Combination therapies using PD1-LAG3 bispecific antibodies and CD20 t cell bispecific antibodies

Abstract

To provide a combination therapy using a PD1-LAG3 bispecific antibody and a CD20 T cell activating bispecific antibody, use of the combination therapy for cancer treatment, and methods for using combination therapies.SOLUTION: Provided is an anti-CD20 / anti-CD3 bispecific antibody for use in the treatment of CD20-expressing cancer, where the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody, where the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to a programmed cell death protein 1 (PD1) and a second antigen binding domain that specifically binds to lymphocyte activating gene 3 (LAG3), where the first antigen binding domain that specifically binds to PD1 comprises a VH domain comprising a specific amino acid sequence and a VL domain comprising a specific amino acid sequence.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to combination therapies using PD1-LAG3 bispecific antibodies and CD20 T cell activating bispecific antibodies, the use of these combination therapies for the treatment of cancer, and methods of using the combination therapies. [Background technology]

[0002] background B-cell proliferative disorders describe a heterogeneous group of malignancies, including both leukemias and lymphomas. Lymphomas arise from lymphocytes and include two major categories: Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). In the United States, lymphomas of B-cell origin comprise approximately 80-85% of all non-Hodgkin's lymphoma cases, and there is considerable heterogeneity within B-cell subsets based on genotypic and phenotypic expression patterns in the B-cell of origin. For example, B-cell lymphoma subsets include slow-growing, indolent, and incurable diseases such as follicular lymphoma (FL) or chronic lymphocytic leukemia (CLL), as well as more aggressive subtypes such as mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Despite the availability of various drugs for the treatment of B-cell proliferative disorders, there is a continuing need to develop safe and effective therapies to prolong remission and improve cure rates in patients.

[0003] Anti-CD20 / anti-CD3 bispecific antibodies are molecules that target CD20 expressed on B cells and the CD3 epsilon chain (CD3ε) present on T cells. Simultaneous binding leads to T cell activation and T cell-mediated killing of B cells. +In the presence of B cells, whether circulating or in tissues, pharmacologically active doses of CD20-CD3 bispecific antibodies cause T cell activation and associated cytokine release. Paralleling B cell depletion in peripheral blood, CD20 T cell-activating bispecific antibodies result in a transient decrease in T cells in peripheral blood and a peak in cytokine release within 24 hours after the first administration, followed by rapid T cell recovery and a return of cytokine levels to baseline within 72 hours. Two major reported escape mechanisms during treatment with T cell-activating bispecific antibodies include the activation of regulatory T cells (T reg These include increased frequency of PD-L1 expression on precursor B cells and increased expression of PD-L1 on precursor B cells. reg suppresses the activation of effector T cells through CTLA4 and other mechanisms. However, even when T cells are fully activated, upregulation of PD1 will result in inhibitory signaling after binding to PD-L1 expressed by tumor cells. These mechanisms lead to suppression and exhaustion of effector T cells, or dysfunction, which can be treated by checkpoint blockade.

[0004] Exhausted T cells are characterized by persistent expression of the inhibitory molecule PD-1 (programmed cell death protein 1), and it has been found that blocking the PD-1 and PD-L1 (PD-1 ligand) interaction can reverse T cell exhaustion and restore antigen-specific T cell responses. However, targeting the PD-1-PD-L1 pathway alone does not always result in the reversal of T cell exhaustion, possibly due to tolerance mechanisms, the immunosuppressive activity of MDSCs, and / or regulatory T cells.

[0005] Lymphocyte activation gene-3 (LAG3 or CD223) was first discovered in experiments designed to selectively isolate molecules expressed in IL-2-dependent NK cell lines (Triebel F et al., Cancer Lett. 235 (2006), 147-153). LAG3 is a unique transmembrane protein with structural homology to CD4 and four extracellular immunoglobulin superfamily-like domains (D1-D4). This membrane-distal IgG domain contains a short amino acid sequence (a so-called extra loop) not found in other IgG superfamily proteins. The intracellular domain contains a unique amino acid sequence (KIEELE, SEQ ID NO: 103) required for LAG3 to negatively affect T cell function. LAG3 can be cleaved at the connecting peptide (CP) by metalloproteases to generate a soluble form that is detectable in serum. Like CD4, the LAG3 protein binds to MHC class II molecules, but with higher affinity and at a site distinct from that of CD4 (Huard et al. Proc. Natl. Acad. Sci. USA 94 (1997), 5744-5749). LAG3 is expressed on T cells, B cells, NK cells, and plasmacytoid dendritic cells (pDCs) and is upregulated following T cell activation. LAG3 regulates T cell function and T cell homeostasis. A subset of immunologically unresponsive or dysfunctional conventional T cells expresses LAG3. LAG3 + T cells are abundant at tumor sites and during chronic viral infections (Sierro et al. Expert Opin. Ther. Targets 15 (2011), 91-101). LAG3 has been shown to play a role in CD8 T cell exhaustion (Blackburn et al. Nature Immunol. 10 (2009), 29-37). Therefore, there is a need for antibodies that can be used to antagonize the activity of LAG3 and generate and restore immune responses against tumors.

[0006] By targeting both PD-1 and LAG-3 on dysfunctional tumor-specific T lymphocytes, PD1-LAG3 aims to restore effective antitumor immune responses and provide a survival benefit to more cancer patients than currently available checkpoint inhibitors. By preferentially targeting PD-1 / LAG-3 co-expressing dysfunctional T cells and potentially reducing targeting of LAG-3-expressing Tregs in the tumor microenvironment, PD1-LAG3 BsAb may avoid reactivating Treg-mediated immunosuppressive effects while restoring antitumor immune responses.

[0007] Although effective CD20-expressing cancer treatments exist, suboptimal response, relapsed refractory disease, and / or resistance to one or more therapeutic agents remain challenges. Furthermore, patients with higher risk and cytogenetic abnormalities continue to have suboptimal responses to approved therapies, as well as shorter response durations and progression-free survival times. Therefore, there is a need for more effective, safe, and durable targeted combination therapies for the treatment of hematological malignancies. Summary of the Invention

[0008] The present invention relates to combination therapy using an anti-CD20 / anti-CD3 bispecific antibody and a bispecific antibody comprising a first antigen-binding domain that specifically binds programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds lymphocyte activation gene 3 (LAG3). The anti-PD1 / anti-LAG3 bispecific antibodies described herein have been found to be advantageous over anti-PD1 antibodies because they offer better selectivity and efficacy. These anti-PD1 / anti-LAG3 bispecific antibodies are further characterized by a reduced sink effect (as indicated by reduced internalization by T cells). These bispecific antibodies preferentially bind to conventional T cells over Tregs, can spare T cell effector function from Treg suppression, and exhibit increased tumor-specific T cell effector function and increased tumor eradication in vivo. Based on these properties, they are advantageous for use in combination with T cell bispecific antibodies, particularly anti-CD20 / anti-CD3 bispecific antibodies.

[0009] Described herein are anti-CD20 / anti-CD3 bispecific antibodies for use in methods of treating cancer, particularly CD20-expressing cancers, where the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody.

[0010] The present invention relates to an anti-CD20 / anti-CD3 bispecific antibody for use in a method as defined herein above, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene 3 (LAG3), wherein the first antigen-binding domain that specifically binds to PD1 is (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; a VL domain comprising

[0013] An anti-CD20 / anti-CD3 bispecific antibody is provided, comprising:

[0011] In one aspect, there is provided an anti-CD20 / anti-CD3 bispecific antibody for use in a method of treating a CD20-expressing cancer, wherein the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered together in a single composition or separately in two or more different compositions.

[0012] Further provided is an anti-CD20 / anti-CD3 bispecific antibody for use in a method for treating a CD20-expressing cancer, wherein the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody, the anti-PD1 / anti-LAG3 bispecific antibody comprising an Fc domain that is an IgG Fc domain, particularly an IgG1 Fc domain or an IgG4 Fc domain, and the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor. More particularly, the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain of the human IgG1 subclass with amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0013] In one embodiment an anti-CD20 / anti-CD3 bispecific antibody for use in the methods described herein above, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; a VL domain comprising contains, or (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 24; and a VL domain comprising

[0023] An anti-CD20 / anti-CD3 bispecific antibody is provided, comprising a second antigen-binding domain comprising:

[0014] In another aspect, there is provided an anti-CD20 / anti-CD3 bispecific antibody for use in the methods disclosed herein, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds PD1, the first antigen-binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10.

[0015] In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody for use as described herein, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26

[0023] An anti-CD20 / anti-CD3 bispecific antibody is provided, comprising a second antigen-binding domain comprising:

[0016] In an additional embodiment, there is provided an anti-CD20 / anti-CD3 bispecific antibody for use in the methods described herein, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 27 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28; or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30; or (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 31 and a VL domain comprising the amino acid sequence of SEQ ID NO: 32; or (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 33 and a VL domain comprising the amino acid sequence of SEQ ID NO: 34

[0023] An anti-CD20 / anti-CD3 bispecific antibody is provided, comprising a second antigen-binding domain comprising:

[0017] Further provided herein is an anti-CD20 / anti-CD3 bispecific antibody for use in the methods disclosed herein, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises: a first antigen-binding domain that specifically binds to PD1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; a second antigen-binding domain that specifically binds to LAG3, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18;

[0013] An anti-CD20 / anti-CD3 bispecific antibody is provided, comprising:

[0018] In a further aspect there is provided an anti-CD20 / anti-CD3 bispecific antibody for use in a method of treating a CD20-expressing cancer, the bispecific antibody comprising an Fab fragment that specifically binds to PD1 and an Fab fragment that specifically binds to LAG3. In one aspect the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1, and the variable domains VL and VH are swapped for each other such that VL is part of the heavy chain and VH is part of the light chain.

[0019] In another aspect, there is provided an anti-CD20 / anti-CD3 bispecific antibody for use in the methods disclosed herein above, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises monovalent binding to PD-1 and monovalent binding to LAG3.

[0020] In a further aspect, there is provided an anti-CD20 / anti-CD3 bispecific antibody for use in the methods disclosed herein above, wherein the anti-PD1 / anti-LAG3 bispecific antibody is a humanized or chimeric antibody. In particular, the anti-PD1 / anti-LAG3 bispecific antibody is a humanized antibody. Further provided is an anti-PD1 / anti-LAG3 bispecific antibody described herein above, comprising an Fc domain containing a modification that promotes association of the first and second subunits of the Fc domain. In one aspect, there is provided an anti-PD1 / anti-LAG3 bispecific antibody, wherein the first subunit of the Fc domain comprises a knob and the second subunit of the Fc domain comprises a hole, according to the knob-into-hole approach. In particular, the first subunit of the Fc domain contains the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain contains the amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index).

[0021] In certain embodiments, an anti-CD20 / anti-CD3 bispecific antibody for use in a method of treating a CD20-expressing cancer, wherein the anti-PD1 / anti-LAG3 bispecific antibody is (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35, a first light chain comprising the amino acid sequence of SEQ ID NO: 36, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37, and a second light chain comprising the amino acid sequence of SEQ ID NO: 38; or (b) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35, a first light chain comprising the amino acid sequence of SEQ ID NO: 36, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 39, and a second light chain comprising the amino acid sequence of SEQ ID NO: 40.

[0013] An anti-CD20 / anti-CD3 bispecific antibody is provided, comprising:

[0022] More particularly, the anti-PD1 / anti-LAG3 bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35, a first light chain comprising the amino acid sequence of SEQ ID NO: 36, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37, and a second light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0023] Further provided is an anti-CD20 / anti-CD3 bispecific antibody for use in a method of treating a CD20-expressing cancer, wherein the anti-CD20 / anti-CD3 bispecific antibody is for use in combination with an anti-PD1 / anti-LAG3 bispecific antibody, and the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V H CD3) and the light chain variable region (V L The first antigen-binding domain contains a heavy chain variable region (V H CD20) and the light chain variable region (V L and a second antigen-binding domain comprising the CDR-H1 sequence of SEQ ID NO: 41, the CDR-H2 sequence of SEQ ID NO: 42, and the CDR-H3 sequence of SEQ ID NO: 43. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 41, the CDR-H2 sequence of SEQ ID NO: 42, and the CDR-H3 sequence of SEQ ID NO: 43. H a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 44, the CDR-L2 sequence of SEQ ID NO: 45, and the CDR-L3 sequence of SEQ ID NO: 46; L More particularly, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47. HCD3), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 48 L In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody for use in the method of treating a CD20-expressing cancer comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 49, the CDR-H2 sequence of SEQ ID NO: 50, and the CDR-H3 sequence of SEQ ID NO: 51. H CD20), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 52, the CDR-L2 sequence of SEQ ID NO: 53, and the CDR-L3 sequence of SEQ ID NO: 54. L In particular, the second antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 55. H CD20), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 56 L In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody for use in the method of treating a CD20-expressing cancer comprises a third antigen-binding domain that binds to CD20. In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises an Fc domain comprising one or more amino acid substitutions that reduce Fc receptor binding and / or effector function.

[0024] In one particular embodiment, the anti-CD20 / anti-CD3 bispecific antibody for use in the method of treating a CD20-expressing cancer is glofitamab. In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody for use in the method of treating a CD20-expressing cancer is mosunetuzumab.

[0025] In a further aspect, there is provided an anti-CD20 / anti-CD3 bispecific antibody for use in a method of treating a CD20-expressing cancer, wherein the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody, and the combination is for administration at intervals of about 1 week to 3 weeks.

[0026] In yet another embodiment, the anti-CD20 / anti-CD3 bispecific antibody is for use in a method of treating a CD20-expressing cancer, wherein prior treatment with a type II anti-CD20 antibody, preferably obinutuzumab, precedes the combination treatment, and the period between the prior treatment and the combination treatment is sufficient to allow for B cell depletion in the individual in response to the type II anti-CD20 antibody. Preferably, the type II anti-CD20 antibody is obinutuzumab.

[0027] In a further embodiment, there is provided a composition comprising an anti-PD1 / anti-LAG3 bispecific antibody for use in treating a CD20-expressing cancer, the treatment comprising the administration of said composition comprising an anti-PD1 / anti-LAG3 bispecific antibody in combination with a composition comprising an anti-CD20 / anti-CD3 bispecific antibody, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds lymphocyte activation gene 3 (LAG3), and wherein the first antigen-binding domain that specifically binds PD1 is: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; a VL domain comprising A composition is provided comprising:

[0028] In one embodiment, the composition comprises an anti-PD1 / anti-LAG3 bispecific antibody comprising a first antigen-binding domain that specifically binds PD1, wherein the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10. In a further embodiment, the composition is an anti-PD1 / anti-LAG3 bispecific antibody comprising a second antigen-binding domain that specifically binds LAG3, (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; a VL domain comprising contains, or (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 24; and a VL domain comprising and a second antigen-binding domain comprising:

[0029] In one embodiment, the composition is an anti-PD1 / anti-LAG3 bispecific antibody comprising an antigen-binding domain that specifically binds to LAG3, (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26 and anti-PD1 / anti-LAG3 bispecific antibodies, comprising:

[0030] In one particular embodiment, the composition is an anti-PD1 / anti-LAG3 bispecific antibody a first Fab fragment that specifically binds to PD1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; a second Fab fragment that specifically binds to LAG3, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18; and and an anti-PD1 / anti-LAG3 bispecific antibody comprising:

[0031] Further provided is a composition comprising an anti-PD1 / anti-LAG3 bispecific antibody for use in treating a CD20-expressing cancer, wherein the treatment comprises administering a composition comprising an anti-PD1 / anti-LAG3 bispecific antibody in combination with a composition comprising an anti-CD20 / anti-CD3 bispecific antibody, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V H CD3) and the light chain variable region (V L The first antigen-binding domain contains a heavy chain variable region (V H CD20) and the light chain variable region (V L and a second antigen-binding domain comprising the CDR-H1 sequence of SEQ ID NO: 41, the CDR-H2 sequence of SEQ ID NO: 42, and the CDR-H3 sequence of SEQ ID NO: 43. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 41, the CDR-H2 sequence of SEQ ID NO: 42, and the CDR-H3 sequence of SEQ ID NO: 43. H a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 44, the CDR-L2 sequence of SEQ ID NO: 45, and the CDR-L3 sequence of SEQ ID NO: 46; L More particularly, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47. H CD3), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 48 LIn one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 49, the CDR-H2 sequence of SEQ ID NO: 50, and the CDR-H3 sequence of SEQ ID NO: 51. H CD20), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 52, the CDR-L2 sequence of SEQ ID NO: 53, and the CDR-L3 sequence of SEQ ID NO: 54. L In particular, the second antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 55. H CD20), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 56 L In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a third antigen-binding domain that binds to CD20. In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises an Fc domain comprising one or more amino acid substitutions that reduce Fc receptor binding and / or effector function. In one particular embodiment, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab. In another particular embodiment, the anti-CD20 / anti-CD3 bispecific antibody is mosunetuzumab.

[0032] In yet another aspect, there is provided a composition comprising an anti-PD1 / anti-LAG3 bispecific antibody for use in treating a CD20-expressing cancer, the treatment comprising administering a composition comprising an anti-PD1 / anti-LAG3 bispecific antibody in combination with a composition comprising an anti-CD20 / anti-CD3 bispecific antibody, wherein prior treatment with a type II anti-CD20 antibody, preferably obinutuzumab, precedes the combination treatment, and the period between the prior treatment and the combination treatment is sufficient to deplete the individual's B cells in response to the type II anti-CD20 antibody. Preferably, the type II anti-CD20 antibody is obinutuzumab.

[0033] In a further aspect, there is provided a pharmaceutical product comprising: (A) a first composition comprising an anti-CD20 / anti-CD3 bispecific antibody as an active ingredient and a pharmaceutically acceptable carrier; and (B) a second composition comprising an anti-PD1 / anti-LAG3 bispecific antibody as an active ingredient and a pharmaceutically acceptable carrier for use in the combination, sequential or simultaneous treatment of a disease, in particular a CD20-expressing cancer.

[0034] In another aspect, a pharmaceutical composition is provided comprising a combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody for use in the combined, sequential, or simultaneous treatment of a disease, particularly a CD20-expressing cancer, in particular a disease selected from the group consisting of B-cell proliferative disorders, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin's lymphoma (HL).

[0035] In another aspect, the use of a combination of an anti-CD20 / CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody in the manufacture of a medicament for treating or delaying the progression of a proliferative disease, in particular for treating a CD20-expressing cancer, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds lymphocyte activation gene 3 (LAG3), and the first antigen-binding domain that specifically binds PD1 is (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; a VL domain comprising Uses are provided, including

[0036] In a further embodiment, the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; a VL domain comprising contains, or (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 24; and a VL domain comprising and a second antigen-binding domain comprising:

[0037] In another aspect, there is provided the use of a combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody in the manufacture of a medicament for treating or delaying the progression of a proliferative disease, in particular for treating a CD20-expressing cancer, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first Fab fragment that specifically binds to PD1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10, and a second Fab fragment that specifically binds to LAG3, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.

[0038] In yet another aspect, there is provided the use of a combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody in the manufacture of a medicament for treating or delaying the progression of a proliferative disease, in particular for treating a CD20-expressing cancer, wherein prior treatment with a type II anti-CD20 antibody, preferably obinutuzumab, precedes the combination treatment, and the period between prior treatment and the combination treatment is sufficient for depletion of B cells in the individual in response to the type II anti-CD20 antibody. Preferably, the type II anti-CD20 antibody is obinutuzumab.

[0039] In a further aspect, there is provided a method for treating a CD20-expressing cancer in a subject, comprising administering to the subject an effective amount of an anti-CD20 / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 bispecific antibody, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds lymphocyte activation gene 3 (LAG3), and wherein the first antigen-binding domain that specifically binds PD1 is: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; a VL domain comprising A method is provided, comprising:

[0040] In one embodiment, the method comprises: (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a VH domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; a VL domain comprising contains, or (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 24; and a VL domain comprising A method is provided, comprising:

[0041] In another aspect, a method is provided, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first Fab fragment that specifically binds PD1, wherein the first Fab fragment comprises a VH domain comprising the amino acid sequence of SEQ ID NO:9 and a VL domain comprising the amino acid sequence of SEQ ID NO:10, and a second Fab fragment that specifically binds LAG3, wherein the VH domain comprises the amino acid sequence of SEQ ID NO:17 and a VL domain comprising the amino acid sequence of SEQ ID NO:18.

[0042] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V H CD3) and the light chain variable region (V L The first antigen-binding domain contains a heavy chain variable region (V H CD20) and the light chain variable region (V L and a second antigen-binding domain comprising the CDR-H1 sequence of SEQ ID NO: 41, the CDR-H2 sequence of SEQ ID NO: 42, and the CDR-H3 sequence of SEQ ID NO: 43. H a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 44, the CDR-L2 sequence of SEQ ID NO: 45, and the CDR-L3 sequence of SEQ ID NO: 46; L More particularly, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47. H CD3), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 48 L In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 49, the CDR-H2 sequence of SEQ ID NO: 50, and the CDR-H3 sequence of SEQ ID NO: 51. H CD20), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 52, the CDR-L2 sequence of SEQ ID NO: 53, and the CDR-L3 sequence of SEQ ID NO: 54. L In particular, the second antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 55.H CD20), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 56 L In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a third antigen-binding domain that binds to CD20. In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises an Fc domain comprising one or more amino acid substitutions that reduce Fc receptor binding and / or effector function. In one particular embodiment, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab. In another particular embodiment, the anti-CD20 / anti-CD3 bispecific antibody is mosunetuzumab.

[0043] In yet another aspect, there is provided a method of treating a CD20-expressing cancer in a subject, comprising administering to the subject an effective amount of an anti-CD20 / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 bispecific antibody, wherein prior treatment with a type II anti-CD20 antibody, preferably obinutuzumab, occurs before the combination treatment, and the period of time between the prior treatment and the combination treatment is sufficient to deplete the individual's B cells in response to the type II anti-CD20 antibody. Preferably, the type II anti-CD20 antibody is obinutuzumab.

[0044] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered together in a single composition or separately in two or more different compositions. In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered intravenously or subcutaneously. In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody is administered simultaneously with the anti-PD1 / anti-LAG3 bispecific antibody, before the anti-PD1 / anti-LAG3 bispecific antibody, or after the anti-PD1 / anti-LAG3 bispecific antibody.

[0045] In any of the above aspects, the individual is preferably a mammal, particularly a human. [Brief explanation of the drawings]

[0046] [Figure 1]Figures 1A and 1B are schematic diagrams of a specific anti-PD1 / anti-LAG3 bispecific antibody (Figure 1A) and a specific anti-CD20 / anti-CD3 bispecific antibody (Figure 1B) used in the Examples. These molecules are described in further detail in Examples 2 and 1, respectively. Figure 1A shows an anti-PD1 / anti-LAG3 bispecific antibody in a 1+1 format, in which the PD1-binding domain comprises a crossFab (VH / VL domain swap) and the LAG3-binding domain comprises a CH1 domain and a CK domain with amino acid mutations to aid in correct pairing ("charge variant"). The Fc portion contains knob-into-hole mutations (indicated by black arrows) and amino acid mutations L234A, L235A, and P329G, which almost completely abolish Fcγ receptor binding of the human IgG1 Fc domain. Figure 1B shows an exemplary bispecific anti-CD20 / anti-CD3 antibody in a 2+1 format (designated CD20 TCB). [Figure 2] Figure 2 shows the effect of an anti-PD1 / anti-LAG3 bispecific antibody (PD1-LAG3 BsAb) in combination with CD20 TCB on cytotoxic granzyme B release by human CD4 T cells cocultured with a B cell lymphoblastoid cell line (ARH77). PD1-LAG3 BsAb is compared to PD-1 antibodies (nivolumab, pembrolizumab, and parental PD-1 antibodies). [Figure 3] Figure 3 shows the protocol for an in vivo efficacy study of PD1-LAG3 BsAb versus PD1 antibody in combination with CD20 TCB in WSU-DLCL2-bearing fully humanized NSG mice. The table below defines the subgroups of mice that received the different combinations. The experiment is described in Example 4. [Figure 4]The results of the study are shown in Figure 4. 1.5 x 10 WSU-DLCL2 cells expressing CD20 were injected subcutaneously into humanized NSG mice. After tumors reached a mean volume of approximately 350–400 mm (day 14), mice were randomized into six groups to receive: A) phosphate-buffered saline (PBS; vehicle) as control; B) CD20-TCB (0.15 mg / kg, once weekly, iv); C) CD20-TCB (0.15 mg / kg, once weekly, iv) + nivolumab (1.5 mg / kg, once weekly, iv); D) CD20-TCB (0.15 mg / kg, once weekly, iv) + nivolumab (1.5 mg / kg, once weekly, ip) + anti-LAG3 (1.5 mg / kg, once weekly, iv); E) CD20-TCB (0.15 mg / kg, once weekly, iv) + PD1-LAG3 B) CD20-TCB (0.15 mg / kg, iv) + PD1-LAG3 BsAb (1.5 mg / kg, iv) once a week; F) CD20-TCB (0.15 mg / kg, iv) + PD1-LAG3 BsAb (3 mg / kg, iv) once a week. Tumor volumes were measured three times a week with digital calipers. Data are shown as mean tumor volume and standard error of the mean (+ / - SEM). [Figure 5] In Figures 5A-5F, measurements of tumor volume (mm3 + / - SEM) over the period from day 14 to day 45 are shown for each individual animal, demonstrating the uniformity of the anti-tumor response in the PD1-LAG3 BsAb-treated groups. Tumor growth curves are shown in Figure 5A for the vehicle group, Figure 5B for CD20 CD3 TCB alone (0.15 mg / kg), Figure 5C for the combination of CD20 CD3 TCB with nivolumab (1.5 mg / kg), Figure 5D for the combination of CD20 CD3 TCB with nivolumab and LAG3 (1.5 mg / kg), and Figures 5E (1.5 mg / kg) and 5F (3 mg / kg PD1 / LAG3 BsAb) for the combination of CD20 CD3 TCB with PD1 / LAG3 BsAb. [Figure 6]Figure 6 shows that the combination of 3 mg / kg CD20 CD3 TCB and PD1 / LAG3 BsAb resulted in statistically significant tumor protection compared with treatment with nivolumab or nivolumab plus anti-LAG3. For this analysis, tumor volume data were transformed to introduce a new endpoint: we assessed whether the final observed tumor volume for each animal was less than 800 mm3, providing a binary readout and the proportion of small tumors. This endpoint was then subjected to pairwise group comparisons based on the Chi2 test. [Figure 7] Figure 7 shows the protocol for an in vivo efficacy study of CD20 TCB in combination with PD1-LAG3 BsAb or pembrolizumab + anti-LAG3 in fully humanized NSG mice bearing OCI-Ly18. The table below defines the subgroups of mice that received the different combinations. The experiment is described in Example 5. [Figure 8] Figure 8 shows the results of the study. Humanized NSG mice were injected subcutaneously with CD20-expressing OCI-Ly18 lymphoma cells. After tumors reached an average volume of approximately 200 mm (day 10), mice were randomized and injected with therapeutic agents. Measurements of tumor volume (mm + / - SEM) are shown as the average volume within a group of mice. Tumor size was measured until there were at least 6 (vehicle) or 7 (treatment) mice per group per time point. Vehicle was followed until day 26, and treatment until day 35. Data are shown as the mean tumor volume and standard error of the mean (+ / - SEM). [Figure 9] In Figures 9A-9D, measurements of tumor volume (mm3 + / - SEM) are shown for each individual animal over the period from day 10 to day 35. Tumor growth curves are shown in Figure 9A for the vehicle group, in Figure 9B for CD20 CD3 TCB alone, in Figure 9C for the combination of CD20 CD3 TCB with PD1-LAG3 BsAb, and in Figure 9D for the combination of CD20 CD3 TCB with pembrolizumab and anti-LAG3. [Figure 10]Figure 10 shows the protocol for an in vivo efficacy study of CD20 TCB alone compared to the combination with PD1-LAG3 BsAb in fully humanized NSG mice bearing OCI-Ly18, when pre-treatment with obitunutuzumab was used. The table below defines the subgroups of mice that received the different combinations. The experiment is described in Example 6. [Figure 11] Figure 11 shows the results of the study. Humanized NSG mice were subcutaneously injected with CD20-expressing OCI-Ly18 lymphoma cells. After tumors reached an average volume of approximately 400 mm (day 17), mice were randomized and treatments were injected according to the experimental layout. Measurements of tumor volume (mm + / - SEM) are shown as the mean volume within mouse groups. Tumor sizes were measured up to day 35 for treatment groups and up to day 26 for vehicle groups. [Figure 12] In Figures 12A-12C, measurements of tumor volume (mm + / - SEM) are shown for each individual animal over the period from day 17 to day 35. Tumor growth curves are shown for the vehicle group in Figure 12A, obinutuzumab and CD20 CD3 TCB in Figure 12B, and the combination of obinutuzumab with CD20 CD3 TCB and PD1-LAG3 BsAb in Figure 12C. DETAILED DESCRIPTION OF THE INVENTION

[0047] Detailed Description of the Invention definition Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa.

[0048] The term "antibody" 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.

[0049] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each individual antibody in the population is identical and / or binds to the same epitope, except for possible variant antibodies containing, for example, naturally occurring mutations or mutations that arise during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0050] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each binding to the same epitope of the same antigen. The term "bispecific" means that the antibody can specifically bind to at least two distinct antigenic determinants, for example, two binding sites formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), each binding to different antigens or different epitopes on the same antigen. Such bispecific antibodies are in a 1+1 format. Other bispecific antibody formats are the 2+1 format (two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or the 2+2 format (two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). Typically, bispecific antibodies contain two antigen-binding sites, each specific for a different antigenic determinant.

[0051] The term "valency," as used herein, refers to the presence of a specific number of binding domains in an antigen-binding molecule. In this context, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two binding domains, four binding domains, and six binding domains, respectively, in an antigen-binding molecule. Bispecific antibodies of the present invention are at least "bivalent," and may also be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent"). In certain embodiments, antibodies of the present invention have two or more binding sites and are bispecific. That is, antibodies may be bispecific even when more than two binding sites are present (i.e., the antibody is trivalent or multivalent).

[0052] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody. A "native antibody" refers to a naturally occurring immunoglobulin molecule having a variety of structures. For example, native IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy domain or a heavy chain variable domain) followed by three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light domain or a light chain variable domain) followed by a light chain constant domain (CL) (also called a light chain constant region). Antibody heavy chains may be divided into one of five types called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), several of which may be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0053] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, triabodies, tetrabodies, cross-Fab fragments, linear antibodies, single-chain antibody molecules (e.g., scFv), multispecific antibodies made from antibody fragments, and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Plueckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. For a description of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having extended in vivo half-lives, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments containing two antigen-binding domains that may be bivalent or bispecific; see, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, Massachusetts; see, e.g., U.S. Pat. No. 6,248,516 B1).Additionally, antibody fragments comprise a single polypeptide chain characterized by a VH domain (i.e., capable of assembling with a VL domain) or characterized by a VL domain (i.e., capable of assembling with a VH domain into a functional antigen-binding site), thereby conferring antigen-binding properties of a full-length antibody. Antibody fragments may be produced 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.

[0054] Papain digestion of an intact antibody produces two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains, 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 containing the VL domain and constant domain of the light chain (CL), and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment, which contains two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0055] The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. Two possible chain compositions of crossover Fab molecules are possible and are included in the bispecific antibodies of the present invention. On the other hand, the variable regions of the Fab heavy and light chains are swapped, i.e., the crossover Fab molecule contains a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). This crossover Fab molecule is called CrossFab. (VLVH) On the other hand, when the constant regions of the Fab heavy and light chains are replaced, the crossover Fab molecule contains a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL), and a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1). This crossover Fab molecule is called CrossFab. (CLCH1) It is also called.

[0056] A "single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, where the antibody domains and linker are arranged in the following order from N-terminus to C-terminus: (a) VH-CH1-linker-VL-CL, (b) VL-CL-linker-VH-CH1, (c) VH-CL-linker-VL-CH1, or (d) VL-CH1-linker-VH-CL; and the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab molecules may be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to the Kabat numbering).

[0057] A "crossover single-chain Fab fragment" or "x-scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following orders, from N- to C-terminus: (a) VH-CL-linker-VL-CH1; or (b) VL-CH1-linker-VH-CL, where VH and VL together form an antigen-binding domain that specifically binds to an antigen, and the linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules may be further stabilized by the creation of an interchain disulfide bond through the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain, according to the Kabat numbering system).

[0058] A "single-chain variable fragment (scFv)" is a fragment of an antibody heavy chain (V) connected using a short linker peptide of 10 to about 25 amino acids. H ) and light chain (V L ) variable region fusion proteins. The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility, and H N-terminus and V L The scFv antibody can be linked to the C-terminus of the full-length antibody or vice versa. This protein retains the specificity of the original antibody, although the constant regions have been removed and a linker has been introduced. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, antibody fragments contain a single polypeptide chain characterized by a VH domain (i.e., capable of assembling with a VL domain) or a VL domain (i.e., capable of assembling with a VH domain into a functional antigen-binding site), thereby conferring the antigen-binding properties of a full-length antibody.

[0059] "Scaffold antigen-binding proteins" are known in the art; for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen-binding domains. See, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13:695-701 (2008). In one embodiment of the invention, the scaffold antigen binding protein is selected from the group consisting of CTLA-4 (e.g., cytochrome P456), lipocalin (anticalin), protein A derived molecules such as the Z-domain (affibody), A-domain (avimer / maxibody) of protein A, serum transferrin (transbody); engineered ankyrin repeat proteins (DARPins), variable domains of antibody light or heavy chains (single domain antibodies, sdAb), variable domains of antibody heavy chains (nanobodies, aVH), V NAR Fragments, fibronectin (adnectin), C-type lectin domain (tetranectin); variable domain of novel antigen receptor beta-lactamase (V NARThe antibody may be selected from the group consisting of human γ-crystallin or ubiquitin fragments, human gamma-crystallin or ubiquitin (affilin molecules); Kunitz-type domains of human protease inhibitors, microbodies, e.g., proteins from the knottin family, peptide aptamers, and fibronectin (adnectins). CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is a CD28 family receptor expressed primarily on CD4+ T cells. Its extracellular domain has a variable domain-like Ig fold. Loops corresponding to the CDRs of an antibody may be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as ebibodies (e.g., U.S. Pat. No. 7,166,697 B1). Ebibodies are approximately the same size as the isolated variable region of an antibody (e.g., a domain antibody). For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have a rigid beta-sheet secondary structure with many loops at the open end of the conical structure, which can be engineered to bind to different target antigens. Anticalins are 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000), U.S. Patent No. 7,250,297 B1, and U.S. Patent Application Publication No. 20070224633. Affibodies are scaffolds derived from Staphylococcus aureus protein A that can be engineered to bind antigens. Domains consist of three helical bundles of approximately 58 amino acids. Libraries have been created by randomization of surface residues. For further details see Protein Eng. Des. Sel. 2004, 17, 455-462 and EP 1 641 818 A1. Avimers are multi-domain proteins derived from the A-domain scaffold family. Natural domains of approximately 35 amino acids conform to defined disulfide-bonded structures.Diversity is generated by shuffling the natural variation exhibited by the A-domain family. For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind different target antigens by inserting peptide sequences into permissive surface loops. Examples of engineered transferrin scaffolds include transbodies. For further details, see J. Biol. Chem 274, 24066-24073 (1999). Designed ankyrin repeat proteins (DARPins) are derived from ankyrins, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha helices and a beta turn. A single ankyrin repeat can be engineered to bind to different target antigens by randomizing residues in the first alpha helix and beta turn of each repeat. The binding interface can be increased by increasing the number of modules (affinity maturation method). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003), and J. Mol. Biol. 369, 1015-1028 (2007), and U.S. Patent Application Publication No. 20040132028 A1.

[0060] Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single domains were derived from the variable domain of antibody heavy chains from camelids (nanobodies or V H Furthermore, the term single domain antibody refers to an antibody that contains an autonomous human heavy chain variable domain (aVH) or a shark-derived VH. NARThese include fragments. Fibronectin is a scaffold that can be engineered to bind to antigens. Adnectins consist of a backbone with the native amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the beta-sandwich can be engineered to enable Adnectins to specifically recognize therapeutic targets of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), U.S. Patent Application Publication No. 20080139791, WO2005056764, and U.S. Patent No. 6,818,418 B1. Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop that is inserted into the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from naturally occurring microproteins that are 25-50 amino acids long and contain 3-4 cysteine ​​bridges; examples of microproteins include KalataBI, conotoxins, and knottins. Microproteins have loops that can be engineered to contain up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knottin domains, see WO2008098796.

[0061] 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 competitive assay; conversely, the reference molecule blocks the binding of the antigen-binding molecule to its antigen by 50% or more in a competitive assay.

[0062] As used herein, the term "antigen-binding domain" or "antigen-binding site" refers to a portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a portion of an antibody that specifically binds to and is complementary to part or all of an antigen. When an antigen is large, an antigen-binding molecule may bind only to a specific portion of the antigen, which portion is called an epitope. An antigen-binding domain may, for example, be provided by one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one embodiment, the antigen-binding domain is capable of binding to the antigen and blocking or partially blocking its function. Antigen-binding domains that specifically bind to PD1 and LAG3 include antibodies and fragments thereof, as further defined herein. In addition, the antigen-binding domain may comprise a binding domain based on a scaffold antigen-binding protein, such as an engineered repeat protein or an engineered repeat domain (see, for example, WO 2002 / 020565).

[0063] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to the site on a polypeptide polymer to which an antigen-binding moiety binds (e.g., a contiguous stretch of amino acids or a conformational structure composed of different regions of non-contiguous amino acids), forming an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise specified, proteins useful as antigens herein can be proteins in any naturally occurring form from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. When a particular protein is referred to herein, the term encompasses the "full-length," unprocessed protein and any form of the protein obtained by intracellular processing. The term also encompasses naturally occurring protein variants, e.g., splice variants or allelic variants.

[0064] "Specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or nonspecific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques known in the art, such as surface plasmon resonance (SPR) technology (analyzed with a BIAcore device) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the binding of the antigen-binding molecule to the antigen, as measured, for example, by SPR. In certain embodiments, molecules that bind to an antigen have a 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 -7 M or less, e.g., 10-7 M~10 -13 M, e.g., 10 -9 M~10 -13 M).

[0065] "Affinity" or "binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its binding partner Y can generally be expressed by the dissociation constant (Kd), which is the ratio of the detachment rate constant to the dissociation rate constant (koff and kon, respectively). Thus, equivalent affinities can involve different rate constants as long as the ratio of the rate constants is the same. Affinity can be measured by methods common in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0066] As used herein, the term "high affinity" for an antibody refers to an antibody with a Kd of 10 or greater for a target antigen. -9 M or less, and more specifically 10 -10 A "low affinity" antibody has a Kd of 10 -8 This refers to the above antibodies.

[0067] An "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) relative to a parent antibody that does not possess such alterations, which alterations improve the affinity of the antibody for antigen.

[0068] "CD20" refers to the B lymphocyte antigen CD20, also known as B lymphocyte surface antigen B1 or leukocyte surface antigen Leu-16, and 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), unless otherwise specified. The amino acid sequence of human CD20 is set forth in Uniprot accession number P11836 (version 149, SEQ ID NO: 61). CD20 is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD 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 emerging protein family are characterized by common structural features and similar intron / exon splice boundaries and display unique expression patterns between hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that plays a role in the development and differentiation of B cells into plasma cells. This family member is localized to 11q12 within a cluster of family members. Alternative splicing of this gene results in two transcript variants that encode the same protein. The term "CD20" encompasses "full-length," unprocessed CD20 and any form of CD20 resulting from processing within the cell. The term also encompasses naturally occurring variants of CD20, such as splice variants or allelic variants.

[0069] The terms "anti-CD20 antibody" and "antibody that binds to CD20" refer to an antibody that is capable of binding to CD20 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD20. In one embodiment, the binding of an anti-CD20 antibody to an unrelated, non-CD20 protein is less than about 10% of the binding of the antibody to CD20 as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD20 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 ~10 -13 M, e.g. 10 -9 M~10 -13 In certain embodiments, the anti-CD20 antibody binds to an epitope of CD20 that is conserved among CD20 from different species.

[0070] By "type II anti-CD20 antibody" is meant an anti-CD20 antibody having the binding characteristics and biological activity of type II anti-CD20 antibodies described in Cragg et al., Blood 103 (2004) 2738-2743; Cragg et al., Blood 101 (2003) 1045-1052, Klein et al., mAbs 5 (2013), 22-33, and summarized in Table 1 below. [Table A] * For IgG1 isotype

[0071] Examples of Type II anti-CD20 antibodies include, for example, obinutuzumab (GA101), tositumumab (B1), humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in WO 2005 / 044859), 11B8 IgG1 (as disclosed in WO 2004 / 035607), and AT80 IgG1.

[0072] In one embodiment, the type II anti-CD20 antibody has the heavy chain variable region sequence of SEQ ID NO: 55 (V H CD20) and the light chain variable region sequence of SEQ ID NO: 56 (V L In another embodiment, the type II anti-CD20 antibody is modified to increase the proportion of nonfucosylated oligosaccharides in the Fc region compared to the unmodified antibody. In one embodiment, at least about 40% of the N-linked oligosaccharides in the Fc region of the type II anti-CD20 antibody are nonfucosylated.

[0073] In a specific embodiment, the type II anti-CD20 antibody is obinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453). As used herein, obinutuzumab is synonymous with GA101. Trade names are GAZYVA® or GAZYVARO®. This replaces all previous versions (e.g., Vol. 25, No. 1, 2011, pp. 75-76) and was formerly known as afutuzumab (recommended INN, WHO Drug Information, Vol. 23, No. 2, 2009, p. 176; Vol. 22, No. 2, 2008, p. 124). In one embodiment, the type II anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 and a light chain comprising the amino acid sequence of SEQ ID NO: 63. In one embodiment, the type II anti-CD20 antibody is tositumomab.

[0074] Examples of Type I anti-CD20 antibodies include, for example, rituximab, ofatumumab, veltuzumab, caratuzumab, ocrelizumab, PRO131921, brituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (disclosed in WO 2005 / 103081), 2F2 IgG1 (disclosed in WO 2004 / 035607 and WO 2005 / 103081), and 2H7 IgG1 (disclosed in WO 2004 / 056312).

[0075] The term "humanized B-Ly1 antibody" refers to the humanized B-Ly1 antibody disclosed in WO 2005 / 044859 and WO 2007 / 031875, which was obtained from the mouse monoclonal anti-CD20 antibody B-Ly1 (variable region of mouse heavy chain (VH): SEQ ID NO: 64; variable region of mouse light chain (VL): SEQ ID NO: 65; see Poppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131-139) by chimerization with human constant domains derived from IgG1 and subsequent humanization (see WO 2005 / 044859 and WO 2007 / 031875). These "humanized B-Ly1 antibodies" are disclosed in detail in WO 2005 / 044859 and WO 2007 / 031875.

[0076] The term "decrease" (and grammatical variations thereof, such as "reduce" or "reducing"), e.g., a decrease in the number of B cells or cytokine release, refers to a decrease in the respective amount as measured by an appropriate method known in the art. For clarity, the term also includes a decrease to zero (or below the detection limit of the analytical method), i.e., complete disappearance or elimination. Conversely, "increased" refers to an increase in the respective amount.

[0077] As used herein, "T cell antigen" refers to an antigenic determinant displayed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes.

[0078] 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. Further 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, such as CD20 or CD19.

[0079] As used herein, "activating T cell antigen" refers to an antigenic determinant expressed by T lymphocytes, particularly cytotoxic T lymphocytes, which can induce or enhance T cell activation upon interaction with an antigen-binding molecule. Specifically, interaction of an antigen-binding molecule with a T cell activation antigen can induce T cell activation by triggering a signal transduction cascade of the T cell receptor complex. An exemplary activating T cell antigen is CD3.

[0080] Unless otherwise specified, 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 and any form of CD3 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is set forth in UniProt (www.uniprot.org) accession number P07766 (version 144) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. See also SEQ ID NO: 66. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank number BAB71849.1. See also SEQ ID NO: 67.

[0081] The terms "a bispecific antibody comprising a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3," "a bispecific antibody that specifically binds PD1 and LAG3," "a bispecific antigen-binding molecule specific for PD1 and LAG3," or "anti-PD1 / anti-LAG3 antibody" are used interchangeably herein and refer to a bispecific antibody that is capable of binding to PD1 and LAG3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting PD1 and LAG3.

[0082] The term "PD1," also known as programmed cell death protein 1, is a 288-amino acid type I membrane protein first described in 1992 (Ishida et al., EMBO J., 11 (1992), 3887-3895). PD-1 is a member of the extended CD28 / CTLA-4 family of T cell regulatory factors and has two ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The protein structure comprises an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located within an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, suggesting that PD-1 negatively regulates TCR signaling. This is consistent with the binding of SHP-1 and SHP-2 phosphatases to the cytoplasmic tail of PD-1 upon ligand binding. PD-1 is not expressed on naive T cells but is upregulated following T cell receptor (TCR)-mediated activation and is observed on both activated and exhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772). These exhausted T cells have a dysfunctional phenotype and are unable to respond appropriately. PD-1 has a relatively broad expression pattern, but its most important role may be as a co-inhibitory receptor for T cells (Chinai et al., Trends in Pharmacological Sciences 36 (2015), 587-595). Therefore, current therapeutic approaches focus on blocking the interaction of PD-1 with its ligand to enhance T cell responses. The terms "programmed death 1," "programmed cell death 1," "protein PD-1," "PD-1," "PD1," "PDCD1," "hPD-1," and "hPD-I" can be used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-1 that share at least one epitope in common with PD-1. The amino acid sequence of human PD1 is set forth in UniProt (www.uniprot.org) accession number Q15116 (SEQ ID NO: 68).

[0083] The terms "anti-PD1 antibody" and "antibody comprising an antigen-binding domain that binds to PD1" refer to an antibody that is capable of binding to PD1 (particularly a PD1 polypeptide expressed on the cell surface) with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting PD1. In one embodiment, the degree of binding of the anti-PD1 antibody to an unrelated, non-PD1 protein is less than about 10% of the binding of the antibody to PD1, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance assay using a biosensor system such as a Biacore® system. In particular embodiments, the antigen-binding protein that binds to human PD1 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 K, the binding affinity of M) for binding to human PD1 D In a preferred embodiment, the binding affinities each have a K D The value is determined for PD1 binding affinity using the extracellular domain (ECD) of human PD1 (PD1-ECD) in a surface plasmon resonance assay. The term "anti-PD1 antibody" also encompasses bispecific antibodies capable of binding to PD1 and a second antigen.

[0084] In certain embodiments, the anti-PD1 antibody is selected from the group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), PDR001 (spartalizumab), SHR1210 (canrelizumab), MEDI-0680 (AMP-514), REGN2810, and BGB-108. In one particular embodiment, the anti-PD1 antibody is pembrolizumab, or an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:75 and a light chain comprising the amino acid sequence of SEQ ID NO:76. Pembrolizumab (Merck), also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO 2009 / 114335 (CAS Registry Number 1374853-91-4). In a particular embodiment, the anti-PD1 antibody is nivolumab, or an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 77 and a light chain comprising the amino acid sequence of SEQ ID NO: 78. Nivolumab (CAS Registry Number: 946414-94-4, Bristol-Myers Squibb / Ono), also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO 2006 / 121168 (CAS Registry Number 946414-94-4). In another specific embodiment, the anti-PD-1 antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8, or a humanized variant thereof. In a specific embodiment, the anti-PD-1 antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 10.

[0085] The term "LAG3" or "Lag-3" or "lymphocyte activation gene-3" or "CD223," as used herein, unless otherwise specified, refers to any native LAG3 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed LAG3 and any form of LAG3 resulting from cellular processing. The term also encompasses naturally occurring variants of LAG3, e.g., splice variants or allelic variants. In a preferred embodiment, the term "LAG3" refers to human LAG3. The amino acid sequence of an exemplary processed (signal sequence-free) LAG3 is set forth in SEQ ID NO: 69. The amino acid sequence of an exemplary extracellular domain (ECD) LAG3 is set forth in SEQ ID NO: 70.

[0086] The terms "anti-LAG3 antibody" and "antibody that binds to LAG3" refer to an antibody that is capable of binding to LAG3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting LAG3. In one aspect, the degree of binding of an anti-LAG3 antibody to an unrelated, non-LAG3 protein is less than about 10% of the binding of the antibody to LAG3 as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to LAG3 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 In a particular aspect, the anti-LAG3 antibody binds to an epitope of LAG3 that is conserved among LAG3s from different species. In a preferred embodiment, an "anti-LAG3 antibody," an "antibody that specifically binds to human LAG3," and an "antibody that binds to human LAG3" have a dissociation constant (Kd) of 1.0 x 10 -8 K in mol / l or less D value, in one embodiment 1.0× -9 K in mol / l or less D value, in one embodiment 1.0×10-9 mol / l ~ 1.0 × 10 -13 K in mol / l D "anti-LAG3 antibody" refers to an antibody that specifically binds to the human LAG3 antigen or its extracellular domain (ECD) with a binding affinity of a certain value. In this respect, binding affinity is determined using standard binding assays, such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden), e.g., using the LAG3 extracellular domain. The term "anti-LAG3 antibody" also encompasses bispecific antibodies capable of binding to LAG3 and a second antigen. In one embodiment, the anti-LAG3 antibody is leratolimab or BMS-986016, or an antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 28.

[0087] "Blocking" or "antagonist" antibodies are antibodies that inhibit or reduce the biological activity of the antigen to which they bind. In some embodiments, blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. For example, the bispecific antibodies of the invention block signaling by PD-1 and LAG3, thereby restoring a functional response (e.g., proliferation, cytokine production, target cell death) by T cells from a dysfunctional state to antigenic stimulation.

[0088] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of an antigen-binding molecule to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th, W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0089] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain, e.g., "complementarity-determining regions" (CDRs), that are hypervariable in sequence and determine antigen-binding specificity. Generally, antibodies contain six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) Hypervariable loops occurring at amino acid residues 26–32 (L1), 50–52 (L2), 91–96 (L3), 26–32 (H1), 53–55 (H2), and 96–101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901–917 (1987); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0090] Unless otherwise specified, CDRs are determined according to Kabat et al., supra. One skilled in the art will understand that CDR designations can be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0091] The terms "Kabat-like variable domain residue numbering" or "Kabat-like amino acid position numbering" and variations thereof refer to the numbering system used for the heavy chain variable domain or light chain variable domain of the antibody compilation of Kabat et al. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or HVR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with the "standard" Kabat-numbered sequence at the regions of homology. Generally, native four-chain antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).

[0092] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0093] For purposes herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0094] 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.

[0095] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0096] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant regions have been further modified or altered from those of the original antibody to create properties according to the invention, particularly in terms of C1q binding and / or Fc receptor (FcR) binding.

[0097] A "human" antibody is one having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0098] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each individual antibody in the population is identical and / or binds to the same epitope, excluding possible variant antibodies, including, for example, naturally occurring mutations or mutations that arise during production of the monoclonal antibody preparation. Such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0099] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In particular, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Although the amino acid sequence of the heavy chain is always present with a C-terminal lysine, variants that do not contain a C-terminal lysine are included in the present invention.

[0100] The IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain. The "CH2 domain" of a human IgG Fc region typically extends from an amino acid residue at approximately amino acid position 231 to an amino acid residue at approximately amino acid position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain of the present invention may be a native-sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises an extension of residues C-terminal to the CH2 domain in the Fc region (i.e., from an amino acid residue at approximately amino acid position 341 to an amino acid residue at approximately amino acid position 447 of IgG). The CH3 region of the present invention may be a native-sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "bulge" ("knob") in one chain and an introduced corresponding "cavity" ("hole") in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains as described herein. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0101] The "knob-into-hole" technique is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protuberance can be positioned within the cavity to promote heterodimer formation and discourage homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protuberance is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protuberance and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In a further specific embodiment, the subunit of the Fc domain containing the knob modification further comprises the amino acid substitution S354C, and the subunit of the Fc domain containing the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of disulfide bridges between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0102] A "region equivalent to the Fc region of an immunoglobulin" is intended to include allelic variants of the Fc region of naturally occurring immunoglobulins and variants having alterations that result in substitutions, additions, or deletions, but that do not substantially reduce the ability of the immunoglobulin to mediate effector function (e.g., antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art (see, e.g., Bowie, JU et al., Science 247:1306-10 (1990)) to have minimal effect on activity.

[0103] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody and varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0104] An "activating Fc receptor" is an Fc receptor that, upon ligation by the Fc region of an antibody, triggers signaling events that stimulate the receptor-bearing cell to exert effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt Accession No. P08637, version 141).

[0105] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, ((G4S) n, (SG4) n , or G4 (SG4) n is a peptide linker, where "n" is generally a number between 1 and 10, typically 2 to 4, and in particular 2. Particularly interesting peptide linkers are (G4S) (SEQ ID NO: 71), (G4S)2 or GGGGSGGGGS (SEQ ID NO: 72), (G4S)3 (SEQ ID NO: 73) and (G4S)4 (SEQ ID NO: 74), more particularly (G4S)2 or GGGGSGGGGS (SEQ ID NO: 72).

[0106] "Fused to" or "connected to" means that the components (e.g., antigen binding domain and Fc domain) are linked by a peptide bond directly or via one or more peptide linkers.

[0107] As used in this application, the term "amino acid" refers to the group of naturally occurring carboxy α-amino acids, including alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0108] "Percent (%) amino acid sequence identity" to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence when these sequences are aligned, gaps are introduced as necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, and ALIGN. It can also be achieved using SAWI or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 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 its source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it may be written as a given amino acid sequence A having or containing a particular percent amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y In this case, X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A differs from the length of amino acid sequence B, the % amino acid sequence identity of A to B will differ from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​as used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0109] In certain embodiments, amino acid sequence variants of the bispecific antibodies of the invention provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the bispecific antibody. Amino acid sequence variants of bispecific antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, 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 possesses the desired properties, e.g., antigen binding. Sites of interest for substitutional mutagenesis include HVRs and framework regions (FRs). 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)-(6). Amino acid substitutions can be introduced into the molecule of interest, and the products screened for the desired activity, e.g., retention / improvement of antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table B]

[0110] Amino acids can be classified according to general side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0111] Non-conservative substitutions would involve exchanging a member of one of these classes for another class.

[0112] The term "amino acid sequence variant" includes substantial variants in which amino acid substitutions are present in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing will have altered (e.g., improved) specific biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antigen-binding molecule and / or will substantially retain specific biological properties of the parent antigen-binding molecule. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently generated using, for example, the phage display-based affinity maturation techniques described herein. Briefly, variant antigen-binding molecules in which one or more HVR residues are mutated, phage-displayed, and screened for a specific biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in HVRs. A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and antigen is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antigen complex may be used to identify contact points between the antibody and antigen. Such contact and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they possess desired properties.

[0113] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is a bispecific antibody with an N-terminal methionyl residue. Other insertional variants of the molecule include N- or C-terminal fusions to polypeptides that increase the serum half-life of the bispecific antibody.

[0114] In certain aspects, the bispecific antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation variants of the molecule may be conveniently obtained by altering the amino acid sequence to create or remove one or more glycosylation sites. For example, the carbohydrate attached to the Fc domain may be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, commonly attached via an N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications in the bispecific antibodies of the invention may be performed to generate variants with specific improved properties. In one aspect, bispecific antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. Such fucosylation variants may have improved ADCC function; see, e.g., U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) or U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Further variants of the bispecific antibodies of the invention include those with bisected oligosaccharides, e.g., those in which the biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, see, e.g., WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), and U.S. Pat. App. Pub. No. 2005 / 0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.Such antibody variants may have improved CDC function and are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.).

[0115] In certain aspects, it may be desirable to create cysteine-engineered variants of the bispecific antibodies of the invention, e.g., "thioMAbs," in which one or more residues of the molecule are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the molecule. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antigen-binding molecules may be created, for example, as described in U.S. Pat. No. 7,521,541.

[0116] In certain embodiments, the bispecific antibodies provided herein may be modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable sites for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous during manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, the polymers can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved and whether the antibody derivative will be used in therapeutic treatment under defined conditions.

[0117] In another aspect, a conjugate of an antibody and a nonproteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam, N. Wet et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can be of any wavelength, including but not limited to, wavelengths that are not harmful to normal cells but that heat the nonproteinaceous moiety to temperatures that kill cells proximal to the antibody-nonproteinaceous moiety.

[0118] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including but not limited to, a cytotoxic agent.

[0119] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), viral-derived RNA, or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0120] By "isolated" nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA, or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain the polynucleotide molecule, but where the polynucleotide molecule is present extrachromosomally or at a chromosomal location that differs from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive- and negative-stranded forms and double-stranded forms. Furthermore, isolated polynucleotides or nucleic acids of the present invention include such molecules produced synthetically. In addition, polynucleotides or nucleic acids may be or include regulatory elements, such as a promoter, ribosome binding site, or transcription terminator.

[0121] A nucleic acid or polynucleotide having a nucleotide sequence at least, e.g., 95% "identical" to a reference nucleotide sequence of the present invention is intended to be identical to the reference sequence, except that the nucleotide sequence of the polynucleotide may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with alternative nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. Such alterations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, and may be interspersed individually among residues in the reference sequence or interspersed in one or more contiguous groups within the reference sequence. In practical terms, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be conventionally determined using known computer programs, such as those described above for polypeptides (e.g., ALIGN-2).

[0122] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide with a set of specific nucleic acid elements that allows transcription of a specific nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector comprises, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In a specific embodiment, an expression cassette of the invention comprises a polynucleotide sequence encoding a bispecific antigen-binding molecule of the invention, or a fragment thereof.

[0123] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce and induce expression of a specific gene to which it is operably linked in a target cell. This term includes a vector as a self-replicating nucleic acid structure and a vector that has been integrated into the genome of a host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows for the stable transcription of large amounts of mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antibody of the present invention or a fragment thereof.

[0124] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parent cell and may contain mutations. Included herein are progeny mutants that have the same function or biological activity as screened or selected for in the originally transformed cell. Host cells are any type of cell line that can be used to produce the bispecific antigen-binding molecules of the invention. In particular, host cells are prokaryotic or eukaryotic host cells. Host cells include cultured cells, e.g., cultured mammalian cells, such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, to name just a few, yeast cells, insect cells and plant cells, but also cells contained in transgenic animals, transgenic plants or cultured plant or animal tissue.

[0125] An "effective amount" of a drug refers to the amount necessary to cause a physiological change in a cell or tissue to which the drug is administered.

[0126] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the side effects of a disease.

[0127] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0128] The term "pharmaceutical composition" refers to a preparation in a form such that the biological activity of the active ingredient contained therein is effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0129] A "pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, or preservatives.

[0130] The term "package insert" is used to refer to instructions customarily included in the commercial packaging of a therapeutic product, which contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or precautions regarding the use of that therapeutic product.

[0131] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed prophylactically or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological consequence of the disease, prevention of metastasis, slowing the rate of disease progression, remission or palliation of symptoms, and improved or improved prognosis. In some embodiments, the molecules of the invention are used to delay the onset of disease or slow the progression of the disease.

[0132] As used herein, the term "cancer" includes lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar carcinoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, The cancer may be a CD20-expressing tumor, ...

[0133] The term "CD20 expression" is intended to refer to a significant level of expressed CD20 on the cell surface of cells, preferably T cells or B cells, more preferably B cells, from a tumor or cancer, respectively, preferably a non-solid tumor. Patients with "CD20-expressing cancer" can be determined by standard assays known in the art. For example, CD20 antigen expression can be measured using immunohistochemistry (IHC) detection, FACS, or via PCR-based detection of corresponding mRNA.

[0134] As used herein, the term "CD20-expressing cancer" refers to any cancer in which cancer cells exhibit expression of the CD20 antigen. Preferably, as used herein, CD20-expressing cancer refers to lymphoma (preferably B-cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemia. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small non-cleaved cell lymphoma / Burkitt lymphoma (including regional Burkitt lymphoma, sporadic Burkitt lymphoma, and non-Burkitt lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B-cell lymphoma, and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B-cell diffuse large cell lymphoma, f) hairy cell lymphoma; g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia; h) acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia; i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma; j) Hodgkin's disease.

[0135] In one embodiment, the CD20-expressing cancer is B-cell non-Hodgkin's lymphoma (NHL). In another embodiment, the CD20-expressing cancer is selected from the group consisting of mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell diffuse large cell lymphoma (DLCL), Burkitt's lymphoma, hairy cell leukemia, follicular lymphoma, multiple myeloma, marginal zone lymphoma, post-transplant lymphoproliferative disorder (PTLD), HIV-associated lymphoma, Waldenstrom's macroglobulinemia, or primary CNS lymphoma.

[0136] "B cell proliferative disorder" refers to 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 a disease in which an increased number of B cells is the cause or characteristic of the disease. A "CD20-positive B cell proliferative disorder" is a B cell proliferative disorder in which B cells, particularly malignant B cells, express CD20 (in addition to normal B cells). Exemplary B cell proliferative disorders include non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and some types of multiple myeloma (MM) and Hodgkin's lymphoma (HL).

[0137] The terms "method of treating," "therapeutic method," or equivalents thereof, when applied to, for example, cancer, refer to a procedure or course of action designed to reduce or eliminate the number of cancer cells in a patient or alleviate the symptoms of cancer. A "method of treating" cancer or another proliferative disorder does not necessarily mean that cancer cells or other disorders are actually eliminated, that the number of cellular disorders is actually reduced, or that the symptoms of the cancer or other disorder are actually alleviated. In many cases, a method of treating cancer is performed even when the likelihood of success is low, but given the patient's medical history and estimated survival expectancy, it is nevertheless believed to induce an overall beneficial course of action.

[0138] The terms "combination," "co-administration," or "co-administering" refer to the administration of an anti-CD20 / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody as two separate formulations (or as a single formulation). Co-administration can be simultaneous or sequential, preferably with a period of time during which both (or all) active agents simultaneously exert their biological activity. The anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are co-administered either simultaneously or sequentially (e.g., intravenously (iv) by continuous infusion) (one anti-CD20 / anti-CD3 bispecific antibody and one anti-PD1 / anti-LAG3 bispecific antibody). When both therapeutic agents are co-administered sequentially, the doses are administered on the same day in two separate administrations, or one of the agents is administered on day 1 and the second is co-administered on days 2-7, preferably days 2-4. Thus, the term "sequential" refers to the sequential administration of the first component (anti-CD20 / anti-CD3 bispecific antibody or anti-PD1 / anti-LAG3 bispecific antibody). The term "concurrently" refers to the same time, and refers to the administration of the first component within seven days of the administration of the second component (anti-CD20 / anti-CD3 bispecific antibody), preferably within four days of the administration of the third component. The term "concurrently administered" with respect to maintenance doses of anti-CD20 / anti-CD3 bispecific antibody and anti-PD1 / anti-LAG3 bispecific antibody means that the maintenance doses can be co-administered simultaneously, e.g., weekly, if the treatment cycle is appropriate for both drugs. Alternatively, the anti-PD1 / anti-LAG3 bispecific antibody can be administered, e.g., every two weeks, and the anti-CD20 / anti-CD3 bispecific antibody can be administered every three weeks. Alternatively, the maintenance doses can be co-administered sequentially, either within one day or within several days.

[0139] Exemplary anti-CD20 / anti-CD3 bispecific antibodies for use in the present invention The present invention relates to anti-CD20 / anti-CD3 bispecific antibodies and their use in combination with anti-PD1 / anti-LAG3 bispecific antibodies, particularly in methods of treating or delaying the progression of CD20-expressing cancers, more particularly in methods of treating or delaying the progression of B-cell proliferative disorders. As used herein, anti-CD20 / anti-CD3 bispecific antibodies are bispecific antibodies comprising a first antigen-binding domain that binds to CD3 and a second antigen-binding domain that binds to CD20. Thus, they target CD20-expressing B cells.

[0140] Thus, as used herein, an anti-CD20 / anti-CD3 bispecific antibody refers to a heavy chain variable region (V H CD3) and the light chain variable region (V L The first antigen-binding domain contains a heavy chain variable region (V H CD20) and the light chain variable region (V L and a second antigen-binding domain comprising a nucleotide sequence encoding ...

[0141] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody for use in combination comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 41, the CDR-H2 sequence of SEQ ID NO: 42, and the CDR-H3 sequence of SEQ ID NO: 43. H a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 44, the CDR-L2 sequence of SEQ ID NO: 45, and the CDR-L3 sequence of SEQ ID NO: 46; L More particularly, the anti-CD20 / anti-CD3 bispecific comprises a first antigen-binding domain comprising a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 47. H CD3), and / or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 48 L In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47. HCD3), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 48 L CD3).

[0142] In one embodiment, the antibody that specifically binds to CD3 is a full-length antibody. In one embodiment, the antibody that specifically binds to CD3 is an antibody of the human IgG class, particularly an antibody of the human IgG1 class. In one embodiment, 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 a particular embodiment, the antibody that specifically binds to CD3 is a crossover Fab molecule in which the variable or constant domains of the Fab heavy chain and the Fab light chain are exchanged (i.e., replaced with each other). In one embodiment, the antibody that specifically binds to CD3 is a humanized antibody.

[0143] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 49, the CDR-H2 sequence of SEQ ID NO: 50, and the CDR-H3 sequence of SEQ ID NO: 51. H CD20), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 52, the CDR-L2 sequence of SEQ ID NO: 53, and the CDR-L3 sequence of SEQ ID NO: 54. L More particularly, the anti-CD20 / anti-CD3 bispecific comprises a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 55. H CD20), and / or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 56. L In a further embodiment, the anti-CD20 / anti-CD3 bispecific comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 55. H CD20), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 56 L It contains a second antigen-binding domain, including CD20.

[0144] In another particular embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a third antigen-binding domain that binds to CD20. In particular, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 49, the CDR-H2 sequence of SEQ ID NO: 50, and the CDR-H3 sequence of SEQ ID NO: 51. H CD20), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 52, the CDR-L2 sequence of SEQ ID NO: 53, and the CDR-L3 sequence of SEQ ID NO: 54 L More particularly, the anti-CD20 / anti-CD3 bispecific comprises a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 55. H CD20), and / or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 56. L In a further embodiment, the anti-CD20 / anti-CD3 bispecific comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 55. H CD20), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 56 L It contains a third antigen-binding domain, which includes a nucleotide sequence similar to that of the nucleotide sequence of the target antigen (e.g., CD20).

[0145] In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a bispecific antibody, wherein the first antigen-binding domain is a cross-Fab molecule in which the variable or constant domains of the Fab heavy and Fab light chains have been exchanged, and the second and third antigen-binding domains, if present, are conventional Fab molecules.

[0146] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a bispecific antibody in which (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain, the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0147] Multiple Fab molecules can be fused to the Fc domain or to each other directly or via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) (SEQ ID NO: 71), (G4S)2 or GGGGSGGGGS (SEQ ID NO: 72), (G4S)3 (SEQ ID NO: 73), and (G4S)4 (SEQ ID NO: 74), more particularly (G4S)2 or GGGGSGGGGS (SEQ ID NO: 72). A particularly suitable peptide linker for fusing the Fab light chains of the first and second Fab molecules to each other is (G4S)2. Another suitable linker comprises the sequence (G4S)4(G4S)4 (SEQ ID NO: 74). Additionally, the linker can comprise (part of) an immunoglobulin hinge region. In particular, when a Fab molecule is fused to the N-terminus of an Fc domain subunit, the fusion may be via an immunoglobulin hinge region or part thereof, with or without an additional peptide linker.

[0148] In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises an Fc domain comprising one or more amino acid substitutions that reduce Fc receptor binding and / or effector function. In particular, the anti-CD20 / anti-CD3 bispecific antibody comprises an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A, and P329G (according to EU numbering).

[0149] In particular aspects, the anti-CD20 / anti-CD3 bispecific antibody comprises a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 57, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 58, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 59, and a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 60. In further particular embodiments, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 57, the polypeptide sequence of SEQ ID NO: 58, the polypeptide sequence of SEQ ID NO: 59 and the polypeptide sequence of SEQ ID NO: 60 (CD20 TCB).

[0150] In a particular embodiment, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

[0151] Glofitamab (Proposed INN: List 121 WHO Drug Information, Vol. 33, No. 2, 2019; also known as CD20-TCB, RO7082859, or RG6026) is a novel T cell-engaging bispecific full-length antibody with a 2:1 molecular configuration for bivalent binding to CD20 on B cells and monovalent binding to CD3, specifically the CD3 epsilon chain (CD3e), on T cells. Its CD3-binding domain is fused to one of the CD20-binding domains in a head-to-tail fashion via a flexible linker. This configuration gives glofitamab superior in vitro potency compared to other CD20-CD3 bispecific antibodies with a 1:1 configuration and produces significant antitumor effects in preclinical DLBCL models. CD20 bivalency maintains this potency in the presence of competing anti-CD20 antibodies, providing the opportunity for pretreatment or cotreatment with these agents. Glofitamab contains an engineered heterodimeric Fc region that completely abolishes binding to FcgR and C1q. By simultaneously binding to CD3ε of the T cell receptor (TCR) complex on human CD20-expressing tumor cells and T cells, it induces tumor cell lysis, as well as 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 simultaneous binding (crosslinking) of T cells to CD20-expressing cells. In addition to killing, T cells are activated by CD3 crosslinking, as detected by increased 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.

[0152] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody for use in combination comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 83, the CDR-H2 sequence of SEQ ID NO: 84, and the CDR-H3 sequence of SEQ ID NO: 85. H and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 86, the CDR-L2 sequence of SEQ ID NO: 87, and the CDR-L3 sequence of SEQ ID NO: 88. LMore particularly, the anti-CD20 / anti-CD3 bispecific comprises a first antigen-binding domain comprising a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 89. H CD3), and / or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 90. L In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 89. H CD3), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 90 L CD3).

[0153] In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 91, the CDR-H2 sequence of SEQ ID NO: 92 and the CDR-H3 sequence of SEQ ID NO: 93. H CD20), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 94, the CDR-L2 sequence of SEQ ID NO: 95, and the CDR-L3 sequence of SEQ ID NO: 96 L More particularly, the anti-CD20 / anti-CD3 bispecific comprises a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 97. H CD20), and / or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 98. L In a further embodiment, the anti-CD20 / anti-CD3 bispecific comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 97. H CD20), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 98 L It contains a second antigen-binding domain, including CD20.

[0154] In particular aspects, the anti-CD20 / anti-CD3 bispecific antibody comprises a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 99, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 100, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 101, and a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 102. In further particular embodiments, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 99, the polypeptide sequence of SEQ ID NO: 100, the polypeptide sequence of SEQ ID NO: 101 and the polypeptide sequence of SEQ ID NO: 102.

[0155] In a specific embodiment, the anti-CD20 / anti-CD3 bispecific antibody is mosunetuzumab. Mosunetuzumab (RO7030816; also known as BTCT4465A) is a humanized, full-length anti-CD20 / CD3 T-cell-dependent bispecific (TDB) antibody of the human IgG1 class, containing the amino acid substitution N297G (according to EU numbering) in the fragment crystallizable (Fc) region. This substitution results in an aglycosylated heavy chain that minimally binds to the Fc gamma (Fc-γ) receptor, resulting in reduced Fc effector function. Mosunetuzumab's mechanism of action involves CD3-mediated engagement of T cells with CD20-expressing cells, resulting in T cell activation and T cell-mediated cytolysis of CD20-expressing cells. Based on its structure as a full-length antibody and preclinical data, the pharmacokinetic (PK) properties of mosunetuzumab, similar to those of other monoclonal antibodies, allow for intermittent dosing in clinical settings.

[0156] Certain bispecific antibodies are described in PCT Publication Nos. WO 2016 / 020309 A1 or WO 2015 / 095392 A1.

[0157] In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody may also comprise a bispecific T cell engager (BiTE®). In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody is XmAb® 13676. In another embodiment, the bispecific antibody is REGN1979. In another embodiment, the bispecific antibody is FBTA05 (Lymphomun).

[0158] Exemplary Bispecific Anti-PD1 / Anti-LAG3 Antibodies for Use in the Invention The combinations provided herein utilize novel bispecific antibodies comprising a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene-3 (LAG3), and which have particularly advantageous properties, such as productivity, stability, binding affinity, bioactivity, specific targeting of certain T cells, targeting efficiency, and reduced toxicity. Particular bispecific anti-PD1 / anti-LAG3 antibodies for use herein are described in WO 2018 / 185043 A1.

[0159] In certain embodiments, bispecific antibodies are provided that contain a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3, which exhibit reduced internalization upon binding to the T cell surface. Internalization represents an important sink of molecules that can be degraded within hours, while their target receptors are rapidly re-expressed on the cell surface ready to inhibit TCR signaling. In a further embodiment, bispecific antibodies are provided that contain a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3, which preferentially bind to conventional T cells over Tregs. This is advantageous because targeting LAG-3 on Tregs with blocking antibodies can exacerbate their suppressive function by increasing their suppressive function, ultimately masking the positive blocking effect on other T cells. In a further embodiment, bispecific antibodies are provided that contain a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3, which can protect T cell effector function from Treg suppression. In another embodiment, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3, which is capable of inducing granzyme B secretion by CD4 T cells when cultured with the tumor cell line ARH77, as shown in the assays presented herein. In a further embodiment, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3, which exhibits increased tumor-specific T cell effector function and / or enhances the cytotoxic effect of T cells. In another embodiment, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3, which exhibits increased tumor eradication in vivo.

[0160] In one aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, wherein the first antigen-binding domain that specifically binds to PD1 is: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; a VL domain comprising Includes:

[0161] In one embodiment, the bispecific antibody comprises an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain, which Fc domain has reduced or even abolished effector function, in particular the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors, in particular Fcγ receptors.

[0162] In a further aspect, there is provided a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises an Fc domain that is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain, and wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor.

[0163] In another embodiment, a bispecific antibody comprises a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, wherein the second antigen-binding domain that specifically binds to LAG3 is (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a VH domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; a VL domain comprising contains, or (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 24; a VL domain comprising

[0023] A bispecific antibody is provided, comprising:

[0164] In certain further embodiments, a bispecific antibody is provided, comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, wherein the first antigen-binding domain that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10.

[0165] In another embodiment, a bispecific antibody comprises a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, wherein the second antigen-binding domain that specifically binds to LAG3 is (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26

[0023] A bispecific antibody is provided, comprising:

[0166] In a further embodiment, a bispecific antibody comprises a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, wherein the second antigen-binding domain that specifically binds to LAG3 is: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 27 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28; or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30; or (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 31 and a VL domain comprising the amino acid sequence of SEQ ID NO: 32; or (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 33 and a VL domain comprising the amino acid sequence of SEQ ID NO: 34

[0023] A bispecific antibody is provided, comprising:

[0167] In another embodiment, a bispecific antibody is provided, comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, wherein the second antigen-binding domain that specifically binds to LAG3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 81 and a VL domain comprising the amino acid sequence of SEQ ID NO: 82.

[0168] In certain embodiments, a bispecific antibody, comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3; the first antigen-binding domain that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; Provided is a bispecific antibody, wherein the second antigen-binding domain that specifically binds to LAG3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18, or a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0169] In one embodiment, a bispecific antibody of the invention comprises a first antigen-binding domain that specifically binds to PD1, wherein the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10, and a second antigen-binding domain that specifically binds to LAG3, wherein the second antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.

[0170] In a further embodiment, the bispecific antibody of the invention comprises a first antigen-binding domain that specifically binds to PD1, wherein the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10, and a second antigen-binding domain that specifically binds to LAG3, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0171] In a further embodiment, the bispecific antibody comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3 is a human antibody, a humanized antibody, or a chimeric antibody. In particular, the bispecific antibody is a humanized antibody or a chimeric antibody.

[0172] In one embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3 is bivalent, meaning that the bispecific antibody comprises one antigen-binding domain that specifically binds to PD1 and one antigen-binding domain that specifically binds to LAG3 (1+1 format).

[0173] In one embodiment, a bispecific antibody is provided, comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises an Fc domain, a first Fab fragment comprising the antigen-binding domain that specifically binds to PD1, and a second Fab fragment comprising the antigen-binding domain that specifically binds to LAG3. In a particular embodiment, in one of the Fab fragments, the variable domains VL and VH are swapped with each other such that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In a particular embodiment, in the first Fab fragment comprising the antigen-binding domain that specifically binds to PD1, the variable domains VL and VH are swapped with each other.

[0174] In certain embodiments, a bispecific antibody comprises a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises: (a) a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 35; and a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 36; a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 37 and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 38; or (b) a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 35, and a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 36; a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 39, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 40.

[0023] A bispecific antibody is provided, comprising:

[0175] More particularly, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35, a first light chain comprising the amino acid sequence of SEQ ID NO: 36, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37, and a second light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0176] Fc domain modifications that reduce Fc receptor binding and / or effector function In certain embodiments, anti-PD1 / anti-LAG3 bispecific antibodies are provided, which comprise an Fc domain comprising one or more amino acid modifications that reduce binding to Fc receptors, particularly Fcγ receptors, and reduce or eliminate effector function.

[0177] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0178] The following sections describe preferred embodiments of bispecific antigen-binding molecules of the present invention that contain Fc domain modifications that reduce Fc receptor binding and / or effector function. In one embodiment, the present invention relates to an anti-PD1 / anti-LAG3 bispecific antibody in which the Fc domain contains one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors. In particular, the Fc domain is a human IgG1 subclass Fc domain with the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0179] The Fc domain confers desirable pharmacokinetic properties to the bispecific antibodies of the invention, including a long serum half-life and a desirable tissue-to-blood distribution ratio, which contribute to favorable accumulation in target tissues. However, at the same time, it may cause undesirable targeting of the bispecific antibodies of the invention to cells expressing Fc receptors rather than cells bearing the desired antigen. Thus, in a specific embodiment, the Fc domain of the bispecific antibodies of the invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to native IgG Fc domains, particularly IgG1 Fc domains or IgG4 domains. More particularly, the Fc domain is an IgG1 Fc domain.

[0180] In one such embodiment, the Fc domain (or the bispecific antigen-binding molecule of the present invention comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to an Fc receptor compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule of the present invention comprising a native IgG1 Fc domain), and / or exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule of the present invention comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or the bispecific antigen-binding molecule of the present invention comprising said Fc domain) does not substantially bind to an Fc receptor and / or does not induce effector function. In a particular embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly human FcγRIIIa. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In a particular embodiment, the Fc receptor is an inhibitory human Fcγ receptor, more particularly human FcγRIIB. In one embodiment, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the bispecific antigen-binding molecule of the invention comprising said Fc domain) exhibits a binding affinity for FcRn that is greater than about 70%, particularly greater than about 80%, and more particularly greater than about 90%, compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule of the invention comprising a native IgG1 Fc domain).

[0181] In certain embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function compared to a non-engineered Fc domain. In certain embodiments, the Fc domain of a bispecific antigen-binding molecule of the present invention comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor. In another embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least two-fold, at least five-fold, or at least ten-fold. In one embodiment, a bispecific antigen-binding molecule of the present invention comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity to an Fc receptor compared to a bispecific antibody of the present invention comprising a non-engineered Fc domain. In a certain embodiment, the Fc receptor is an Fcγ receptor. In another embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In a specific embodiment, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complement components (specifically, binding affinity to C1q) is also reduced. In one embodiment, binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn (i.e., preservation of the binding affinity of the Fc domain to the receptor) is achieved when the Fc domain (or a bispecific antigen-binding molecule of the invention comprising the Fc domain) exhibits a binding affinity to FcRn that is greater than about 70% of the binding affinity of an unengineered form of the Fc domain (or a bispecific antigen-binding molecule of the invention comprising this unengineered form of Fc).The Fc domain, or a bispecific antigen-binding molecule of the present invention comprising the Fc domain, may exhibit greater than about 80%, or even greater than about 90%, of such affinity. In certain embodiments, the Fc domain of a bispecific antigen-binding molecule of the present invention is engineered to have reduced effector function compared to an unengineered Fc domain. Reduced effector function includes, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0182] Antibodies with reduced effector function include those containing one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Pat. No. 6,737,056). Such Fc mutants include those with 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. Pat. No. 7,332,581). Specific antibody variants with improved or reduced binding to FcRs have been described (e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields, R. et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0183] In one embodiment of the invention, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331, and P329. In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A ("LALA"). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and further amino acid substitutions selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a further specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor binding of a human IgG1 Fc domain, as described in PCT Application WO 2012 / 130831 A1. That document also describes methods for preparing such mutant Fc domains and for determining their properties, such as Fc receptor binding or effector function. Such antibodies are IgG1s with the mutations L234A and L235A, or with the mutations L234A, L235A, and P329G (numbering according to the EU index of Kabat et al., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0184] In one embodiment the anti-PD1 / anti-LAG3 bispecific antibody comprises (i) a homodimeric Fc region of the human IgG1 subclass, optionally with the mutations P329G, L234A and L235A, or (ii) a homodimeric Fc region of the human IgG4 subclass, optionally with the mutations P329G, S228P and L235E, or (iii) a homodimeric Fc region of the human IgG4 subclass, optionally with the mutations P329G, L234A, L235A, I253A, H310A and H435A, or optionally with the mutations P329G, L234A, L235A, I253A, H310A and H435A. or (iv) a homodimeric Fc region of the human IgG1 subclass having the mutations T366W and the other Fc region polypeptide having the mutations T366S, L368A, Y407V, and S354C; or (v) a homodimeric Fc region of the human IgG1 subclass having the mutations T366W and Y349C and the other Fc region polypeptide having the mutations T366S, L368A, Y407V, and S354C; or (v) a heterodimeric Fc region in which one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C, or (v) both Fc region polypeptides comprise the mutations P329G, L234A and L235A, and one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A and Y407V, or one The Fc region polypeptide comprises the mutations T366W and Y349C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C (all positions according to the EU index of Kabat).

[0185] In one aspect, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising the amino acid substitutions L235E, S228P, and P329G. This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).

[0023] Thus, in one aspect there is provided a bispecific antibody comprising a heterodimeric Fc region of the human IgG4 subclass, wherein both Fc-region polypeptides comprise the mutations P329G, S228P and L235E, and one Fc-region polypeptide comprises the mutations T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or one Fc-region polypeptide comprises the mutations T366W and Y349C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or one Fc-region polypeptide comprises the mutations T366W and S354C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C (all positions according to EU index of Kabat).

[0186] Antibodies with increased half-life and improved binding to neonatal Fc receptors are responsible for the transfer of mature IgG to the fetus (Guyer, RR et al., J. Immunol. 117 (1976) 587-593 and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434) and are described in U.S. Patent Application Publication No. 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include variants having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan, A.R., and Winter, G., Nature 322 (1988) 738-740; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0187] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment, such as a BIAcore instrument (GE Healthcare), and Fc receptors that can be obtained, for example, by recombinant expression. Suitable such binding assays are described herein. Alternatively, the binding affinity of an Fc domain or a cell-activating bispecific antigen-binding molecule comprising an Fc domain to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor). The effector function of an Fc domain or a bispecific antibody of the present invention comprising an Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362, Hellstrom et al., Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985), U.S. Patent No. 5,821,337, Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (e.g., the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0188] The following sections describe preferred embodiments of bispecific antibodies of the invention that contain Fc domain modifications that reduce Fc receptor binding and / or effector function. In one embodiment, an anti-PD1 / anti-LAG3 bispecific antibody is provided in which the Fc domain contains one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fcγ receptor, particularly to an Fc receptor. In another embodiment, an anti-PD1 / anti-LAG3 bispecific antibody is provided in which the Fc domain contains one or more amino acid substitutions that reduce effector function. In a specific embodiment, the Fc domain is a human IgG1 subclass Fc domain with the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0189] Fc domain modifications that promote heterodimerization The bispecific antigen-binding molecules described herein comprise different antigen-binding domains fused to one or the other of the two subunits of the Fc domain, and thus the two subunits of the Fc domain may be contained in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. To increase the yield and purity of the bispecific antibodies of the present invention in recombinant production, it is advantageous to introduce modifications to the Fc domain of the bispecific antigen-binding molecules disclosed herein that promote the desired association of the polypeptides.

[0190] Thus, in certain embodiments, anti-PD1 / anti-LAG3 bispecific antibodies are provided, in which the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain. The most extensive site of protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0191] In a specific embodiment, the modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. Thus, the present invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding site that specifically binds to LAG3, wherein, according to the knob-into-hole method, the first subunit of the Fc domain comprises the knob and the second subunit of the Fc domain comprises the hole. In a specific embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0192] Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protuberance can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protuberance is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (eg, alanine or threonine).

[0193] Thus, in one embodiment, in the CH3 domain of a first subunit of the Fc domain of a bispecific antigen-binding molecule of the invention, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance in the CH3 domain of the first subunit that can be repositioned within a cavity in the CH3 domain of the second subunit, and in the CH3 domain of a second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit into which the protuberance in the CH3 domain of the first subunit can be repositioned. The protuberance and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the (CH3 domain of the) second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0194] In yet a further embodiment, the first subunit of the Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C), and the second subunit of the Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C). Introduction of these two cysteine ​​residues results in the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter (2001), J Immunol Methods 248, 7-15). In a particular embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0195] However, other knob-in-hole techniques can be used instead or in addition, as described in EP 1 870 459. In one embodiment, the multispecific antibody comprises the mutations R409D and K370E in the CH3 domain of the "knob chain" and the mutations D399K and E357K in the CH3 domain of the "hole chain" (numbering according to Kabat EU index).

[0196] In one embodiment the bispecific antibody comprises a T366W mutation in the CH3 domain of the "knob chain", the mutations T366S, L368A and Y407V in the CH3 domain of the "hole" chain, and further comprises the mutations R409D and K370E in the CH3 domain of the "knob chain" and the mutations D399K and E357K in the CH3 domain of the "hole" chain (numbering according to Kabat EU index).

[0197] In one embodiment the bispecific antibody comprises in one of the two CH3 domains the mutations Y349C and T366W and in the other of the two CH3 domains the mutations S354C, T366S, L368A and Y407V, or the multispecific antibody comprises in one of the two CH3 domains the mutations Y349C and T366W and in the other of the two CH3 domains the mutations S354C, T366S, L368A and Y407V, and further comprises in the CH3 domain of the "knob strand" the mutations R409D and K370E and in the CH3 domain of the "hole strand" the mutations D399K and E357K (numbering according to Kabat EU index).

[0198] In another embodiment, the modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates an electrostatic steering effect, e.g., as described in PCT Publication WO 2009 / 089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with a charged amino acid residue, such that homodimer formation is electrostatically unfavorable, but heterodimerization is electrostatically favored.

[0199] Besides the "knob-into-hole technique", other techniques for modifying the CH3 domain of the heavy chain of a multispecific antibody to promote heterodimerization are known in the art. These techniques, in particular those described in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, and WO 2013 / 096291, are envisaged herein as alternatives to "knobs-into-holes" in combination with bispecific antibodies.

[0200] In one embodiment, in bispecific antibodies, the approach described in EP 1 870 459 is used to support heterodimerization of the first and second heavy chains of a multispecific antibody, which is based on the introduction of oppositely charged amino acids at specific amino acid positions in the CH3 / CH3 domain interface between the first and second heavy chains.

[0201] Thus, in this embodiment, in the tertiary structure of the multispecific antibody, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface present between the respective antibody CH3 domains, and the amino acid sequence of each of the CH3 domains of the first heavy chain and the second heavy chain each comprises a series of amino acids located within that interface in the tertiary structure of the antibody, wherein from the series of amino acids located at the interface, a first amino acid in the CH3 domain of one heavy chain is substituted by a positively charged amino acid, and from the series of amino acids located at the interface, a second amino acid in the CH3 domain of the other heavy chain is substituted by a negatively charged amino acid. Bispecific antibodies of this embodiment are also referred to herein as "CH3(+ / -) engineered bispecific antibodies" (wherein the abbreviation "+ / -" represents amino acids of the opposite charge introduced into the respective CH3 domains).

[0202] In one embodiment, in the CH3(+ / -) engineered bispecific antibody, the positively charged amino acids are selected from K, R and H and the negatively charged amino acids are selected from E or D.

[0203] In one embodiment, in the CH3(+ / -) engineered bispecific antibody, the positively charged amino acids are selected from K and R and the negatively charged amino acids are selected from E or D.

[0204] In one embodiment, the positively charged amino acid is K and the negatively charged amino acid is E in the CH3(+ / -) engineered bispecific antibody.

[0205] In one embodiment in the CH3(+ / -) engineered bispecific antibody, in the CH3 domain of one heavy chain the amino acid R at position 409 is substituted by D and the amino acid K at position 409 is substituted by E, and in the CH3 domain of the other heavy chain the amino acid D at position 399 is substituted by K and the amino acid E at position 357 is substituted by K (numbering according to Kabat EU index).

[0206] In one aspect, the approach described in WO 2013 / 157953 is used to support heterodimerization of the first and second heavy chains of a multispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted by K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index). In another embodiment, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted by K, and the amino acid L at position 351 is substituted by K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index).

[0207] In another embodiment, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted by K, and the amino acid L at position 351 is substituted by K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index). Additionally, at least one of the following substitutions is contained in the CH3 domain of the other heavy chain: the amino acid Y at position 349 is substituted by E, the amino acid Y at position 349 is substituted by D, and the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index). In one embodiment, the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index).

[0208] In one embodiment, the approach described in WO 2012 / 058768 is used to support heterodimerization of the first and second heavy chains of a multispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, the amino acid L at position 351 is substituted by Y and the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is substituted by A and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In another embodiment, in addition to the substitutions mentioned above, in the CH3 domain of the other heavy chain, at least one of the amino acids at positions 411 (originally T), 399 (originally D), 400 (originally S), 405 (originally F), 390 (originally N), and 392 (originally K) is substituted (numbering according to Kabat EU index). Preferred substitutions are as follows: - substitution of the amino acid T at position 411 with an amino acid selected from N, R, Q, K, D, E and W (numbering according to the Kabat EU index), - substitution of the amino acid D at position 399 with an amino acid selected from R, W, Y and K (numbering according to the Kabat EU index), - substitution of amino acid D at position 400 with an amino acid selected from E, D, R and K (numbering according to the Kabat EU index), - substitution of the amino acid F at position 405 with an amino acid selected from I, M, T, S, V and W (numbering according to the Kabat EU index), - substitution of the amino acid N at position 390 with an amino acid selected from R, K and D (numbering according to the Kabat EU index), - the amino acid K at position 392 is substituted with an amino acid selected from V, M, R, L, F and E (numbering according to the Kabat EU index).

[0209] In another aspect, the bispecific antibody is engineered according to WO 2012 / 058768), i.e., in the CH3 domain of one heavy chain, the amino acid L at position 351 is substituted by Y and the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is substituted by V and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In another embodiment of the multispecific antibody, in the CH3 domain of one heavy chain, the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is substituted by A and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In the latter above-mentioned embodiment, in the CH3 domain of the other heavy chain, the amino acid K at position 392 is substituted by E, the amino acid T at position 411 is substituted by E, the amino acid D at position 399 is substituted by R, and the amino acid S at position 400 is substituted by R (numbering according to the Kabat EU index).

[0210] In one embodiment, the techniques described in WO 2011 / 143545 are used to support heterodimerization of the first and second heavy chains of a multispecific antibody. In one embodiment, amino acid modifications in the CH3 domains of both heavy chains are introduced at positions 368 and / or 409 (numbering according to the Kabat EU index).

[0211] In one embodiment, the technique described in WO 2011 / 090762 is used to support heterodimerization of the first and second heavy chains of a bispecific antibody. WO 2011 / 090762 relates to amino acid modifications according to the "knob-into-hole" (KiH) technique. In one embodiment, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted with W, and in the CH3 domain of the other heavy chain, the amino acid Y at position 407 is substituted with A (numbering according to the Kabat EU index). In another embodiment, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted with Y, and in the CH3 domain of the other heavy chain, the amino acid Y at position 407 is substituted with T (numbering according to the Kabat EU index).

[0212] In one aspect, the approach described in WO 2009 / 089004 is used to support heterodimerization of the first and second heavy chains of a bispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, the amino acid K or N at position 392 is substituted by a negatively charged amino acid (in one embodiment, by E or D, in a preferred embodiment, by D), and in the CH3 domain of the other heavy chain, the amino acid D at position 399, the amino acid E or D at position 356, or the amino acid E at position 357 is substituted by a positively charged amino acid (in one embodiment, by K or R, in a preferred embodiment, by K; in a preferred embodiment, the amino acid at position 399 or 356 is substituted by K) (numbering according to the Kabat EU index). In a further embodiment, in addition to the above substitutions, in the CH3 domain of one heavy chain, the amino acid K or R at position 409 is substituted by a negatively charged amino acid (in one embodiment by E or D, in a preferred embodiment by D) (numbering according to Kabat EU index). In a still further aspect, in addition to or instead of the above substitutions, in the CH3 domain of one heavy chain, the amino acid K at position 439 and / or the amino acid K at position 370 are / are substituted, independently of one another, by a negatively charged amino acid (in one embodiment by E or D, in a preferred embodiment by D) (numbering according to Kabat EU index).

[0213] In one aspect, the approach described in WO 2007 / 147901 is used to support heterodimerization of the first and second heavy chains of a multispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, the amino acid K at position 253 is substituted by E, the amino acid D at position 282 is substituted by K, and the amino acid K at position 322 is substituted by D, and in the CH3 domain of the other heavy chain, the amino acid D at position 239 is substituted by K, the amino acid E at position 240 is substituted by K, and the amino acid K at position 292 is substituted by D (numbering according to Kabat EU index).

[0214] The C-terminus of the heavy chain of the bispecific antibody reported herein may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may also be a shortened C-terminus in which one or two of the C-terminal amino acid residues have been removed. In a preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus ending with PG.

[0215] In one aspect of all aspects reported herein, a bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index). In one embodiment of all aspects reported herein, a bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, numbering according to Kabat EU index).

[0216] Modifications within the Fab domain In one aspect, an anti-PD1 / anti-LAG3 bispecific antibody is provided in which either the variable domains VH and VL or the constant domains CH1 and CL have been exchanged in one of the Fab fragments. The bispecific antibody is prepared according to the crossmab technology.

[0217] Multispecific antibodies with domain replacement / swapping in one binding arm (CrossMabVH-VL or CrossMabCH-CL) are described in WO 2009 / 080252, WO 2009 / 080253, and Schaefer, W. et al., PNAS, 108 (2011) 11187-1191. These multispecific antibodies clearly reduce by-products caused by mismatches between a light chain for one antigen and an incorrect heavy chain for a second antigen (compared to approaches without such domain replacement).

[0218] In certain embodiments, anti-PD1 / anti-LAG3 bispecific antibodies are provided, in which in one of the Fab fragments 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. In certain embodiments, the bispecific antibody is a bispecific antibody in which the variable domains VL and VH are replaced with each other in a first Fab fragment that comprises an antigen-binding domain that specifically binds to PD1.

[0219] In another embodiment, to further improve correct pairing, the anti-PD1 / anti-LAG3 bispecific antibody can contain differently charged amino acid substitutions (so-called "charged residues"). These modifications can be introduced into the crossed or non-crossed CH1 and CL domains. These modifications are described, for example, in WO 2015 / 150447, WO 2016 / 020309, and PCT / EP2016 / 073408.

[0220] In a particular embodiment, an anti-PD1 / anti-LAG3 bispecific antibody is provided, wherein in one of the Fab fragments, in the constant domain CL, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU index), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index). In a particular embodiment, the bispecific antibody is a second Fab fragment comprising an antigen-binding domain that specifically binds to TIM3, wherein in the constant domain CL the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU index), and in the constant domain CH1 the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0221] In a particular embodiment, an anti-PD1 / anti-LAG3 bispecific antibody is provided, in which in one of the CL domains the amino acid at position 123 (EU numbering) is replaced by arginine (R) and the amino acid at position 124 (EU numbering) is replaced by lysine (K), and in one of the CH1 domains the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are replaced by glutamic acid (E). In a particular embodiment, the bispecific antibody is a bispecific antibody in which in a Fab fragment comprising an antigen-binding domain that specifically binds to LAG3, the amino acid at position 123 (EU numbering) is replaced by arginine (R) and the amino acid at position 124 (EU numbering) is replaced by lysine (K), and in one of the CH1 domains the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are replaced by glutamic acid (E).

[0222] In a further embodiment, the bispecific antibody is a bivalent antibody, (a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen; (b) a second light chain and a second heavy chain that specifically bind to a second antigen; wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced by each other.

[0223] The antibody of a) does not contain the modifications reported in b), and the heavy and light chains of a) are isolated chains.

[0224] In antibody (b), in the light chain, the variable light domain VL is replaced by the variable heavy domain VH of the antibody, and in the heavy chain, the variable heavy domain VH is replaced by the variable light domain VL of the antibody.

[0225] and in the constant domain CHI of the first heavy chain of (a), the amino acid at position 147 or the amino acid at position 213 (numbering according to Kabat EU index) is substituted by a negative amino acid; or (ii) in the constant domain CL of the second light chain of (b), the amino acid at position 124 (numbering according to Kabat) is substituted by a positive amino acid; and in the constant domain CHI of the second heavy chain of (b), the amino acid at position 147 or the amino acid at position 213 (numbering according to Kabat EU index) is substituted by a negative amino acid.

[0226] In another aspect, (i) in the constant domain CL of the first light chain of (a) the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat) (in a preferred embodiment, independently substituted by lysine (K) or arginine (R)); and in the constant domain CHI of the first heavy chain of (a) the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat). or (ii) in the constant domain CL of the second light chain of (b), the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat) (in one preferred embodiment, independently by lysine (K) or arginine (R)); and in the constant domain CHI of the second heavy chain of (b), the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0227] In one embodiment, in the constant domain CL of the second heavy chain, the amino acids at positions 124 and 123 are substituted by K (numbering according to Kabat EU index).

[0228] In one embodiment, in the constant domain CL of the second heavy chain, the amino acid at position 123 is substituted by R and the amino acid at position 124 is substituted by K (numbering according to Kabat EU index).

[0229] In one embodiment, in the constant domain CH1 of the second light chain, the amino acids at positions 147 and 213 are substituted by E (numbering according to Kabat EU index).

[0230] In one embodiment, in the constant domain CL of the first light chain the amino acids at positions 124 and 123 are substituted by K and in the constant domain CH1 of the first heavy chain the amino acids at positions 147 and 213 are substituted by E (numbering according to Kabat EU index).

[0231] In one embodiment, in the constant domain CL of the first light chain the amino acid at position 123 is substituted by R and the amino acid at position 124 is substituted by K; and in the constant domain CH1 of the first heavy chain the amino acids at positions 147 and 213 are both substituted by E (numbering according to Kabat EU index).

[0232] In one embodiment in the constant domain CL of the second heavy chain the amino acids at positions 124 and 123 are substituted by K, and in the constant domain CHI of the second light chain the amino acids at positions 147 and 213 are substituted by E, in the variable domain VL of the first light chain the amino acid at position 38 is substituted by K, and in the variable domain VH of the first heavy chain the amino acid at position 39 is substituted by E, in the variable domain VL of the second heavy chain the amino acid at position 38 is substituted by K and in the variable domain VH of the second light chain the amino acid at position 39 is substituted by E (numbering according to Kabat EU index).

[0233] In one embodiment, the bispecific antibody is a bivalent antibody, (a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen; (b) a second light chain and a second heavy chain that specifically bind to a second antigen; wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced with each other, and the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced with each other.

[0234] The antibody of (a) does not contain the modifications reported in (b), and the heavy and light chains of (a) are isolated chains. In the antibody of (b), within the light chain, the variable light domain VL is replaced by the variable heavy domain VH of the antibody, and the constant light domain CL is replaced by the constant heavy domain CH1 of the antibody, and within the heavy chain, the variable heavy domain VH is replaced by the variable light domain VL of the antibody, and the constant heavy domain CH1 is replaced by the constant light domain CL of the antibody.

[0235] In one embodiment, the bispecific antibody is a bivalent antibody, (a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen; (b) a second light chain and a second heavy chain that specifically bind to a second antigen; wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced by each other.

[0236] The antibody of (a) does not contain the modifications reported in (b), and the heavy and light chains of (a) are isolated chains. In the antibody of (b), in the light chain, the constant light chain domain CL is replaced by the constant heavy chain domain CH1 of the antibody, and in the heavy chain, the constant heavy chain domain CH1 is replaced by the constant light chain domain CL of the antibody.

[0237] In one embodiment, the bispecific antibody comprises: (a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; (b) one, two, three, or four single-chain Fab fragments that specifically bind to a second antigen; a bispecific antibody comprising: The single-chain Fab fragment of (b) is fused to the full-length antibody of (a) via a peptide linker at the C-terminus or N-terminus of the heavy chain or light chain of the full-length antibody.

[0238] In one embodiment, one or two identical single-chain Fab fragments that bind to a second antigen are fused to a full-length antibody via a peptide linker at the C-terminus of the heavy or light chain of the full-length antibody.

[0239] In one embodiment, one or two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of the heavy chain of the full-length antibody via a peptide linker.

[0240] In one embodiment, one or two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of the light chain of the full-length antibody via a peptide linker.

[0241] In one embodiment, two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to a full-length antibody via a peptide linker at the C-terminus of each of the heavy or light chains of the full-length antibody.

[0242] In one embodiment, two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of each heavy chain of the full-length antibody via a peptide linker.

[0243] In one embodiment, two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of each light chain of the full-length antibody via a peptide linker.

[0244] In one embodiment, the bispecific antibody comprises: a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; b) (ba) an antibody heavy chain variable domain (VH), or (bb) a first polypeptide consisting of an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1), a first polypeptide fused at the N-terminus of its VH domain to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; (c) (ca) an antibody light chain variable domain (VL), or (cb) a second polypeptide consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), the second polypeptide is fused to the N-terminus of the VL domain via a peptide linker to the C-terminus of the other of the two heavy chains of the full-length antibody; The antibody heavy chain variable domain (VH) of the first polypeptide and the antibody light chain variable domain (VL) of the second polypeptide together form an antigen-binding domain that specifically binds to a second antigen.

[0245] In one embodiment, the antibody heavy chain variable domain (VH) of the polypeptide of (b) and the antibody light chain variable domain (VL) of the polypeptide of (c) are connected and stabilized via an interchain disulfide bridge by introducing a disulfide bond between the following positions: (i) from position 44 in the heavy chain variable domain to position 100 in the light chain variable domain; or (ii) from position 105 of the heavy chain variable domain to position 43 of the light chain variable domain; or (iii) from position 101 in the heavy chain variable domain to position 100 in the light chain variable domain (numbering always according to the Kabat EU index).

[0246] Techniques for introducing non-natural disulfide bridges for stabilization are described in WO 94 / 029350, Rajagopal, V., et al., Prot. Eng. (1997) 1453-1459; Kobayashi, H., et al., Nucl. Med. Biol. 25 (1998) 387-393, and Schmidt, M., et al., Oncogene 18 (1999) 1711-1721. In one embodiment, the optional disulfide bond between the variable domains of polypeptides (b) and (c) is between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, the optional disulfide bond between the variable domains of polypeptides (b) and (c) is between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (numbering always according to the Kabat EU index). In one embodiment, trivalent bispecific antibodies without any disulfide stabilization between the variable domains VH and VL of the single-chain Fab fragments are preferred.

[0247] In one embodiment, the bispecific antibody is a triabody or a tetrabody; (a) a first light chain and a first heavy chain of a full-length antibody that specifically binds to a first antigen; (b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to a second antigen, wherein the variable domains VL and VH have been replaced with each other and / or the constant domains CL and CH1 have been replaced with each other; (c) One to four antigen-binding domains that specifically bind to one or two additional antigens (i.e., a third and / or fourth antigen) are fused to the C-terminus or N-terminus of the light chain or heavy chain of (a) and / or (b) via a peptide linker.

[0248] The antibody in (a) does not contain the modifications reported in (b), and the heavy and light chains in (a) are isolated chains.

[0249] In one embodiment, the triabody or tetrabody comprises in (c) one or two antigen-binding domains that specifically bind to one or two additional antigens.

[0250] In one embodiment, the antigen binding domain is selected from the group consisting of an scFv fragment and an scFab fragment.

[0251] In one embodiment, the antigen binding domain is an scFv fragment.

[0252] In one embodiment, the antigen binding domain is an scFab fragment.

[0253] In one embodiment, the antigen binding domain is fused to the C-terminus of the heavy chain of (a) and / or (b).

[0254] In one embodiment, the triabody or tetrabody comprises in (c) one or two antigen-binding domains that specifically bind to one additional antigen.

[0255] In one aspect, the triabody or tetrabody comprises two identical antigen-binding domains in (c) that specifically bind to a third antigen. In a preferred embodiment, such two identical antigen-binding domains are both fused to the C-terminus of the heavy chains of (a) and (b) via the same peptide linker. In a preferred embodiment, the two identical antigen-binding domains are scFv or scFab fragments.

[0256] In one aspect, the triabody or tetrabody comprises two antigen-binding domains in (c) that specifically bind to a fourth antigen. In one embodiment, the two antigen-binding domains are both fused to the C-terminus of the heavy chains of (a) and (b) via the same peptide connector. In a preferred embodiment, the two antigen-binding domains are scFv or scFab fragments.

[0257] In one embodiment, the bispecific antibody is a bispecific tetravalent antibody, (a) two light chains and two heavy chains (and two Fab fragments) of an antibody that specifically binds to a first antigen; (b) two additional Fab fragments of an antibody that specifically binds to a second antigen; and wherein the additional Fab fragments are both fused to either the C-terminus or the N-terminus of the heavy chain of (a) via a peptide linker; In the Fab fragment, the following modifications were made: (i) in both Fab fragments of (a) or in both Fab fragments of (b), the variable domains VL and VH are replaced with each other, and / or the constant domains CL and CH1 are replaced with each other; or (ii) in both Fab fragments of (a), the variable domains VL and VH are replaced by one another and the constant domains CL and CH1 are replaced by one another, and in both Fab fragments of (b), the variable domains VL and VH are replaced by one another or the constant domains CL and CH1 are replaced by one another; or (iii) in both Fab fragments of (a), the variable domains VL and VH are replaced by one another or the constant domains CL and CH1 are replaced by one another, and in both Fab fragments of (b), the variable domains VL and VH are replaced by one another and the constant domains CL and CH1 are replaced by one another; or (iv) in both Fab fragments of (a), the variable domains VL and VH are replaced with each other, and in both Fab fragments of (b), the constant domains CL and CH1 are replaced with each other; or (v) In both Fab fragments of (a), the constant domains CL and CH1 are replaced with each other, and in both Fab fragments of (b), the variable domains VL and VH are replaced with each other.

[0258] In one embodiment, the additional Fab fragments are both fused to either the C-terminus of the heavy chain of (a) or the N-terminus of the heavy chain of (a) via a peptide linker.

[0259] In one embodiment, the additional Fab fragments are both fused to the C-terminus of the heavy chain of (a) via a peptide linker.

[0260] In one embodiment, the additional Fab fragments are both fused to the N-terminus of the heavy chain of (a) via a peptide linker.

[0261] In one embodiment, the following modifications are made in the Fab fragments: in both Fab fragments of (a), or in both Fab fragments of (b), the variable domains VL and VH are replaced with each other, and / or the constant domains CL and CH1 are replaced with each other.

[0262] In one embodiment, the bispecific antibody is a tetravalent antibody, (a) a (modified) heavy chain of a first antibody, which specifically binds to a first antigen and comprises a first VH-CH1 domain pair, and the N-terminus of a second VH-CH1 domain pair of the first antibody is fused to the C-terminus of the heavy chain via a peptide linker; (b) two light chains of the first antibody of (a); and (c) a (modified) heavy chain of a second antibody, which specifically binds to a second antigen and comprises a first VH-CL domain pair, and the N-terminus of a second VH-CL domain pair of the second antibody is fused to the C-terminus of the heavy chain via a peptide linker; and (d) two (modified) light chains of the second antibody of (c), each containing a CL-CH1 domain; and Includes:

[0263] In one embodiment, the bispecific antibody comprises: (a) heavy and light chains of a first full-length antibody that specifically binds to a first antigen; (b) a heavy chain and a light chain of a second full-length antibody that specifically binds to a second antigen, and in which the N-terminus of the heavy chain is connected to the C-terminus of the light chain via a peptide linker; Includes:

[0264] The antibody in (a) does not contain the modifications reported in (b), and the heavy and light chains are isolated chains.

[0265] In one embodiment, the bispecific antibody comprises: (a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; (b) an Fv fragment that specifically binds to a second antigen, comprising a VH2 domain and a VL2 domain; wherein both domains are connected to each other via a disulfide bridge, Either the VH2 domain or the VL2 domain alone is fused via a peptide linker to the heavy or light chain of a full-length antibody that specifically binds to a first antigen.

[0266] In a bispecific antibody, the heavy and light chains of (a) are separate chains.

[0267] In one embodiment, the other of the VH2 domain or the VL2 domain is not fused via a peptide linker to the heavy or light chain of the full-length antibody that specifically binds to the first antigen.

[0268] In all embodiments reported herein, the first light chain comprises a VL domain and a CL domain, and the first heavy chain comprises a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

[0269] In one embodiment, the bispecific antibody comprises: (a) two Fab fragments that specifically bind to a first antigen; (b) one CrossFab fragment that specifically binds to a second antigen and in which the CH1 and CL domains are replaced by each other; (c) a trivalent antibody comprising one Fc region comprising a heavy chain of a first Fc region and a heavy chain of a second Fc region; The C-terminus of the CH1 domains of the two Fab fragments is connected to the N-terminus of the heavy chain Fc region polypeptide, and the C-terminus of the CL domain of the CrossFab fragment is connected to the N-terminus of the VH domain of one of the Fab fragments.

[0270] In one embodiment, the bispecific antibody comprises: (a) two Fab fragments that specifically bind to a first antigen; (b) one CrossFab fragment that specifically binds to a second antigen and in which the CH1 and CL domains are replaced by each other; (c) a trivalent antibody comprising one Fc region comprising a heavy chain of a first Fc region and a heavy chain of a second Fc region; The C-terminus of the CH1 domain of the first Fab fragment is connected to the N-terminus of one of the heavy chain Fc region polypeptides, the C-terminus of the CL domain of the CrossFab fragment is connected to the N-terminus of the other heavy chain Fc region polypeptide, and the C-terminus of the CH1 domain of the second Fab fragment is connected to the N-terminus of the VH domain of the first Fab fragment or the N-terminus of the VH domain of the CrossFab fragment.

[0271] In one embodiment, the bispecific antibody comprises: (a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; (b) a Fab fragment that specifically binds to a second antigen, comprising a heavy chain fragment and a light chain fragment comprising a VH2 domain and a VL2 domain, wherein in the light chain fragment the variable light chain domain VL2 is replaced by the variable heavy chain domain VH2 of the antibody, and in the heavy chain fragment the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of the antibody; and Including, The heavy chain Fab fragment is inserted between the CH1 domain of one of the heavy chains of the full-length antibody and the respective Fc region of the full-length antibody, and the N-terminus of the light chain Fab fragment is conjugated to the C-terminus of the light chain of the full-length antibody that pairs with the heavy chain of the full-length antibody, into which the heavy chain Fab fragment is inserted.

[0272] In one embodiment, the bispecific antibody comprises: (a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; (b) a Fab fragment that specifically binds to a second antigen, comprising a VH2 domain and a VL2 domain, the VH2 domain comprising a heavy chain fragment and a light chain fragment, wherein in the light chain fragment the variable light chain domain VL2 is replaced by the variable heavy chain domain VH2 of the antibody, and in the heavy chain fragment the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of the antibody, the C-terminus of the heavy chain fragment of the Fab fragment is conjugated to the N-terminus of one of the heavy chains of the full-length antibody, and the C-terminus of the light chain fragment of the Fab fragment is conjugated to the N-terminus of the light chain of the full-length antibody that pairs with the heavy chain of the full-length antibody, whereas the heavy chain fragment of the Fab fragment is conjugated to the N-terminus of Includes:

[0273] Polynucleotides Further provided are isolated polynucleotides encoding the bispecific antibodies or fragments thereof described herein.

[0274] The terms "nucleic acid molecule" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by their base sequence, whereby the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, and both single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides, including those containing derivatized sugar or phosphate backbone linkages or chemically modified residues, include modified nucleotide bases. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for directing the expression of antibodies of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo. (See, for example, Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0275] An "isolated" polynucleotide refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated polynucleotides include nucleic acid molecules that are contained in cells that originally contained the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0276] Isolated polynucleotides encoding the bispecific antibodies of the present invention may be expressed as a single polynucleotide encoding the entire antigen-binding molecule, or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides expressed together may associate, for example, via disulfide bonds or other means, to form a functional antigen-binding molecule. For example, the light chain portion of an immunoglobulin may be encoded by a polynucleotide separate from the heavy chain portion of the immunoglobulin. When co-expressed, the heavy chain polypeptides associate with the light chain polypeptides to form an immunoglobulin.

[0277] In some embodiments, the isolated polynucleotide encodes a polypeptide comprised in a bispecific antibody according to the invention described herein.

[0278] In one aspect, an isolated polynucleotide encoding an anti-PD1 / anti-LAG3 bispecific antibody is provided, wherein the first antigen-binding domain that specifically binds to PD1 comprises a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0279] Preparation of bispecific antibodies for use in the present invention Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816, 567. For these methods, one or more isolated nucleic acid(s) encoding the antibody are provided.

[0280] In the case of a native antibody or native antibody fragment, two nucleic acids are required: one for the light chain or fragment thereof and one for the heavy chain or fragment thereof. Such nucleic acid(s) encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chain(s) of the antibody). These nucleic acids may be on the same expression vector or on different expression vectors. In the case of a particular bispecific antibody having heterodimeric heavy chains, four nucleic acids are required: one for the first light chain, one for the first heavy chain comprising a heteromonomeric Fc-region polypeptide, one for the second light chain, and one for the second heavy chain comprising a second heteromonomeric Fc-region polypeptide. The four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors. For example, such nucleic acid(s) encode an amino acid sequence comprising a first VL and / or an amino acid sequence comprising a first VH comprising a first heteromonomeric Fc region and / or an amino acid sequence comprising a second VL and / or an amino acid sequence comprising a second VH comprising a second heteromonomeric Fc region of an antibody (e.g., a first and / or second light chain and / or a first and / or second heavy chain of an antibody). These nucleic acids may be on the same or different expression vectors; typically, these nucleic acids are located on two or three expression vectors, i.e., one vector may contain two or more of these nucleic acids. Examples of these bispecific antibodies are CrossMab and T cell bispecific antibodies (see, e.g., Schaefer, W. et al., PNAS, 108 (2011) 11187-1191). For example, one of the heteromonomer heavy chains contains a so-called "knob mutation" (T366W and, optionally, one of S354C or Y349C) and the other contains a so-called "hole mutation" (T366S, L368A and Y407V and, optionally, Y349C or S354C) (see, e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73).

[0281] In one aspect, an isolated nucleic acid encoding a bispecific antibody described herein is provided. Such a nucleic acid encodes an amino acid sequence comprising the VL and / or the VH of an antigen-binding domain that specifically binds to PD1 and LAG3 (e.g., the light chain and / or the heavy chain of an antibody). In a further aspect, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further aspect, a host cell comprising such a nucleic acid is provided. In some such aspects, the host cell comprises (e.g., has been transformed with): (1) a first vector comprising a first nucleic acid pair encoding amino acid sequences, one of which comprises a first VL of an antibody and the other of which comprises a first VH, and a second vector comprising a second nucleic acid pair encoding amino acid sequences, one of which comprises a second VL of an antibody and the other of which comprises a second VH, or (2) a first vector comprising a first nucleic acid encoding amino acid sequences, one of which comprises a variable domain (preferably a light chain variable domain), and a nucleic acid pair encoding amino acid sequences, one of which comprises a light chain variable domain and the other of which comprises a first heavy chain variable domain. and a third vector comprising a pair of nucleic acids encoding amino acid sequences, one of the amino acid sequences comprising the light chain variable domain of the other of the second vectors and the other of the amino acid sequences comprising the second heavy chain variable domain, or (3) a first vector comprising nucleic acids encoding amino acid sequences, one of the amino acid sequences comprising a first VL of the antibody, a second vector comprising nucleic acid encoding an amino acid sequence comprising a first VH of the antibody, a third vector comprising nucleic acid encoding an amino acid sequence comprising a second VL of the antibody, and a fourth vector comprising nucleic acid encoding an amino acid sequence comprising a second VH of the antibody. In one embodiment, the host cell is a eukaryote, e.g., a Chinese hamster ovary (CHO) cell or a lymphocytic cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method for producing a bispecific antibody is provided, the method comprising culturing a host cell comprising nucleic acids encoding the antibody under conditions suitable for expression of the antibody, as provided above, and, optionally, recovering the antibody from the host cell (or host cell culture medium).

[0282] For recombinant production of the anti-CD20 / anti-CD3 or anti-PD1 / anti-LAG3 bispecific antibodies described herein, nucleic acids encoding the bispecific antibodies, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0283] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0284] In addition to prokaryotes, eukaryotic organisms such as filamentous fungi and yeast are suitable cloning or expression hosts for antibody-encoding vectors, including bacterial and yeast strains that have been "humanized" in their glycosylation pathways, resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414, and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0285] Suitable host cells for the expression of (glycosylated) antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many strains of baculovirus have been identified that may be used in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0286] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0287] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells, as described in Graham, Flett et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells, as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT060562); TRI cells (e.g., Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cells suitable for antibody production, see, for example, Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0288] Assay The bispecific antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0289] 1. Affinity Assay The affinity of the bispecific antigen-binding molecules, antibodies, and antibody fragments provided herein for their corresponding antigens can be determined by surface plasmon resonance (SPR) using standard equipment such as a Biacore® instrument (GE Healthcare), receptors, or target proteins such as those obtainable by recombinant expression, according to the methods described in the Examples. Specific exemplary and illustrative embodiments for measuring binding affinity are described in Examples 2, 8, or 11 of WO 2018 / 185043. In one aspect, K D is measured by surface plasmon resonance at 25° C. using a BIACORE® T100 machine (GE Healthcare).

[0290] 2. Binding Assays and Other Assays In one embodiment, the bispecific antibodies of the invention are tested for their antigen binding activity by known methods, such as ELISA, Western blot, etc. Binding of the anti-PD1 / anti-LAG3 bispecific antibodies provided herein to the corresponding recombinant antigen or antigen-expressing cells can be assessed by ELISA as described in Examples 8 or 11 of WO 2018 / 185043. In a further embodiment, fresh peripheral blood mononuclear cells (PBMCs) can be used in binding assays to demonstrate binding to different PBMCs, such as monocytes, NK cells, and T cells.

[0291] In another embodiment, a cellular dimerization assay was used to demonstrate the dimerization or eventual binding / interaction of two different receptors, PD1 and LAG3, which are cytoplasmically fused with two fragments of an enzyme upon cleavage or crosslinking using a bispecific antibody against both targets. Here, only one receptor exhibits no enzymatic activity. For this specific interaction, the cytosolic C-termini of both receptors are individually fused to heterologous subunits of a reporter enzyme. Only one enzyme subunit exhibits no reporter activity. However, simultaneous binding to both receptors is expected to result in local cellular accumulation of both receptors, complementation of the two heterologous enzyme subunits, and ultimately the formation of a specific, functional enzyme that hydrolyzes the substrate, thereby generating a chemiluminescent signal (Example 11 of WO 2018 / 185043).

[0292] 3. Activity Assay In one embodiment, an assay is provided for identifying anti-PD1 / anti-LAG3 bispecific antibodies with biological activity. Biological activities may include, for example, the ability to enhance activation and / or proliferation of different immune cells, particularly T cells; secretion of immunomodulatory cytokines, such as IFNγ or TNF-alpha; blockade of the PD1 pathway; blockade of the LAG3 pathway; and tumor cell killing. Antibodies with such biological activity in vivo and / or in vitro are also provided. In certain embodiments, antibodies of the present invention are tested for such biological activity. In one embodiment, an immune cell assay is provided that measures activation of lymphocytes from one individual (donor X) to lymphocytes from another individual (donor Y). The use of a mixed lymphocyte reaction (MLR) can demonstrate the effect of blocking the PD1 pathway on lymphocyte effector cells. T cells in this assay were tested for activation and their IFN-γ secretion in the presence or absence of a bispecific antibody of the present invention. This assay is described in more detail in Example 9 of WO 2018 / 185043.

[0293] Pharmaceutical Compositions, Formulations, and Routes of Administration In a further aspect, the present invention provides pharmaceutical compositions comprising an anti-CD20 / anti-CD3 antibody and an anti-PD1 / anti-LAG3 antibody provided herein, e.g., for use in any of the following methods of treatment. In one embodiment, the pharmaceutical composition comprises an anti-CD20 / anti-CD3 antibody and an anti-PD1 / anti-LAG3 antibody provided herein and at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises any of the bispecific antibodies provided herein and at least one additional therapeutic agent (e.g., those described below).

[0294] Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more bispecific antibodies dissolved or dispersed in a pharmaceutically acceptable excipient. The phrase "pharmaceutically acceptable or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic, or other untoward reactions when administered as needed to an animal, e.g., a human. The preparation of pharmaceutical compositions comprising at least one antibody and, optionally, multiple additional active ingredients is known in the art in view of the present disclosure, as exemplified in Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, incorporated herein by reference. In particular, the compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable excipients" include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, salts, stabilizers, and combinations thereof, as would be known to one of skill in the art.

[0295] Parenteral compositions include those designed for administration by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, the antigen-binding molecules of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solutions may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the fusion protein may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the fusion protein of the present invention in the required amount in the appropriate solvent, with various other ingredients, as required, as listed below. Sterilization can be readily accomplished, for example, by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and / or other ingredients. In the case of sterile powders for preparing sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered liquid medium. The liquid medium should be appropriately buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose prior to injection. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. It is understood that endotoxin contamination should be kept to a minimum at a safe level, e.g., less than 0.5 ng / mg protein.Suitable pharmaceutically acceptable excipients include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins Examples of suitable suspensions include proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension (e.g., sodium carboxymethylcellulose, sorbitol, dextran, etc.). Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.

[0296] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may also be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, for example, films or microcapsules. In certain embodiments, sustained absorption of injectable compositions may be brought about by the use in the compositions of agents delaying absorption (e.g., aluminum monostearate, gelatin, or combinations thereof).

[0297] Exemplary pharmaceutically acceptable excipients of the present invention further include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases).

[0298] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0299] In addition to the compositions already described, bispecific antibodies may be formulated as a depot preparation. Such sustained-release preparations can be administered by implantation (e.g., subcutaneously or intramuscularly) or intramuscular injection. Thus, for example, the fusion protein may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0300] Pharmaceutical compositions containing the bispecific antigen-binding molecules of the present invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate the processing of proteins into pharmaceutically usable preparations. The appropriate formulation depends on the selected route of administration.

[0301] The bispecific antibodies disclosed herein may be formulated in compositions in free acid or free base, neutral, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. Pharmaceutically acceptable salts include acid addition salts formed with free amino groups of the proteinaceous composition, or with inorganic acids such as hydrochloric acid or phosphoric acid, or with 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, potassium, ammonium, calcium, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0302] The compositions of the present invention may contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other, and such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0303] In one embodiment, a pharmaceutical composition is provided comprising an anti-CD20 / anti-CD3 bispecific antibody and a pharmaceutically acceptable carrier, and a second medicament comprising an anti-PD1 / anti-LAG3 antibody described herein. In one embodiment, the pharmaceutical composition is for use in treating a CD20-expressing cancer. In a particular embodiment, the pharmaceutical composition is for use in treating a disease selected from the group consisting of B-cell proliferative disorders, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin's lymphoma (HL).

[0304] Formulations to be used for in vivo administration are generally sterile, which may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0305] Administration of anti-CD20 / anti-CD3 bispecific antibody and anti-PD1 / anti-LAG3 antibody Both the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody (both referred to herein as agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if desired for localized treatment, intralesional administration. However, the methods described herein are particularly useful with respect to therapeutic agents administered parenterally, particularly by intravenous infusion.

[0306] Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time periods, bolus administration, and pulse infusion. In one embodiment, the therapeutic agent is administered parenterally, particularly intravenously. In a particular embodiment, the agent is administered by intravenous infusion. In another embodiment, the agent is administered subcutaneously.

[0307] Both the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to physicians. Both the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of therapeutic agent present in the formulation, the type of disorder or treatment, and the other factors discussed above. These will generally be used in the same dosages and by the same routes of administration as those described herein, or about 1-99% of the dosages described herein, or at any dosage and by any route empirically / clinically determined to be appropriate.

[0308] The appropriate dosage of an anti-CD20 / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 antibody (when used in combination, or in combination with one or more other additional therapeutic agents) for disease prevention or treatment depends on the type of disease being treated, the type of anti-CD20 / anti-CD3 bispecific antibody, the severity and course of the disease, whether both agents are administered for prophylactic or therapeutic purposes, previous therapies, the patient's medical history and response to the therapeutic agents, and the discretion of the attending physician. Each agent is appropriately administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, an initial candidate dosage for administration to a subject may be about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg), whether by one or more separate administrations or by continuous infusion. Depending on the above factors, a typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more. For repeated administrations over several days or longer, treatment is typically continued until a desired suppression of disease symptoms occurs, depending on the condition. One exemplary dosage of a bispecific antibody ranges from about 0.005 mg / kg to about 10 mg / kg. In other examples, dosages may also include about 1 μg / kg body weight, about 5 μg / kg body weight, about 10 μg / kg body weight, about 50 μg / kg body weight, about 100 μg / kg body weight, about 200 μg / kg body weight, about 350 μg / kg body weight, about 500 μg / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 350 mg / kg body weight, about 500 mg / kg body weight, to about 1000 mg / kg body weight, or more, or any range derivable therebetween. Examples of ranges derivable from the numbers recited herein include about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc., which may be administered based on the above numbers. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to a patient.Such doses may be administered intermittently, for example every week or every three weeks (e.g., so that the patient receives from about 2 to about 20, or for example about 6, doses of the antibody). An initial higher loading dose, followed by one or more lower doses, may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0309] In one embodiment, the administration of both the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody is a single dose. In certain embodiments, the administration of the therapeutic agent is two or more doses. In one such embodiment, the agent is administered weekly, every two weeks, or every three weeks, particularly every two weeks. In one embodiment, the agent is administered in a therapeutically effective amount. In one embodiment, the agent is administered at a dose of about 10 μg / kg, about 100 μg / kg, about 200 μg / kg, about 300 μg / kg, about 400 μg / kg, about 500 μg / kg, about 600 μg / kg, about 700 μg / kg, about 800 μg / kg, about 900 μg / kg, or about 1000 μg / kg. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is administered at a higher dose than the dose of the anti-CD20 / anti-CD3 bispecific antibody in a corresponding treatment regimen without administration of an anti-PD1 / anti-LAG3 antibody. In one aspect, the administration of the anti-CD20 / anti-CD3 bispecific antibody comprises an initial administration of a first dose of the anti-CD20 / anti-CD3 bispecific antibody, and one or more subsequent administrations of a second dose of the anti-CD20 / anti-CD3 bispecific antibody, wherein the second dose is higher than the first dose. In one aspect, the administration of the anti-CD20 / anti-CD3 bispecific antibody comprises an initial administration of a first dose of the anti-CD20 / anti-CD3 bispecific antibody, and one or more subsequent administrations of a second dose of the anti-CD20 / anti-CD3 bispecific antibody, wherein the first dose is not lower than the second dose.

[0310] In one embodiment, administration of an anti-CD20 / anti-CD3 bispecific antibody in a treatment regimen according to the invention is the first administration of an anti-CD20 / anti-CD3 bispecific antibody to a subject (at least within the same course of treatment). In one embodiment, administration of an anti-PD1 / anti-LAG3 antibody is not administered to a subject prior to administration of an anti-CD20 / anti-CD3 bispecific antibody. In another embodiment, the anti-PD1 / anti-LAG3 antibody is administered prior to administration of an anti-CD20 / anti-CD3 bispecific antibody.

[0311] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody is for use in combination with an anti-PD1 / anti-LAG3 antibody, wherein prior treatment with a type II anti-CD20 antibody, preferably obinutuzumab, precedes the combination treatment, and the period between the prior treatment and the combination treatment is sufficient to allow for B-cell depletion in the individual in response to the type II anti-CD20 antibody, preferably obinutuzumab.

[0312] T cell activation can lead to severe cytokine release syndrome (CRS). In a phase 1 study conducted by TeGenero (Suntharalingam et al., N Engl J Med (2006) 355, 1018-1028), all six healthy volunteers experienced rapid, near-fatal severe cytokine release syndrome (CRS) after infusion of an inappropriately dosed T cell stimulating super-agonist anti-CD28 monoclonal antibody. Cytokine release associated with administration of T cell activating therapeutic agents, such as anti-CD20 / anti-CD3 bispecific antibodies, to subjects can be significantly reduced by pre-treatment of the subjects with type II anti-CD20 antibodies, such as obinutuzumab. The use of GAZYVA® pretreatment (Gpt) should support rapid depletion of B cells in both peripheral blood and secondary lymphoid organs, reducing the risk of highly relevant adverse events (AEs) from strong systemic T cell activation by T cell-activating therapeutics (e.g., CRS), while supporting sufficiently high T cell-activating therapeutic exposure levels from the start of dosing to mediate tumor cell elimination. To date, the safety profile of obinutuzumab (including cytokine release) has been evaluated and managed in hundreds of patients in ongoing obinutuzumab clinical trials. Finally, in addition to supporting the safety profile of T cell-activating therapeutics such as anti-CD20 / anti-CD3 bispecific antibodies, Gpt should also help prevent the formation of anti-drug antibodies (ADAs) against these specific molecules.

[0313] In the present invention, the combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 antibody can be used in combination with one or more other agents in a therapeutic method. For example, at least one additional therapeutic agent can be co-administered. In certain embodiments, the additional therapeutic agent is an immunotherapeutic agent.

[0314] Such combination therapy, as described above, encompasses combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, where administration of a therapeutic agent can occur before, simultaneously with, and / or after administration of the additional agent or agents. In one embodiment, administration of a therapeutic agent and administration of an additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0315] Therapeutic methods and compositions CD20 is expressed on most B cells (pan-B-cell marker) except stem cells and plasma cells, and is frequently expressed on most human B-cell malignancies (tumor-associated antigen), such as lymphomas and leukemias except multiple myeloma, including non-Hodgkin's lymphoma and acute lymphoblastic leukemia.

[0316] In one aspect, a method is provided for treating or delaying progression of a CD20-expressing cancer in a subject, comprising administering to the subject an effective amount of an anti-CD20 / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 antibody.

[0317] In one such aspect, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In a further embodiment, provided herein is a method of depleting B cells comprising administering to a subject an effective amount of an anti-CD20 / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 antibody. An "individual" or "subject" according to any of the above aspects is preferably a human.

[0318] In a further aspect, a composition for use in cancer immunotherapy is provided, comprising an anti-CD20 / anti-CD3 antibody and an anti-PD1 / anti-LAG3 antibody. In a specific embodiment, a composition is provided comprising an anti-CD20 / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 antibody for use in a method of cancer immunotherapy.

[0319] In a further aspect, provided herein is the use of a composition comprising an anti-CD20 / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 antibody in the manufacture or preparation of a medicament. In one aspect, the medicament is for the treatment of a CD20-expressing cancer. In one aspect, the medicament is for the treatment of a B-cell proliferative disorder. In a further aspect, the medicament is for use in a method of treating a B-cell proliferative disorder, comprising administering an effective amount of the medicament to an individual having a B-cell proliferative disorder. In one such aspect, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. In a further aspect, the medicament is for depleting B cells. The B-cell proliferative disorder is selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin's lymphoma (HL). In a particular embodiment, the B-cell cancer is non-Hodgkin's lymphoma or acute lymphoblastic leukemia.

[0320] In a further aspect, provided herein is a method for treating a B-cell cancer. In one embodiment, the method comprises administering to an individual having such a B-cell cancer an effective amount of an anti-PD1 / anti-LAG3 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. The "individual" according to any of the above embodiments may be a human. In particular, the B-cell cancer is a B-cell lymphoma or a B-cell leukemia. In one aspect, the B-cell cancer is a non-Hodgkin's lymphoma or an acute lymphoblastic leukemia.

[0321] The above-mentioned combination therapy encompasses combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, where administration of an anti-PD1 / anti-LAG3 bispecific antibody as reported herein can occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s). In one embodiment, administration of an effective amount of an anti-CD20 / anti-CD3 bispecific antibody, administration of an effective amount of an anti-PD1 / anti-LAG3 antibody, and administration of the additional therapeutic agent(s) occurs within about 1 month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0322] Both the anti-CD20 / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 antibodies (and any additional therapeutic agents) described herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses over various time periods, a bolus dose, or pulse infusion.

[0323] As reported herein, both the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to physicians. The antibody is optionally, but need not be, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These will generally be used in the same dosages and via the same routes of administration as those described herein, or about 1-99% of the dosages described herein, or at any dosage and via any route empirically / clinically determined to be appropriate.

[0324] Those skilled in the art will readily recognize that in many cases, a bispecific molecule may not be a cure, but may provide only a partial benefit. In some embodiments, any physiological change that has some benefit is also considered therapeutically beneficial. Thus, in some aspects, the amount of bispecific antibody that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount."

[0325] Both the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody defined herein are suitably administered to a patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of bispecific antibody may be an initial candidate dosage for administration to a patient, whether by one or more separate administrations or by continuous infusion, for example. Depending on the factors described above, a typical daily dosage may range from about 1 μg / kg to 10 mg / kg or more. For repeated administrations over several days or longer, treatment is typically continued until a desired suppression of disease symptoms occurs, depending on the condition. An exemplary dosage for an anti-CD20 / anti-CD3 bispecific antibody would be in the range of about 0.05 μg / kg to about 1000 μg / kg. For anti-PD1 / anti-LAG3 antibodies, dosages can also include about 0.01 mg / kg body weight, about 0.05 mg / kg body weight, about 2 mg / kg body weight, about 4 mg / kg body weight, about 10 mg / kg body weight, about 20 mg / kg body weight, about 30 mg / kg body weight, about 40 mg / kg body weight, about 45 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 300 mg / kg body weight, about 400 mg / kg body weight, about 500 mg / kg body weight, about 600 mg / kg body weight, about 800 mg / kg body weight, about 1000 mg / kg body weight, up to about 1200 mg / kg body weight or more, and any range derivable therein. Examples of ranges derivable from the numbers recited herein include about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 0.05 μg / kg / body weight to about 500 mg / kg / body weight, etc., which may be administered based on the above numbers. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody may be administered to a patient at a dose of about 0.01 mg, 2.5 mg to about 10 mg, or about 20 mg or about 30 mg. Such dosages may be administered intermittently, e.g., weekly or every three weeks (e.g., the patient receives about two to about 20, or, e.g., about six, doses of the fusion protein). An initial lower loading dose, followed by one or more higher doses, may be administered. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.In one embodiment, anti-PD1 / anti-LAG3 may be administered to a patient at a dose of about 100 mg to about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg.

[0326] Bispecific antibodies comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3 as defined herein will generally be used in an amount effective to achieve its intended purpose. For use in treating or preventing disease symptoms, the bispecific antibodies of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0327] For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, e.g., cell culture assays. The IC 50 A dose may also be formulated in animal models to achieve a circulating concentration range including 100 mg / kg / day. Such information can be used to more accurately determine useful doses in humans.

[0328] Initial dosages can also be estimated from in vivo data, e.g., from animal models, using techniques well known in the art. Those skilled in the art will readily be able to optimize administration to humans based on the animal data.

[0329] Dosage and dosing intervals may be adjusted individually to provide plasma concentrations of the bispecific antibody sufficient to maintain therapeutic efficacy. Typical patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Plasma levels can be measured, for example, by HPLC.

[0330] In cases of local administration or selective uptake, the effective local concentration of the bispecific antibody may not be related to plasma concentration. One skilled in the art can optimize a therapeutically effective local dosage without undue experimentation.

[0331] Therapeutically effective dosages of the bispecific antibodies described herein generally provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the fusion proteins can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (lethal dose for 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 The therapeutic index can be expressed as a ratio. Bispecific antibodies that exhibit large therapeutic indices are preferred. In one embodiment, the bispecific antibodies of the invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a dosage range appropriate for human use. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending on various factors, such as the dosage form employed, the route of administration utilized, and the condition of the subject. The exact formulation, route of administration, and dosage can be selected by the individual physician in consideration of the patient's condition (see, e.g., Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).

[0332] The attending physician of a patient being treated with a bispecific antibody of the invention will know how and when to discontinue, interrupt, or adjust administration due to toxicity, organ failure, etc. Conversely, the attending physician will also know to adjust treatment to higher levels if the clinical response is not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary depending on the severity of the condition being treated, the route of administration, etc. The severity of the condition may, for example, be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dosage, and perhaps frequency of administration, will also vary according to the age, weight, and response of the individual patient.

[0333] Such other agents are suitably present in combination in amounts effective for the intended purpose. The effective amount of such other agents will depend on the amount of fusion protein used, the type of disorder or treatment, and other factors discussed above. Bispecific antibodies are generally used in the same dosages and by the routes of administration described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0334] Such combination therapy as described above encompasses combined administration (wherein two or more therapeutic agents are contained in the same or separate compositions) and separate administration, where administration of the bispecific antibody may occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s) and / or adjuvant(s).

[0335] H.Manufactured products In another aspect of the present invention, an article of manufacture is provided containing materials useful for the treatment, prevention, and / or diagnosis of the disorders described above. The article of manufacture comprises a container and a label or package insert inserted into or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a compound, alone or in combination with other compositions, effective for the treatment, prevention, and / or diagnosis of a condition and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the anti-PD1 / anti-LAG3 antibody described herein above.

[0336] The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture may comprise (a) a first container containing a composition, the composition comprising an anti-CD20 / anti-CD3 bispecific antibody, and (b) a second container containing a composition, the composition comprising an anti-PD1 / anti-LAG3 antibody. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a particular condition.

[0337] Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. [Table C] TIFF2025148352000005.tif252170TIFF2025148352000006.tif248170TIFF2025148352000007.tif249170TIFF20251483520 00008.tif252170TIFF2025148352000009.tif248170TIFF2025148352000010.tif249170TIFF2025148352000011.tif247170 TIFF2025148352000012.tif248170TIFF2025148352000013.tif252170TIFF2025148352000014.tif249170TIFF20251483520 00015.tif252170TIFF2025148352000016.tif250170TIFF2025148352000017.tif250170TIFF2025148352000018.tif201170

[0338] General information relating to 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 ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of antibody chains are numbered and referenced according to the numbering system according to Kabat (Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) as defined above.

[0339] Aspects of the invention Some aspects of the present invention are listed below.

[0340] 1. An anti-CD20 / anti-CD3 bispecific antibody for use in a method of treating a CD20-expressing cancer, wherein the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody.

[0341] 2. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to item 1, wherein the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered together in a single composition or separately in two or more different compositions.

[0342] 3. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to paragraph 1 or 2, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain that is an IgG Fc domain, particularly an IgG1 Fc domain or an IgG4 Fc domain, and the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor.

[0343] 4. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of Items 1 to 3, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises an Fc domain of the human IgG1 subclass having the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0344] 5. The anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene 3 (LAG3), wherein the first antigen-binding domain that specifically binds to PD1 is: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; a VL domain comprising Item 5. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of Items 1 to 4, comprising:

[0345] 6. The anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain is (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a VH domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; a VL domain comprising contains, or (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19; and (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 24; a VL domain comprising Item 5. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of Items 1 to 4, comprising:

[0346] 7. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of Items 1 to 6, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds to PD1, the first antigen-binding domain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 9 and the VL domain comprising the amino acid sequence of SEQ ID NO: 10.

[0347] 8. The anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain is (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26 8. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of Items 1 to 7, comprising:

[0348] 9. The anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain is (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 27 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28; or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30; or (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 31 and a VL domain comprising the amino acid sequence of SEQ ID NO: 32; or (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 33 and a VL domain comprising the amino acid sequence of SEQ ID NO: 34 Item 8. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of Items 1 to 5 or 7.

[0349] 10. The anti-PD1 / anti-LAG3 bispecific antibody a first antigen-binding domain that specifically binds to PD1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; a second antigen-binding domain that specifically binds to LAG3, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18; Item 10. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of Items 1 to 9, comprising:

[0350] 11. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of items 1 to 10, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1 and a Fab fragment that specifically binds to LAG3.

[0351] 12. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of items 1 to 11, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1, and the variable domains VL and VH are swapped with each other such that VL is part of the heavy chain and VH is part of the light chain.

[0352] 13. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of items 1 to 12, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises monovalent binding to PD-1 and monovalent binding to LAG3.

[0353] 14. The anti-PD1 / anti-LAG3 bispecific antibody (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35, a first light chain comprising the amino acid sequence of SEQ ID NO: 36, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37, and a second light chain comprising the amino acid sequence of SEQ ID NO: 38; or (b) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35, a first light chain comprising the amino acid sequence of SEQ ID NO: 36, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 39, and a second light chain comprising the amino acid sequence of SEQ ID NO: 40. Item 14. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of Items 1 to 13, comprising:

[0354] 15. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of items 1 to 14, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35, a first light chain comprising the amino acid sequence of SEQ ID NO: 36, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37, and a second light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0355] 16. The anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V H CD3) and the light chain variable region (V L The first antigen-binding domain contains a heavy chain variable region (V H CD20) and the light chain variable region (V L Item 16. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of Items 1 to 15, comprising: a second antigen-binding domain comprising a CD20 antibody;

[0356] 17. The first antigen-binding domain comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 41, the CDR-H2 sequence of SEQ ID NO: 42, and the CDR-H3 sequence of SEQ ID NO: 43. H a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 44, the CDR-L2 sequence of SEQ ID NO: 45, and the CDR-L3 sequence of SEQ ID NO: 46; L Item 17. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of Items 1 to 16, comprising an anti-CD20 / anti-CD3 bispecific antibody.

[0357] 18. The second antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47. H CD3) and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 48 L Item 18. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of Items 1 to 17, comprising an anti-CD20 / anti-CD3 bispecific antibody.

[0358] 19. The second antigen-binding domain comprises a heavy chain variable region (VH1) comprising the CDR-H1 sequence of SEQ ID NO: 49, the CDR-H2 sequence of SEQ ID NO: 50, and the CDR-H3 sequence of SEQ ID NO: 51. H CD20), and / or a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 52, the CDR-L2 sequence of SEQ ID NO: 53, and the CDR-L3 sequence of SEQ ID NO: 54. L Item 19. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of Items 1 to 18, comprising an anti-CD20 / anti-CD3 bispecific antibody.

[0359] 20. The second antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 55. H CD20) and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 56 L 20. An anti-CD20 / anti-CD3 bispecific antibody for use in the method according to any one of items 1 to 19, comprising an anti-CD20 / anti-CD3 bispecific antibody.

[0360] 21. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of items 1 to 20, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises a third antigen-binding domain that binds to CD20.

[0361] 22. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of items 1 to 21, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises an Fc domain that comprises one or more amino acid substitutions that reduce Fc receptor binding and / or effector function.

[0362] 23. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of items 1 to 22, wherein the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody, and the combination is for administration at intervals of about 1 to 3 weeks.

[0363] 24. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of clauses 1 to 23, wherein prior treatment with a type II anti-CD20 antibody, preferably obinutuzumab, occurs before said combination treatment, and the period between said prior treatment and said combination treatment is sufficient to allow for a depletion of B cells in the individual in response to said type II anti-CD20 antibody, preferably obinutuzumab.

[0364] 25. A pharmaceutical composition comprising a combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody for use in the combined, sequential or simultaneous treatment of a disease, in particular a CD20-expressing cancer.

[0365] 26. A pharmaceutical composition comprising an anti-CD20 / anti-CD3 bispecific antibody and a pharmaceutically acceptable carrier, and a second medicament comprising an anti-PD1 / anti-LAG3 bispecific antibody.

[0366] 27. The pharmaceutical composition according to item 26, for use in treating a CD20-expressing cancer, particularly a blood cancer selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin's lymphoma (HL).

[0367] 28. Use of a combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody in the manufacture of a medicament for treating a CD20-expressing cancer.

[0368] 29. A method for treating a CD20-expressing cancer in a subject, comprising administering to the subject an effective amount of an anti-CD20 / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 bispecific antibody.

[0369] 30. The method of paragraph 29, wherein the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered together in a single composition or separately in two or more different compositions.

[0370] 31. The method of paragraph 29 or 30, wherein the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered intravenously or subcutaneously.

[0371] 32. The method of any one of items 29 to 31, wherein the anti-CD20 / anti-CD3 bispecific antibody is administered simultaneously with, before, or after the anti-PD1 / anti-LAG3 bispecific antibody. [Example]

[0372] Recombinant DNA Technology Standard methods were used to manipulate DNA as described by Sambrook et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 91-3242.

[0373] DNA sequencing The DNA sequence was determined by double-strand sequencing.

[0374] Gene synthesis Desired gene segments were generated by PCR using appropriate templates or synthesized by automated gene synthesis from synthetic oligonucleotides and PCR products by Geneart AG (Regensburg, Germany). In cases where the exact gene sequence was unavailable, oligonucleotide primers were designed based on the sequence of the closest homolog, and the gene was isolated by RT-PCR from RNA derived from the appropriate tissue. Gene segments flanked by unique restriction endonuclease cleavage sites were cloned into standard cloning / sequencing vectors. Plasmid DNA was purified from transformed bacteria, and the concentration was determined by UV spectroscopy. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene segments were designed with appropriate restriction sites to enable subcloning into the respective expression vectors. All constructs were designed with a 5'-terminal DNA sequence encoding a leader peptide that targets the protein for secretion in eukaryotic cells.

[0375] Cell culture technology Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds.), John Wiley & Sons, Inc.

[0376] Protein purification Proteins were purified from filtered cell culture supernatants according to standard protocols. Briefly, the antibody was applied to a Protein A Sepharose column (GE Healthcare) and washed with PBS. Antibody elution was achieved at pH 2.8, immediately followed by neutralization of the sample. Aggregated proteins were separated from monomeric antibody by size-exclusion chromatography (Superdex 200, GE Healthcare) in PBS or 20 mM histidine, 150 mM NaCl (pH 6.0). Monomeric antibody fractions were pooled and concentrated (if necessary) using, for example, a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, and stored frozen at -20°C or -80°C. Portions of these samples were submitted for subsequent protein analysis and analytical characterization, for example, by SDS-PAGE, size-exclusion chromatography (SEC), or mass spectrometry.

[0377] SDS-PAGE The NuPAGE® Pre-Cast Gel System (Invitrogen) was used according to the manufacturer's instructions, specifically, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and NuPAGE® MES (reducing gels, supplemented with NuPAGE® antioxidant running buffer supplement) or MOPS (non-reducing gels) running buffer.

[0378] Analytical Size Exclusion Chromatography Size-exclusion chromatography (SEC) to determine the aggregation and oligomeric state of the antibody was performed by HPLC chromatography. Briefly, Protein A-purified antibody was applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH2PO4 / K2HPO4 (pH 7.5) on an Agilent HPLC 1100 system, or to a Superdex 200 column (GE Healthcare) in 2x PBS on a Dionex HPLC-System. Eluted protein was quantified by UV absorbance and peak area integration. BioRad Gel Filtration Standard 151-1901 was used as the standard.

[0379] Determination of binding and binding affinity of multispecific antibodies to their respective antigens using surface plasmon resonance (SPR) (BIACORE). The binding of the generated antibodies to their respective antigens was monitored by surface plasmon resonance using a BIACORE instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurements, goat anti-human IgG, JIR 109-005-098 antibody, was immobilized on a CM5 chip via amine coupling for display of the antibody against the respective antigen. Binding is measured in HBS buffer (HBS-P (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, pH 7.4) at 25°C (or alternatively, 37°C). Antigen (either R&D Systems or in-house purified) is added at various concentrations in solution. Association is measured by 80 s–3 min antigen injection, and dissociation is measured by washing the chip surface with HBS buffer for 3–10 min. KD values ​​are estimated using a 1:1 Langmuir binding model. Negative control data (e.g., buffer curve) are subtracted from sample curves to correct for system-specific baseline drift and reduce noise signals. The respective Biacore Evaluation Software is used to analyze sensorgrams and calculate affinity data.

[0380] Example 1 Preparation, purification and characterization of T cell bispecific (TCB) antibodies The TCB molecules were prepared according to the method described in WO 2016 / 020309 A1.

[0381] The anti-CD20 / anti-CD3 bispecific antibody (CD20 CD3 TCB or CD20 TCB) used in the experiments corresponds to molecule B described in Example 1 of WO 2016 / 020309 A1. Molecule B is a "2+1 IgG CrossFab" antibody composed of two distinct heavy chains and two distinct light chains. To facilitate assembly of the two distinct heavy chains, point mutations ("knob-into-hole") were introduced into the CH3 domain. Following the method described in WO 2012 / 130831, Pro329Gly, Leu234Ala, and Leu235Ala mutations were introduced into the constant regions of the knob and hole heavy chains to abolish Fcγ receptor binding. To facilitate correct assembly of the two distinct light chains, the VH and VL domains of the CD3-binding Fab were swapped, and point mutations were made in the CH and CL domains of the CD20-binding Fab. 2+1 means that the molecule has two antigen-binding domains specific for CD20 and one antigen-binding domain specific for CD3.

[0382] CD20 TCB comprises the amino acid sequences of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60. A schematic diagram of a bispecific antibody in a 2+1 format is shown in Figure 1B.

[0383] This molecule is further characterized in Example 1 of WO 2016 / 020309 A1.

[0384] Example 2 Preparation, purification and characterization of bispecific anti-PD1 / anti-LAG3 antibodies VH / VL domain swap / replacement (CrossMAb) in one binding arm Vh-VLA bispecific antibody binding to human PD1 and human LAG3 via 1+1 was generated as described in Example 10.1 of WO 2018 / 185043. Vh-Vl The preparation of the antibodies is also described in WO 2009 / 080252. The bispecific antibodies were expressed using expression plasmids containing nucleic acids encoding the amino acid sequences shown in Table 1. 1+1 CrossMAb Vh-Vl A schematic structure of a bispecific antibody is shown in Figure 1A. [Table 1]

[0385] For all constructs, the knob-into-hole heterodimerization technique was used with a typical knob (T366W) substitution in the first CH3 domain and the corresponding hole substitution (T366S, L368A, and Y410V) in the second CH3 domain (and two additional introduced cysteine ​​residues S354C / Y349C) (included in the respective corresponding heavy chain (HC) sequences described above). Following the method described in WO 2012 / 130831, Pro329Gly, Leu234Ala, and Leu235Ala mutations were introduced into the constant regions of the knob and hole heavy chains to abolish binding to Fcγ receptors. To improve correct pairing, additional amino acid substitutions were introduced into the CH and CL domains of the conventional Fab (charge variant).

[0386] The bispecific antibodies expressed as above were purified from the supernatant by a combination of Protein A affinity and size exclusion chromatography. The resulting products were characterized for their identity by mass spectrometry and for analytical properties such as purity, monomer content, and stability by SDS-PAGE.

[0387] The parental PD1 antibody PD1(0376)IgG1 used for comparison comprises a VH domain comprising the amino acid sequence of SEQ ID NO:9 and a VL domain comprising the amino acid sequence of SEQ ID NO:10.

[0388] Example 3 Effect of PD-1 / LAG-3 bispecific antibody in combination with CD20 CD3 TCB on cytotoxic granzyme B release by human CD4 T cells cocultured with a B-cell lymphoblastoid cell line (ARH77) To investigate the combinatorial potential of PD-1 / LAG-3 bispecific antibodies with CD20-TCB, we developed an assay in which freshly purified CD4 T cells were co-cultured for 5 days in the presence of an EBV-immortalized B-cell lymphoblastoid tumor cell line (ARH77). We selected the ARH77 cell line because of its intermediate expression levels of the PD-1 ligand, PD-L1, and high levels of the LAG-3 ligand, MHC-II, allowing us to assess the contribution of LAG-3 blockade in addition to PD-1.

[0389] CD4 T cells were obtained from 10 healthy donors. 8 PBMCs were enriched using a microbead kit (Miltenyi Biotec). Before culture, CD4 T cells were labeled with 5 μM carboxy-fluorescein succinimidyl ester (CFSE). Then, 10 5 10 CD4 T cells were treated with a blocking anti-PD1 antibody (either parent anti-PD-1, nivolumab, or pembrolizumab), or 10 -7 The PD-1 / LAG-3 bispecific antibody PD1 / LAG3 0927 (PD1-LAG3 BsAb) was plated in a 96-well plate (5:1) with B cell lines in the presence or absence of a fixed concentration of CD20-TCB (66 pM) at concentrations between 10 μg / ml. After 5 days, for the final 5 hours of incubation, we added Golgi-plug and Golgi-stop to block protein trafficking and allow intracellular accumulation of cytokines.

[0390] Interestingly, we observed a dose-dependent effect of PD-1 blocking antibodies in combination with CD20-TCB on CD4 T cell secretion of granzyme B (see Figure 2). However, equimolar PD1-LAG3 BsAb was more potent and effective than PD-1 blocking antibodies in increasing granzyme B secreted by CD4 T cells in a dose-dependent manner (E max ), was a suitable combination partner for CD20-TCB. The corresponding EC50 values ​​are shown in Table 2 below: [Table 2]

[0391] Example 4 Potent antitumor effects of combination therapy with PD1 / LAG3 bispecific antibody and CD20 CD3 TCB in vivo in a WSU-DLCL2 xenograft model in humanized NSG mice The antitumor activity of the PD-1 / LAG-3 bispecific antibody PD1 / LAG3 0927 (PD1-LAG3 BsAb) in combination with CD20 CD3 TCB (CD20 TCB) was evaluated in vivo in HSC-NSG mice implanted with the human diffuse large B-cell lymphoma model WSU-DLCL2 subcutaneously. The efficacy of this combination was compared with that of CD20 CD3 TCB as a monotherapy and its combination with nivolumab or nivolumab plus an anti-LAG3 reference antibody.

[0392] a) Experimental materials and methods Preparation of WSU-DLCL2 cell line: WSU-DLCL2 cells (human diffuse large B-cell lymphoma) were originally obtained from the European Collection of Cell Cultures (ECACC) and, after expansion, deposited in the Roche Glycart internal cell bank. Cells were cultured in RPMI containing 10% FCS and 1x Glutamax. Cells were cultured at 37°C in a water-saturated atmosphere with 5% CO2. 1.5x10 cells per animal were cultured in RPMI cell culture medium (Gibco) and GFR Matrigel (1:1, total volume 100µl) with a viability of 98.6%. 6 cells (in vitro passage P13) were injected subcutaneously per mouse.

[0393] Generation of fully humanized mice: Female NSG mice (Jackson Laboratory), 4-5 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12-hour light / 12-hour dark cycle in accordance with official guidelines (GV-Solas; Felasa; TierschG). This experimental research protocol was compiled and approved by the local government (P 2011 / 128). After arrival, the animals were maintained for one week to acclimate to their new environment and to be observed. Continuous health monitoring was performed regularly. NSG mice were intraperitoneally injected with 15 mg / kg busulfan, and one day later, 1 x 10 guinea pigs isolated from umbilical cord blood were injected. 5 Human hematopoietic stem cells were intravenously injected. 14–16 weeks after stem cell injection, humanized immunodeficient mice (HSC-NSG) were subjected to tongue bleeding, and blood was analyzed by flow cytometry for successful humanization. Efficiently engrafted mice were randomized into different treatment groups according to human T cell frequency.

[0394] Efficacy experiments: Fully humanized HSC-NSG mice were inoculated with 1.5 x 10 cells on day 0 in the presence of Matrigel at a 1:1 ratio. 6 The tumors were subcutaneously challenged with 100 WSU-DLCL2 cells (human diffuse large B-cell lymphoma expressing CD20). Tumors were measured three times a week using calipers throughout the entire experimental period. On day 14 (tumors averaged approximately 350-400 mm), tumors were measured three times a week using calipers. 3), mice were randomized into seven groups (Figure 3) and given the first treatment. Weekly scheduled treatments were initiated: Group A received vehicle (phosphate-buffered saline, PBS), Group B received CD20 TCB (0.15 mg / kg, once per week, iv), Group C received CD20 TCB (0.15 mg / kg, once per week, iv) + nivolumab (1.5 mg / kg, once per week, iv), Group D received CD20 TCB (0.15 mg / kg, once per week, iv) + nivolumab (1.5 mg / kg, once per week, iv) + anti-LAG3 (BMS-986016, 1.5 mg / kg, once per week, iv), Group E received CD20 TCB (0.15 mg / kg, once per week, iv) + PD1-LAG3 BsAb (1.5 mg / kg, once per week, iv), and Group F received CD20 They received TCB (0.15 mg / kg, once a week, iv) + PD1-LAG3 BsAb (3 mg / kg, once a week, iv). Treatment was administered by intraperitoneal injection of up to 400 μl. Tumor growth was measured three times a week using calipers, and tumor volume was calculated as follows: T v :(W 2 / 2)×L (W: width, L: length)

[0395] The study ended on day 45.

[0396] Therefore, the impact of treatment was assessed by measuring tumor size and expressed as tumor growth over time, either as an average (Figure 4) or as tumor growth over time for each single mouse (Figures 5A-5F). For statistical analysis, the last observed tumor volume for each animal was used as the endpoint, assessing whether it was less than 800 mm. This endpoint was then subjected to pairwise group comparisons based on the Chi2 test (Figure 6).

[0397] b) Results In this setting, treatment of WSU-DCLC2-bearing mice with CD20 TCB was found to mediate potent tumor growth inhibition from day 30 compared with vehicle (Figure 4). Because activation with TCB is known to induce PD1 and LAG3 expression on T cells, we attempted to further improve efficacy by combining CD20 TCB with either nivolumab or nivolumab plus anti-LAG3 antibody. However, such combinations did not induce a statistically significant reduction in tumor growth compared with single treatments (Figures 4 and 6). In contrast, treatment with both 1.5 mg / kg and 3 mg / kg of PD1-LAG3 BsAb in combination with CD20 TCB resulted in strong ...

Claims

1. 1. An anti-CD20 / anti-CD3 bispecific antibody for use in a method of treating a CD20-expressing cancer, wherein the anti-CD20 / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody, the anti-PD1 / anti-LAG3 bispecific antibody comprising a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene 3 (LAG3), wherein the first antigen-binding domain that specifically binds to PD1 is: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; and a VL domain comprising:

1. An anti-CD20 / anti-CD3 bispecific antibody comprising:

2. 10. The method of claim 1, wherein the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered together in a single composition or separately in two or more different compositions.

3. 3. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of claim 1 or 2, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain that is an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain, and wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, in particular an Fcγ receptor.

4. the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, the second antigen-binding domain comprising: (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; and and a VL domain comprising: contains, or (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; and and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 24; and and a VL domain comprising:

4. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of claims 1 to 3, comprising:

5. 5. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of claims 1 to 4, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds to PD1, the first antigen-binding domain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 9 and the VL domain comprising the amino acid sequence of SEQ ID NO:

10.

6. the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, the second antigen-binding domain comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18; or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26 6. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of claims 1 to 5, comprising:

7. the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, the second antigen-binding domain comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 27 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28; or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30; or (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 31 and a VL domain comprising the amino acid sequence of SEQ ID NO: 32; or (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 33 and a VL domain comprising the amino acid sequence of SEQ ID NO: 34 6. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of claims 1 to 3 or 5, comprising:

8. the anti-PD1 / anti-LAG3 bispecific antibody a first antigen-binding domain that specifically binds to PD1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; a second antigen-binding domain that specifically binds to LAG3, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18; and 7. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of claims 1 to 6, comprising:

9. 9. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of claims 1 to 8, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1 and a Fab fragment that specifically binds to LAG3.

10. 10. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of claims 1 to 6 or 8 or 9, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35, a first light chain comprising the amino acid sequence of SEQ ID NO: 36, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37, and a second light chain comprising the amino acid sequence of SEQ ID NO:

38.

11. The anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (V Ｈ CD3) and light chain variable region (V Ｌ a first antigen-binding domain comprising a heavy chain variable region (V Ｈ CD20) and light chain variable region (V Ｌ and a second antigen-binding domain comprising the antibody (anti-CD20 / anti-CD3) (anti-CD20).

12. The first antigen-binding domain comprises a heavy chain variable region (V) comprising a CDR-H1 sequence of SEQ ID NO: 41, a CDR-H2 sequence of SEQ ID NO: 42, and a CDR-H3 sequence of SEQ ID NO:

43. Ｈ a light chain variable region (V) comprising the CDR-L1 sequence of SEQ ID NO: 44, the CDR-L2 sequence of SEQ ID NO: 45, and the CDR-L3 sequence of SEQ ID NO: 46; Ｌ 12. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of claims 1 to 11, comprising an anti-CD20 / anti-CD3 antibody.

13. 13. An anti-CD20 / anti-CD3 bispecific antibody for use in the method of any one of claims 1 to 12, wherein prior treatment with a type II anti-CD20 antibody, preferably obinutuzumab, occurs before said combination treatment, and the period between said prior treatment and said combination treatment is sufficient to allow for B-cell depletion in the individual in response to said type II anti-CD20 antibody, preferably obinutuzumab.

14. 1. A composition comprising an anti-PD1 / anti-LAG3 bispecific antibody for use in treating a CD20-expressing cancer, said treatment comprising administering a composition comprising an anti-PD1 / anti-LAG3 bispecific antibody in combination with a composition comprising an anti-CD20 / anti-CD3 bispecific antibody, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene 3 (LAG3), and wherein the first antigen-binding domain that specifically binds to PD1 is: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and and a VH domain comprising: A VL domain, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; and a VL domain comprising: A composition comprising:

15. 14. The composition of claim 13, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds to PD1, the first antigen-binding domain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 9 and the VL domain comprising the amino acid sequence of SEQ ID NO:

10.

16. the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, the second antigen-binding domain comprising: (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; and and a VL domain comprising: contains, or (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; and and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 24; and and a VL domain comprising:

16. The composition of claim 14 or 15, comprising:

17. the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, the second antigen-binding domain comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18; or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26 The composition according to claims 14 to 16, comprising:

18. the anti-PD1 / anti-LAG3 bispecific antibody a first Fab fragment that specifically binds to PD1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; and a second Fab fragment that specifically binds to LAG3, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18; and The composition according to claims 14 to 17, comprising:

19. The composition of claims 14 to 18, wherein the anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

20. The composition of claims 14 to 18, wherein the anti-CD20 / anti-CD3 bispecific antibody is mosunetuzumab.

21. 1. A pharmaceutical composition comprising a combination of an anti-CD20 / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody for use in the combined, sequential or simultaneous treatment of a disease, particularly a CD20-expressing cancer.

22. 22. The pharmaceutical composition of claim 21 for use in the treatment of CD20-expressing cancers, particularly hematological cancers selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin's lymphoma (HL).

23. 1. Use of a combination of an anti-CD20 / CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody in the manufacture of a medicament for treating a CD20-expressing cancer, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene 3 (LAG3), and the first antigen-binding domain that specifically binds to PD1 is: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; and a VL domain comprising: Including, use.

24. the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, the second antigen-binding domain comprising: (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; and and a VL domain comprising: contains, or (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; and and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 24; and and a VL domain comprising:

24. The use according to claim 23, comprising:

25. the anti-PD1 / anti-LAG3 bispecific antibody a first Fab fragment that specifically binds to PD1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; and a second Fab fragment that specifically binds to LAG3, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18; and 25. The use according to claim 23 or 24, comprising:

26. 1. A method for treating a CD20-expressing cancer in a subject, comprising administering to the subject an effective amount of an anti-CD20 / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 bispecific antibody, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene 3 (LAG3), wherein the first antigen-binding domain that specifically binds to PD1 is: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; and a VL domain comprising: A method comprising:

27. the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, the second antigen-binding domain comprising: (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; and and a VL domain comprising: contains, or (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; and and a VH domain comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; and (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 24; and and a VL domain comprising:

27. The method of claim 26, comprising:

28. the anti-PD1 / anti-LAG3 bispecific antibody a first Fab fragment that specifically binds to PD1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; and a second Fab fragment that specifically binds to LAG3, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18; and 28. The method of claim 26 or 27, comprising:

29. The method of any one of claims 26 to 28, wherein the anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

30. 30. The method of any one of claims 26 to 29, wherein the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered together in a single composition or separately in two or more different compositions.

31. 31. The method of any one of claims 26 to 30, wherein the anti-CD20 / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered intravenously or subcutaneously.

32. 32. The method of any one of claims 26 to 31, wherein the anti-CD20 / anti-CD3 bispecific antibody is administered simultaneously with the anti-PD1 / anti-LAG3 bispecific antibody, before the anti-PD1 / anti-LAG3 bispecific antibody, or after the anti-PD1 / anti-LAG3 bispecific antibody.

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