonist targeting CD19 comprises: a first 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:18; a second 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:19; 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:20; 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. More specifically, the 4-1BB agonist targeting CD19 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.

[0028] In a further aspect, there is provided a combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use as disclosed above, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises: a first antigen-binding domain comprising a heavy chain variable region (V H CD3) and a light chain variable region (V L CD3); and a second antigen-binding domain comprising a heavy chain variable region (V H CD20) and a light chain variable region (V L CD20). In one aspect, the first antigen-binding domain comprises: a heavy chain variable region (V H CD3) that comprises 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; and / or a light chain variable region (V LCD3), which comprises 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. In one aspect, the first antigen-binding domain comprises: a heavy-chain variable region (V H CD3), which comprises the amino acid sequence of SEQ ID NO:28; and / or a light-chain variable region (V L CD3), which comprises the amino acid sequence of SEQ ID NO:29. In one aspect, the second antigen-binding domain comprises: a heavy-chain variable region (V H CD20), which comprises the CDR-H1 sequence of SEQID NO:30, the CDR-H2 sequence of SEQ ID NO:31, and the CDR-H3 sequence of SEQ ID NO:32; and / or a light-chain variable region (V L CD20), which comprises 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. In one aspect, the second antigen-binding domain comprises: a heavy-chain variable region (V H CD20), which comprises the amino acid sequence of SEQ ID NO:36; and / or a light-chain variable region (V L CD20), which comprises the amino acid sequence of SEQ IDNO:37.

[0029] In a further aspect, there is provided a combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody as disclosed above for use with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises a third antigen-binding domain that binds to CD20. In a particular aspect, the anti-CD20 / anti-CD3 bispecific antibody comprises: a first 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: 38; a second 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: 39; 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: 40; and fourth and fifth polypeptides, each 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. 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, each comprising the amino acid sequence of SEQ ID NO: 41. More particularly, the anti-CD20 / anti-CD3 bispecific antibody is gefapixant.

[0030] In one aspect, there is provided a combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody as disclosed above for use with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19, wherein the anti-CD19 / anti-CD28 bispecific antibody comprises: a first antigen-binding domain comprising a heavy chain variable region (V H CD28) and a light chain variable region (V L CD28); and a second antigen-binding domain comprising a heavy chain variable region (V H CD19) and a light chain variable region (V L CD19). In one aspect, the anti-CD19 / anti-CD28 bispecific antibody comprises a first antigen-binding domain that comprises: a heavy chain variable region (V HCD28), which comprises a CDR-H1 sequence of SEQ ID NO:42, a CDR-H2 sequence of SEQ ID NO:43, and a CDR-H3 sequence of SEQ ID NO:44; and / or a light chain variable region (V L CD20), which comprises a CDR-L1 sequence of SEQ ID NO:45, a CDR-L2 sequence of SEQ ID NO:46, and a CDR-L3 sequence of SEQ ID NO:47. In one aspect, the anti-CD19 / anti-CD28 bispecific antibody comprises a first antigen-binding domain, which first antigen-binding domain comprises: a heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO:48; and / or a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO:49. In one aspect, the anti-CD19 / anti-CD28 bispecific antibody comprises a second antigen-binding domain, which second antigen-binding domain comprises: a heavy chain variable region (V H CD19), which comprises a CDR-H1 sequence of SEQ ID NO:10, a CDR-H2 sequence of SEQ ID NO:11, and a CDR-H3 sequence of SEQ ID NO:12; and / or a light chain variable region (V L CD19), which comprises a CDR-L1 sequence of SEQ ID NO:13, a CDR-L2 sequence of SEQ ID NO:14, and a CDR-L3 sequence of SEQ ID NO:15. In one aspect, the anti-CD19 / anti-CD28 bispecific antibody comprises a second antigen-binding domain, which second antigen-binding domain comprises: a heavy chain variable region (V H CD19), which comprises the amino acid sequence of SEQ ID NO:16; and / or a light chain variable region (V L CD19), which comprises the amino acid sequence of SEQ ID NO:17.

[0031] In one aspect, there is provided a combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody as described above for use with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19, 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 Fc receptors and / or effector functions. More particularly, the anti-CD19 / anti-CD28 bispecific antibody comprises an IgG1 Fc domain that comprises the amino acid substitutions L234A, L235A and P329G (EU numbering according to Kabat).

[0032] In a particular aspect, the anti-CD19 / anti-CD28 bispecific antibody comprises: a first 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:50; a second 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:51; 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:52; 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. 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.

[0033] In a further aspect, the present invention provides a combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody as described above for use with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19, wherein the combination therapy is administered at intervals of from about one week to about three weeks.

[0034] In one aspect, there is provided a combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody as disclosed above for use with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19, wherein the B cell proliferative disorder is selected from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM) and Hodgkin lymphoma (HL). In a particular aspect, the B cell proliferative disorder is diffuse large B cell lymphoma (DLBCL).

[0035] In a further aspect, the present invention provides a combination, purposes, method, kit or medicine of an anti-CD20 / anti-CD3 bispecific antibody for use and an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 as disclosed above, wherein anti-CD20 / anti-CD3 bispecific antibody, anti-CD19 / anti-CD28 bispecific antibody and 4-1BB (CD137) agonist targeting CD19 are administered intravenously. On the other hand, anti-CD20 / anti-CD3 bispecific antibody, anti-CD19 / anti-CD28 bispecific antibody and 4-1BB (CD137) agonist targeting CD19 are administered subcutaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figures 1A to 1C Schematic diagram of a specific CD19-4-1BBL antigen binding molecule, a specific anti-CD20 / anti-CD3 bispecific antibody, and a specific anti-CD19 / anti-CD28 bispecific antibody as used in the examples. These molecules are described in more detail in Examples 1, 2, and 3, respectively. Thick black dots represent knob-in-hole structural modifications. * represents amino acid modifications in the CH1 and CL domains (so-called charged variants). Figure 1A A monovalent CD19 4-1BBL trimer is shown, which contains a modified antigen binding molecule in which the CH1 and CL domains near the 4-1BBL dimer and 4-1BBL monomer are modified. This molecule is designated herein as CD19-4-1BBL. Figure 1B An exemplary bispecific anti-CD20 / anti-CD3 antibody (designated as CD20-TCB or Glofitamab) in a 2+1 format is shown in FIG. Figure 1C An exemplary bispecific anti-CD19 / anti-CD28 antibody in a 1+1 crossfab format is shown in . The VH and VL domains of the CD19 antigen binding domain are exchanged such that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

[0037] Figures 2A to 2D The results 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 in patients undergoing malignant splenectomy for stage IVB B-cell lymphoma compared to CD20-TCB treatment alone. Figure 2A )、IFNγ( Figure 2B )、IL-8( Figure 2C ) and IL-2 (Fig. Figure 2D )’s release.

[0038] Figure 3Shows the study design of an efficacy study to evaluate the triple combination effect of CD20-TCB with CD19-4-1BBL and CD19-CD28 in the human OCI-Ly18 xenograft in humanized NSG mice. Shown is the design for different treatment groups A to G (10 mice per group), i.e., various injections at different time points.

[0039] Figures 4A to 4G Shows the results of the efficacy study of the OCI-Ly18 xenograft in humanized NSG mice. Shown is the growth of tumors in individual mice plotted on the y-axis for seven treatment groups. Figure 4A Shows the tumor growth in each individual mouse in the vehicle group, Figure 4B are the mice treated with CD20-TCB, Figure 4C are the mice treated with CD20-TCB and CD19-CD28, and Figure 4D are the mice treated with CD20-TCB and CD19-4-1BBL. Figure 4E Shows the tumor growth of mice that first received combination treatment with CD20-TCB and CD19-4-1BBL and switched the treatment to the combination of CD20-TCB and CD19-CD28 on day 66. Figure 4F Shows the tumor growth of mice that first received combination treatment with CD20-TCB and CD19-CD28 and subsequently (on day 66) received combination treatment with CD20-TCB and CD19-4-1BBL. It can be seen that among all treated animals, the group that received the alternating treatment regimen of starting with CD19-CD28 for the first four cycles followed by CD19-4-1BBL combination treatment until the end of the study achieved complete control of the tumor within 120 days, while the alternating treatment regimen of starting with CD19-4-1BBL for the first four cycles followed by CD19-CD28 combination treatment did not completely inhibit tumor growth. Figure 4G Shows the tumor growth of mice receiving concomitant triple combination treatment with CD20-TCB, CD19-4-1BBL, and CD19-CD28. Interestingly, compared to treatment with CD20-TCB and CD19-4-1BBL alone, simultaneous administration of CD20-TCB, CD19-CD28, and CD19-4-1BBL did not improve tumor growth control.

[0040] Figure 5 Shows the time-to-event analysis of the triple combination efficacy study. Using 1500m 3Tumor volume cut-off value. The survival probability (in %) was plotted against time (in days). Mice treated first with the combination of CD20-TCB and CD19-CD28 and then with the combination of CD20-TCB and CD19-4-1BBL had the highest survival chance (100%).

[0041] Figure 6 The study design of an efficacy study is shown to evaluate the triple combination effect of CD20-TCB with CD19-4-1BBL and CD19-CD28 in the human OCI-Ly18 xenograft in humanized BRGS-CD47 mice. Shown is the design for different treatment groups A to G (10 mice per group), i.e., various injections at different time points. The combination treatment started one week earlier compared to the first humanized NSG mouse study.

[0042] Figures 7A to 7G The results of an efficacy study of the OCI-Ly18 xenograft in humanized BRGS-CD47 mice are shown. Shown is the growth of tumors in individual mice plotted on the y-axis for seven treatment groups. Figure 7A The tumor growth of each individual mouse in the vehicle group is shown, Figure 7B which are mice treated with CD20-TCB only, Figure 7C which are mice treated with CD20-TCB and CD19-CD28, and Figure 7D which are mice treated with CD20-TCB and CD19-4-1BBL. The combination treatment started on day 27. Figure 7E The tumor growth of mice that first received the combination treatment of CD20-TCB and CD19-4-1BBL and switched the treatment to the combination of CD20-TCB and CD19-CD28 on day 55 is shown. Figure 7F The tumor growth of mice that first received the combination treatment of CD20-TCB and CD19-CD28 and then (on day 55) received the combination treatment of CD20-TCB and CD19-4-1BBL is shown. It can be seen that in most of the treated animals, the group that received the alternating treatment regimen of starting with CD19-CD28 for the first four cycles followed by CD19-4-1BBL combination treatment until the end of the study achieved better control of the tumor within 94 days, while the alternating treatment regimen of starting with CD19-4-1BBL for the first four cycles followed by CD19-CD28 combination treatment did not completely inhibit tumor growth. Figure 7GTumor growth in mice receiving concomitant triple combination therapy of CD20-TCB, CD19-4-1BBL, and CD19-CD28 is shown. In this experiment, tumor control in the concomitant group was stronger than the combination of CD20-TCB and CD19-4-1BBL, which may be due to the earlier start of combination therapy. The group receiving an alternating treatment regimen of 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 of concomitant administration within 94 days.

[0043] Figures 8A to 8C Comparison between the corresponding treatment regimens in two studies is shown. Shown are the differences in tumor growth of mice that first received the combination therapy of CD20-TCB and CD19-4-1BBL and received the combination therapy of CD20-TCB and CD19-CD28 on day 55 ( Figure 8A ), the differences in tumor growth of mice that first received the combination therapy of CD20-TCB and CD19-CD28 and thereafter (on day 55) received the combination therapy of CD20-TCB and CD19-4-1BBL ( Figure 8B ), and the differences in tumor growth of mice receiving concomitant triple combination therapy of CD20-TCB, CD19-4-1BBL, and CD19-CD28 ( Figure 8C ), indicating that starting combination therapy one week earlier can better control tumors than starting combination therapy later. Detailed Description

[0044] Definitions

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the field to which this invention belongs. For the purpose of interpreting this specification, the following definitions will be applied, and where appropriate, terms used in the singular will also include the plural and vice versa.

[0046] As used herein, the term "antigen-binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are antibodies, antibody fragments, and scaffold antigen-binding proteins. The term "antibody" as used herein 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 substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or arising during the production of the monoclonal antibody preparation, such variants typically being present in minor amounts). In contrast to polyclonal antibody preparations, which generally include 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 denotes an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. The term "bispecific" refers to an antigen-binding molecule that is capable of specifically binding to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule comprises two antigen-binding sites, each of which is specific for a different antigenic determinant. However, a bispecific antigen-binding molecule may also comprise additional antigen-binding sites that bind to other antigenic determinants. In some aspects, a bispecific antigen-binding molecule is capable of binding two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells or on the same cell. Thus, the term "bispecific" according to the present disclosure may also include trispecific molecules, such as a bispecific molecule comprising a CD28 antibody and two antigen-binding domains directed against two different target cell antigens.

[0047] As used herein, the term "antigen-binding domain that binds to a B cell surface antigen" or "portion capable of specifically binding to a B cell surface antigen" refers to a polypeptide molecule that specifically binds to an antigenic determinant on the surface of a B cell. In one aspect, the antigen-binding domain is capable of activating signal transduction through its target cell antigen. In certain aspects, the antigen-binding domain is capable of directing an entity attached thereto (e.g., a CD28 agonist) to a target site (e.g., 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. Additionally, antigen-binding domains capable of specifically binding to a B cell surface antigen include scaffold antigen-binding proteins as further defined herein, such as binding domains based on designed repeat proteins or designed repeat domains (see, e.g., WO 2002 / 020565).

[0048] As used herein, the term "valence" refers to the presence of a specific number of binding sites specific for a unique antigenic determinant in an antigen-binding molecule specific for a unique antigenic determinant. Thus, the terms "bivalent", "tetravalent" and "hexavalent" indicate the presence of two, four and six binding sites specific for a particular antigenic determinant, respectively, in the antigen-binding molecule. In certain aspects of the invention, the bispecific antigen-binding molecules according to the invention can be monovalent for a particular antigenic determinant, meaning that they have only one binding site for said antigenic determinant, or can be bivalent or tetravalent for a particular antigenic determinant, meaning that they have two or four binding sites for said antigenic determinant, respectively.

[0049] The terms "full-length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody. A "native antibody" refers to an immunoglobulin molecule that occurs naturally with a distinct structure. For example, a native IgG class antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also referred to as a variable heavy domain or heavy chain variable domain), followed by three constant domains (CH1, CH2, and CH3) (also referred to as heavy chain constant regions). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) (also referred to as a variable light domain or light chain variable domain), followed by a light chain constant domain (CL) (also referred to as a light chain constant region). The heavy chains of an antibody can be assigned to one of five types, which are designated alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), some of which can be further divided into subtypes, such as gamma1 (IgG1), gamma2 (IgG2), gamma3 (IgG3), gamma4 (IgG4), alpha1 (IgA1), and alpha2 (IgA2). The light chains of an antibody can be assigned to one of two types based on the amino acid sequence of their constant domain, which are designated kappa (κ) and lambda (λ).

[0050] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2; bispecific antibodies, trispecific antibodies, tetraspecific antibodies, crossFab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives, see U.S. Patent No. 5,869,046. Bispecific antibodies are antibody fragments that have two antigen-binding sites and can be bivalent or bispecific, see, e.g.: EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Trispecific and tetraspecific antibodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that comprise all or part of the heavy-chain variable domain or all or part of the light-chain variable domain of an antibody. In certain embodiments, the single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1). Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies, and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0051] Papain digestion of a complete antibody produces two identical antigen-binding fragments called "Fab" fragments, each "Fab" fragment containing a variable heavy chain domain and a variable light chain domain, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, as used herein, the term "Fab fragment" refers to an antibody fragment that contains a light chain fragment containing a variable light chain (VL) domain and the constant domain of the light chain (CL), as well as a variable heavy chain (VH) domain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that the Fab' fragment has additional residues added at the carboxyl terminus of the heavy chain CH1 domain, which residues include one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine residue of the constant domain has a free sulfhydryl group. Pepsin treatment yields F(ab') 2 fragments, which have two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0052] The so-called "crossed" Fab molecule (also referred to as "Crossfab") means a Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., replaced with each other), i.e., the crossed Fab molecule contains a peptide chain composed of a variable light chain domain VL and a constant heavy chain domain 1CH1 (VL-CH1, in the N-terminal to C-terminal direction), and a peptide chain composed of a variable heavy chain domain VH and a constant light chain domain CL (VH-CL, in the N-terminal to C-terminal direction). For clarity, in a crossed Fab molecule in which the variable domain of the Fab light chain and the variable domain of the Fab heavy chain are exchanged, the peptide chain containing the constant heavy chain domain 1CH1 is referred to herein as the "heavy chain" of the (crossed) Fab molecule. Conversely, in a crossed Fab molecule in which the constant domain of the Fab light chain and the constant domain of the Fab heavy chain are exchanged, the peptide chain containing the variable heavy chain domain VH is referred to herein as the "heavy chain" of the (crossed) Fab molecule.

[0053] In contrast, the so-called "conventional" Fab molecule means a Fab molecule in its native form, i.e., containing a heavy chain composed of a variable and a constant heavy chain domain (VH-CH1, in the N-terminal to C-terminal direction), and a light chain composed of a variable and a constant light chain domain (VL-CL, in the N-terminal to C-terminal direction).

[0054] "Single-chain variable fragment (scFv)" is the variable region of the heavy chain of an antibody (V H ) and the variable region of the light chain (V L) fusion protein, linked by a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine to obtain flexibility and rich in serine or threonine to obtain solubility, and can connect the N-terminus of V H to the C-terminus of V L , or vice versa. Although the constant region is removed and a linker is introduced, the protein retains the specificity of the original antibody. scFv antibodies are described, for example, in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96. Additionally, an antibody fragment comprises a single-chain polypeptide characterized by having a VH domain, i.e., capable of assembling with a VL domain into a functional antigen-binding site; or characterized by having a VL domain, i.e., capable of assembling with a VH domain into a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0055] An "antigen-binding molecule that binds to the same epitope" as a reference molecule refers to an antigen-binding molecule that blocks the binding of the reference molecule to its antigen by 50% or more in a competition assay, and conversely, the reference molecule blocks the binding of the antigen-binding molecule to its antigen by 50% or more in a competition assay.

[0056] The term "antigen-binding domain" refers to a part of an antigen-binding molecule that contains a region that specifically binds and is complementary to a part or all of an antigen. In the case of a large antigen, the antigen-binding molecule may bind only to a specific part of the antigen, which is called an epitope. The antigen-binding domain can be provided by, for example, 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).

[0057] As used herein, the term "epitope" is synonymous with "antigen" and "epitope", and refers to a site on a polypeptide macromolecule (e.g., a continuous stretch of amino acids or a conformational configuration consisting of different regions of non - contiguous amino acids) to which an antigen - binding moiety binds to form an antigen - binding moiety - antigen complex. Useful epitopes can be found, for example, on the surface of tumor cells, on the surface of virus - infected cells, on the surface of other diseased cells, on the surface of immune cells, in free substances in serum and / or in the extracellular matrix (ECM). Unless otherwise specified, a protein used as an antigen herein can be any native form of a protein from any vertebrate source, which vertebrate sources include mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a particular embodiment, the antigen is a human protein. When referring to a specific protein herein, the term encompasses "full - length", unprocessed proteins, as well as any form of protein produced by intracellular processing. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants.

[0058] "Specifically binds" means that the binding is selective for the antigen and can be distinguished from unwanted or non - specific 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 (e.g., surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323 - 329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217 - 229 (2002)). In one embodiment, for example, as measured by SPR, the degree of binding of an antigen - binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen - binding molecule to the antigen. In certain embodiments, a molecule that binds to an antigen has a dissociation constant (Kd) 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 lower, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0059] "Affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between the members of the binding pair (such as an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by the 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 include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0060] As used herein, "B cell surface antigen" refers to an antigenic determinant presented on the surface of B lymphocytes, particularly malignant B lymphocytes (in which case the antigen is also referred to as a "malignant B cell surface antigen"). In the context of immunotherapy for hematological malignancies, several B cell surface antigens are of interest. In one aspect, the B cell surface antigen is selected from the group consisting of CD19, CD79b, CD20, CD22, and CD37.

[0061] The term "CD19" refers to B-lymphocyte antigen CD19, also known as B-lymphocyte surface antigen B4 or T-cell surface antigen Leu-12, and unless otherwise indicated, the term includes any native CD19 from any vertebrate source, which vertebrate source includes mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human CD19 is shown in Uniprot accession number P15391 (version 160, SEQ ID NO:54). The term encompasses "full-length" unprocessed human CD19 as well as any form of human CD19 produced by processing in cells, provided that an antibody as reported herein binds thereto. CD19 is a structurally distinct cell surface receptor expressed on the surface of human B cells, which human B cells include but are not limited to pre-B cells, early developing B cells (i.e., immature B cells), mature B cells that terminally differentiate into plasma cells, and malignant B cells. CD19 is expressed by most pre-B acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, B-cell chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, hairy cell leukemia, common acute lymphocytic leukemia, and some null-acute lymphocytic leukemia. Expression of CD19 on plasma cells further suggests that it may be expressed on differentiated B-cell tumors such as multiple myeloma. Thus, the CD19 antigen is a target for immunotherapy for the treatment of non-Hodgkin lymphoma, chronic lymphocytic leukemia, and / or acute lymphocytic leukemia.

[0062] "CD20" refers to the B lymphocyte antigen CD20, also known as B lymphocyte surface antigen B1 or leukocyte surface antigen Leu-16, and unless otherwise specified, the term includes any native CD20 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human CD20 is shown in Uniprot accession number P11836 (version 149, SEQ ID NO:55). CD20 is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD and is expressed on 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 nascent protein family are characterized by common structural features and similar intron / exon splicing boundaries and exhibit unique expression patterns in 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. Members of this family are localized to 11q12 in a cluster of family members. Alternative splicing of this gene produces two transcript variants that encode the same protein. The term "CD20" encompasses the "full-length" unprocessed CD20, as well as any form of CD20 produced by processing in cells. The term also encompasses naturally occurring variants of CD20, such as splice variants or allelic variants.

[0063] The terms "anti-CD20 antibody" and "antibody that binds CD20" refer to an antibody that is capable of binding CD20 with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent that targets CD20. In one embodiment, the degree of binding of an anti-CD20 antibody to an unrelated non-CD20 protein is less than about 10% of the degree of binding of the antibody to CD20, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the antibody that binds CD20 is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or lower, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the anti-CD20 antibody binds to an epitope of CD20 that is conserved among CD20s from different species.

[0064] "Type II anti-CD20 antibody" refers to an anti-CD20 antibody having the binding characteristics and biological activities of a Type II anti-CD20 antibody, such as those 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.

[0065] Table A. Properties of type I and type II anti-CD20 antibodies

[0066] Type I anti-CD20 antibody Type II anti-CD20 antibody Binds to class I CD20 epitope Binds to class II CD20 epitope Localizes CD20 to lipid rafts Does not localize CD20 to lipid rafts High CDC* Low CDC* ADCC activity* ADCC activity* Full binding ability to B cells Approximately half-binding ability to B cells Weak homotypic aggregation Homotypic aggregation Low cell death induction Strong cell death induction

[0067] * If IgG 1 isotype

[0068] Examples of Type II anti-CD20 antibodies include, for example, obinutuzumab (GA101), tositumomab (B1), humanized B-Ly1 antibody IgG1 (such as the chimeric humanized IgG1 antibody disclosed in WO 2005 / 044859), 11B8 IgG1 (such as that disclosed in WO 2004 / 035607), and AT80 IgG1.

[0069] In one aspect, the Type II anti-CD20 antibody comprises the heavy chain variable region sequence of SEQ ID NO:36 (V H CD20) and / or the light chain variable region sequence of SEQ ID NO:37 (V L CD20). In another aspect, compared to non-engineered antibodies, the Type II anti-CD20 antibody is engineered to have an increased proportion of non-fucosylated oligosaccharides in the Fc region. In one aspect, at least about 40% of the N-linked oligosaccharides in the Fc region of the Type II anti-CD20 antibody are non-fucosylated. In a particular aspect, the Type II anti-CD20 antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:56 and the light chain comprising the amino acid sequence of SEQ ID NO:57. The antibody is named GA101 or obinutuzumab (Proposed INN, WHO Drug Information, Volume 26, Issue 4, 2012, page 453).

[0070] Trade name or

[0071] Examples of type I anti-CD20 antibodies include, for example, rituximab, ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab, PRO131921, ublituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in WO 2005 / 103081), 2F2 IgG1 (as disclosed in WO 2004 / 035607 and WO 2005 / 103081), and 2H7 IgG1 (as disclosed in WO 2004 / 056312).

[0072] The term "reduce" (and its grammatical variants such as "reduce" or "reducing"), e.g., a reduction in the number of B cells or a reduction in the amount of cytokine release, refers to a decrease in the corresponding amount as measured by an appropriate method known in the art. For clarity, the term also includes a reduction to zero (or below the limit of detection of the assay method), i.e., complete removal or elimination. In contrast, "increased" refers to an increase in the corresponding amount.

[0073] As used herein, "T cell antigen" refers to an antigenic determinant present on the surface of T lymphocytes, particularly cytotoxic T lymphocytes.

[0074] As used herein, a "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. Other examples include chimeric antigen receptors (CARs) that comprise a T cell activation domain and an antigen binding portion that specifically binds to a target cell antigen (e.g., CD20 or CD19).

[0075] As used herein, an "activating T cell antigen" refers to an antigenic determinant expressed by T lymphocytes, particularly cytotoxic T lymphocytes, that is capable of inducing or enhancing T cell activation upon interaction with an antigen binding molecule. Specifically, the interaction of the antigen binding molecule with the activating T cell antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. An exemplary activating T cell antigen is CD3.

[0076] Unless otherwise indicated, the term "CD3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses "full-length" unprocessed CD3, as well as any form of CD3 produced by processing in cells. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, CD3 is human CD3, particularly the ε subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown by UniProt (www.uniprot.org) accession number P07766 (version 144) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. See also SEQ ID NO:58. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank accession number BAB71849.1. See also SEQ ID NO:59.

[0077] Unless otherwise indicated, the term "CD28" (cluster of differentiation 28, Tp44) refers to any CD28 protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). CD28 is expressed on T cells and provides co-stimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), stimulation of T cells through CD28 can provide effective signals for the production of various interleukins. CD28 is a 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 shown in UniProt (www.uniprot.org) accession number P10747 (SEQ ID NO:60).

[0078] An "agonistic antibody" refers to an antibody that contains the agonistic function against a given receptor. Generally, when an agonist ligand (factor) binds to a receptor, the tertiary structure of the receptor protein changes and the receptor is activated (when the receptor is a membrane protein, usually transducing cell growth signals, etc.). If the receptor is of the dimer formation type, an agonistic antibody can dimerize the receptor at an appropriate distance and angle, thus acting similarly to the ligand. A suitable anti-receptor antibody can mimic the receptor dimerization carried out by the ligand and thus can become an agonistic antibody.

[0079] "CD28 agonistic antibody" or "CD28 conventional agonistic antibody" is an antibody that mimics the role of the natural ligands of CD28 (CD80 or CD86) in enhancing T cell activation in the presence of T cell receptor signals ("signal 2"). T cells require two signals to be fully activated. Under physiological conditions, "signal 1" is generated by the interaction of the T cell receptor (TCR) molecule with the peptide / major histocompatibility complex (MHC) complex on the antigen-presenting cell (APC), and "signal 2" is provided by co-stimulatory receptors such as CD28. A CD28 agonistic antigen-binding molecule can co-stimulate T cells (signal 2). It can also jointly induce T cell proliferation and cytokine secretion with a molecule specific for the TCR complex, but a CD28 agonistic antigen-binding molecule cannot fully activate T cells without additional stimulation of the TCR. However, there is a subclass of CD28-specific antigen-binding molecules, namely the so-called CD28 superagonistic antigen-binding molecules. A "CD28 superagonistic antibody" is a CD28 antibody that can fully activate T cells without additional stimulation of the TCR. A CD28 superagonistic antibody can induce T cell proliferation and cytokine secretion without prior activation of T cells (signal 1). An example of a CD28 superagonistic antibody is TGN1412 (disclosed in WO 2006 / 050949).

[0080] 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 such that the antibody can be used as a diagnostic agent and / or therapeutic agent in targeting CD28. In one embodiment, as measured, for example, by radioimmunoassay (RIA) or fluorescence-activated cell sorting (FACS), the degree of binding of the anti-CD28 antibody to an unrelated non-CD28 protein is less than about 10% of the degree of binding of the antibody to CD28. In certain embodiments, the dissociation constant (K D ) of the antibody that binds to CD28 is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -6 M or less, e.g., 10 -6 M to 10 -13 M, e.g., 10 -8 M to 10 -10 M).

[0081] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that participates in the binding of an antigen-binding molecule to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of a natural antibody typically have similar structures, each containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. As used herein, "Kabat numbering" with respect to a variable region sequence refers to the numbering system set forth by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0082] As used herein, the amino acid positions of all constant regions and constant domains of the heavy and light chains are numbered according to the Kabat numbering system described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "according to Kabat numbering" or "Kabat numbering." Specifically, the Kabat numbering system (see pages 647 to 660 of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of κ and λ isotypes, and the Kabat EU index numbering system (see pages 661 to 723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3), which is further clarified herein by referring to this case as "according to Kabat EU index numbering."

[0083] As used herein, the term "hypervariable region" or "HVR" refers to each region within an antibody variable domain that is highly variable in sequence and determines antigen binding specificity, such as "complementary determining regions" ("CDRs"). Generally, an antibody contains six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include:

[0084] (a) hypervariable loops 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));

[0085] (b) CDRs 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

[0086] (c) antigen - contacting points 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)).

[0087] Unless otherwise specified, CDRs are determined according to the method described by Kabat et al. (supra). Those skilled in the art will understand that CDR designations can also be determined according to the methods described by Chothia (supra), McCallum (supra), or any other scientifically - accepted nomenclature system.

[0088] As used herein, the term "affinity matured" in the context of an antigen binding molecule (e.g., an antibody) refers to an antigen binding molecule derived from a reference antigen binding molecule, e.g., by mutation, that binds to the same antigen as the reference antibody, preferably to the same epitope; and has a higher affinity for the antigen than the reference antigen binding molecule. Affinity maturation generally involves modification of 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 initial reference antigen binding molecule.

[0089] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain is usually composed of the following four FR domains: FR1, FR2, FR3 and FR4. Therefore, HVR and FR sequences usually appear in the following sequence in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0090] For the purposes of this article, "acceptor human framework" is a framework that includes an 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. The acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may include an amino acid sequence identical to the human immunoglobulin framework or a human consensus framework, or it may include 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 to the VL human immunoglobulin framework sequence or a human consensus framework sequence in sequence.

[0091] 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, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0092] The "class" of an antibody refers to the type of constant domain or region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG. 1 IgG 2 IgG 3 IgG 4 , IgA 1 and IgA 2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0093] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will generally comprise all of at least one, and usually two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody in "humanized form", e.g., a non-human antibody, refers to an antibody that has been humanized. Other forms of "humanized antibodies" encompassed by the present invention are antibodies wherein, relative to the original antibody, the constant region has been additionally modified or altered to produce the properties according to the present invention, particularly with regard to C1q binding and / or Fc receptor (FcR) binding properties.

[0094] A "human" antibody is an antibody that has an amino acid sequence corresponding to an antibody produced by a human or a human cell, or an amino acid sequence derived from an antibody of non-human origin using a human antibody library or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0095] The term "CH1 domain" refers to the portion of the antibody heavy chain polypeptide that generally extends from EU position 118 to EU position 215 (according to the EU numbering system of Kabat). In one aspect, the CH1 domain has the amino acid sequence ASTKGPSVFP LAPSSKSTSGGTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPSNTKVDKKV (SEQ ID NO:61). Typically, a fragment having the amino acid sequence EPKSC (SEQ ID NO:62) follows to link the CH1 domain to the hinge region.

[0096] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that links the CH1 domain and the CH2 domain in a wild-type antibody heavy chain, e.g., from approximately position 216 to approximately position 230 according to the Kabat EU numbering system, or from approximately position 226 to approximately position 230 according to the Kabat EU numbering system. The hinge regions of other IgG subclasses can be determined by alignment with the hinge region cysteine residues of the IgG1 subclass sequence. The hinge region is typically a dimer molecule composed of two polypeptides with identical amino acid sequences. The hinge region typically 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: the upper hinge region, the middle hinge region, and the lower hinge region (see, e.g., Roux et al., J. Immunol. 161 (1998) 4083).

[0097] The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region contains the IgG CH2 domain and the IgG CH3 domain.

[0098] The "CH2 domain" of the human IgG Fc region typically extends from the amino acid residue at approximately EU position 231 to the amino acid residue at approximately EU position 340 (according to the Kabat EU numbering system). In one aspect, the CH2 domain has the amino acid sequence APELLGGPSVFLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDWLNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO:63). The CH2 domain is unique in that its pairing with another domain is not tight. Instead, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of the intact native Fc region. It is speculated that the carbohydrate may provide an alternative for domain-domain pairing and contribute to the stabilization of the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206. In one embodiment, the carbohydrate chain is linked to the CH2 domain. The CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain.

[0099] The "CH3 domain" includes the extension of the C-terminus of the residues in the Fc region to the CH2 domain, representing the portion of the antibody heavy chain polypeptide that extends generally from position EU 341 to position EU 446 (according to the EU numbering system of Kabat). In one aspect, the CH3 domain has the amino acid sequence of GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO:64). The CH3 region herein can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "bump" ("stalk") in one of its chains and a corresponding introduced "cavity" ("socket") in its other chain; see U.S. Patent No. 5,821,333, which is hereby expressly incorporated by reference). Such variant CH3 domains can be used to facilitate the heterodimerization of two different antibody heavy chains as described herein. In one embodiment, the 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 or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0100] The "mortar and pestle structure" technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., ProtEng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("pestle") at the interface of a first polypeptide and a corresponding cavity ("mortar") in the interface of a second polypeptide such that the protrusion can be positioned within the cavity to facilitate heterodimer formation and impede homodimer formation. The protrusion is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (such as tyrosine or tryptophan). A compensatory cavity 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 amino acid side chains (such as alanine or threonine). The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis. In a specific embodiment, the pestle modification comprises the amino acid substitution T366W in one of the two subunits comprising the Fc domain, while the mortar modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits comprising the Fc domain. In another specific embodiment, the subunit comprising the pestle-modified Fc domain additionally comprises the amino acid substitution S354C, while the subunit comprising the mortar-modified Fc domain additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bond between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0101] "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 modified variants having the ability to produce substitutions, additions, or deletions but substantially not reducing immunoglobulin-mediated effector functions such as antibody-dependent cytotoxicity. By way of 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 are selected according to general rules known in the art so as to have a minimal impact on activity (see, for example, Bowie, J.U. et al., Science 247: 1306-10 (1990)).

[0102] The term "wild-type Fc domain" refers to an amino acid sequence that is identical to the amino acid sequence of the Fc domain found in nature. Wild-type human Fc domains include the native human IgG1 Fc region (non-A and A allotypes), the native human IgG2 Fc region, the native human IgG3 Fc region, and the native human IgG4 Fc region, as well as naturally occurring variants thereof. The human IgG1 Fc region is represented in SEQ ID NO:65.

[0103] The term "variant (human) Fc domain" refers to an amino acid sequence that differs from the amino acid sequence of the "wild-type" (human) Fc domain by at least one "amino acid mutation". In one aspect, the variant Fc region has at least one amino acid mutation compared to the native Fc region, such as from about one to about ten amino acid mutations in the native Fc region, and in one aspect, from about one to about five amino acid mutations. In one aspect, the (variant) Fc region has at least about 95% homology with the wild-type Fc region.

[0104] The term "effector function" refers to those biological activities that can be attributed to the Fc region of an antibody and that vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, antigen uptake by immune complex-mediated antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0105] Fc receptor-binding dependent effector functions can be mediated through the interaction of the Fc region of an antibody with an Fc receptor (FcR), which are specific cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of red blood cells and various other cell targets (e.g., tumor cells) coated with the corresponding antibody by antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., 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: Fc receptors for IgG antibodies are called FcγRs. Fc receptor binding is described, for example, in: Ravetch, J.V. and Kinet, J.P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P.J. et al., Immunomethods 4 (1994) 25-34; de Haas, M. et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J.E. et al., Ann. Hematol. 76 (1998) 231-248.

[0106] Cross-linking of the Fc region receptor of IgG antibodies (FcγR) triggers multiple effector functions, including phagocytosis, antibody-dependent cytotoxicity, release of inflammatory mediators, and regulation of immune complex clearance and antibody production. Three classes of FcγRs have been identified in humans, including:

[0107] -FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils. Modification of at least one residue in the Fc region of IgG at amino acid residues E233-G236, P238, D265, N297, A327, and P329 (EU index numbering according to Kabat) reduces binding to FcγRI. Substitution of the IgG2 residues at positions 233-236 with IgG1 and IgG4 results in a 103-fold reduction in binding to FcγRI and abolishes the response of human monocytes to antibody-sensitized red blood cells (Armour, K.L. et al., Eur. J. Immunol. 29 (1999) 2613–2624).

[0108] -FcγRII (CD32) binds IgG complexes with moderate to low affinity and is widely expressed. This receptor can be divided into two subtypes, namely FcγRIIA and FcγRIIB. FcγRIIA is present in many cells involved in killing (such as macrophages, monocytes, neutrophils), and seems to be able to activate the killing process. FcγRIIB seems to play a role in the inhibitory process and is present in B cells, macrophages, as well as mast cells and eosinophils. On B cells, it seems to play a role in inhibiting further production of immunoglobulins and isotype switching to, for example, the IgE class. On macrophages, FcγRIIB is used to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B type may contribute to the inhibition of activation of these cells through the binding of IgE to its individual receptor. Antibodies are found, for example, (including IgG Fc regions with mutations in at least one amino acid residue E233 - G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (EU index number according to Kabat)) with reduced binding affinity for FcγRIIA.

[0109] -FcγRIII (CD16) binds IgG with moderate to low affinity and includes two types. FcγRIIIA is present on NK cells, macrophages, eosinophils, as well as some monocytes and T cells, and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. Antibodies are found, for example, (including IgG Fc regions with mutations in at least one amino acid residue E233 - G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (EU index number according to Kabat)) with reduced binding affinity for FcγRIIIA.

[0110] Shields, R.L. et al. J. Biol. Chem. 276 (2001) 6591 - 6604 describes the localization of the binding sites to Fc receptors on human IgG1, the above - mentioned mutation sites, and the method for measuring binding to FcγRI and FcγRIIA.

[0111] The term "ADCC" or "antibody-dependent cell cytotoxicity" is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. The target cell is a cell that specifically binds to an antibody or a derivative thereof that contains an Fc region, and this specific binding is typically through the protein moiety at the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as a decrease in the number of target cells lysed by the ADCC mechanism as defined above at a given antibody concentration in the culture medium surrounding the target cells over a given time period, and / or an increase in the antibody concentration required to achieve lysis of a given number of target cells by the ADCC mechanism in the culture medium surrounding the target cells over a given time period. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cell but not engineered, using the same standard production, purification, formulation, and storage methods (which are known to those skilled in the art). For example, the reduction in ADCC mediated by an antibody containing an amino acid substitution in the Fc domain that reduces ADCC is relative to the ADCC mediated by the same antibody without that amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, PCT Publication No. WO 2006 / 082515 or PCT Publication No. WO 2012 / 130831). For example, the ability of an antibody to induce the initial steps of ADCC-mediated lysis is investigated by measuring the binding of the antibody to cells expressing Fcγ receptors (e.g., cells recombinantly expressing FcγRI and / or FcγRIIA or NK cells (which inherently express FcγRIIIA)). In particular, the binding to FcγR on NK cells is measured.

[0112] An "activating Fc receptor" is an Fc receptor that, upon engagement of the Fc region of an antibody, causes a signaling event that stimulates the cell carrying the receptor to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activating Fc receptor is human FcγRIIIa (see UniProt accession number P08637, version 141).

[0113] The term "effector function" refers to those biological activities that are attributable to the Fc region of an antibody and that vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, antigen uptake by immune complex-mediated antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0114] As used herein, the term "effector cell" refers to a population of lymphocytes that display on their surface effector moiety receptors (such as cytokine receptors) and / or Fc receptors through which they bind effector moieties (such as cytokines) and / or the Fc region of antibodies and contribute to the destruction of target cells, such as tumor cells. Effector cells can, for example, mediate cytotoxicity or phagocytosis. Effector cells include, but are not limited to, effector T cells, such as CD8 + cytotoxic T cells, CD4 + helper T cells, γδ T cells, NK cells, lymphokine-activated killer (LAK) cells, and macrophages / monocytes.

[0115] The "extracellular domain" is the domain of a membrane protein that extends into the extracellular space (i.e., the space outside the target cell). The extracellular domain is typically the part of the protein that initiates surface contact leading to signal transduction. Thus, the extracellular domain of 4-1BBL as defined herein refers to the part of 4-1BBL that extends into the extracellular space (extracellular domain), but also includes the shorter part or fragment thereof responsible for trimerization and for binding to the corresponding receptor 4-1BB. Thus, the term "extracellular domain or fragment thereof of 4-1BBL" refers to the extracellular domain of 4-1BBL that forms the extracellular domain or the part thereof that is still capable of binding to the receptor (receptor-binding domain).

[0116] "4-1BBL", "4-1BB ligand", or "CD137L" is a co-stimulatory TNF ligand family member that can co-stimulate T cell proliferation and cytokine production. Co-stimulatory TNF family ligands can co-stimulate TCR signaling upon interaction with their corresponding TNF receptors, and interaction with their receptors leads to the recruitment of TNFR-associated factors (TRAF), thereby initiating a signaling cascade that results in T cell activation. 4-1BBL is a type II transmembrane protein. A full-length or full-length 4-1BBL having the amino acid sequence of SEQ ID NO:66 has been described to form trimers on the cell surface. The formation of trimers is facilitated by specific motifs in the extracellular domain of 4-1BBL. The motifs are designated herein as "trimerization regions". Amino acids 50-254 of the human 4-1BBL sequence (SEQ ID NO:9) form the extracellular domain of 4-1BBL, but even fragments thereof are capable of forming trimers. In a specific embodiment of the invention, the term "extracellular domain or fragment thereof of 4-1BBL" refers to a polypeptide having an amino acid sequence selected from SEQ ID NO:4 (amino acids 52-254 of human 4-1BBL), SEQ ID NO:1 (amino acids 71-254 of human 4-1BBL), SEQ ID NO:3 (amino acids 80-254 of human 4-1BBL), SEQ ID NO:2 (amino acids 85-254 of human 4-1BBL), SEQ ID NO:5 (amino acids 71-248 of human 4-1BBL), SEQ ID NO:6 (amino acids 85-248 of human 4-1BBL), SEQ ID NO:7 (amino acids 80-248 of human 4-1BBL), SEQ ID NO:8 (amino acids 52-248 of human 4-1BBL), and SEQ ID NO:9 (amino acids 50-254 of human 4-1BBL), but other fragments of the extracellular domain capable of trimerization are also included herein.

[0117] Unless otherwise specified, the term "4-1BB" or "CD137" as used herein refers to any native 4-1BB from any vertebrate source, including mammals such as primates (e.g., human) and rodents (e.g., mouse and rat). The term encompasses "full-length" unprocessed 4-1BB, as well as any form of 4-1BB produced by processing in cells. 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 shown in SEQ ID NO:67 (Uniprot accession number Q07011).

[0118] The term "peptide linker" refers to a peptide comprising one or more amino acids (usually about 2 to 20 amino acids). Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides are, for example, (G 4 S) 2 (SEQ ID NO:68).

[0119] As used herein, the term "amino acid" refers to the group of naturally occurring carboxy α-amino acids comprising the following: 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).

[0120] "Fusion" or "linkage" means that components (e.g., the extracellular domain of a polypeptide and 4-1BBL) are joined by a peptide bond, directly or via one or more peptide linkers.

[0121] "Percent amino acid sequence identity (%)" relative to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the amino acid residues in the candidate sequence with the amino acid residues in the reference polypeptide sequence and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. The alignment for determining the percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN.SAWI or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes herein, the sequence comparison computer program ALIGN-2 is used to generate the percent amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office, Washington D.C., 20559, where it 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 the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are invariant. In the case of performing amino acid sequence comparison using ALIGN-2, the percent amino acid sequence identity of a given amino acid sequence A with or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain percent amino acid sequence identity with or against a given amino acid sequence B) is calculated as follows:

[0122] 100 times the fraction X / Y

[0123] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that in the case where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A with B will not be equal to the percent amino acid sequence identity of B with A. Unless otherwise specifically indicated, all values of percent amino acid sequence identity used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.

[0124] 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 the antigen-binding molecule can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct, provided that the final construct has the desired characteristics, such as antigen binding. Sites of interest for substitution mutagenesis include the HVRs and framework (FR). Conservative substitutions are provided 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 the target molecule and the product screened for the desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0125] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. The target cell is a cell to which an antibody or a fragment thereof containing an Fc region specifically binds, usually via the protein moiety at the N-terminus of the Fc region. As used herein, the term “increased / reduced ADCC” is defined as an increase / decrease in the number of target cells lysed by the ADCC mechanism as defined above at a given antibody concentration in the culture medium surrounding the target cells over a given time period, and / or a decrease / increase in the antibody concentration required to effect lysis of a given number of target cells by the ADCC mechanism over a given time period in the culture medium surrounding the target cells. The increase / reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cell that has not been engineered, using the same standard production, purification, formulation, and storage methods (which are known to those of skill in the art). For example, relative to the ADCC mediated by the same antibody produced by the same type of unengineered host cell, the ADCC mediated by an antibody produced by a host cell engineered by the methods disclosed herein to have an altered glycosylation pattern (e.g., to express a glycosyltransferase, GnTIII, or other glycosyltransferase) is increased.

[0126] Antibodies with reduced effector function include those having substitutions in one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). Certain antibody variants with improved or reduced binding to FcR are described. (See, e.g., U.S. Patent 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 having 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.

[0127] In certain aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In one aspect, the substitutions are L234A and L235A (LALA). In certain aspects, the antibody variant further comprises D265A and / or P329G in the Fc region derived from the human IgG1 Fc region. In one aspect, in the Fc region derived from the human IgG1 Fc region, the substitutions are L234A, L235A, and P329G (LALA-PG). (See, e.g., WO2012 / 130831). In another aspect, in the Fc region derived from the human IgG1 Fc region, the substitutions are L234A, L235A, and D265A (LALA-DA).

[0128] In some embodiments, for example, as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000), alterations are made in the Fc region, resulting in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC).

[0129] Antibodies having an extended half-life and improved neonatal Fc receptor (FcRn) binding, which is responsible for the transfer of maternal IgG to the fetus (Guyer, R.L. et al., J. Immunol. 117:587 (1976), and Kim, J.K. et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934 (Hinton et al.). Those antibodies contain an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include Fc variants having substitutions at one or more of the following 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, such as a substitution at Fc region residue 434 (see, e.g., U.S. Patent No. 7,371,826; Dall'Acqua, W.F. et al., J. Biol. Chem. 281 (2006) 23514-23524).

[0130] In some aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253, and / or 310, and / or 435 of the Fc region (EU numbering of residues). In some aspects, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310, and 435. In one aspect, in an Fc region derived from a human IgG1 Fc region, the substitutions are I253A, H310A, and H435A. See, e.g., Grevys, A. et al., J. Immunol. 194 (2015) 5497-5508.

[0131] In another aspect, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 310, and / or 433, and / or 436 of the Fc region (EU numbering of residues). In some aspects, the antibody variant comprises an Fc region having amino acid substitutions at positions 310, 433, and 436. In one aspect, in an Fc region derived from a human IgG1 Fc region, the substitutions are H310A, H433A, and Y436A. (See, e.g., WO 2014 / 177460 A1).

[0132] An "effective amount" of an agent is an amount sufficient to produce a physiological change in the cells or tissues to which it is administered.

[0133] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical composition) is an amount effective to achieve the desired therapeutic or prophylactic result at the required dosage and for the required period of time. A therapeutically effective amount of an agent, for example, abolishes, reduces, delays, minimizes or prevents the adverse effects of a disease.

[0134] 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.

[0135] The term "pharmaceutical composition" refers to a formulation that is in a form that permits the bioactive ingredients contained therein to be biologically active and that contains no additional components that are unacceptably toxic to the subject to which the formulation is to be administered.

[0136] A "pharmaceutically acceptable carrier" is a component of a pharmaceutical composition that is non-toxic to the subject in addition to the active ingredient. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers or preservatives.

[0137] The term "package insert" is used to refer to the instructions that are typically included in the commercial packaging of a therapeutic product and that contain information regarding indications, usage, dosage, administration, combination therapy, contraindications and / or warnings relating to the use of such therapeutic product.

[0138] As used herein, "treatment" (and its grammatical variants such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated and that may be prophylactic or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and relieving or improving the prognosis. In some embodiments, the molecules of the invention are used to delay the development of a disease or to slow the progression of a disease.

[0139] As used herein, the term "cancer" refers to a proliferative disease such as lymphoma or lymphocytic leukemia or melanoma.

[0140] "B cell proliferative disorder" means a disease in which the number of B cells in a patient is increased compared to the number of B cells in a healthy subject, particularly where the increase in the number of B cells is a cause or hallmark of the disease. "CD20-positive B cell proliferative disorder" is 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 diseases include non-Hodgkin lymphoma (NHL), acute lymphoblastic 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 lymphoma (HL). Particular B cell proliferative conditions are non-Hodgkin lymphoma (NHL) or diffuse large B cell lymphoma (DLBCL). In certain aspects, the B cell proliferative condition is diffuse large B cell lymphoma (DLBCL).

[0141] The present invention relates to a combination of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a CD19-targeted 4-1BB (CD137) agonist for use in combination therapy for treating B-cell proliferative disorders. Scheduling studies conducted with obinutuzumab and CD19-CD28 in humanized NSG mice showed that a safe and potent treatment regimen is to pre-treat with Gazyva and then stagger the infusion of obinutuzumab and the anti-CD19 / anti-CD28 bispecific antibody CD19-CD28 at three-day intervals during the first treatment cycle. In huNSG mice bearing a refractory disseminated WSU-DLCL2 DLBCL tumor model, combination treatment with obinutuzumab and CD19-CD28 led to the formation of tumor-free animals compared to the corresponding monotherapy groups. In vivo mode of action studies revealed that for both CD4+ and CD8+ T-cell subsets, obinutuzumab-mediated T-cell infiltration was strongly enhanced without any evidence of increased regulatory T-cell activity. Second-line treatment with CD19-CD28 was able to prolong the duration of the obinutuzumab response and delay tumor recurrence in vivo in the subcutaneous OCI-Ly18 DLBCL model. Interestingly, the alternation of CD19-CD28 with a CD19-targeted 4-1BB (CD137) agonist (CD19-4-1BBL), giving CD19-CD28 during the first treatment cycle and then CD19-4-1BBL in subsequent cycles, completely prevented tumor recurrence during more than 120 days of obinutuzumab treatment. Finally, CD19-CD28 enhanced obinutuzumab-mediated cytokine secretion and T-cell activation in ex vivo DLBCL patient samples, validating its activity against T cells from both healthy donors and patients. Collectively, the preclinical data show that there is good reason to combine CD19-CD28 with CD20 TCB in r / r NHL patients to deepen and further prolong the treatment response, however the optimal scheduling involves alternating combination therapy with CD20 TCB (obinutuzumab) and a CD19-targeted 4-1BB (CD137) agonist (CD19-4-1BBL).

[0142] Exemplary anti-CD20 / anti-CD3 bispecific antibodies for use in the present invention

[0143] As used herein, an anti-CD20 / anti-CD3 bispecific antibody is a bispecific antibody comprising a first antigen-binding domain that binds to CD3 and a second antigen-binding domain that binds to CD20.

[0144] Thus, an anti-CD20 / anti-CD3 bispecific antibody as used herein comprises: comprising a heavy-chain variable region (V H CD3) and a light-chain variable region (VL a first antigen-binding domain that binds to CD3, and a heavy chain variable region (V H CD20) and a light chain variable region (V L CD20) of a second antigen-binding domain.

[0145] In one particular aspect, the anti-CD20 / anti-CD3 bispecific antibody used in combination comprises a first antigen-binding domain that comprises: a heavy chain variable region (V H CD3) that comprises 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; and / or a light chain variable region (V L CD3) that comprises 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. More particularly, the anti-CD20 / anti-CD3 bispecific antibody comprises a first antigen-binding domain that comprises a heavy chain variable region (V H CD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:28 and / or a light chain variable region (V L CD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:29. In a further aspect, the anti-CD20 / anti-CD3 bispecific antibody comprises: a heavy chain variable region (V H CD3) that comprises the amino acid sequence of SEQ ID NO:28; and / or a light chain variable region (V L CD3) that comprises the amino acid sequence of SEQ ID NO:29.

[0146] In one aspect, the antibody that specifically binds to CD3 is a full-length antibody. In one aspect, the antibody that specifically binds to CD3 is a human IgG class antibody, particularly a human IgG 1 class antibody. In one aspect, the antibody that specifically binds to CD3 is an antibody fragment, particularly a Fab molecule or an scFv molecule, more particularly a Fab molecule. In one particular aspect, the antibody that specifically binds to CD3 is a crossed Fab molecule, wherein the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., replaced with each other). In one aspect, the antibody that specifically binds to CD3 is a humanized antibody.

[0147] In another aspect, the anti-CD20 / anti-CD3 bispecific antibody comprises a second antigen-binding domain that comprises a heavy chain variable region (V H CD20) and / or a light chain variable region (VL CD20), the heavy chain variable region contains 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, and the light chain variable region contains 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. More specifically, the anti-CD20 / anti-CD3 bispecific antibody contains a second antigen-binding domain, which contains a heavy chain variable region (V H CD20) and / or a light chain variable region (V L CD20) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:37. H CD20). In a further aspect, the anti-CD20 / anti-CD3 bispecific contains a second antigen-binding domain, which includes a heavy chain variable region (V L CD20) and / or a light chain variable region (V

[0148] In another specific aspect, the anti-CD20 / anti-CD3 bispecific antibody contains a third antigen-binding domain that binds to CD20. In particular, the anti-CD20 / anti-CD3 bispecific antibody contains a third antigen-binding domain, which contains a heavy chain variable region (V H CD20) and / or a light chain variable region (V L CD20), the heavy chain variable region contains 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, and the light chain variable region contains 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. More particularly, the anti-CD20 / anti-CD3 bispecific antibody contains a third antigen-binding domain, which contains a heavy chain variable region (V H CD20) and / or a light chain variable region (V LCD20). In another aspect, the anti-CD20 / anti-CD3 bispecific antibody comprises a third antigen-binding domain, which 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).

[0149] In another aspect, 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 are exchanged, and the second and third (if present) antigen-binding domains are conventional Fab molecules.

[0150] In another aspect, the anti-CD20 / anti-CD3 bispecific antibody is a bispecific antibody, wherein (i) the second antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain at the C-terminus of the Fab heavy chain, the first antigen-binding domain is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and the third antigen-binding domain is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain, or (ii) the first antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain at the C-terminus of the Fab heavy chain, the second antigen-binding domain is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and the third antigen-binding domain is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

[0151] The Fab molecule can be fused to the Fc domain directly or via a peptide linker, which comprises one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, (G 4 S) 2 peptide linker (SEQ ID NO:68). Additionally, the linker can comprise (a portion of) an immunoglobulin hinge region. In particular, in the case of fusion of the Fab molecule to the N-terminus of the Fc domain subunit, the fusion can be via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

[0152] In another aspect, the anti-CD20 / anti-CD3 bispecific antibody comprises an Fc domain, which comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or decrease effector function. In particular, the anti-CD20 / anti-CD3 bispecific antibody comprises an IgG1 Fc domain, which comprises the amino acid substitutions L234A, L235A and P329G (according to the Kabat EU index number).

[0153] In certain aspects, the anti-CD20 / anti-CD3 bispecific antibody comprises: a first 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: 38; a second 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: 39; 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: 40; and fourth and fifth polypeptides, both 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. In a further specific embodiment, 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 twice the polypeptide sequence of SEQ ID NO: 41 (CD20 TCB). In one particular aspect, the anti-CD20 / anti-CD3 bispecific antibody is gefapixant.

[0154] Glofitamab (WHO Drug Information (International Nonproprietary Name 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, p. 276, also known as CD20-TCB, RO7082859, or RG6026; CAS No.: 2229047-91-8) is a T cell-engaging bispecific (TCB) full-length antibody with a 2:1 molecular configuration for bivalent binding to CD20 on B cells and monovalent binding to CD3 on T cells (specifically the CD3ε chain (CD3e)). Its CD3-binding region is fused via a flexible linker to one of the head-to-tail CD20-binding regions. This structure confers Glofitamab with superior in vitro potency compared to CD20-CD3 bispecific antibodies with other 1:1 configurations and produces significant anti-tumor efficacy in preclinical DLBCL models. CD20 bivalency retains this potency in the presence of competing anti-CD20 antibodies, providing opportunities for pretreatment or combination therapy with these drugs. Glofitamab contains an engineered heterodimeric Fc region with complete removal of binding to FcgR and C1q. By simultaneously binding human CD20-expressing tumor cells and CD3e of the T cell receptor (TCR) complex on T cells, it induces tumor cell lysis in addition to T cell activation, proliferation, and cytokine release. Glofitamab-mediated B cell lysis is CD20-specific and does not occur in the absence of CD20 expression or when T cells are not co-bound (cross-linked) to CD20-expressing cells. In addition to killing, T cells are activated due to CD3 cross-linking, which can be detected by an increase in T cell activation markers (CD25 and CD69), cytokine release (IFNγ, TNFα, IL-2, IL-6, IL-10), cytotoxic granule release (granzyme B), and T cell proliferation. A schematic picture of the molecular structure of Glofitamab is depicted in Figure 1B in.

[0155] Certain other bispecific antibodies are described in PCT Publication No. WO 2016 / 020309 A1 or WO 2015 / 095392 A1. In a further aspect, the antibody is Moxetumomab.

[0156] In another aspect, the anti-CD20 / anti-CD3 bispecific antibody may further comprise a bispecific T cell engager In another aspect, the anti-CD20 / anti-CD3 bispecific antibody is 13676. In another aspect, the bispecific antibody is REGN1979. In another aspect, the bispecific antibody is FBTA05 (Lymphomun).

[0157] Exemplary 4-1BB agonists for use in the present invention

[0158] In particular, a CD19-targeted 4-1BB (CD137) agonist for use in combination with an anti-CD20 / anti-CD3 bispecific antibody is a molecule comprising 4-1BBL. In particular, the 4-1BB agonist for use in the present invention comprises three extracellular domains of 4-1BBL or a fragment thereof.

[0159] In certain aspects, the CD19-targeted 4-1BB (CD137) agonist is a molecule comprising three extracellular domains of 4-1BBL or a fragment thereof, and wherein the extracellular domain 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, particularly the amino acid sequence of SEQ ID NO:5.

[0160] It has been shown that 4-1BB agonists comprising at least one antigen-binding domain capable of specifically binding to CD19 are not internalized into B cells by CD19 and thus do not lose their ability to interact with the tumor microenvironment. In one aspect, a 4-1BB agonist is provided that is not internalized in B cells, thereby maintaining its activity.

[0161] In another aspect, the CD19-targeted 4-1BB (CD137) agonist is an antigen-binding molecule comprising three extracellular domains of 4-1BBL or a fragment thereof and at least one moiety capable of specifically binding to CD19, wherein the antigen-binding domain capable of specifically binding to CD19 is cynomolgus monkey cross-reactive, i.e., the antigen-binding domain capable of specifically binding to CD19 specifically binds to both human and cynomolgus monkey CD19.

[0162] In a further aspect, the CD19-targeted 4-1BB (CD137) agonist is an antigen-binding molecule comprising three extracellular domains of 4-1BBL or a fragment thereof and at least one moiety capable of specifically binding to CD19, wherein the antigen-binding domain capable of specifically binding to CD19 comprises a heavy chain variable region (V H CD19) and a light chain variable region (V LCD19), wherein the heavy chain variable region comprises: (i) CDR-H1, which comprises the amino acid sequence of SEQ ID NO:10, (ii) CDR-H2, which comprises the amino acid sequence of SEQ ID NO:11, and (iii) CDR-H3, which comprises the amino acid sequence of SEQ ID NO:12, and the light chain variable region comprises: (iv) CDR-L1, which comprises the amino acid sequence of SEQ ID NO:13, (v) CDR-L2, which comprises the amino acid sequence of SEQ ID NO:14, and (vi) CDR-L3, which comprises the amino acid sequence of SEQ ID NO:15.

[0163] In a further aspect, the CD19-targeting 4-1BB (CD137) agonist is an antigen-binding molecule comprising three extracellular domains 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 H CD19) and a light chain variable region (V L CD19), the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:16, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:17

[0164] In another aspect, the CD19-targeting 4-1BB (CD137) agonist is an antigen-binding molecule further comprising an Fc domain composed of a first and a second subunit capable of stable association. In one aspect, the CD19-targeting 4-1BB (CD137) agonist is an antigen-binding molecule comprising an IgG Fc domain, particularly an IgG1 Fc domain or an IgG4 Fc domain. In particular, the CD19-targeting 4-1BB (CD137) agonist is an antigen-binding molecule comprising an Fc domain comprising one or more amino acid substitutions that reduce binding to Fc receptors and / or effector functions. In a specific aspect, the CD19-targeting 4-1BB (CD137) agonist is an antigen-binding molecule comprising an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A, and P329G.

[0165] In one aspect, the CD19-targeting 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 polypeptide and a second polypeptide, the first polypeptide and the second polypeptide being linked to each other by a disulfide bond,

[0166] The first polypeptide comprises two extracellular domains of 4-1BBL or fragments thereof linked to each other by a peptide linker, and is characterized in that the second polypeptide comprises one extracellular domain of 4-1BBL or a fragment thereof.

[0167] In another aspect, the CD19-targeted 4-1BB agonist comprises

[0168] (a) a first polypeptide comprising: (a1) a first extracellular domain of 4-1BBL or a fragment thereof, which is fused to the N-terminus of a second extracellular domain of 4-1BBL or a fragment thereof at the C-terminus, (a2) a second extracellular domain of 4-1BBL or a fragment thereof, which is fused to the N-terminus of a CL domain at the C-terminus, (a3) a CL domain, which is fused to the N-terminus of one of the subunits (e.g., the first subunit) of the Fc domain at the C-terminus, and (a4) one of the subunits (e.g., the first subunit) of the Fc domain;

[0169] (b) a second polypeptide comprising: (b1) a third extracellular domain of 4-1BBL or a fragment thereof, which is fused to the N-terminus of a CH1 domain at the C-terminus, and (b2) a CH1 domain;

[0170] (c) a third polypeptide comprising: (c1) the heavy chain of a Fab molecule that binds to CD19, which is fused to the N-terminus of the other subunit (e.g., the second subunit) of the Fc domain at the C-terminus, and (c2) the other subunit (e.g., the second subunit) of the Fc domain; and

[0171] (d) a fourth polypeptide comprising the light chain of a Fab molecule that binds to CD19.

[0172] In a particular aspect, the CD19-targeted 4-1BB agonist comprises: a first 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: 18; a second 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: 19; 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: 20; 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. 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. In a particular aspect, the CD19-targeted 4-1BB agonist is the CD19-targeted 4-1BB ligand (CD19-4-1BBL) or englumafusp alfa.

[0173] Englumafusp alfa (WHO Drug Information (International Nonproprietary Name for the Substance of a Pharmaceutical), Recommended INN: List 89, 2023, Volume 37, Issue 1, Page 97, also known as CD19-4-1BBL, RO7227166, RG6076; CAS number: 2417199-08-5) is a CD19-targeted 4-1BB ligand (CD19-4-1BBL). The potential mode of action of this molecule is to enhance the effector functions of tumor-infiltrating T cells or NK cells after activation by crosslinking 4-1BB-positive activated effector cells with CD19-positive tumor targets, either through a tumor-targeted T cell bispecific (TCB) antibody or antibody-dependent cell cytotoxicity (ADCC), respectively. Crosslinking of 4-1BB results in co-stimulation of immune cells, namely enhanced effects or functions (e.g., proliferation and production of interferon-γ and IL2), protection of cells from death (e.g., upregulation of anti-apoptotic pathways), and promotion of the development of immune memory and the creation of a durable immune response. By selectively promoting the immune response in the microenvironment of tumors expressing CD19, the risk of off-target immune responses is limited. The safety of englumafusp alfa is further enhanced by eliminating the interaction of the Fcγ receptor (FcγR) and C1q complex with the IgG1 antibody moiety to prevent the triggering of ADCC and antibody-dependent cell phagocytosis (ADCP) by inhibiting the co-activation of FcγR-mediated innate immune effector cells such as NK cells or macrophages / monocytes. A schematic picture of the molecular structure of englumafusp alfa is depicted in Figure 1A in.

[0174] In a further aspect, the 4-1BB agonist is an anti-CD19 / anti-4-1BB bispecific antibody.

[0175] Exemplary anti-CD28 bispecific antibodies for use in the present invention

[0176] As used herein, an anti-CD28 bispecific antibody is a bispecific agonistic CD28 antibody that comprises: 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 composed of first and second subunits that can stably associate, the Fc domain comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for Fc receptors. In one aspect, the bispecific agonistic CD28 antibody described herein is characterized by monovalent binding to CD28. In a further aspect, the bispecific agonistic CD28 antibody described herein is characterized by monovalent binding to a B cell surface antigen. In particular, the B cell surface antigen is CD19.

[0177] In one aspect, a bispecific agonistic CD28 antibody as defined above is provided, wherein the Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain. In a particular aspect, the Fc domain consisting of a first subunit and a second subunit capable of stable association is an IgG1 Fc domain. In particular, the Fc domain contains one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor. In one aspect, the Fc domain contains the amino acid substitutions L234A and L235A (according to the Kabat EU index numbering). In one aspect, the Fc domain belongs to the human IgG1 subclass and contains the amino acid mutations L234A, L235A, and P329G (according to the Kabat EU index numbering).

[0178] In one aspect, the anti-CD19 / anti-CD28 bispecific antibody as used herein comprises: a first antigen-binding domain comprising a heavy-chain variable region (V H CD28) and a light-chain variable region (V L CD28); and a second antigen-binding domain comprising a heavy-chain variable region (V H CD19) and a light-chain variable region (V L CD19).

[0179] In a further aspect, the anti-CD19 / anti-CD28 bispecific antibody comprises a first antigen-binding domain that comprises: a heavy-chain variable region (V H CD28) that comprises 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; and / or a light-chain variable region (V L CD28) that comprises 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. In a further aspect, the anti-CD19 / anti-CD28 bispecific antibody comprises a first antigen-binding domain that comprises: a heavy-chain variable region (V H CD28) that comprises 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; and / or a light-chain variable region (V LCD28), which comprises 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. In particular, the anti-CD19 / anti-CD28 bispecific antibody comprises a first antigen-binding domain that comprises: a heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 48; and / or a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 49.

[0180] In one aspect, the anti-CD19 / anti-CD28 bispecific antibody comprises a second antigen-binding domain that comprises: a heavy chain variable region (V H CD19), which comprises 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; and / or a light chain variable region (V L CD19), which comprises 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. In one aspect, the anti-CD19 / anti-CD28 bispecific antibody comprises a second antigen-binding domain that comprises: a heavy chain variable region (V H CD19), which comprises 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; and / or a light chain variable region (V L CD19), which comprises 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. In one aspect, the anti-CD19 / anti-CD28 bispecific antibody comprises a second antigen-binding domain that comprises: a heavy chain variable region (V H CD19), which comprises the amino acid sequence of SEQ ID NO: 16; and / or a light chain variable region (V L CD19), which comprises the amino acid sequence of SEQ ID NO: 17.

[0181] In a particular aspect, the anti-CD19 / anti-CD28 bispecific antibody comprises: a first 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:50; a second 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:51; 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:52; 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. 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. A schematic picture of the molecular structure of the anti-CD19 / anti-CD28 bispecific antibody is depicted in Figure 1C In.

[0182] Preparation of the bispecific antibody for use in the present invention

[0183] In certain aspects, the therapeutic agents used in the combination include multispecific antibodies, such as bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different sites. In certain aspects, the binding specificities are directed against different antigens. In certain aspects, the binding specificities are directed against different epitopes on the same antigen. The bispecific antibody can be made as a full-length antibody or an antibody fragment.

[0184] Techniques for preparing 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 "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic manipulation effects to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004A1); 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 produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to prepare bispecific antibody fragments segment (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

[0185] Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576 A1).

[0186] The antibodies or fragments herein also include "dual-acting FAbs" or "DAFs", which comprise antigen binding sites that bind to two different antigens (see, e.g., US2008 / 0069820). Also included herein are "Crossmab" antibodies (see, e.g., WO2009 / 080251, WO 2009 / 080252, WO2009 / 080253, or WO2009 / 080254).

[0187] Another technology used to prepare bispecific antibody fragments is "bispecific T cell engager" or Method (see, e.g., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 119567). The method utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain comprises two single-chain Fv (scFv) fragments, each having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker, the length of the polypeptide linker being sufficient to permit intramolecular association between the two domains. The single polypeptide further comprises a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes can be specific for different cell types, such that when each scFv engages its cognate epitope, cells of two different cell types are brought into close proximity or bound together. A particular embodiment of the method comprises an scFv that recognizes a cell surface antigen expressed by an immune cell (e.g., the CD3 polypeptide on a T cell), the scFv being linked to another scFv that recognizes a cell surface antigen expressed by a target cell, such as a malignant or tumor cell).

[0188] Because it is a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic cell expression system known in the art (e.g., the CHO cell line). However, specific purification techniques may be required (see, e.g., EP1691833) to separate the monomeric bispecific T cell engager from other multimeric species that may have biological activities different from the expected activity of the monomer. In an exemplary purification protocol, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted with a gradient of imidazole concentration. The eluate is further purified using anion exchange chromatography, and the polypeptide is eluted with a gradient of sodium chloride concentration. Finally, the eluate is subjected to size exclusion chromatography to separate the monomer from the multimer. In one aspect, the bispecific bispecific antibody of the invention is composed of a single polypeptide chain that comprises two single-chain FV fragments (scFV) fused to each other by a peptide linker.

[0189] Fc domain modifications that reduce Fc receptor binding and / or effector function

[0190] The Fc domain of the antigen-binding molecule of the invention consists of a pair of polypeptide chains comprising the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other.

[0191] The Fc domain confers favorable pharmacokinetic properties to the antigen-binding molecules of the present invention, including a long serum half-life and a favorable tissue-blood distribution ratio that contribute to good accumulation in target tissues. However, at the same time, it may cause the bispecific antibodies of the present invention to undesirably target cells expressing Fc receptors rather than the preferred antigen-carrying cells. Thus, in certain aspects, the Fc domain of the antigen-binding molecules of the present invention exhibits a reduced binding affinity for Fc receptors and / or reduced effector functions compared to the native IgG1 Fc domain. In one aspect, the Fc binds substantially not to Fc receptors and / or does not induce effector functions. In a particular aspect, the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically human FcγRIIIa. In one aspect, the Fc domain does not induce effector functions. Reduced effector functions can include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cell phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake by immune complex-mediated antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0192] In certain aspects, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants. The Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0193] In a particular aspect, the present invention provides an antibody wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors, particularly binding to Fcγ receptors.

[0194] In one aspect, the Fc domain of the antibody of the invention comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain for Fc receptors and / or effector functions. 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 position 329 (EU numbering) of the IgG heavy chain. More specifically, there is provided an antibody according to the invention, which comprises an Fc domain having amino acid substitutions L234A, L235A and P329G ("P329G LALA", EU numbering) in the IgG heavy chain. The amino acid substitutions L234A and L235A refer to the so-called LALA mutations. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor binding of the human IgG1 Fc domain and is described in International Patent Application Publication No. WO 2012 / 130831 A1, which also describes methods for preparing such mutant Fc domains and methods for determining their properties such as Fc receptor binding or effector functions.

[0195] Fc domains with reduced Fc receptor binding and / or effector functions also include those having substitutions of one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc domain (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0196] In another aspect, the Fc domain is an IgG4 Fc domain. Compared with IgG1 antibodies, IgG4 antibodies exhibit reduced binding affinity for Fc receptors and reduced effector functions. In a more specific aspect, the Fc domain is an IgG4 Fc domain that comprises an amino acid substitution at position S228 (Kabat numbering), specifically the amino acid substitution S228P. In a more specific aspect, the Fc domain is an IgG4 Fc domain that comprises amino acid substitutions L235E, S228P and P329G (EU numbering). Such IgG4 Fc domain mutants and their Fcγ receptor binding properties are also described in WO 2012 / 130831.

[0197] Mutant Fc domains can be prepared by genetic or chemical methods well-known in the art, through amino acid deletion, substitution, insertion or modification. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, by sequencing.

[0198] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instruments such as a BIAcore instrument (GE Healthcare), and Fc receptors can be obtained, for example, by recombinant expression. Alternatively, cell lines known to express specific Fc receptors (e.g., human NK cells expressing the FcγIIIa receptor) can be used to evaluate the binding affinity of an Fc domain or an Fc domain-containing cell-activating antibody for the Fc receptor.

[0199] The effector functions of the Fc domain, or the antibodies of the invention containing the 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 evaluating the ADCC activity of a target molecule 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 used (see, for example, ACTI TM for flow cytometry; CellTechnology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a target molecule can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95,652-656 (1998).

[0200] In some aspects, the binding of the Fc domain to complement components, particularly C1q, is reduced. Thus, in some embodiments, the Fc domain is engineered to have reduced effector function, which includes reduced CDC. A C1q binding assay can be performed to determine whether the bispecific antigen-binding molecule of the invention is capable of binding C1q and thus has CDC activity (see, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402). To assess complement activation, a CDC assay can be performed (see, e.g., 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)).

[0201] Fc domain modifications that promote heterodimerization

[0202] The bispecific antigen-binding molecule of the invention comprises different antigen-binding sites fused to one or the other of the two subunits of the Fc domain, such that the two subunits of the Fc domain can be contained in two different polypeptide chains. The recombinant co-expression of these polypeptides and subsequent dimerization result in several possible combinations of the two polypeptides. To increase the yield and purity of the bispecific antibody of the invention in recombinant production, it would thus be advantageous to introduce modifications in the Fc domain of the bispecific antigen-binding molecule of the invention that promote the association of the desired polypeptides.

[0203] In certain aspects, the Fc domain comprises modifications that promote the association of the first and second subunits of the Fc domain. The most extensive protein-protein interaction sites between the two subunits of the human IgG Fc domain are in the CH3 domain. Thus, in one aspect, the modifications are in the CH3 domain of the Fc domain.

[0204] In one specific aspect, the modification that promotes the association of the first and second subunits of the Fc domain is the so-called "knob-into-hole" modification, which includes a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. The knob-into-hole technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") in the interface of the second polypeptide such that the protrusion can be positioned in the cavity to promote the formation of heterodimers and impede the formation of homodimers. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (such as tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (such as alanine or threonine).

[0205] Thus, in some aspects, the amino acid residues in the CH3 domain of the first subunit of the Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit that can be positioned in a cavity within the CH3 domain of the second subunit, and the amino acid residues in the CH3 domain of the second subunit of the Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit into which the protrusion within the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and the cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0206] In a specific such aspect, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), and optionally the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (according to the Kabat EU index number). In another aspect, in the first subunit of the Fc domain, in addition, the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (especially the serine residue at position 354 is replaced with a cysteine residue), and in the second subunit of the Fc domain, in addition, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (according to the Kabat EU index number). In a preferred aspect, the first subunit of the Fc domain contains the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain contains the amino acid substitutions Y349C, T366S, L368A and Y407V (according to the Kabat EU index number).

[0207] The C-terminus of the heavy chain of the bispecific antibody reported herein can be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain can be a shortened C-terminus in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect among all aspects reported herein, the bispecific antibody containing a heavy chain with a C-terminal CH3 domain as specified herein contains a C-terminal glycine-lysine dipeptide (G446 and K447, according to the Kabat EU index number). In an embodiment of all aspects reported herein, the bispecific antibody containing a heavy chain with a C-terminal CH3 domain as specified herein contains a C-terminal glycine residue (G446, according to the Kabat EU index number).

[0208] Modifications in the Fab domain

[0209] In one aspect, the molecule used herein is a bispecific antibody in which in one of the Fab fragments, the variable domains VH and VL or the constant domains CH1 and CL are exchanged. The bispecific antibody is prepared according to the Crossmab technology.

[0210] Multispecific antibodies with domain permutation / exchange in one binding arm (CrossMab VH-VL or CrossMab CH-CL) are described in detail in WO2009 / 080252 and Schaefer, W. et al., PNAS, 108 (2011) 11187-1191. They significantly reduce by-products resulting from the mismatch of the light chain against the first antigen and the wrong heavy chain against the second antigen (compared to methods without such domain exchange). In certain aspects, the additional Fab fragment is a Fab fragment in which the variable domains VL and VH are replaced with each other such that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

[0211] In one aspect, the present invention relates to a bispecific agonistic CD28 antigen-binding molecule, characterized by monovalent binding to CD28, the antigen-binding molecule comprising: (a) an 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 first and second subunits capable of stable association, the Fc domain comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for Fc receptors, wherein in the Fab fragment capable of specifically binding to a 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.

[0212] In another aspect, and to further improve correct pairing, the bispecific antibodies used herein (e.g., bispecific agonistic CD28 antibodies characterized by monovalent binding to CD28) comprise: (a) a Fab fragment capable of specifically binding to CD28, (b) a Fab domain capable of specifically binding to CD19, and (c) an Fc domain composed of first and second subunits capable of stable association, the Fc domain comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for Fc receptors, and may contain different charged amino acid substitutions (so-called "charged residues"). These modifications are introduced into the CH1 and CL domains, either crossed or non-crossed. In a particular aspect, the invention relates to a bispecific agonistic CD28 antigen-binding molecule, wherein in one of the CL domains, the amino acid at position 123 (EU numbering) has been replaced by arginine (R) and the amino acid at position 124 (EU numbering) has been replaced by lysine (K), and wherein in one of the CH1 domains, the amino acids at positions 147 (EU numbering) and 213 (EU numbering) have been replaced by glutamate (E). In a specific aspect, in the CL domain of the Fab fragment capable of specifically binding to CD28, the amino acid at position 123 (EU numbering) has been replaced by arginine (R), the amino acid at position 124 (EU numbering) has been replaced by lysine (K), and in the CH1 domain of the Fab fragment capable of specifically binding to CD28, the amino acids at positions 147 (EU numbering) and 213 (EU numbering) have been replaced by glutamate (E).

[0213] More particularly, the bispecifics used herein may comprise a Fab in which the amino acid at position 123 (EU numbering) in the CL domain has been replaced by arginine (R) and the amino acid at position 124 (EU numbering) has been replaced by lysine (K), and in which the amino acids at positions 147 (EU numbering) and 213 (EU numbering) in the CH1 domain adjacent to a TNF ligand family member have been replaced by glutamate (E).

[0214] Pharmaceutical compositions, medicaments, formulations and routes of administration

[0215] In a further aspect, there is provided a pharmaceutical composition or medicament comprising an anti-CD20 / anti-CD3 antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19, for use in any of the following methods of treatment, for example. In one aspect, the pharmaceutical composition comprises an antibody provided herein and at least one pharmaceutically acceptable excipient. In another aspect, the pharmaceutical composition comprises an antibody provided herein and at least one additional therapeutic agent, as described, for example, below.

[0216] 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 phrase "pharmaceutically or pharmacologically acceptable" means that the molecular entities and compositions are generally non-toxic to the recipient at the dosages and concentrations employed, i.e., they do not produce adverse, allergic, or other untoward reactions when administered to an animal (such as a human, as the case may be). The preparation of pharmaceutical compositions containing at least one antibody and, optionally, additional active ingredients will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, which is incorporated herein by reference. In particular, the compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable excipients" include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, salts, stabilizers, and combinations thereof, as known to the ordinary skilled person in the art.

[0217] Pharmaceutical compositions comprising the bispecific antigen-binding molecules disclosed herein can be produced by conventional mixing, dissolving, emulsifying, encapsulating, entrapping, or lyophilization processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries that facilitate processing the protein into a pharmaceutically usable formulation. The appropriate formulation depends on the chosen route of administration.

[0218] Bispecific antibodies can be formulated as compositions in the 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 pharmaceutically acceptable salts include acid addition salts, such as those formed with the free amino groups of proteinaceous compositions, or those formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; or organic bases, such as isopropylamine, trimethylamine, histidine, or procaine. Compared with the corresponding free base forms, pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents.

[0219] The compositions herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combinations effective for the intended purpose.

[0220] Preparations to be used for in vivo administration are usually sterile. For example, sterility can be readily achieved by filtration through sterile filtration membranes.

[0221] Administration of Bispecific Antibodies

[0222] Anti-CD20 / anti-CD3 bispecific antibodies, anti-CD19 / anti-CD28 bispecific antibodies, and a CD19-targeted 4-1BB (CD137) agonist (collectively referred to herein as the agents) can be administered by any suitable means, including parenterally, intranasally, and intratracheally, and, if desired for local treatment, by intralesional administration. However, the methods disclosed herein are particularly useful for therapeutic agents administered parenterally, particularly by intravenous infusion.

[0223] Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be effected by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, partly depending on whether the administration is to be short-term or long-term. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulsed infusion. In one aspect, the therapeutic agent is administered parenterally, particularly by intravenous administration. In a specific aspect, the agent is administered by intravenous infusion. In another aspect, the agent is administered subcutaneously.

[0224] Anti-CD20 / anti-CD3 bispecific antibodies, anti-CD19 / anti-CD28 bispecific antibodies, and a CD19-targeted 4-1BB (CD137) agonist will be formulated, dosed, and administered in a manner that is in keeping with good medical practice. Factors to be considered 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 to which the agent is to be delivered, the method of administration, the dosing schedule, and other factors known to the medical practitioner. Anti-CD20 / anti-CD3 bispecific antibodies, anti-CD19 / anti-CD28 bispecific antibodies, and a CD19-targeted 4-1BB (CD137) agonist need not be one or more of the agents currently used to prevent or treat the disorder under discussion, but may optionally be formulated with such agents. The effective amount of such other agents depends on the amount of the therapeutic agent present in the formulation, the type of disorder or treatment, and the other factors discussed above. These are generally used at the same dose and by the same route of administration as described herein, or at about 1% to 99% of the doses described herein, or at any dose and by any route determined to be appropriate empirically / clinically.

[0225] The anti-CD20 / anti-CD3 bispecific antibody can be administered by any suitable means, including parenterally, intraluminally, and intranasally, and if local treatment is desired, it can be administered intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be carried out by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. In one aspect, the anti-CD20 / anti-CD3 bispecific antibody is administered parenterally, particularly intravenously, such as by intravenous infusion. In one aspect, the infusion rate of the anti-CD20 / anti-CD3 bispecific antibody, particularly gefolimomab, is at least 4 hours. In one aspect, the infusion time of the anti-CD20 / anti-CD3 bispecific antibody can be reduced or extended. In one aspect, in the absence of infusion-related adverse events, the infusion time of gefolimomab in subsequent cycles is reduced to 2 hours ± 15 minutes. In one aspect, for subjects at high risk of experiencing cytokine release syndrome (CRS), the infusion time is increased to up to 8 hours. In one aspect, for example, for patients in whom the risk of CRS may increase, patients who experienced IRR or CRS in their previous dose of gefolimomab, or patients in whom the risk of recurrence of IRR / CRS in subsequent doses is increased, the infusion time of gefolimomab is extended to up to 8 hours.

[0226] In a particular aspect, the present disclosure relates to a combination, use, method, kit, or medicament of an anti-CD20 / anti-CD3 bispecific antibody as described herein for use with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19, wherein the combination therapy comprises the concomitant administration of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a 4-1BB (CD137) agonist targeting CD19. In certain aspects, the concomitant administration is for one or more treatment cycles, particularly 3 to 8 treatment cycles. The length of the treatment cycle corresponds to 7 days or 14 days or 21 days, particularly 7 days or 14 days.

[0227] Particularly, the present disclosure also relates to a combination, use, method, kit, or medicament of an anti-CD20 / anti-CD3 bispecific antibody as described herein for use with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19, wherein the combination therapy comprises a first treatment regimen with a combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-CD19 / anti-CD28 bispecific antibody and a second treatment regimen with a combination of an anti-CD20 / anti-CD3 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19.

[0228] In one aspect, the first treatment regimen with a combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-CD19 / anti-CD28 bispecific antibody is a single administration (one treatment cycle). In certain aspects, the administration of the first treatment regimen with a combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-CD19 / anti-CD28 bispecific antibody is two or more treatment cycles. In one aspect, the first treatment regimen comprises 1 to 5 treatment cycles, and the second treatment regimen starts from the next treatment cycle. In another aspect, the first treatment regimen comprises 3 to 5 treatment cycles, and the second treatment regimen starts from the next treatment cycle. In one aspect, the first treatment regimen comprises 4 treatment cycles, and the second treatment regimen starts from treatment cycle 5. In one aspect, 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., 7 days). In another aspect, the anti-CD19 / anti-CD28 bispecific antibody is administered 2 days later than the anti-CD20 / anti-CD3 bispecific antibody, but within the same treatment cycle (e.g., 7 days or 14 days or 21 days).

[0229] In one aspect, the second treatment regimen with a combination of an anti-CD20 / anti-CD3 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 is a single administration (one treatment cycle). In certain aspects, the administration of the second treatment regimen with a combination of an anti-CD20 / anti-CD3 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 is two or more treatment cycles. In one aspect, the second treatment regimen comprises 1 to 5 treatment cycles. In another aspect, the second treatment regimen comprises 3 to 5 treatment cycles. In one aspect, the second treatment regimen comprises 2 or more treatment cycles, and then the first treatment regimen will be repeated. In one aspect, the repeated first treatment regimen starts from the next treatment cycle. In one aspect, the repeated first treatment regimen will subsequently be the repeated second treatment regimen (alternating administration).

[0230] In one such aspect, the substance is administered weekly, bi-weekly or tri-weekly, particularly bi-weekly. In one aspect, the anti-CD20 / anti-CD3 bispecific antibody is administered in a therapeutically effective amount. In one aspect, 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 aspect, 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 the corresponding treatment regimen, without administering the anti-CD19 / anti-CD28 bispecific antibody and the CD19-targeted 4-1BB (CD137) agonist. In one aspect, the administration of the anti-CD20 / anti-CD3 bispecific antibody includes 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 aspect, the administration of the anti-CD20 / anti-CD3 bispecific antibody includes 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.

[0231] In one aspect, after one to three treatment cycles of single administration of the anti-CD20 / anti-CD3 bispecific antibody, the first treatment regimen with the combination of the anti-CD20 / anti-CD3 bispecific antibody and the anti-CD19 / anti-CD28 bispecific antibody will be initiated. In one aspect, the anti-CD20 / anti-CD3 bispecific antibody will be administered weekly. One week after the last single administration of the anti-CD20 / anti-CD3 bispecific antibody alone, the first treatment regimen with the combination of the anti-CD20 / anti-CD3 bispecific antibody and the anti-CD19 / anti-CD28 bispecific antibody will be initiated.

[0232] In another aspect, the administration of the anti-CD20 / anti-CD3 bispecific antibody in the treatment regimen according to the present invention is the first administration (at least within the same treatment course) of the anti-CD20 / anti-CD3 bispecific antibody to the subject. In one aspect, the anti-CD19 / anti-CD28 bispecific antibody is not administered to the subject before the administration of the anti-CD20 / anti-CD3 bispecific antibody. In another aspect, the anti-CD19 / anti-CD28 bispecific antibody is administered before the administration of the anti-CD20 / anti-CD3 bispecific antibody.

[0233] In all these aspects, the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use in combination therapy, wherein prior to initiation of the combination therapy, the treatment regimen starts with administration of the anti-CD20 / anti-CD3 bispecific antibody alone and continues for one or more treatment cycles, particularly three to five treatment cycles. In a particular aspect, the anti-CD20 / anti-CD3 bispecific antibody is administered at increasing doses (escalated dosing) in each single treatment cycle.

[0234] In one aspect, the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19, wherein prior to the combination therapy, pre-treatment is carried out with a type II anti-CD20 antibody, preferably obinutuzumab, wherein the time period between the pre-treatment and the combination therapy is sufficient to reduce B cells in individuals responsive to the type II anti-CD20 antibody, preferably obinutuzumab. In a particular aspect, obinutuzumab is administered 7 days prior to the first treatment with the anti-CD20 / anti-CD3 bispecific antibody. In a particular aspect, obinutuzumab is administered 7 days prior to the first treatment with the anti-CD20 / anti-CD3 bispecific antibody, and the anti-CD20 / anti-CD3 bispecific antibody is administered on day 1 of the first treatment cycle, and subsequently, prior to the combination therapy starting from the second treatment cycle, on days 3 and 8 of the first treatment cycle, two progressively higher dose levels of the anti-CD20 / anti-CD3 bispecific antibody are administered.

[0235] Activation of T cells 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 6 healthy volunteers rapidly experienced near-fatal severe cytokine release syndrome (CRS) after infusion of an improperly dosed T cell-stimulating superagonist anti-CD28 monoclonal antibody. By pre-treating subjects with a type II anti-CD20 antibody such as obinutuzumab, cytokine release associated with administration of a T cell-activating therapeutic agent (such as an anti-CD20 / anti-CD3 bispecific antibody) to the subjects can be significantly reduced. Using Pre-treatment (Gpt) should help rapidly deplete B cells in peripheral blood and secondary lymphoid organs, thereby reducing the risk of highly related adverse events (AE) (such as CRS) due to strong systemic T cell activation by T cell activating therapeutics, while supporting an exposure level of the T cell activating therapeutic that is high enough from the start of dosing to mediate tumor cell elimination. To date, in ongoing obinutuzumab clinical trials, the safety profile of obinutuzumab (including cytokine release) has been evaluated and managed in hundreds of patients. Finally, in addition to supporting the safety of T cell activating therapeutics such as anti-CD20 / anti-CD3 bispecific antibodies, Gpt should help prevent the formation of anti-drug antibodies (ADA) against these unique molecules.

[0236] Such combination therapies as described above encompass combination administration (wherein two or more therapeutics are included in the same or separate formulations) and separate administration, in which case the administration of the therapeutics can be before, at the same time as, and / or after the administration of another therapeutic or agent. In one embodiment, the administration of the therapeutic and the administration of the other therapeutic are carried out within about 1 month of each other, or within about 1 week, 2 weeks, or 3 weeks, or within about 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days.

[0237] Methods of treatment and compositions

[0238] CD20 and CD19 are expressed on most B cells (pan-B cell markers) except for stem cells and plasma cells, and are often expressed on most human B cell malignancies, such as lymphomas and leukemias, for example, expressed in non-Hodgkin 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.

[0239] In one aspect, provided is a method of treating a B cell cancer in an individual in need thereof, the method comprising administering to the individual a combination therapy comprising a combination of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a 4-1BB (CD137) agonist targeting anti-CD19.

[0240] 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 for depleting B cells, the method comprising administering to the subject an effective amount of an anti-CD20 / anti-CD3 antibody and an anti-CD19 / anti-CD28 bispecific antibody and / or a 4-1BB (CD137) agonist targeting CD19. The "individual" or "subject" according to any of the above aspects is preferably human.

[0241] In a further aspect, there is provided a composition for use in cancer immunotherapy, the composition comprising an anti-CD20 / anti-CD3 antibody and an anti-CD19 / anti-CD28 bispecific antibody and / or a 4-1BB (CD137) agonist targeting CD19. In certain aspects, there is provided a composition for use in a method of cancer immunotherapy, the composition comprising an anti-CD20 / anti-CD3 antibody and an anti-CD19 / anti-CD28 bispecific antibody and / or a 4-1BB (CD137) agonist targeting CD19.

[0242] In a further aspect, there is provided herein the use of a composition comprising an anti-CD20 / anti-CD3 antibody and an anti-CD19 / anti-CD28 bispecific antibody and / or a 4-1BB (CD137) agonist targeting CD19 in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of B-cell proliferative disorders. In a further embodiment, the medicament is for use in a method of treating a B-cell proliferative disorder, the method comprising administering to an individual suffering from a B-cell proliferative disorder an effective amount of the medicament. 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. B-cell proliferative disorders are selected from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin lymphoma (HL). In a particular aspect, the B-cell cancer is non-Hodgkin lymphoma or diffuse large B-cell lymphoma (DLBCL).

[0243] In another aspect, there is provided herein a method of treating B-cell cancer. In one embodiment, the method comprises administering to an individual suffering from such B-cell cancer a combination of an effective amount of an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described hereinafter. The "individual" according to any of the above embodiments may be a human. In one embodiment, the B-cell cancer is B-cell lymphoma or B-cell leukemia. In one embodiment, the B-cell cancer is non-Hodgkin lymphoma or acute lymphoblastic leukemia.

[0244] The above combination therapies encompass combination administration (wherein two or more therapeutic agents are included in the same or separate formulations) and separate administration, and in the case of separate administration, administration of the antibodies reported herein can be carried out before, simultaneously with, and / or after administration of additional therapeutic agents or medicaments. In one embodiment, administration of the combination of an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19, and administration of additional therapeutic agents, are carried out within about one month of each other, or within about one week, two weeks, or three weeks of each other, or within about one day, two days, three days, four days, five days, or six days of each other.

[0245] The combination of an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 (and any additional therapeutic agents) reported herein can be administered by any suitable means, including parenterally, intraluminally, and intranasally, and, if desired for local treatment, by intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be effected by any suitable route, for example by injection, such as intravenous or subcutaneous injection, partly depending on whether the administration is short-term or long-term. A variety of dosing schedules are contemplated herein, including but not limited to single or multiple administrations, bolus administration, and pulsed infusion at various time points.

[0246] The combination of an anti-CD20 / anti-CD3 bispecific antibody with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 reported herein will be formulated, dosed, and administered in a manner that is in keeping with good medical practice. Factors to be considered 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 to which the medicament is to be delivered, the method of administration, the timing of administration, and other factors known to the practicing physician. The antibodies are not required, but are optionally co-formulated with one or more currently used formulations for the prevention or treatment of the disorder under discussion. The effective amount of such other medicaments depends on the amount of antibody present in the formulation used, the type of disorder or treatment, and the other factors discussed above. These are generally used at the same dose and by the same route of administration as described herein, or at about 1% to 99% of the doses described herein, or at any dose and by any route determined to be appropriate empirically / clinically.

[0247] In another aspect, the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use in combination therapy, wherein prior to the combination treatment, pre-treatment is carried out with a type II anti-CD20 antibody, preferably obinutuzumab, wherein the time period between the pre-treatment and the combination treatment is sufficient to reduce B cells in individuals responsive to the type II anti-CD20 antibody, preferably obinutuzumab.

[0248] Activation of T cells 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 6 healthy volunteers rapidly experienced near-fatal severe cytokine release syndrome (CRS) after infusion of an inappropriate dose of the T cell-stimulating superagonist anti-CD28 monoclonal antibody. By pre-treating subjects with a type II anti-CD20 antibody such as obinutuzumab, cytokine release associated with administration of a T cell-activating therapeutic agent (e.g., an anti-CD20 / anti-CD3 bispecific antibody) to the subjects can be significantly reduced. Using Pre-treatment (Gpt) should help rapidly deplete B cells in peripheral blood and secondary lymphoid organs, thereby reducing the risk of highly associated adverse events (AEs) (e.g., CRS) resulting from intense systemic T cell activation by the T cell-activating therapeutic agent, while supporting an exposure level of the T cell-activating therapeutic agent that is high enough from the start of administration to mediate tumor cell elimination. To date, in ongoing obinutuzumab clinical trials, the safety profile of obinutuzumab (including cytokine release) has been evaluated and managed in hundreds of patients. Finally, in addition to supporting the safety of T cell-activating therapeutic agents such as anti-CD20 / anti-CD3 bispecific antibodies, Gpt should also help prevent the formation of anti-drug antibodies (ADA) against these unique molecules.

[0249] Other agents and treatments

[0250] The antigen-binding molecules of the present invention can be administered in combination with one or more other agents in a treatment. For example, the fusion proteins of the present invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent that can be administered to treat the symptoms or diseases of an individual in need of such treatment. Such additional therapeutic agents can comprise any active ingredient suitable for the specific indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is another anti-cancer agent.

[0251] Such other agents are present in a combined amount effective for the intended purpose. The effective amount of such other agents depends on the amount of the fusion protein employed, the type of disorder or treatment, and other factors discussed above. The antigen-binding molecule is generally used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and by a route empirically / clinically determined to be appropriate.

[0252] Such combination therapies as described above encompass the administration of the combination (wherein two or more therapeutic agents are included in the same composition or separate compositions), as well as separate administration, in which case the administration of the antigen-binding molecule of the present invention can be carried out before, simultaneously with, and / or after the administration of an additional therapeutic agent and / or adjuvant.

[0253] Article (kit)

[0254] In another aspect, there is provided a kit containing materials useful for treating, preventing, and / or diagnosing the disorders described above. The kit includes at least one container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition, which alone or in combination with another composition effective for treating, preventing, and / or diagnosing the disorder, and the container can have a sterile inlet (for example, the container can be an intravenous injection solution bag or a vial with a stopper pierceable by a hypodermic needle). At least three active agents in the kit are an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a 4-1BB (CD137) agonist targeting CD19.

[0255] In a particular aspect, there is provided a kit for treating or delaying cancer progression in a subject, the kit comprising a package containing: (A) 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 as an active ingredient and a pharmaceutically acceptable carrier; (C) a third composition comprising a 4-1BB (CD137) agonist targeting CD19 as an active ingredient and a pharmaceutically acceptable carrier; and instructions for using these compositions in combination therapy.

[0256] Labels or package inserts indicate how the compositions are used to treat selected conditions and provide instructions for using the compositions in combination therapies. Additionally, the kit can include: (a) a first container containing a composition that includes the anti-CD20 / anti-CD3 bispecific antibody of the invention; (b) a second container containing a composition that includes an anti-CD19 / anti-CD28 bispecific antibody; and (c) a third container containing a composition that includes a 4-1BB (CD137) agonist targeting CD19. Additionally, the kit can include one or more other containers that contain other active ingredients that can be used in combination. The article of this embodiment of the invention can further include a package insert that indicates that these compositions can be used to treat a specific condition.

[0257] Alternatively or additionally, the kit can further include a second (or third) container that contains a pharmaceutical buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It can also include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0258] Drug

[0259] In another aspect, there is provided a drug comprising an anti-CD20 / anti-CD3 bispecific antibody for treating B-cell proliferative disorders, wherein the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19.

[0260] In one aspect, there is provided a drug comprising an anti-CD20 / anti-CD3 bispecific antibody for treating B-cell proliferative disorders, wherein the bispecific antibody is used in combination with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19. In one aspect, the anti-CD20 / anti-CD3 bispecific antibody, the anti-CD19 / anti-CD28 bispecific antibody, and the 4-1BB (CD137) agonist targeting CD19 are administered in a single composition or separately in two or more different compositions. In a particular aspect, the anti-CD20 / anti-CD3 bispecific antibody, the anti-CD19 / anti-CD28 bispecific antibody, and the 4-1BB (CD137) agonist targeting CD19 are administered separately in three different compositions.

[0261] Table B (sequences):

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271] General information on the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of the antibody chains are numbered and referred to according to the Kabat (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)) numbering system as defined above.

[0272] ***

[0273] Examples

[0274] The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments can be practiced given the general description provided above.

[0275] Example 1

[0276] Preparation, purification and characterization of CD19-41BBL antigen-binding molecules

[0277] Prepare an Fc fusion antigen-binding molecule (Construct 4.5) containing a trimer of the 4-1BB ligand targeting CD19 as described in International Patent Application Publication No. WO 2016 / 075278 A1, particularly Example 7.2.7.

[0278] For preparation, a polypeptide comprising a dimeric 4-1BB ligand fused to the human CL domain was co-subcloned with the human IgG1 heavy chain CH2 and CH3 domains on the pestle in-frame. A polypeptide comprising one extracellular domain of the 4-1BB ligand was fused to the human IgG1-CH1 domain. To improve correct pairing, additional amino acid mutations were introduced in the crossed CH-CL (charged variants), CL domain E123R and Q124K, and CH1 domain K147E and K213E (EU numbering according to Kabat).

[0279] The variable regions of the heavy and light chain DNA sequences encoding the CD19 antibody clone 8B8-2B11 were co-subcloned in-frame with the constant heavy chain of the mortar or the constant light chain of human IgG1. According to the method described in WO 2012 / 130831, Pro329Gly, Leu234Ala, and Leu235Ala mutations were introduced into the constant regions of the pestle and mortar heavy chains to eliminate binding to Fcγ receptors. The combination of the dimeric ligand-Fc pestle chain containing the S354C / T366W mutation, the monomeric CH1 fusion, the targeted anti-CD19-Fc mortar chain containing the Y349C / T366S / L368A / Y407V mutations, and the anti-CD19 light chain allowed the generation of a heterodimer that includes an assembled trimeric 4-1BB ligand and a CD19-binding Fab. This molecule is referred to herein as CD19-4-1BBL.

[0280] CD19-4-1BBL contains the amino acid sequences of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60. Figure 1A A schematic diagram of an Fc fusion antigen-binding molecule containing a trimeric 4-1BB ligand targeting CD19 is shown.

[0281] The generation and characterization of Fc fusion antigen-binding molecules containing trimeric 4-1BB ligands targeting and not targeting CD19 were described in detail in WO 2016 / 075278, Example 7.4, and Examples 8 to 11, respectively.

[0282] Example 2

[0283] Preparation, purification, and characterization of a T cell bispecific (TCB) antibody

[0284] TCB molecules have been prepared according to the method described in WO 2016 / 020309 A1.

[0285] The anti-CD20 / anti-CD3 bispecific antibody (CD20 CD3 TCB or CD20 TCB or gefapixant) used in the experiments corresponds to molecule B as described in Example 1 of WO 2016 / 020309 A1. Molecule B is a "2+1IgG CrossFab" antibody and includes two different heavy chains and two different light chains. Point mutations ("knobs-into-holes structure") are introduced into the CH3 domain to facilitate the assembly of the two different heavy chains. According to the method described in WO 2012 / 130831, Pro329Gly, Leu234Ala, and Leu235Ala mutations are introduced into the constant regions of the knob and hole heavy chains to eliminate binding to Fcγ receptors. Exchange of the VH and VL domains in the CD3-binding Fab and point mutations in the CH and CL domains in the CD20-binding Fab are performed to facilitate the correct assembly of the two different light chains. 2+1 means that the molecule has two antigen-binding domains specific for CD20 and one antigen-binding domain specific for CD3.

[0286] CD20 TCB contains the amino acid sequences of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60. Figure 1B A schematic diagram of the bispecific antibody in 2+1 form is shown.

[0287] This molecule is further characterized in Example 1 of WO 2016 / 020309 A1.

[0288] Example 3

[0289] Preparation, purification, and characterization of CD19-CD28 bispecific antibody

[0290] The CD19-CD28 bispecific antibody was prepared as described in International Patent Application Publication No. WO 2020 / 127618 A1.

[0291] More particularly, the generation and production are described in Example 18. For the generation of the corresponding 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 corresponding constant regions pre-inserted in the corresponding receptor mammalian expression vectors. In Figure 1CA schematic description of the resulting molecule is shown. It is a "1+1 IgG1 CrossFab" antibody and is composed of two different heavy chains and two different light chains. Point mutations ("knob-into-hole structure") are introduced into the CH3 domain to facilitate the assembly of the two different heavy chains. According to the method described in WO 2012 / 130831, the Pro329Gly, Leu234Ala, and Leu235Ala mutations are introduced into the constant regions of the knob and hole heavy chains to eliminate binding to Fcγ receptors. Exchange of the VH and VL domains in the CD19-binding Fab and point mutations in the CH and CL domains in the CD28-binding Fab are performed to facilitate the correct assembly of the two different light chains.

[0292] 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.

[0293] Example 4

[0294] Ex vivo combination therapies of CD19-CD28, CD19-4-1BBL, and CD20 TCB

[0295] The hypothesis of the present inventors is that CD19-CD28 and CD19-4-1BBL can cooperate with CD20-TCB for T cell activation. To test this, the present inventors digested malignant splenectomy specimens from stage IVB B cell lymphoma patients and incubated the cells with either CD20-TCB alone (25 pM) or one of the two costimulators CD19-CD28 or CD19-4-1BBL (1 nM) or a combination of TCB and costimulator. After 3 days, a CBA kit was used to determine cytokine release in the supernatant (flow cytometric bead array, BD Biosciences). Figures 2A to 2D It is shown that CD20-TCB induces cytokine release (GzB, IFNg, IL-2, and IL-8), and CD19-CD28 and CD19-4-1BBL can further boost CD20-TCB-induced cytokine release (especially IFNg and IL-2, see respectively Figure 2B and Figure 2D ). Compared with the dual combinations of CD20-TCB and CD19-CD28 or CD19-4-1BBL, the triple combinations of CD20-TCB and the two costimulators (using 0.5 nM or 1 nM of each costimulator) showed a further increased cytokine release (especially IFNg and IL-2).

[0296] Example 5

[0297] Efficacy study to evaluate the triple combination effect of CD20-TCB with CD19-4-1BBL and CD19-CD28 in humanized NSG mice

[0298] The efficacy study described herein was designed to evaluate the potential triple combination effect of CD20-TCB with CD19-CD28 and CD19-41BBL in a CD19 / CD20-positive human lymphoma model in fully humanized NSG mice.

[0299] Human OCI-Ly18 (diffuse large B-cell lymphoma; DLBCL) was originally obtained from ATCC and stored in the Roche Glycart in-house cell bank after expansion. Cells were cultured in RPMI containing 10% FCS and 1x Glutamax. Cells were cultured at 37 °C in a water-saturated atmosphere with 5% CO 2 A 50 μl cell suspension (5x10 6 NALM6 cells) mixed with 50 μl Matrigel was subcutaneously injected into the flanks of anesthetized mice using a 22G to 30G needle.

[0300] Female NSG mice (bred at The Jackson Laboratory) at 4-5 weeks of age at the start of the experiment were maintained under specific pathogen-free conditions according to the prescribed guidelines (GV-Solas; Felasa; TierschG), with a 12 h light / 12 h dark day cycle. The experimental study protocol was reviewed and approved by the local government (ZH183 / 2020). Upon arrival, the animals were maintained for one week to acclimatize to the new environment and observed. Continuous health status monitoring was performed regularly. Mice were intraperitoneally injected with 15 mg / kg of busulfan, followed one day later by intravenous injection of 1x10 5 human hematopoietic stem cells isolated from umbilical cord blood. At 14-16 weeks after stem cell injection, mice were bled sublingually and blood was analyzed by flow cytometry for successful humanization. Mice with effective engraftment were randomized into different treatment groups according to their human T cell frequency. At that time, the mice were subcutaneously injected with tumor cells as described ( Figure 3 ), and when the tumor size reached approximately 250 mm 3 (on day 12), they were treated with the compound or histidine buffer (vehicle; group A). All mice were injected i.v. with 200 μl of the appropriate solution. To obtain the appropriate amount of compound per 200 μl, the stock solution was diluted with histidine buffer as needed (Table 1). For combination treatments (groups C to G), the antibodies were mixed and co-injected. All combination treatments started on day 38 after tumor cell injection following GAZYVA and three cycles of CD20-TCB. As Figure 3As shown, Group C received CD19-CD28 combination therapy, and Group D received CD19-4-1BBL combination therapy. Group G received concomitant injection of the triple combination starting from Day 38. Groups E and F received an alternating treatment regimen, where treatment started with CD19-CD28 (Group F) or CD19-4-1BBL (Group E) for four cycles, followed by injection of the other costimulatory molecule for the remainder of all treatment cycles.

[0301] Table 1: Compositions used in this experiment

[0302]

[0303]

[0304] Table 2: Groups and their treatment regimens

[0305]

[0306] Tumor growth was measured three times a week using calipers, and tumor volume was calculated using the following formula:

[0307] T v : (W 2 / 2) × L (W: width, L: length)

[0308] After a total of 15 cycles of treatment, the study was terminated on Day 120.

[0309] Figures 4A to 4G Tumor growth was shown as the individual tumor growth kinetics for each group and each mouse. As described herein, in all treated animals, CD20-TCB monotherapy ( Figure 4B ) initially induced strong tumor growth inhibition, followed by tumor recurrence. Treatment with the CD19-CD28 combination ( Figure 4C ) induced a mild delay in tumor outgrowth in several mice. The CD19-41BBL combination ( Figure 4D ) showed a more uniform delay in tumor recurrence. However, most animals reached the termination criteria (tumor volume greater than 2000 mm 3 ) before the end of the study. The treatment group receiving the concomitant injection of the triple combination starting from Day 38 ( Figure 4G ) did not show any further improvement in terms of the duration of increased treatment activity compared to the combination arm with only CD19-4-1BBL. Interestingly, in all treated animals, the group receiving the alternating treatment regimen starting with CD19-CD28 for the first four cycles followed by the CD19-4-1BBL combination treatment until the end of the study ( Figure 4F) achieved complete control of the tumor within 120 days. In contrast, the alternating regimen starting with CD19-4-1BBL followed by CD19-CD28 did not show such tumor control( Figure 4E ).

[0310] Using a tumor volume cutoff of 1500m 3 time-to-event analysis( Figure 5 ) showed that gefitinib had a strong synergistic effect with the combination of CD19-CD28 and CD19-4-1BBL when CD19-CD28 was administered in the first 4 treatment cycles and CD19-4-1BBL was administered in the subsequent treatment cycles.

[0311] Example 6

[0312] A potency study evaluating the triple combination effect of CD20-TCB with CD19-4-1BBL and CD19-CD28 in humanized BRGS-CD47 mice

[0313] A second potency study was conducted in humanized BRGS-CD47 mice and aimed to evaluate the potential triple combination effect of CD20-TCB with CD19-CD28 and CD19-41BBL in a CD19 / CD20-positive human lymphoma model when combination treatment was started one week earlier.

[0314] Human OCI-Ly18 (diffuse large B-cell lymphoma; DLBCL) was originally obtained from ATCC and stored in the Roche Glycart in-house cell bank after expansion. Cells were cultured in RPMI containing 10% FCS and 1x Glutamax. Cells were cultured at 37 °C in a water-saturated atmosphere under 5% CO 2 . A 50 μL cell suspension (5x10 6 NALM6 cells) mixed with 50 μL Matrigel was subcutaneously injected into the flanks of anesthetized mice using a 22G to 30G needle.

[0315] Female humanized BRGS-CD47 mice were generated at The Jackson Laboratory by injecting human hematopoietic stem cells at 4 - 5 weeks of age. After confirmation of engraftment, the humanized mice were shipped to Roche at 15 - 16 weeks of age. After arrival, the mice were maintained for one week to acclimatize to the new environment and observed. Continuous health monitoring was performed regularly and, according to the specified guidelines (GV-Solas; Felasa; TierschG), the mice were maintained under specific pathogen-free conditions with a 12h light / 12h dark daily cycle. The experimental study protocol was reviewed and approved by the local government (ZH181 / 2020). Mice were subcutaneously injected with tumor cells as described Figure 6), and when the tumor size reached approximately 250 mm 3 (on day 10), the mice were treated with the compound or histidine buffer (vehicle; group A). All mice were injected i.v. with 200 μl of the appropriate solution. To obtain the appropriate amount of compound per 200 μl, the stock solution was diluted with histidine buffer as needed (Table 1). For combination therapies (groups C to G), the antibodies were mixed and co-injected. All combination therapies were initiated on day 27 after tumor cell injection following receipt of GAZYVA and two cycles of CD20-TCB. As Figure 6 shown, group C received CD19-CD28 combination therapy, and group D received CD19-4-1BBL combination therapy. Group G received co-injected triple combination starting from day 27. Groups E and F received an alternating treatment regimen where treatment started with CD19-CD28 (group F) or CD19-4-1BBL (group E) for four cycles, followed by injection of the other costimulatory molecule for the remainder of all treatment cycles.

[0316] Table 3: Compositions used in this experiment

[0317]

[0318] Table 4: Groups and their treatment regimens

[0319]

[0320]

[0321] Tumor growth was measured three times a week using calipers, and tumor volume was calculated using the following formula:

[0322] T v : (W 2 / 2) × L (W: width, L: length)

[0323] The study was terminated on day 94 after a total of 13 cycles of treatment.

[0324] Figures 7A to 7G Tumor growth was shown as the individual tumor growth kinetics for each group and each mouse. As described herein, in all treated animals, CD20-TCB monotherapy ( Figure 7B ) initially induced strong tumor growth inhibition, followed by tumor recurrence. Treatment with the CD19-CD28 combination ( Figure 7C ) induced a delay in tumor outgrowth in several mice and tumor control in a few mice. The CD19-41BBL combination ( Figure 7D) showed a similar delay in tumor recurrence. Compared with the combination arms of CD19-4-1BBL alone and CD19-CD28, the treatment group ([ Figure 7G ) receiving the triple combination with concomitant injection starting from day 27 showed superior tumor control in terms of the duration of increased treatment activity. Due to the earlier start of the combination treatment, the tumor control in this concomitant group might be stronger in this experiment. The group ([ Figure 7F ) receiving the alternating treatment regimen of starting with CD19-CD28 for the first four cycles followed by the CD19-4-1BBL combination treatment until the end of the study achieved tumor control similar to that with concomitant administration within 94 days. In contrast, the alternating regimen starting with CD19-4-1BBL followed by CD19-CD28 did not show such obvious tumor control ([ Figure 7E ).

[0325] ***

Claims

1. A combination of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a 4-1BB (CD137) agonist targeting CD19, for use in combination therapy for treating B-cell proliferative disorders.

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

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

4. A kit for use in combination therapy, the kit 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 4-1BB (CD137) agonist targeting CD19, and optionally further comprising a package insert comprising instructions for administering the combination of the first medicament and the second medicament for treating cancer in an individual.

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

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

7. The combination, use, method, kit, or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody, and a 4-1BB (CD137) agonist targeting CD19 according to claim 5, wherein the first treatment regimen comprises 1 to 5 treatment cycles, and the second treatment regimen starts from the next treatment cycle.

8. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use, wherein the first treatment regimen comprises 4 treatment cycles and the second treatment regimen starts from the 5th treatment cycle.

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

10. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use according to any one of the preceding claims, wherein prior to the combination therapy with the anti-CD19 / anti-CD28 bispecific antibody and the 4-1BB (CD137) agonist targeting CD19, pre-treatment is carried out with a type II anti-CD20 antibody, preferably obinutuzumab, and wherein the time period between the pre-treatment and the combination therapy is sufficient to reduce B cells in the individual responsive to the type II anti-CD20 antibody, preferably obinutuzumab.

11. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use according to any one of the preceding claims, wherein the 4-1BB agonist targeting CD19 comprises three extracellular domains of 4-1BBL, each extracellular domain 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, particularly the amino acid sequence of SEQ ID NO:

5.

12. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use according to any one of the preceding claims, wherein the 4-1BB agonist targeting CD19 comprises an Fc domain, particularly an IgG1 or IgG4 Fc domain, the Fc domain comprising one or more amino acid substitutions that reduce or eliminate binding to Fc receptors and / or effector functions.

13. The anti-CD20 / anti-CD3 bispecific antibody for use, the combination, use, method, kit or drug with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 according to any one of the preceding claims, wherein the 4-1BB agonist targeting CD19 comprises an antigen-binding domain capable of specifically binding to CD19, and the antigen-binding domain comprises a heavy-chain variable region (V H CD19) and a light-chain variable region (V L CD19), the heavy-chain variable region comprises: (i) CDR-H1, which comprises the amino acid sequence of SEQ ID NO:10, (ii) CDR-H2, which comprises the amino acid sequence of SEQ ID NO:11, and (iii) CDR-H3, which comprises the amino acid sequence of SEQ ID NO:12, and the light-chain variable region comprises: (iv) CDR-L1, which comprises the amino acid sequence of SEQ ID NO:13, (v) CDR-L2, which comprises the amino acid sequence of SEQ ID NO:14, and (vi) CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

15.

14. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody for use, an anti-CD19 / anti-CD28 bispecific antibody and a CD19-targeted 4-1BB (CD137) agonist according to any one of the preceding claims, wherein the CD19-targeted 4-1BB agonist comprises an antigen-binding domain capable of specifically binding to CD19, and the antigen-binding domain comprises a heavy-chain variable region (V H CD19) and a light-chain variable region (V L CD19), the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO:16, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO:

17.

15. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use as claimed in any one of the preceding claims, wherein the 4-1BB agonist targeting CD19 comprises (a) a first polypeptide comprising: (a1) a first extracellular domain of 4-1BBL or a fragment thereof, which is fused at its C-terminus to the N-terminus of a second extracellular domain of 4-1BBL or a fragment thereof; (a2) the second extracellular domain of 4-1BBL or a fragment thereof, which is fused at its C-terminus to the N-terminus of a CL domain; (a3) a CL domain, which is fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain (such as the first subunit); and (a4) one of the subunits of the Fc domain (such as the first subunit); (b) a second polypeptide comprising: (b1) a third extracellular domain of 4-1BBL or a fragment thereof, which is fused at its C-terminus to the N-terminus of a CH1 domain; and (b2) the CH1 domain; (c) a third polypeptide comprising: (c1) the heavy chain of a Fab molecule that binds to CD19, which is fused at its C-terminus to the N-terminus of the other subunit of the Fc domain (such as the second subunit); and (c2) the other subunit of the Fc domain (such as the second subunit); and (d) a fourth polypeptide comprising the light chain of a Fab molecule that binds to CD19.

16. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use as claimed in any one of the preceding claims, wherein the 4-1BB agonist targeting CD19 comprises: a first 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: 18; a second 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: 19; 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: 20; 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. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 for use as claimed in any one of the preceding claims, wherein the 4-1BB agonist targeting CD19 is enlumafusp alfa.

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

19. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody for use, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 according to any one of the preceding claims, wherein the first antigen-binding domain comprises: a heavy-chain variable region (V H CD3), which comprises 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; and / or a light-chain variable region (V L CD3), which comprises 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).

20. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody for use, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 according to any one of the foregoing claims, wherein the first antigen-binding domain comprises: a heavy chain variable region (V H CD3), which comprises the amino acid sequence of SEQ ID NO:28; and / or a light chain variable region (V L CD3), which comprises the amino acid sequence of SEQ ID NO:

29.

21. The combination, use, method, kit or medicament of the anti-CD20 / anti-CD3 bispecific antibody for use, the anti-CD19 / anti-CD28 bispecific antibody and the 4-1BB (CD137) agonist targeting CD19 according to any one of the preceding claims, wherein the second antigen-binding domain comprises: a heavy-chain variable region (V H CD20), which comprises 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; and / or a light-chain variable region (V L CD20), which comprises the CDR-L1 sequence of SEQID NO:33, the CDR-L2 sequence of SEQ ID NO:34 and the CDR-L3 sequence of SEQID NO:

35.

22. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody for use, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 according to any one of the preceding claims, wherein the second antigen-binding domain comprises: a heavy-chain variable region (V H CD20), which comprises SEQ ID The amino acid sequence of NO:36; and / or the variable region of the light chain (V L CD20), which comprises SEQ ID The amino acid sequence of NO:

37.

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

24. The combination, use, method, kit or drug of the anti-CD20 / anti-CD3 bispecific antibody for use, anti-CD19 / anti-CD28 bispecific antibody and 4-1BB (CD137) agonist targeting CD19 according to any one of the preceding claims, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises: a first 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:38; a second 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:39; 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:40; and a fourth polypeptide and a fifth polypeptide, both of which comprise 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.

25. The combination, use, method, kit or drug of the anti-CD20 / anti-CD3 bispecific antibody for use, anti-CD19 / anti-CD28 bispecific antibody and 4-1BB (CD137) agonist targeting CD19 according to any one of the preceding claims, wherein the anti-CD20 / anti-CD3 bispecific antibody is gefapixant.

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

27. A combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody for use with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 according to any one of the preceding claims, wherein the anti-CD19 / anti-CD28 bispecific antibody comprises a first antigen-binding domain, and the first antigen-binding domain comprises: a heavy chain variable region (V H CD28), which comprises 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; and / or a light chain variable region (V L CD28), which comprises 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).

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

49.

29. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody for use, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 according to any one of the preceding claims, wherein the anti-CD19 / anti-CD28 bispecific antibody comprises a second antigen-binding domain, and the second antigen-binding domain comprises: a heavy-chain variable region (V H CD19), which comprises 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; and / or a light-chain variable region (V L CD19), which comprises 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.

30. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody for use, an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 according to any one of the preceding claims, wherein the anti-CD19 / anti-CD28 bispecific antibody comprises a second antigen-binding domain, and the second antigen-binding domain comprises: a heavy chain variable region (V H CD19), which comprises the amino acid sequence of SEQ ID NO:16; and / or a light chain variable region (V L CD19), which comprises the amino acid sequence of SEQ ID NO:

17.

31. The combination, use, method, kit or drug of the anti-CD20 / anti-CD3 bispecific antibody for use, anti-CD19 / anti-CD28 bispecific antibody and 4-1BB (CD137) agonist targeting CD19 according to any one of the preceding claims, wherein the anti-CD19 / anti-CD28 bispecific antibody comprises an Fc domain, particularly an IgG1 or IgG4 Fc domain, and the Fc domain comprises one or more amino acid substitutions that reduce or eliminate binding to Fc receptors and / or effector functions.

32. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody for use with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 as described in any one of the preceding claims, wherein the anti-CD19 / anti-CD28 bispecific antibody comprises: a first 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:50; a second 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:51; 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:52; 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. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody for use with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 as described in any one of the preceding claims, wherein the combination therapy is administered at intervals of from about one week to about three weeks.

34. The combination, use, method, kit or medicament of an anti-CD20 / anti-CD3 bispecific antibody for use with an anti-CD19 / anti-CD28 bispecific antibody and a 4-1BB (CD137) agonist targeting CD19 as described in any one of the preceding claims, wherein the B-cell proliferative disorder is selected from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM) and Hodgkin lymphoma (HL).

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

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