WO2020169698A1

WO2020169698A1 - Sensitization of cancer cells to tnf by bet inhibition

Info

Publication number: WO2020169698A1
Application number: PCT/EP2020/054413
Authority: WO
Inventors: Marina Bacac; Tanja Andrea FAUTI; Simon John Hogg; Ricky Wayne Johnstone; Astrid Alexandra RUEFLI-BRASSE; Daniel Alan ROHLE; Lisa Christina WELLINGER
Original assignee: F Hoffmann La Roche AG; Hoffmann La Roche Inc
Current assignee: F Hoffmann La Roche AG; Hoffmann La Roche Inc
Priority date: 2019-02-21
Filing date: 2020-02-20
Publication date: 2020-08-27
Anticipated expiration: 2021-08-21

Abstract

The invention relates to a method of sensitizing a cancer cell to TNF induced cell death comprising the administration of a BET inhibitor to a patient in need thereof, in particular in a patient undergoing a therapy with an immune activating agent.

Description

Sensitization of cancer cells to TNF by BET inhibition

The present invention relates to the use of a BET inhibitor for sensitizing a cancer cell to TNF induced cell death.

The field of cancer immunotherapy (CIT) has been rapidly evolving with the approval of anti-CTLA4, anti -PD 1, and anti-PD-Ll in solid tumors. These approvals were a watershed moment in cancer therapy. However, the majority of patients show limited therapeutic benefit or show a lack of durable response. Upon activation through CIT agents, cytotoxic T cells release pro-inflammatory cytokines such as IFNy, TNF and Granzyme B leading to direct cancer cell cytolysis at the immunological synapse and bystander cancer cell death in the surrounding tumor microenvironment. However, rapid genetic and epigenetic tumor evolution can lead to immune escape and clinical resistance against CIT agents. Improving the response to CIT has become a field of intense research and identifying the right combination strategy is required.

Bromodomain and extra-terminal family (BET) proteins, which include BRD2, BRD3, BRD4, and the testis specific BRDT, bind to acetylated lysine residues including the histone tails within nucleosomes. Bromodomains act as readers by binding to acetylated lysines and recruiting transcriptional protein machinery to specific chromatin sites regulating gene expression. BET proteins have been shown to regulate oncogenic transcription factor expression e.g. cMYC leading to the development of BET inhibitors (BETi) for the treatment of a range of cancers. Besides regulating oncogenic transcription factors, it has been established that BETi can modulate anti-tumor immunity by regulating expression of PD-L1. Combining anti -PD 1 with BETi has shown to be synergistic in preclinical mouse models of cancer (Cell Rep. 2017 Feb 28; 18(9): 2162-2174; Cancer Immunol Res. 2018 Oct;6(10): 1234-1245). T cell activating bispecific antibodies are a novel class of cancer therapeutics, designed to engage cytotoxic T cells against tumor cells. The simultaneous binding of such an antibody to CD3 on T cells and to an antigen expressed on the tumor cells will force a temporary interaction between tumor cell and T cell, causing activation of the T cell and subsequent lysis of the tumor cell.

CEA-TCB (RG7802, R06958688, cibisatamab) is a novel T cell activating bispecific antibody targeting CEA on tumor cells and CD3e on T cells. In mouse models, CEA-TCB displays potent anti-tumor activity, leads to increased intratumoral T cell infiltration, increased release of pro-inflammatory cytokines such as IFNy, TNF and Granzyme B, and up-regulates the PD-Ll/PD-1 pathway and its activation. The increase in PD-Ll/PD-1 pathway is a sign of fully activated T cells as it is one of the suppressive pathways that is turned on during T cell activation.

It was surprisingly found that BETi broadly sensitized cancer cells to TNF induced cell death irrespective of their histology or genetic background. TNF derived from cytotoxic lymphocytes through either T cell receptor (TCR) activation or T cell bispecific antibody activation was also able to significantly increase bystander killing of cancer cells. Therefore, the combination of BETi with any CIT agent that stimulates cytotoxic lymphocytes to release TNF will be more than additive, i.e. superior to either agent alone.

Brief description of the Figures:

Figure 1 shows that MKN45 and HCT116 cells’ viability is decreased with TNF and increasing amounts of BETi.

Figures 2-3 show that MKN45 and HCT116 cells’ viability is significantly decreased with TNF and increasing amounts of chemically distinct BETi.

Figure 4 shows that MC38 cells’ cell death is significantly increased with TNF and different BETi’s.

Figure 5 demonstrates that MKN45 cells treated with the combination of a BETi and TNF show induction of cleaved PARP, signifying cell death.

Figure 6 demonstrates that HCT116 and MKN45 cells treated with the combination of BETi’s and TNF show induction of cleaved PARP, signifying cell death.

Figures 7-8 show that the combination of a BETi and TNF induces synergistic cell death in AU565 and MC38 cells.

Figure 9 shows that single-agent TNF is able to affect cell growth in MKN45 and HCT116 cells. Figure 10 shows that chemically distinct BETi’s functionally increase cytotoxicity from T cells towards MC38-Ova tumor cell targets significantly.

Figure 11 shows that neutralization of TNF from T cells reduces cytotoxicity towards MC38-Ova tumor cell targets. Figure 12 shows that a BETi is capable of augmenting the cytotoxic activity of OT-1

T cells towards MLL-AF9-driven acute myeloid leukemia cells independently of perforin.

Figures 13-14 show that cytokines released by CEA-TCB activated T cells is able to synergistically combine with BET inhibition.

Figure 15 shows that TNF is the cytotoxic cytokine from CEA-TCB activated T cells. Figure 16 shows that BET inhibition combined with supernatant from CEA-TCB activated T cell is able to increase PARP cleavage.

Figure 17 shows that supernatant from CEA-TCB 2 activated T cells synergizes with BET inhibition.

Figure 18 shows that supernatant released by CEA-TCB 2 activated T cells induces potent cell death when combined with a BET inhibitor.

Figure 19 shows that T cell receptor (TCR)-independent bystander killing of tumor antigen negative tumor cells is enhanced in the presence of a BET inhibitor.

Figures 20-21 show that CEA-TCB activated T cells are able to kill non CEA expressing cancer cells when combined with a BETi. Figure 22 shows that CEA-TCB activated T cells are able to decrease the number of non CEA expressing cancer cells significantly when combined with a BETi.

Figure 23 shows that CEA-TCB 2 is able to induce bystander killing in the presence of a BET inhibitor.

Figures 24-25 show that a combination of TNF and an increasing concentration of BETi enhance Caspase 3/7 and Caspase 8 activity significantly.

Figures 26-27 show that inhibition of HCT116 cell growth through RG6146 and TNF treatment is partially rescued by Caspase 8 knockdown.

Figure 28 shows that overexpression of cFLIP, but not Bcl-2, is able to rescue induction of cell death induced by RG6146 and TNF in MC38 cells. Figure 29 shows that MC38 cells are less sensitive to RG6146 due to expression of p-gp·

Figure 30 shows that the combination of CEA-TCB and JQ1 induces tumor regression in vivo, which is rescued by TNF blockade. Figure 31 is a different representation of the data from Figure 30 monitoring tumor volume over the time of the study.

Figure 32 shows that BETi’s, among a library of epigenetic small molecule inhibitors, are most effective in decreasing viability of HCT116-NLV cells in a coculture with CMV-specific T cells. Figure 33 shows that BETi’s, among a library of epigenetic small molecule inhibitors, are most effective in enhancing cytotoxicity of T cells towards MC38 tumor targets.

According to the invention, an“immune activating agent” is an agent capable of activating a naive T cell into a cytotoxic T cell against cancer cells. Examples of immune activating agents are cancer immunotherapy agents (also known as immuno-oncology agents), such as for example anti-PD-1 or anti-PD-Ll antibodies like e.g. atezolizumab (TECENTRIQ®), pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®) or durvalumab (IMFIZI®); anti-CD20 antibodies like e.g. rituximab (MABTHERA®), obinutuzumab (GAZYVA®/GAZYVARO®) or ofatumumab (ARZERRA®); anti-CD52 antibodies like e.g. alemtuzumab (CAMPATH-1H®); CAR-T immunotherapy like e.g. tisagenlecleucel (KYMRIAH®) or axicabtagene ciloleucel (YESCARTA®); anti-CTLA4 antibodies like e.g. ipilimumab (YERVOY®); or T cell bispecific antibodies. A particular class of immune activating agents are bispecific CD3 antibodies, i.e. antibodies binding specifically to CD3 and to another antigen determinant. “TNF” refers to tumor necrosis factor, also called e.g. tumor necrosis factor alpha

(TNF a) or TNFa.

“TNF mediated killing” and“TNF induced cell death” refers to the death of a cell, in particular a tumor cell, caused by the application of TNF to said cell.

“Sensitizing” means, in the context of the invention as in its common acceptation, making something sensitive or more sensitive. Therefore, sensitizing a cancer cell to TNF induced cell death means making said cancer cell sensitive or more sensitive to cell death induced by TNF. In other words, after sensitization of a cancer cell to TNF induced cell death according to the invention, this cancer cell will be more susceptible to the action of TNF than before the sensitization. Therefore, after sensitization of cancer cells by a BET inhibitor according to the invention, a higher number of of cancer cells are killed by TNF compared to the number of cancer cells killed by TNF in the absence of the BET inhibitor.

“T cell activation” as used herein refers to one or more cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays to measure T cell activation are known in the art and described herein.

The term“bispecific” means that the antibody is able to specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antibody comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antibody is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.

The term“T cell bispecific (TCB) antibody” refers to a bispecific antibody that has an antigen binding moiety capable of forming an antigen binding moiety-antigen complex with an antigenic determinant found on the surface of T cells.

As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope", and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM).

As used herein, the term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen binding moiety is able to direct the entity to which it is attached (e.g. a second antigen binding moiety) to a target site, for example to a specific type of tumor cell bearing the antigenic determinant. In another embodiment an antigen binding moiety is able to activate signaling through its target antigen, for example a T cell receptor complex antigen. Antigen binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen binding moieties include an antigen binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding moieties may comprise antibody constant regions as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: a, d, e, g, or m. Useful light chain constant regions include any of the two isotypes: k and l.

By "specific binding" is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed e.g. on a BIAcore instrument) (Liljeblad et ah, Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antigen binding moiety to an unrelated protein is less than about 10% of the binding of the antigen binding moiety to the antigen as measured, e.g., by SPR. In certain embodiments, an antigen binding moiety that binds to the antigen, or an antibody comprising that antigen binding moiety, has a dissociation constant (K_D) of < 1 mM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10 ⁸M or less, e.g. from 10 ⁸M to 10 ¹³ M, e.g., from 10 ⁹M to 10 ¹³ M).

“Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless indicated otherwise, as used herein,“binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K_D), which is the ratio of dissociation and association rate constants (k_0ff and k_on, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by well established methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

“CD3” refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed CD3 as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, e.g., splice variants or allelic variants. In one embodiment, CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3e). The amino acid sequence of human CD3e is shown in UniProt (www.uniprot.org) accession no. P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. See also SEQ ID NO: 34. The amino acid sequence of cynomolgus [Macaca fascicularis] CD3e is shown in NCBI GenBank no. BAB71849.1. See also SEQ ID NO: 35. “Carcinoembryonic antigen” or“CEA” (also known as Carcinoembryonic antigen- related cell adhesion molecule 5 (CEACAM5)) refers to any native CEA from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed CEA as well as any form of CEA that results from processing in the cell. The term also encompasses naturally occurring variants of CEA, e.g., splice variants or allelic variants. In one embodiment, CEA is human CEA. The amino acid sequence of human CEA is shown in UniProt (www.uniprot.org) accession no. P06731, or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_004354.2.

As used herein, the terms“first”,“second” or“third” with respect to Fab molecules etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the bispecific antibody unless explicitly so stated.

The term“valent” as used herein denotes the presence of a specified number of antigen binding sites in an antibody. As such, the term“monovalent binding to an antigen” denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the antibody.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

The terms“full length antibody,”“intact antibody,” and“whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and single-domain antibodies. For a review of certain antibody fragments, see Hudson et ak, Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g.

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

The term“variable region” or“variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6^th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. As used herein in connection with variable region sequences, "Kabat numbering" refers to the numbering system set forth by Kabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), referred to as“numbering according to Kabat” or“Kabat numbering” herein. Specifically the Kabat numbering system (see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of kappa and lambda isotype and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CHI, Hinge, CH2 and CH3), which is herein further clarified by referring to“numbering according to Kabat EU index” in this case.

The term“hypervariable region” or“HVR”, as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence

(“complementarity determining regions” or“CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91- 96 (L3), 26-32 (HI), 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 (LI), 50-56 (L2), 89-97 (L3), 31- 35b (HI), 50-65 (H2), and 95-102 (H3) (Rabat et al., Sequences of Proteins of

Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (HI), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732- 745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (HI), 26-35b (HI), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Rabat et al., supra.

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

The“class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgG₃, IgG₄, IgAi, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.

A“Fab molecule” refers to a protein consisting of the VH and CHI domain of the heavy chain (the“Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.

By a“crossover” Fab molecule (also termed“Crossfab”) is meant a Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CHI (VL-CH1, in N- to C-terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in N- to C-terminal direction). For clarity, in a crossover Fab molecule wherein the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1 CHI is referred to herein as the“heavy chain” of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule wherein the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the“heavy chain” of the (crossover) Fab molecule.

In contrast thereto, by a“conventional” Fab molecule is meant a Fab molecule in its natural format, i.e. comprising a heavy chain composed of the heavy chain variable and constant domains (VH-CHl, in N- to C-terminal direction), and a light chain composed of the light chain variable and constant domains (VL-CL, in N- to C-terminal direction).

The term“immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called a (IgA), d (IgD), e (IgE), g (IgG), or m (IgM), some of which may be further divided into subtypes, e.g. gi (IgGi), j2 (IgG2), J3 (IgG3), J4 (IgG4), ai (IgAi) and 012 (IgA₂). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.

The term“Fc domain” or“Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post- translational cleavage of one or more, particularly one or two, amino acids from the C- terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full- length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C- terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447), of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of

Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). A“subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

A“modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational

modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or

electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which might be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding moieties) are not the same. In some embodiments the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a particular embodiment, the modification promoting association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.

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

“Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (1988),“Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W. R. Pearson (1996)“Effective protein sequence comparison” Meth. Enzymol. 266:227- 258; and Pearson et. al. (1997) Genomics 46:24-36, and is publicly available from http://fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml. Alternatively, a public server accessible at

http://fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used to compare the sequences, using the ggsearch (global protein: protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.

An“activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcyRIIIa (CD 16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).

“Reduced binding”, for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity, the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction. By“fused” is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

In one embodiment, the immune activating agent is a bispecific CD3 antibody.

In a particular embodiment, the immune activating agent is a bispecific CD3 antibody capable of specifically binding to CEA (CEA CD3 bispecific antibody). Particular bispecific antibodies directed to CD3 and CEA are described e.g. in PCT publication nos. WO 2014/131712 and WO 2017/055389 (each incorporated herein by reference in its entirety).

The CEA CD3 bispecific antibody thus comprises a first antigen binding moiety that specifically binds to CD3, and a second antigen binding moiety that specifically binds to CEA.

In one embodiment, the first antigen binding moiety comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO:

5 and the LCDR3 of SEQ ID NO: 6.

In one embodiment, the second antigen binding moiety comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 9, the HCDR2 of SEQ ID NO: 10, and the HCDR3 of SEQ ID NO: 11; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 12, the LCDR2 of SEQ ID NO: 13 and the LCDR3 of SEQ ID NO: 14; or (ii) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 17, the HCDR2 of SEQ ID NO: 18, and the HCDR3 of SEQ ID NO: 19; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 20, the LCDR2 of SEQ ID NO: 21 and the LCDR3 of SEQ ID NO: 22.

In a particular embodiment, the CEA CD3 bispecific antibody comprises

(i) a first antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the LCDR3 of SEQ ID NO: 6; and

(ii) a second antigen binding moiety that specifically binds to CEA and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 9, the HCDR2 of SEQ ID NO: 10, and the HCDR3 of SEQ ID NO: 11; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 12, the LCDR2 of SEQ ID NO: 13 and the LCDR3 of SEQ ID NO: 14; or (ii) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 17, the HCDR2 of SEQ ID NO: 18, and the HCDR3 of SEQ ID NO: 19; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 20, the LCDR2 of SEQ ID NO: 21 and the LCDR3 of SEQ ID NO: 22.

In one embodiment, the first antigen binding moiety comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

In one embodiment, the first antigen binding moiety comprises the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8.

In one embodiment, the second antigen binding moiety comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16; or (ii) a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the second antigen binding moiety comprises the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16; or (ii) the heavy chain variable region sequence of SEQ ID NO: 23 and the light chain variable region sequence of SEQ ID NO: 24.

In a particular embodiment, the CEA CD3 bispecific antibody comprises

(i) a first antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8; and (ii) a second antigen binding moiety that specifically binds to CEA and comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16.

In a further particular embodiment, the CEA CD3 bispecific antibody comprises

(i) a first antigen binding moiety that specifically binds to CD3 and comprises the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8; and

(ii) a second antigen binding moiety that specifically binds to CEA and comprises the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

In a further particular embodiment, the CEA CD3 bispecific antibody comprises

(i) a first antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8; and

(ii) a second antigen binding moiety that specifically binds to CEA and comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24.

In a further particular embodiment, the CEA CD3 bispecific antibody comprises

(ii) a second antigen binding moiety that specifically binds to CEA and comprises the heavy chain variable region sequence of SEQ ID NO: 23 and the light chain variable region sequence of SEQ ID NO: 24.

In some embodiments, the first and/or the second antigen binding moiety is a Fab molecule. In some embodiments, the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged. In such embodiments, the second antigen binding moiety preferably is a conventional Fab molecule.

In some embodiments wherein the first and the second antigen binding moiety of the bi specific antibody are both Fab molecules, and in one of the antigen binding moieties (particularly the first antigen binding moiety) the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other, i) in the constant domain CL of the first antigen binding moiety the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CHI of the first antigen binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index); or ii) in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CHI of the second antigen binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index).

The bispecific antibody does not comprise both modifications mentioned under i) and ii). The constant domains CL and CHI of the antigen binding moiety having the VH/VL exchange are not replaced by each other (i.e. remain unexchanged).

In a more specific embodiment, i) in the constant domain CL of the first antigen binding moiety the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI of the first antigen binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index); or ii) in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI of the second antigen binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). In one such embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Rabat), and in the constant domain CHI of the second antigen binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Rabat EU index).

In a further embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is substituted independently by lysine (R), arginine (R) or histidine (H) (numbering according to Rabat), and in the constant domain CHI of the second antigen binding moiety the amino acid at position 147 is substituted

independently by glutamic acid (E), or aspartic acid (D) (numbering according to Rabat EU index).

In a particular embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is substituted independently by lysine (R), arginine (R) or histidine (H) (numbering according to Rabat) and the amino acid at position 123 is substituted independently by lysine (R), arginine (R) or histidine (H) (numbering according to Rabat), and in the constant domain CHI of the second antigen binding moiety the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Rabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Rabat EU index).

In a more particular embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is substituted by lysine (R) (numbering according to Rabat) and the amino acid at position 123 is substituted by lysine (R) (numbering according to Rabat), and in the constant domain CHI of the second antigen binding moiety the amino acid at position 147 is substituted by glutamic acid (E)

(numbering according to Rabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Rabat EU index).

In an even more particular embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is substituted by lysine (R)

(numbering according to Rabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Rabat), and in the constant domain CHI of the second antigen binding moiety the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Rabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Rabat EU index). In particular embodiments, if amino acid substitutions according to the above embodiments are made in the constant domain CL and the constant domain CHI of the second antigen binding moiety, the constant domain CL of the second antigen binding moiety is of kappa isotype.

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

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

In some embodiments, the CEA CD3 bispecific antibody provides monovalent binding to CD3.

In particular embodiments, the CEA CD3 bispecific antibody comprises a single antigen binding moiety that specifically binds to CD3, and two antigen binding moieties that specifically bind to CEA. Thus, in some embodiments, the CEA CD3 bispecific antibody comprises a third antigen binding moiety that specifically binds to CEA. In some embodiments, the third antigen moiety is identical to the first antigen binding moiety (e.g. is also a Fab molecule and comprises the same amino acid sequences).

In particular embodiments, the CEA CD3 bispecific antibody further comprises an Fc domain composed of a first and a second subunit. In one embodiment, the Fc domain is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgGi Fc domain. In another embodiment the Fc domain is an IgG₄ Fc domain. In a more specific embodiment, the Fc domain is an IgG₄ Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG₄ antibodies (see

Stubenrauch et ak, Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment, the Fc domain is a human Fc domain. In a particularly preferred embodiment, the Fc domain is a human IgGi Fc domain. An exemplary sequence of a human IgGi Fc region is given in SEQ ID NO: 33.

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

In particular embodiments, the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain. Thus, in one embodiment said modification is in the CH3 domain of the Fc domain.

In a specific embodiment said modification promoting the association of the first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a“knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain. The knob-into- hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et ah, Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).

Accordingly, in some embodiments, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.

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

In some embodiments, the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.

In a particular embodiment the Fc receptor is an Fey receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. In one embodiment the effector function is one or more selected from the group of complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

Typically, the same one or more amino acid substitution is present in each of the two subunits of the Fc domain. In one embodiment, the one or more amino acid substitution reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the one or more amino acid substitution reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.

In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). In more particular embodiments, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”,“PGLALA” or“LALAPG”). Specifically, in preferred embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and the second subunit of the Fc domain the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain.

In a preferred embodiment, the CEA CD3 bispecific antibody comprises

(i) a first antigen binding moiety that specifically binds to CD3, comprising a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the

HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the LCDR3 of SEQ ID NO: 6, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;

(ii) a second and a third antigen binding moiety that specifically bind to CEA, comprising a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 9, the HCDR2 of SEQ ID NO: 10, and the HCDR3 of SEQ ID NO: 11; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 12, the LCDR2 of SEQ ID NO: 13 and the LCDR3 of SEQ ID NO: 14, wherein the second and third antigen binding moiety are each a Fab molecule, particularly a conventional Fab molecule; (iii) an Fc domain composed of a first and a second subunit, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

In one embodiment, the second and third antigen binding moiety comprise a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16.

In one embodiment, the second and third antigen binding moieties comprise the heavy chain variable region of SEQ ID NO: 15 and the light chain variable region of SEQ ID NO: 16.

The Fc domain according to the above embodiments may incorporate, singly or in combination, all of the features described hereinabove in relation to Fc domains.

In one embodiment, the antigen binding moieties and the Fc region are fused to each other by peptide linkers, particularly by peptide linkers as in SEQ ID NO: 27 and SEQ ID NO: 28. In one embodiment, the bispecific antibody comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 25, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 26, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 27, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 28.

In one embodiment, the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 25, a polypeptide comprising the sequence of SEQ ID NO: 26, a polypeptide comprising the sequence of SEQ ID NO: 27, and a polypeptide comprising the sequence of SEQ ID NO: 28 (CEA-TCB).

In a particularly preferred embodiment, the CEA CD3 bispecific antibody is CEA- TCB (cibisatamab).

In one embodiment the CEA CD3 antibody is a bispecific antibody comprising

(i) a first antigen binding moiety that specifically binds to CD3, comprising a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the LCDR3 of SEQ ID NO: 6, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions, particularly the variable regions, of the Fab light chain and the Fab heavy chain are exchanged;

(ii) a second and a third antigen binding moiety that specifically bind to CEA, comprising a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 17, the HCDR2 of SEQ ID NO: 18, and the HCDR3 of SEQ ID NO: 19; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 20, the LCDR2 of SEQ ID NO: 21 and the LCDR3 of SEQ ID NO: 22, wherein the second and third antigen binding moiety are each a Fab molecule, particularly a conventional Fab molecule;

(iii) an Fc domain composed of a first and a second subunit capable of stable association, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

In one embodiment, the second and third antigen binding moiety comprise a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24. In one embodiment, the second and third antigen binding moieties comprise the heavy chain variable region of SEQ ID NO: 23 and the light chain variable region of SEQ ID NO: 24.

In one embodiment, the antigen binding moieties and the Fc region are fused to each other by peptide linkers, particularly by peptide linkers as in SEQ ID NO: 30 and SEQ ID NO: 31.

In one embodiment, in the constant domain CL of the second and the third Fab molecule under (ii) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat), and in the constant domain CHI of the second and the third Fab molecule under (ii) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

In one embodiment, the bispecific antibody comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 29, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 30, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 31, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 32. In one embodiment, the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 29, a polypeptide comprising the sequence of SEQ ID NO: 30, a polypeptide comprising the sequence of SEQ ID NO: 31, and a polypeptide comprising the sequence of SEQ ID NO: 32 (CEA-TCB 2). The term“BET inhibitor” according to the invention refers to agents that prevent activity of BET proteins with an IC50 of about 0.001 mM to about 2 pM.

In one embodiment, the BET inhibitor is a compound selected from the compounds described in WO 2011/143669 which incorporated herein by reference. Methods of producing said BET inhibitors are also disclosed in WO 2011/143669. Most preferably, the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H- l-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]- acetamide as in the formula below, or a salt thereof. Example JQ35 of WO 2011/143669 describes a method for its preparation.

The preferred BET inhibitor is depicted in the following formula:

The above BET inhibitor is also known as RG6146, JQ35 or TEN-010.

The invention thus relates in particular to:

A BET inhibitor for use in a method of sensitizing a cancer cell to TNF induced cell death; A method of sensitizing a cancer cell to TNF induced cell death comprising the administration of a BET inhibitor to a patient in need thereof;

A BET inhibitor for use in a method of enhancing TNF mediated killing of cancer cells in a cancer patient undergoing a therapy with an immune activating agent, wherein the immune activating agent is capable of causing the release of TNF by T cells; A method of enhancing TNF mediated killing of cancer cells in a cancer patient undergoing a therapy with an immune activating agent comprising the administration of a BET inhibitor to a patient in need thereof, wherein the immune activating agent is capable of causing the release of TNF by T cells; A BET inhibitor for use in a method of increasing the action of TNF-relasing T cells in a cancer patient undergoing a therapy with an immune activating agent, wherein the immune activating agent is capable of causing the release of TNF by T cells;

A BET inhibitor for use in a method of increasing the action of TNF-relasing T cells in a cancer patient undergoing a therapy with an immune activating agent, wherein the immune activating agent is capable of causing the release of TNF by T cells and wherein the cancer cells are sensitive to TNF;

A method of treating of cancer, comprising sensitizing a cancer cell to TNF induced cell death by the administration of a BET inhibitor to a patient in need thereof;

A BET inhibitor for use or a method according to the invention in a cancer patient undergoing a therapy with an immune activating agent, wherein the immune activating agent is capable of causing the release of TNF by T cells;

A BET inhibitor for use or a method according to the invention wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a-tetraaza- cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]-acetamide (RG6146), INCB-054329, INCB-057643, GSK525762, GS-5829, CPI-0610, Birabresib, PLX51107,

ABBV-075, BI 894999, FT-1101, ZEN-3694, GSK-2820151 or BMS-986158;

A BET inhibitor for use or a method according to the invention wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a-tetraaza- cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]-acetamide (RG6146); A BET inhibitor for use or a method according to the invention wherein the immune activating agent is an antibody;

A BET inhibitor for use or a method according to the invention wherein the immune activating agent is a T cell bispecific (TCB) antibody;

A BET inhibitor for use or a method according to the invention wherein the immune activating agent is a CEA CD3 bispecific antibody;

A BET inhibitor for use or a method according to the invention wherein the CEA CD3 bispecific antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the LCDR3 of SEQ ID NO: 6;

A BET inhibitor for use or a method according to the invention wherein the CEA CD3 bispecific antibody comprises

(ii) a second antigen binding moiety that specifically binds to CEA and comprises (i) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 9, the HCDR2 of SEQ ID NO: 10, and the HCDR3 of SEQ ID NO: 11; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 12, the LCDR2 of SEQ ID NO: 13 and the LCDR3 of SEQ ID NO: 14; or (ii) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 17, the HCDR2 of SEQ ID NO: 18, and the HCDR3 of SEQ ID NO: 19; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 20, the LCDR2 of SEQ ID NO: 21 and the LCDR3 of SEQ ID NO: 22;

A BET inhibitor for use or a method according to the invention wherein the immune activating agent is CEA-TCB (cibisatamab) or CEA-TCB 2;

A BET inhibitor for use or a method according to the invention wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a-tetraaza- cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]-acetamide (RG6146) and the immune activating agent is CEA-TCB (cibisatamab) or CEA-TCB 2;

A BET inhibitor and a TCB antibody for use in the treatment of cancer;

A method of treating cancer, comprising administering a BET inhibitor and a TCB antibody to a patient in need thereof;

A pharmaceutical composition comprising a BET inhibitor, a TCB antibody and one or more pharmaceutically acceptable excipients; A kit comprising a BET inhibitor and a TCB antibody for the simultaneous, separate or sequential administration of said BET inhibitor and TCB antibody to a patient in need thereof;

A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical composition or a kit according to the invention wherein the BET inhibitor is 2-[(S)-4-(4- chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-N- [3-(4-methyl-piperazin-l-yl)-propyl]-acetamide (RG6146), INCB-054329, INCB-057643, GSK525762, GS-5829, CPI-0610, Birabresib, PLX51107, ABBV-075, BI 894999, FT- 1101, ZEN-3694, GSK-2820151 or BMS-986158;

A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical composition or a kit according to the invention wherein the BET inhibitor is 2-[(S)-4-(4- chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-N- [3-(4-methyl-piperazin-l-yl)-propyl]-acetamide (RG6146);

A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical composition or a kit according to the invention wherein the TCB antibody is a CEA CD3 antibody;

A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical composition or a kit according to the invention wherein the CEA CD3 bispecific antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the LCDR3 of SEQ ID NO: 6;

A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical composition or a kit according to the invention wherein the CEA CD3 bispecific antibody comprises

A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical composition or a kit according to the invention wherein the TCB antibody is CEA-TCB (cibisatamab) or CEA-TCB 2;

A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical composition or a kit according to the invention wherein the BET inhibitor is 2-[(S)-4-(4- chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-N- [3-(4-methyl-piperazin-l-yl)-propyl]-acetamide (RG6146) and the TCB antibody is CEA- TCB (cibisatamab) or CEA-TCB 2;

The use of a BET inhibitor in the manufacture of a medicament for sensitizing a cancer cell to TNF induced cell death;

The use of a BET inhibitor in the manufacture of medicament for enhancing TNF mediated killing of cancer cells in a cancer patient undergoing a therapy with an immune activating agent, wherein the immune activating agent is capable of causing the release of TNF by T cells;

The use of a BET inhibitor in the manufacture of medicament according to the invention in a cancer patient undergoing a therapy with an immune activating agent, wherein the immune activating agent is capable of causing the release of TNF by T cells;

The use of a BET inhibitor in the manufacture of a medicament for increasing the action of TNF-relasing T cells in a cancer patient undergoing a therapy with an immune activating agent, wherein the immune activating agent is capable of causing the release of TNF by T cells;

The use of a BET inhibitor in the manufacture of a medicament for increasing the action of TNF-relasing T cells in a cancer patient undergoing a therapy with an immune activating agent, wherein the immune activating agent is capable of causing the release of TNF by T cells and wherein the cancer cells are sensitive to TNF.

The use of a BET inhibitor in the manufacture of medicament according to the invention wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l- thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]- acetamide (RG6146), INCB-054329, INCB-057643, GSK525762, GS-5829, CPI-0610, Birabresib, PLX51107, ABBV-075, BI 894999, FT-1101, ZEN-3694, GSK-2820151 or BMS-986158;

The use of a BET inhibitor in the manufacture of medicament according to the invention wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l- thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]- acetamide (RG6146);

The use of a BET inhibitor in the manufacture of medicament according to the invention wherein the immune activating agent is an antibody;

The use of a BET inhibitor in the manufacture of medicament according to the invention wherein the immune activating agent is a T cell bispecific (TCB) antibody;

The use of a BET inhibitor in the manufacture of medicament according to the invention wherein the immune activating agent is a CEA CD3 bispecific antibody;

The use of a BET inhibitor in the manufacture of medicament according to the invention wherein the CEA CD3 bispecific antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the LCDR3 of SEQ ID NO: 6;

The use of a BET inhibitor in the manufacture of medicament according to the invention wherein the CEA CD3 bispecific antibody comprises

The use of a BET inhibitor in the manufacture of medicament according to the invention wherein the immune activating agent is CEA-TCB (cibisatamab) or CEA-TCB 2;

The use of a BET inhibitor in the manufacture of medicament according to the invention wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l- thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]- acetamide (RG6146) and the immune activating agent is CEA-TCB (cibisatamab) or CEA- TCB 2;

The use of a BET inhibitor and a TCB antibody in the manufacture of a medicament for the treatment of cancer;

The use of a BET inhibitor and a TCB antibody in the manufacture of a medicament according to the invention wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9- trimethyl-6H-l-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin- l-yl)-propyl] -acetamide (RG6146), INCB-054329, INCB-057643, GSK525762, GS-5829, CPI-0610, Birabresib, PLX51107, ABBV-075, BI 894999, FT-1101, ZEN-3694, GSK- 2820151 or BMS-986158;

The use of a BET inhibitor and a TCB antibody in the manufacture of a medicament according to the invention wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9- trimethyl-6H-l-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin- 1 -yl)-propyl] -acetamide (RG6146);

The use of a BET inhibitor and a TCB antibody in the manufacture of a medicament according to the invention wherein the TCB antibody is a CEA CD3 antibody; The use of a BET inhibitor and a TCB antibody in the manufacture of a medicament according to the invention wherein the CEA CD3 bispecific antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the LCDR3 of SEQ ID NO: 6;

The use of a BET inhibitor and a TCB antibody in the manufacture of a medicament according to the invention wherein the CEA CD3 bispecific antibody comprises (i) a first antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the LCDR3 of SEQ ID NO: 6; and

The use of a BET inhibitor and a TCB antibody in the manufacture of a medicament according to the invention wherein the TCB antibody is CEA-TCB (cibisatamab) or CEA- TCB 2; and

The use of a BET inhibitor and a TCB antibody in the manufacture of a medicament according to the invention wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9- trimethyl-6H-l-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin- l-yl)-propyl] -acetamide (RG6146) and the TCB antibody is CEA-TCB (cibisatamab) or CEA-TCB 2.

The BET inhibitor according to the invention enhances cytotoxic T cell-mediated killing of tumor cells through sensitization to TNF-mediated killing.

TNF induces cell death when it is in contact with cells that are treated with a BET inhibitor.

BET inhibition is able to sensitize a wide range of cancer cells to TNF.

According to the invention, the cancer can be for example a solid tumor cancer or a lymphoma, in particular colorectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, prostate cancer, skin cancer, muscle cancer, brain cancer, liver cancer, bone cancer, endometrial cancer, connective tissue cancer, uterus cancer, kidney cancer, ovarian cancer, placenta cancer, leukemia or myeloma. The BET inhibitor according to the invention sensitizes cancer cells to T cell activated conditioned media. For example, a supernatant from primary T cells, cancer cells and an immune activating agent, i.e. cibisatamab or CEA-TCB 2, leads to increased cancer cell death when combined with a BET inhibitor according to the invention. The term“enhance” in this context means that a cancer cell becomes sensitive to

TNF mediated killing only in the presence of the BET inhibitor, or that a cancer cell becomes more sensitive to TNF mediated killing once in the presence of the BET inhibitor.

The BET inhibitor according to the invention increases bystander killing, i.e. killing of cancer cells which are not bound to the TCB or to an activated T cell. Tumor heterogeneity suggests that not every cancer cell will express the TCB antibody’s antigen to the same level, e.g. CEA. When using a BET inhibitor according to the invention, cells that express low levels of the TCB antobody’s antigen will become sensitive to TNF release by T cells, leading to increased total tumor targeting and killing. Likewise, cells which are not bound to activated T cells will also become sensitive to TNF release, leading to increased tumor killing.

The invention thus also relates to a BET inhibitor for use in a method of increasing the tumor killing efficacy of a T cell bound to a TCB antibody, in particular by increasing the bystander tumor killing, i.e. the killing of cancer cells not bound to the TCB antibody but which are in the vicinity of the T cell-bound cancer cell. The invention thus also relates to a BET inhibitor for use in a method of increasing the tumor killing efficacy of a T cell bound to a tumor cell, in particular by increasing the bystander tumor killing, i.e. the killing of cancer cells not bound to the T cell.

The bystander killing is independent of the mechanism of T cell activation.

The invention thus also relates to a BET inhibitor for use in a method of increasing the TNF mediated tumor killing by an activated T cell, in particular by increasing the bystander tumor killing by TNF released by said activated T cell.

Table 1: Sequence listing

The invention will now be illustrated by the following examples which have no limiting character.

Examples

Example 1: Increased response to TNF in gastric and colorectal cancer cells when treated with a BET inhibitor

Viability of MKN45 (RPMI+GlutaMAX+20% hiFCS) and HCT116 (RPMI (ATCC modification) + 10%FCS) was measured at 3 days after treatment with increasing concentrations of RG6146 (Roche), JQ1 (Selleck Chemicals #S7110), OTX015 (MK 8628/Birabresib) Selleck Chemicals #S7360), ΪBET151 (ChemScene #CS-0930) and a single concentration of TNF (BioLegend #570104). MKN45 and HCT116 cells were harvested with Trypsin/EDTA and plated at a density of 5000 cells per well in lOOuL of growth media in a 96 well plate (Corning #3917). The cells were allowed to adhere overnight. One plate was removed from the incubator (37°C, 5% C02) and 50uL of CellTiterGlo 2.0 (Promega #) was added. The plate was shaken for 10 minutes at room temperature. It was then read on a plate reader for luminescence to obtain a T=0 measurement. RG6146, JQ1, OTX015 and ΪBET151 were 3 fold serially diluted in DMSO to create a concentration gradient and were added to wells containing cells to give the final working concentration of BET inhibitors in 0.15% DMSO per well. Finally, TNF that had been reconstituted in PBS 0.5%BSA was added to each well to give a final concentration of 5, 15ng/mL for MKN45 or 5, 15, 50ng/mL for HCT116 as well as wells receiving only PBS 0.5%BSA. The plates were returned to the incubator (37°C, 5% C02) for 72 hours. The experimental plates were removed from the incubator and 50uL of CellTiterGlo

2.0was added. The plates were shaken for 10 minutes at room temperature, and then read on a plate reader for luminescence. The raw data was normalized to the DMSO control for each experimental condition. The data was analyzed by GraphPadPrism7.0 to generate graphs of percent viability relative to DMSO. Data represents mean +/- SEM from one or three biologically independent experiments. Using a two-way analysis of variance

(ANOVA) with Tukey’s multiple comparison test for RG6146, and Sidak’s multiple comparison test for JQ1, ΪBET151 and OTX15 it was seen that BET inhibitors in combination with 15ng/ml TNF significantly decrease viability compared to BET inhibitor single agent treatments. For simplicity, significance is only shown for the highest concentration of compound. Significance was defined as *p<0.05, **p<0.01, ***p<0.001, ****r<0.0001. Figure 1 shows data generated in the initial experiments (one biological replicate including two to three technical replicates), while Figures 2-3 show aggregated data of three biological individual experiments (each containing two to three technical replicates). The murine colon adenocarcinoma cell line MC38, and derivatives expressing Ovalbumin (Ova), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin (Gibco) and incubated at 37°C in 10% C02. For in vitro use, RG6146, JQ1, IBET151, IBET762, Y803, RVX-208, and dBETl were dissolved in DMSO to generate a lOmM stock solution. Briefly, MC38-Ova cells were seeded (1.5e5 cells/well) into 48-well plates for >8 hours prior to addition of recombinant TNF and BET inhibitors or DMSO/PBS control. Following addition of BET inhibitors (RG6146, JQ1, IBET151, IBET762, dBET ImM; RVX-208 IOmM) in the presence or absence of TNF (5ng/mL), MC38-Ova cells were incubated for 18 hours. Cells were harvested by centrifugation, washed once in ice-cold flow cytometry buffer (2% FCS in PBS), prior to being resuspended in flow cytometry buffer containing propidium iodide (PI) and assessed for PI positivity. Data were collected on a FACSCanto P flow cytometer (BD Biosciences) and analyzed using FlowJo Software (Version 10.2, Tree Star). Data represents mean +/- SEM from three biologically independent experiments. Statistical significance was calculated in GraphPad Prism using two-way ANOVA comparing cell death induced by each BET inhibitor to the DMSO control in the presence or absence of TNF, respectively. Significance was defined as *p<0.05, **p<0.01, ***p<0.001. The increased cell death of MC38 cells seen with BET inhibitors and TNF is shown in Figure 4.

Example 2: TNF and a BET inhibitor induce cell death as measured by cleaved PARP

The combination of a BET inhibitor and TNF is able to induce apoptosis in cancer cells were either agent alone is not able. MKN45 cells were harvested with Trypsin/EDTA and plated at a density of 250000 cells per well in 2mL of growth media in a 6 well plate. The cells were allowed to adhere overnight. RG6146, JQ1, OTX015, ΪBET151 were diluted in DMSO and aliquoted into the appropriate treatment well for a final working concentration of luM and 2.5uM. The final DMSO concentration in all wells was 0.1%. TNF was diluted to final concentration of 5, 10, 15 and 40ng/mL in the appropriate wells. The plates were returned for 24 hours to the incubator. After 6, 24 and 48 hours, cells were washed with ice cold PBS and harvested in lysis buffer. The lysis buffer contained Cell Lysis Buffer (Cell Signaling Technology #9803), Phosphatase Inhibitor (Cocktail Set P, Calbiochem

#524625) and Protease Inhibitor (Cocktail Set IP, Calbiochem #539134) . The resultant lysates were frozen at -80C for further processing. Samples were thawed on ice and sonicated to disrupt cellular membranes and solubilize proteins. Lysates were cleared through centrifugation and protein concentration was assessed by DC protein assay kit (BioRad #5000112), and samples were normalized to the lowest protein concentration. An equal amount of loading buffer containing BME (AlfaAeser #161337) was added to each sample. The samples were resolved using SDS-PAGE electrophoresis and transferred to a nitrocellulose membrane for blotting with anti-cleaved PARP (Cell Signaling #5625), anti- actin (Cell Signaling #4970) and anti-vinculin (Cell Signaling #13901) antibodies ovemight. Blots were washed with 0.1% Tween-TBS, incubated with a secondary antibody (Jackson Immuno Research #111-035-144) and imaged using ECL and Fusion camera system. The resultant images were assembled and it was seen that the combination of a BET inhibitor and TNF was able to induce PARP cleavage as shown in Figures 5-6.

Example 3: The induction of tumor cell death in response to a BET inhibitor and recombinant TNF is synergistic

The murine colon adenocarcinoma cell line MC38 expressing Ovalbumin was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin (Gibco) and incubated at 37°C in 10% CO2. The human mammary carcinoma cell line AU565 was cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin (Gibco) and incubated at 37°C in 5% CO2. MC38 and AE1565 cells were exposed to increasing concentrations of RG6146 and recombinant TNF in vitro for 18 hours. Cells were harvested by centrifugation, washed once in ice-cold flow cytometry buffer (2% FCS in PBS), prior to being resuspended in flow cytometry buffer containing propidium iodide (PI) and assessed for PI positivity. Data were collected on a FACSCanto P flow cytometer (BD Biosciences) and analyzed using FlowJo Software (Version 10.2, Tree Star). Cell death data was imported into Rstudio (Version 3.3.1) and raw values were first visualized using pheatmap package (1.0.12) and plotted in Figure 7A, 7C and Figure 8 for AU565 cells. Data were analyzed using the Synergyfmder package (Version 3.8). Synergy analysis was performed by first calculating a four-parameter log-logistic model to generate the dose-response curves for each single agent. Drug synergy scoring was then calculated using the ZIP algorithm (Ianevski, Aleksandr, et al. "SynergyFinder: a web application for analyzing drug combination dose- response matrix data." Bioinformatics 33.15 (2017): 2413-2415). The synergy scores are calculated across all the tested concentration combinations, which are subsequently visualized as either a two-dimensional or a three-dimensional interaction surface over the dose matrix. The inflection of the 3D plot indicates the excess % inhibition beyond the expectation by the ZIP model, the‘ZIP score’ is the overall average % inhibition beyond the expectation by the ZIP model over the whole dose-response matrix. This analysis demonstrated that the induction of cell death in response to a BETi and TNF towards MC38 and AU565 cells is synergstic, resulting in significantly more cell death than would be predicted by either single agent (presented in Figures 7B and 7D).

Example 4: The sensitization of cancer cells to TNF by BET inhibition is found across multiple cancer subtypes

The combination of a BETi and TNF leads to profound growth defects in a subset of cancer cell lines. Cell lines were obtained from ATCC, NCI, CLS, and DSMZ cell repositories and maintained in the repository recommended media. Cells were plated in 96 well plates and allowed to adhere for 48 hours. Each cell line was plated at a density predetermined to ensure exponential growth for the duration of the experiment and sub- confluent by the end of the experiment. After 48 hours a measurement was taken for each cell line to determine the To value to identify cytotoxicity and control for errors in plating. Cells were treated with RG6146 diluted in DMSO resulting in a final concentration of 0.1% DMSO in each well. TNF was diluted in PBS to obtain the appropriate concentration. Both RG6146 and TNF were tested in a six points dose range and the combination was tested at a range of concentrations for RG6146 while TNF was fixed at 15ng/mL. The cells were treated for 120 hours. Measurement of cell number was done by measuring total protein. For adherent cells, cells were fixed with 10% trichloracetic acid (TCA) while for suspension cells 50% TCA was used. Cells were then incubated for 1 hour at 4°C and then washed with deionized water and dried. The cells were stained with 0.04% wt/v Sulforhodamine B (SRB) for 30 minutes at room temperature after which the cells were washed six times with 1% acetic acid. After the plates had dried the SRB bound to cells was solubilized for in lOmM Tris base and measured with a Seelux-LED96 plate reader at an optical density of 492, 520, and 560 nm. The average background optical density, consisting of wells containing media without cells, was subtracted from each experimental well on each respective plate. The data was fitted using non-linear curve fitting calculations similar to XLfit algorithm 205. Growth inhibition of 50% (GI50) was calculated at 100 x [(T_f-To)/(Vehicle Control-To)=50 where T_f= the value for the experimental condition. The max growth inhibition (MGI) is the maximum growth inhibition obtained by RG1646, TNF, or the combination. TNF sensitization was calculated as MGI of RG6146 alone subtracted from MGI of RG6146 with TNF. The results are presented in Table 2.

Table 2: Growth IC50s (GI50) and Max Growth Inhibition (MGI) in a large cancel cell panel

Example 5: Cell lines that respond to the combination have some inherent sensitivity to TNF

On average, it has been seen that in order for BETi to sensitize the cells to TNF, the cells have preferably some basal response to TNF. HCT116 and MKN45 cells were tested for the ability of TNF to suppress their growth. The experimental conditions were the same as outlined in Example 1 with the exception of TNF replacing RG6146. TNF was diluted in PBS 0.5%BSA to give the appropriate concentrations and PBS with 0.5%BSA was used as vehicle control. The growth response of MKN45 and HCT116 are shown in Figure 9.

Three technical replicates are shown for each cell line.

Example 6: Structurally distinct BET inhibitors augment the CTL-mediated killing of tumor cells through sensitization to TNF and not due to perforin

The murine colon adenocarcinoma cell line MC38, and derivatives expressing Ovalbumin (Ova), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin (Gibco) and incubated at 37°C in 10% C02. CD8+ T cells from perforin-wild-type (C57B1/6.0T1) OT-1 transgenic mice were activated from spleens with Ova257-264 peptide (SIINFEKL). Activated OT1 T cells were used on days 5-10 post-activation and had a typical effector phenotype

(CD8+CD69+CD25+CD62L-CD44+). T cells were cultured in RPMI-1640 medium supplemented with 10% FCS, L-glutamine, penicillin/streptomycin, nonessential amino acids, sodium pyruvate, Hepes, 2-mercaptoethanol, and interleukin-2 (100 IU/ml). For in vitro use, RG6146, JQ1, IBET151, IBET762, Y803, RVX-208, and dBETl were dissolved in DMSO to generate a lOmM stock solution. CD8 T cell cytotoxicity towards syngeneic solid tumours was assessed by flow cytometry as previously described [Kearney et al. Cell Death Diff 2017 24(10)]. Briefly, MC38-Ova cells were seeded (1.5e5 cells/well) into 48- well plates for >8 hours prior to addition of activated OT1 T cells and/or BET inhibitors or DMSO control. Co-culture assays were set up at a 1 :20 effector (OT1 T cell) to target (MC38-Ova) ratios in the presence or absence of BET inhibitors (RG6146, JQ1, IBET151, IBET762, dBET ImM; RVX-208 IOmM) and incubated for 18 hours. Cells were harvested by centrifugation, washed once in ice-cold flow cytometry buffer (2% FCS in PBS), prior to being resuspended in flow cytometry buffer containing propidium iodide (PI) and assessed for PI positivity. Data were collected on a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo Software (Version 10.2, Tree Star). Data represents mean +/- SEM from three biologically independent experiments. Statistical significance was calculated in GraphPad Prism using two-way ANOVA comparing cell death induced by each BET inhibitor to the DMSO control in the presence or absence of OT1 T cells, respectively. Significance was defined as *p<0.05, **p<0.01, ***p<0.001. The increased cell death seen with BET inhibitors is shown in Figure 10.

OT-1 T cells that recognize the OVA peptide presented by MC38 cells show increased killing that can be blocked by anti-TNF. The murine colon adenocarcinoma cell line MC38, and derivatives expressing Ovalbumin (Ova), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) and

penicillin/streptomycin (Gibco) and incubated at 37°C in 10% C02. CD8+ T cells from perforin-wild-type (C57B1/6.0T1) or perforin-deficient (C57Bl/6.0Tl.Prf-/-) OT-1 transgenic mice were activated from spleens with Ova257-264 peptide (SIINFEKL). Activated OT1 T cells were used on days 5-10 post-activation and had a typical effector phenotype (CD8+CD69+CD25+CD62L-CD44+). T cells were cultured in RPMI-1640 medium supplemented with 10% FCS, L-glutamine, penicillin/streptomycin, nonessential amino acids, sodium pyruvate, Hepes, 2-mercaptoethanol, and interleukin-2 (100 IU/ml). For in vitro use, RG6146 was dissolved in DMSO to generate a lOmM stock solution. Anti-TNF neutralizing antibody (catalogue # 506325) was obtained from Bioloegnd. CD8 T cell cytotoxicity towards syngeneic solid tumours was assessed by flow cytometry as previously described [Kearney et al. Cell Death Diff 2017 24(10)]. Briefly, MC38-Ova cells were seeded (1.5e5 cells/well) into 48-well plates for >8 hours prior to addition of activated OT1 T cells or small molecules/antibodies. Co-culture assays were set up at varying effector (OT1 T cell) to target (MC38-Ova) ratios in the presence or absence of RG6146 (2.5mM) or anti-TNF neutralizing antibody (20pg/mL) and incubated for 18 hours. Cells were harvested by centrifugation, washed once in ice-cold flow cytometry buffer (2% FCS in PBS), prior to being resuspended in flow cytometry buffer containing propidium iodide (PI) and assessed for PI positivity. Data were collected on a FACSCanto P flow cytometer (BD Biosciences) and analyzed using FlowJo Software (Version 10.2, Tree Star). The increased cell death seen with RG6146 is blocked with anti-TNF neutralizing antibody. Data is shown in Figure 11.

Example 7: A BET inhibitor is capable of augmenting the cytotoxic activity of OT-1 T cells towards MLL-AF9-driven acute myeloid leukemia cells, independently of perforin

Murine C57BL/6-derived MLL-AF9-driven acute myeloid leukemia (AML) cells were cultured in in Dulbecco’s modified Eagle’s medium supplemented with 20% fetal calf serum (FCS) and penicillin/streptomycin (Gibco), recombinant interleukin-3 and incubated at 37°C in 10% C02. MLL-AF9 cells were pulsed with recombinant SIINFEKL peptide for 20 minutes at 37°C in 10% C02 prior to being washed once in pre-warmed media and being exposed to increasing ratios of activated OT1 T cells in the presence or absence of 2.5uM RG6146 for 16 hours. Cells were harvested by centrifugation, washed once in ice- cold flow cytometry buffer (2% FCS in PBS), prior to being resuspended in flow cytometry buffer containing propidium iodide (PI) and assessed for PI positivity. Data were collected on a FACSCanto P flow cytometer (BD Biosciences) and analyzed using FlowJo Software (Version 10.2, Tree Star). This data demonstrated that a BETi augments the cytotoxic activity of both perforin expressing (i.e. wild-type) OT1 T cells and perforin- deficient (C57BL/6-Pfp-/-) OT1 T cells as shown in Figure 12.

Example 8: Cytokines released from activated T cells show enhanced killing in the presence of a BET inhibitor

Target cells were harvested using 0.25%Trypsin/EDTA and plated at 25000 cells/well in lOOuL in a 96 well flat bottom plate. The cells were allowed to adhere overnight.

Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #175442). After centrifugation (1000 x g, 10 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (250 x g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 250 x g, 10 minutes). The resulting PBMC population was counted and kept in RPMI1640 medium containing 10% hiFCS and 1% L-alanyl-L-glutamine in cell incubator (37°C, 5% CO2) until further use (no longer than 24 h) or were frozen in -80C until further use. The PBMCs were resuspended at 5 x 10⁶ cells per ml in RPMI1640 (#31870) + Glutamine + 2% hiFCS assay medium (resulting later in an E:T ratio of 10: 1). The CEA-TCB and the appropriate vehicle control were diluted in the assay media to give a final concentration of 20nM CEA-TCB. 50uL of PBMCs and 50uL of CEA-TCB or 50uL of vehicle was added to each well of the MKN45 cells that had been plated the previous day. The assay plates were placed back into the cell incubation (37°C, 5% CO2) for 24 hours. The supernatants from the CEA-TCB PMBCs and the vehicle PBMCs were collected and filtered through a 0.22uM filter. The target cells, MKN45 and HCT116, plated the previous day at a cell density of 5000 cells/ well in a 96-well plate, were removed from the incubator and had their supernatant removed. The conditioned media was added to the target cells. RG6146 was diluted in DMSO in a 3-fold serial dilution to get a final concentration of 30uM to 0.0005uM. RG6146 or 0.15% DMSO control was added to the target cells with the conditioned media and placed back into the cell incubator for 72 hours. The experimental plates were removed from the incubator and 50uL of CellTiterGlo 2.0 (Promega) was added. The plates were shaken for 10 minutes at room temperature, and then read on a plate reader for luminescence. The raw data was normalized to the DMSO control for each experimental condition. The data of one PBMC Donor for HCT116 and three different PBMC Donors for MKN45 cells was analyzed by GraphPadPrism7.0 to generate graphs of percent viability relative to DMSO as shown in Figures 13-14. Each experiment contained two to three technical replicates.

Example 9: A TNF blocking antibody is able to decrease the sensitization of tumor cells by the BETi to the cytokines released by activated CEA-TCB T cells

The release of TNF by CEA-TCB is what synergizes with BETi leading to increased proliferation arrest which can be blocked with anti-TNF blocking antibody. The experimental conditions outlined in Example 8 were used with the following

modifications. The CEA-TCB and the appropriate vehicle control were diluted in the assay media to give a final concentration of 4nM CEA-TCB and was added to the PBMCs and the MKN45 cells. The assay plates were placed back into the cell incubator (37°C, 5% CO2) for 24 hours. The supernatant from the coculture containing CEA-TCB PMBCs and the vehicle PBMCs were collected and filtered through a 0.22uM filter. The anti-TNF (Cell Signaling Tech, #7321) blocking antibody was added to the supernatant vs isotype control (ITC) (Cell Signaling Tech, #3900) and allowed to incubate with rocking for 2 hours. The resultant supernatant was added back to fresh MKN45 cells with increasing concentrations of RG6146. RG6146 was diluted in DMSO in a 3-fold serial dilution to get a final concentration of 30uM to 0.0005uM. RG6146 was added to the target cells with the conditioned media and place back into the incubator for 72 hours. The experimental plates were removed from the incubator and 50uL of CellTiterGlo 2.0 (Promega) was added. The plates were shaken for 10 minutes at room temperature, and then read on a plate reader for luminescence. The raw data was normalized to the DMSO control for each experimental condition. The data of three technical replicates was analyzed by GraphPadPrism7.0 to generate graphs of percent viability relative DMSO as shown in Figure 15.

The experimental conditions outlined in Example 8 were used with the following modifications. The CEA-TCB and the appropriate vehicle control were diluted in the assay media to give a final concentration of 20nM CEA-TCB and was added to the PBMCs and the MKN45 cells. PBMCs were diluted in growth medium containing lOug/ml Anti-TNF (#MA5-23720) or isotype control (ITC) (#MA1-10407) and added to the MKN45 cells.

The assay plates were placed back into the cell incubator (37°C, 5% CO2) for 24 hours.

The supernatant from the CEA-TCB PMBCs and the vehicle PBMCs were collected and filtered through a 0.22uM filter. The supernatant was added back to fresh MKN45 cells with increasing concentrations of RG6146. RG6146 was diluted in DMSO in a 3-fold serial dilution to get a final concentration of 30uM to 0.0005uM or 0.15% DMSO.

RG6146 was added to the target cells with the conditioned media and placed back into the incubator for 72 hours. The experimental plates were removed from the incubator and 50uL of CellTiterGlo 2.0 (Promega) was added. The plates were shaken for 10 minutes at room temperature, and then read on a plate reader for luminescence. The raw data was normalized to the DMSO control for each experimental condition. The data was analyzed by GraphPadPrism7.0 to generate graphs of percent viability relative to DMSO. Data represents mean +/- SEM from three biologically independent experiments (each experiment consisted of two to three technical replicates). Using a two-way ANOVA with Sidak’s multiple comparison test cell death induced by BET -inhibitor in the presence of Anti-TNF was compared to BET-inhibitor treatment in presence of ITC. It was seen that Anti-TNF significantly blocks a decrease in viability induced by RG6146. For simplicity, significance is only shown for the highest treatment concentration of RG6146. Significance was defined as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The results are shown in Figure 15.

Example 10: Cytokines released by CEA-TCB activated T cells is able to induce cell death in the presence of BET inhibition

Western blotting was used to assess induction of apoptosis as measured by cleaved PARP in response to CEA-TCB conditioned media. The experimental conditions were similar to Example 2 except that after the cells were allowed to adhere overnight, the supernatant was replaced with CEA-TCB conditioned media generated in the same experimental fashion as Example 8. RG6146 was diluted in DMSO and added to conditioned media to give a final working concentration of 1 and 2.5 mM. Cell lysates were collect after 24h and resolved as described in Example 2. The addition of BET inhibition with RG6146 and CEA-TCB conditioned media was able to induce cell death that was not seen in the absence of CEA- TCB shown in Figure 16.

Example 11: CEA-TCB 2 induces cytokine release to synergize with BET inhibition

CEA-TCB 2 is a second TCB that recognizes the CEA epitope on cancer cells and induces activation of CTLs. Experimental conditions used in Example 8 were maintained with the following modifications. The CEA-TCB 2 and the appropriate vehicle control were diluted in the assay media to give a final concentration of 4nM CEA-TCB 2. 50uL of PBMCs and 50uL of CEA-TCB 2 or 50uL of vehicle was added to each well of the MKN45 cells that had been plated the previous day. The assay plates, wrapped in parafilm, were placed back into the incubator (37°C, 5% CO2) for 24 hours. The supernatant from the CEA-TCB 2 PMBCs and the vehicle PBMCs were collected and filtered through a 0.22uM filter. The target cells, MKN45, were removed from the incubator and had their supernatant removed. The conditioned media was added to the target cells. RG6146 was diluted in DMSO in a 3-fold serial dilution to get a final concentration of 30uM to 0.0005uM.

RG6146 was added to the target cells with the conditioned media and place back into the cell incubator for 72 hours. The experimental plates were removed from the incubator and 50uL of CellTiterGlo 2.0 (Promega) was added. The plates were shaken for 10 minutes at room temperature, and then read on a plate reader for luminescence. For the MKN45 cells two different donors of PMBCs are shown (each experiment consisted of two to three technical replicates). The raw data was normalized to the DMSO control for each experimental condition. The data was analyzed by GraphPadPrism7.0 to generate graphs of percent viability relative to DMSO as shown in Figure 17.

Example 12: Cytokines released by CEA-TCB 2 activated T cells is able to induce cell death in the presence of BET inhibition

Western blotting was used to assess induction of apoptosis as measured by cleaved PARP in response to CEA-TCB 2 conditioned media. The experimental conditions were similar to Example 2 except that after the cells were allowed to adhere overnight, the supernatant was replaced with CEA-TCB 2 conditioned media generated in the same experimental fashion as Example 11. MKN45 and HCT116 cells were harvested with Trypsin/EDTA and plated at a density of 250000 cells per well in 2mL of growth media in a 6-well plate. RG6146 was diluted in DMSO and added to conditioned media to give a final working concentration of 2.5 and 7 mM. Lysates were collect and resolved as described in Example 2. The addition of BET inhibition with RG6146 and CEA-TCB 2 conditioned media was able to induce cell death that was not seen in the absence of CEA-TCB 2 shown in Figure 18.

Example 13: BET inhibition is able to increase bystander killing due to sensitization to TNF

Bystander killing activity was measured using a standard chromium release assay as previously described [Kearney et al. Science Immunology. 2018] Briefly, MC38 cells were labeled with 100 pCi of ⁵¹Cr (Perkin Elmer) and mixed 50:50 with non-⁵¹Cr-labelled MC38-Ova cells. Activated OT1 T cells were then added to the targets at the indicated E:T ratios in the presence of RG6146 (2.5pM) or DMSO vehicle. The activation of OT1 T cells was done in the same fashion as Example 6. After 18 hours of incubation (37 °C, 10% C02), co-culture supernatants were harvested, and the level of ⁵¹Cr was quantified by a Gamma counter (Wallac Wizard). Percentage specific killing was determined using the formula: (Sample ⁵¹Cr release - Spontaneous background ⁵¹Cr release)/ (Total ⁵¹Cr release - Spontaneous Background ⁵¹Cr release) ^c 100%, and represented as a Michaelis-Menten kinetic trend. All assays were performed using technical triplicate. The increase in bystander killer with TCR activated CTLs is shown in Figure 19.

Example 14: The cytokines released by CEA-TCB activated T cells is able to significantly induce cell killing in cells that do not express CEA TCB activity is linked to expression of the level of antigen on the target cell. CEA-TCB is not able to induce CTL killing in cells that express low levels of CEA as has been shown for HCT116 cells that express no CEA. Target cells consisting of CEA positive (MKN45) and CEA negative (HCT116) were harvested using 0.25% Trypsin/EDTA. HCT116 cells were stained with a 1 : 1000 dilution of CellTrace Violet (Invitrogen) in PBS for 20 min. The HCT116 cells were then washed with FCS, media, and the concentration adjusted. HCT116 and MKN45 cells were mixed together and plated in 50uL in a flat-bottom 96 well plate at 40000 cells for each cell line leading to 80000 cells per well. The cells were allowed to adhere overnight. PBMCs were generated in the same experimental conditions as reported in Example 8. The antibody was diluted in 50uL of growth media to give a final concentration of 40nM. The target cells were removed from the incubator and

PBMCs at an effector target ratio of 1 : 10 in 50uL were added to the wells containing the target cells as well as the CEA-TCB in 50uL. RG6146 was diluted in DMSO in a 3-fold serial dilution to get a final concentration of 15uM to 0.002uM with the Tecan. The plates were placed into the incubator. The plates were removed from the incubator after 72 hours and the cells were harvested with 0.25% Trypsin/EDTA and washed with PBS. The cells were centrifuged and the PBS was removed. ZombieNIR that had been diluted 1 :500 in PBS was added to the cell pellet. Cells were resuspended and incubated for 30 minutes at 4 degree. The cells were then centrifuged and washed with PBS 0.5% BSA 2mM EDTA. Finally, the cells were resuspended in 50uL PBS 0.5% BSA 2mM EDTA and analyzed using the Cytoflex and FlowJo Software (Version 10, Tree Star). Data of two to three technical replicates is shown. The presence of CEA-TCB activated Tcells and increasing concentrations of BET inhibitor, RG6146 was able to increase cell killing in CEA negative cells as seen in Figures 20-21.

TCB activity is linked to expression of the level of antigen on the target cell. CEA-TCB is not able to induce CTL killing in cells that express low levels of CEA as has been shown for HCT116 cells that express no CEA. Target cells consisting of CEA positive (MKN45- RFP) and CEA negative (HCT116-GFP) were harvested using 0.25% Trypsin/EDTA. HCT116-GFP and MKN45-RFP, stabelly expressing GFP and RFP, respectively, were mixed together and plated in 50uL in a 96 well plate (Corning #3904) at 20000 cells for each cell line leading to 40000 cells per well. The cells were allowed to adhere overnight. PBMCs were generated in the same experimental conditions as reported in Example 8. The antibody was diluted in 50uL of growth media to give a final concentration of 20nM. The target cells were removed from the incubator and PBMCs at an effector to target cell ratio of 1 : 10 in 50uL were added to the wells containing the target cells as well as the CEA- TCB in 50uL. RG6146 was diluted in DMSO in a 3-fold serial dilution. The plates were placed into the incubator and pictures were taken every 4h with the IncucyteS3 for the course of one week. The pictures were analyzed with the Incucyte S3 software and

HCT116-GFP (Count per Image) data was normalized to TO. The presence of CEA-TCB activated Tcells and increasing concentrations of RG6146 was able to significantly decrease cell growth in CEA negative HCT116-GFP cells. Data represents mean +/- SEM from three biologically independent experiments (each consisting of three technical replicates). Significance was calculated using a two-way ANOVA with Sidak’s multiple comparison test comparing the cell growth of HCT-116-GFP cells when treated with RG6146 in combination with CEA-TCB to RG6146 single agent treatment. It was seen that RG6146 decreases cell growth of HCT-116-GFP cells significantly when combined with the CEA-TCB. For simplicity, significance is only shown for the highest treatment concentration of RG6146. Significance was defined as *p<0.05, **p<0.01, ***p<0.001, ****r<0.0001. The results are shown in Figure 22.

Example 15: The cytokines released by CEA-TCB 2 activated T cells is able to induce cell killing through a bystander effect

TCB activation is linked to the level of antigen being expressed on the target cell. CEA- TCB 2 is not able to induce CTL killing in cells that express low levels of CEA as has been shown for HCT116 cells that express no CEA. Target cells consisting of CEA positive (MKN45) and CEA negative (HCT116) were harvested using 0.25%

Trypsin/EDTA. HCT116 cells were stained with a 1 : 1000 dilution of CellTrace Violet (Invitrogen) in PBS for 20 min. The HCT116 cells were then washed with FCS, media, and the concentration adjusted. HCT116 and MKN45 cells were mixed together and plated in 50uL in a flat-bottom 96 well plate at 40000 cells for each cell line leading to 80000 cells per well. The cells were allowed to adhere overnight. PBMCs were generated in the same experimental conditions as reported in Example 8. The antibody was diluted in 50uL of growth media to give a final concentration of lOnM CEA-TCB 2. The target cells were removed from the incubator and PBMCs at an effector target ratio of 1 : 10 in 50uL were added to the wells containing the target cells as well as the CEA-TCB 2 in 50uL. RG6146 was diluted in DMSO in a 3 -fold serial dilution to get a final concentration of 15uM to 0.002uM with the Tecan. The plates were placed into the incubator. The plates were removed from the incubator after 72 hours and the cells were harvested with 0.25% Trypsin/EDTA and washed with PBS. The cells were centrifuged and the PBS was aspirated off. ZombieNIR that had been diluted 1 :500 in PBS was added to the cell pellet and the cells were resuspended in the ZombieNIR and incubated for 30 minutes at4degree. The cells were then centrifugedand washed with PBS 0.5% BSA 2mM EDTA. Finally, the cells were resuspended in 50uL PBS 0.5% BSA 2mM EDTA and analyzed using the Cytoflex and FlowJo Software (Version 10, Tree Star). Data of two to three technical replicates is shown for each Donor. The presence of CEA-TCB 2 activated CTLs and increasing concentrations of BET inhibitor, RG6146 was able to increase cell killing in CEA negative cells as seen in Figure 23.

Example 16: The combination of RG6146 and TNF induce the extrinsic apoptosis signaling pathway

RG6146 and TNF have been shown to induce cell death and increase cPARP levels as seen in Example 2. Cell death can be induced through the extrinsic apoptosis pathway mediated through Caspase 8 cleavage or the intrinsic apoptosis pathway initiated through Caspase 9 cleavage. Both pathways eventually lead to the activation of Caspase 3.7 and PARP cleavage. HCT116 and MKN45 cells were seeded at a cell density of 0.04Mio cells/well in 96-well plates (Corning #3917) and placed in the incubator overnight to allow cells enough time to adhere. RG6146 was 3 -fold serially diluted in DMSO to create a concentration gradient and was added to wells containing cells to give the final working concentration of BET -inhibitor in 0.15% DMSO per well. Every sample was tested in duplicate. Finally, TNF that had been reconstituted in PBS 0.5%BSA was added to each well to give a final concentration of 15ng/mL as well as wells receiving only PBS 0.5%BSA. Finally, ImM of Caspase 8 inhibitor Z-IETD-FMK (R&D Systems #FMK007) or DMSO control was added to the corresponding wells. After 8h plates were removed from the incubator and equilibrated to room temperature for 20min. Caspase 8 and Caspase 3,7 Glo reagents were added according to the manufacturers protocol (Promega #G8091 and #G8201) and incubated for lh at room temperature. Luminescence indicating Caspase activity was measured using the PheraStar. Data was normalized to the control. Data represents mean +/- SEM from three biologically independent experiments (each consisting of two technical replicates). Significance was calculated using a two-way ANOVA with Tukey’s multiple comparison test comparing Caspase activity in cells treated with RG6146 and TNF to RG6146 single agent treatment. It was seen that co-treatment induces Caspase3,7 and 8 activity in HCT-116 and MKN45 cells significantly. For simplicity, significance is only shown for the highest treatment concentration of RG6146. Significance was defined as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The Caspase 8 inhibitor blocked Caspase 8 and downstream Caspase 3,7 activation completely (significance not shown) as seen in Figures 24-25.

Example 17: Caspase 8 knock down partially rescues cell growth arrest induced by RG6146 and TNF treatment Since RG6146 and TNF induce apoptosis through the extrinsic apoptosis pathway mediated through Caspase 8, we assessed if Caspase 8 knock down rescues the effect induced by TNF and RG6146 cotreatment. HCT116 cells were reverse transfected according to the siTools Biotech protocol (Lipofectamine RNAiMAX Transfection Reagent #13778150; Opti-MEM #51985026 both ThermoFisher) using 3nM final concentration of siPOOL control and siPOOL targeting Caspase-8 (siTOOLs Biotech). 2Mio HCT116 cells were seeded in PetriDishes containing either control (scr) or siPOOLs targeting Caspase-8 (siCasp8) and placed in the incubator overnight. The cells were seeded at 1500 cells/ well in a 96-well plate and treated with either the combination of 2.5mM RG6146 and 15ng/ml TNF or the control (0.001% DMSO and PBS 0.5%BSA). Growth of HCT116 cells was assessed with the Incucyte S3 cell imaging system by taking pictures every 4-6h for a course of 7days. Data was analyzed with the Incucyte S3 data analysis system and normalized to TO. Data was visualized using GraphPad Prism 7. Knock Down efficacy was visualized by Western Blot similar as described in Example 2 with the following modifications. HCT116 cells were treated with 15ng/ml TNF for 6h before harvest and cell lysis. Membranes were blotted with anti -Caspase 8 (Cell Signaling #4790) or anti-vinculin antibodies. While combination treatment of TNF and RG6146 completely blocked HCT116 cell growth in the scr treated cells, Caspase 8 knockdown rescued cell growth under these treatment conditions partially as seen in Figure 26. For the first experiment, representative still images are shown in Figure 27. The data of three individual experiments is shown each consisting of three to nine technical replicates.

Example 18: Ectopic expression of Caspase-8 inhibitor cFLIP, but not BCL-2, abrogates the cytotoxic effects of TNF and the combinatorial effects of RG6146 and TNF.

Murine colon adenocarcinoma cell line MC38 cells were infected with murine stem cell virus (MSCV) constructs expressing GFP (MSCV-GFP), murine Bcl-2 and GFP (MSCV- Bcl2-GFP), and murine cFLIP and GFP (MSCV-cFLIP-GFP). GFP-expressing cells were isolated by flow cytometry and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin (Gibco) and incubated at 37°C in 10% C02. MC38 cells were seeded (1.5e5 cells/well) into 48-well plates for >8 hours prior to addition of small molecules and/or cytokines. Co-culture assays were set up at varying concentrations of recombinant TNF in the presence or absence of RG6146 (2.5mM) and incubated for 18 hours. Cells were harvested by centrifugation, washed once in ice-cold flow cytometry buffer (2% FCS in PBS), prior to being resuspended in flow cytometry buffer containing propidium iodide (PI) and assessed for PI positivity. Data were collected on a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo Software (Version 10.2, Tree Star). The increased cell death seen with RG6146 and TNF is shown in the context of Bcl-2 overexpression, whereas cFLIP overexpression is sufficient to abrogate the cytotoxic effects of TNF and TNF+RG6146 in Figure 28.

Example 19: Combination treatment of CEA-TCB and JQ1 induced tumor regression in syngeneic recipient mice.

The export transporter p-glycoprotein 1 (p-gpl also known as ABCB1, MDR1) exports small molecules from cells and therefore reduces sensitivity or induces resistance to small molecule treatment including RG6146 when expressed in cells. MC38 cells used for in vivo studies express p-gp 1 and are therefore less sensitive to RG6146 treatment as compared to JQ1. To verify that p-gp 1 is responsible to reduced sensitivity, MC38 cells were seeded at 5000 cells/well in a 96-well plate and placed in the incubator overnight. The next day JQ1 or RG6146 were 3-fold serially diluted in DMSO to create a concentration gradient and were added to wells containing MC38 cells to give the final working concentration of BET-inhibitors in 0.15% DMSO per well. Every sample was tested in triplicate. TNF (BioLegend # that had been reconstituted in PBS 0.5%BSA was added to each well to give a final concentration of 15ng/mL. Finally, 0.5mM of Zosuquidar (TargetMol #T6018), a p-gp 1 inhibitor, or DMSO control was added to the corresponding wells. The plates were returned to the incubator (37°C, 5% C02) for 72 hours. The experimental plates were removed from the incubator and 50uL of CellTiterGlo 2.0 (Promega) was added. The plates were shaken for 10 minutes at room temperature, and then read on a plate reader for luminescence. The raw data was normalized to the DMSO control for each experimental condition. The data was analyzed by GraphPadPrism7.0 to generate graphs of percent viability relative to DMSO as shown in Figure 29. By blocking p-gp 1 with Zosuquidar MC38 cells were sensitized to RG6146 and TNF combination treatment significantly for some concentrations tested, while no change was observed in JQ1 and TNF treated cells. Data represents mean +/- SEM from three biologically independent experiments (each consisting of three technical replicates). Significance was calculated using a two-way ANOVA with Sidak’s multiple comparison test comparing a change in cell viability induced by RG6146 and TNF treatment to RG6146, TNF and Zosuquidar treatment. Significance was defined as *p<0.05, **p<0.01, ***p<0.001, ****r<0.0001. According to these results, JQ1 was used in the in vivo experiments described below.

The in vivo antitumor efficacy of JQ1 in combination with CEA-TCB and anti TNF blocking antibody was evaluated against MC38-huCEA expressing sc tumors.

The MC38 HOMSA CEACAM5 transfectant cell line was generated internally. Tumor cell line was routinely cultured in DMEM high-glucose medium, NEAA, 4 mM glutamine, 2 mM sodium pyruvate, 10% fetal bovine serum, 500 pg/ml G-418 at 37 °C in a water- saturated atmosphere at 5 % C02. Culture passage was performed with trypsin / EDTA lx splitting twice/week and passage 3 used for transplantation. MC38-CEA cell were injected sc at a concentration of 5xl0⁵ together with matrigel.

Female C57/B16 huCEA tg mice, age 5-8 weeks at arrival, maintained under specific- pathogen-free condition with daily cycles of 12h light /12h darkness according to committed guidelines. Experimental study protocol was reviewed and approved by local government. After arrival animals were maintained in animal facility for one week to get accustomed to new environment and for observation. Continuous health monitoring was carried out on regular basis. Diet food and autoclaved water were provided ad libitum. Animals were controlled daily for clinical symptoms and detection of adverse effects. For monitoring throughout the experiment body weight of animals was documented.

Animal treatment started after randomization when median tumor size was about 100 or 130mm³. CEA-TCB antibody was administered as single agent and in combination at 2.5mg/kg iv twice weekly (4x). BETi inhibitor JQ1 ip treatment at 50mg/kg was done as single agent and in combination once daily (14x). Finally, anti TNF alfa Mab was injected iv at 2mg/kg twice weekly as single agent and in combination (4x).

The results are shown as % change in tumor volume at the last day of study compared to the first day of study in Figure 30. Single agent JQ1 or CEA-TCB treatment decreased tumor growth by 50% and 60%, respectively. Importantly, the combination of JQ1 and CEA-TCB induced more profound tumor regression than single agent treatments. In addition, we observed that the neutralization of TNF-alpha (by antibody depletion) abrogated the therapeutic effect induced by the combination treatment.

Significance was calculated using a one-way ANOVA with Tukey’s multiple comparison test. Significance was defined as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

As a different visualization, spider plots are shown in Figure 31 to monitor tumor volume over time of treatment.

Example 20: Small Molecule epigenetic screen reveals that BET-inhibitors are most potent in enhancing T cell mediated killing of human and mouse cancer cells

CMV T cells were expanded from a CMV specific T cell Donor (generated as described in [Claus et al. Science Translational Medicine 2019]) every 4 weeks. PBMCs isolated as described in Example 8 were kept in the incubator for lh to separate from adherent monocytes. Cytokine production was activated through the addition of Lectin from Phaseolus vulgaris (Sigma #L2796) for lh to produce Feeder cells. NLVPMVATV (NLV) peptide (Thinkpeptides) was loaded on HLA-A2 of LCL cells for lh. LCL-NLV and Feeder cells were irradiated at 5000rad and 2500rad, respectively. O.OlMio/well CMV- specific T cells were plated in RPMI + GlutaMax + 10% heat inactivated FCS + 400U/ml IL2 together with LCL-NLV and Feeder cells (ratio 1 :5: 125) in a 96-well plate. Medium was changed every three days and after one week cells were collected and plated in a 24- well plate. After seven days CMV-specific expanded T cells were plated at 1.5Mio cells/ml and a total of 7.5Mio cells/well in a 6 well plate.

HCT116 HLA-A2 positive cells were loaded with either lOnM NLV (Ag+) or GLCTLVAML (EBV (Thinkpeptides)) (Ag-) peptide for lh on a rotating wheel in the incubator. Cells were plated at a cell density of O.OlMio cells/well in a 96 well plate (#3903 or #3917), Corning) in RPMI+GlutaMax+10% heat inactivated FCS and allow time to adhere for 1.5h in the incubator. CMV-specific T cells, recognizing the NLV, but not the EBV peptide, were added at a cell density of 0.01 Mio cells/ well to the HCT116 cells. Coculture was incubated for 30min in the incubator and a library of epigenetic small molecule inhibitors, SMAC mimetics as a positive control or DMSO were added to the cells at a concentration of 2.5uM or 5uM.

Coculture was incubated for 48h in the incubator. Plates were washed once with PBS to remove CMV-specific T cells and IOOmI PBS and 50pl CellTiterGlo2.0 (Promega) were added to the cells and incubated for lOmin shaking at room temperature. Luminescence was measured using the PheraStar, data normalized to DMSO for each experimental condition and visualized with GraphPad Prism 7. The screen shows that the BET-inhibitors including JQ1, RG6146, ΪBET151, Mivebresib, ABBV744 and OTX015 significantly decrease viability to a similar extent as the positive control Birinapant and LCL161. Most other molecules tested did not have such a strong effect. While HDAC-inhibitors had a similar effect as BET-inhibitors, Entinostat, Panobinostat and Vorinostat decreased viability also in the negative control HCT116-EBV coculture indicating toxicity at the tested concentration (Figure 32).

A similar screen was set up in mouse MC38 cancer cells in coculture with OT-1 T cells. Spleens from 6-10 week old C57BL/6.0T-1 mice were manually dissociated through a 70·M sieve and OT-1 T cells were activated and expanded with 20ng/ml of SIINFEKL peptide (Sigma- Aldrich) and 1000 IU/ml recombinant human IL-2 (Biolegend) in supplemented RPMI media (10% FCS, glutamax [2mM], penicillin/streptomycin, non- essential amino acids, sodium pyruvate [ImM], HEPES [lOmM] and 2-mercaptoethanol [50·M]). OT-1 cultures were subsequently incubated for three days at 37_*C with 5% C02, before being passaged into fresh media (IL-2 only, no SIINFEKL), and cultured for an additional day prior to use in killing assays.

MC38-OVA cells expressing GFP were maintained in DMEM medium supplemented with 10% FCS, glutamax (2mM) and penicillin/ streptomycin and incubated at 37_*C with 10% C02. Adherent MC38-OVA maintenance cultures were harvested using trypsin, washed, and 1.5 x 105 cells were placed in each well of a 48-well plate and allowed to adhere for 3- 4 hours. Expanded and activated OT-1 cells were harvested, washed in supplemented DMEM, and 5 x 104 OT-1 cells were added to MC38 -containing wells. Control wells, containing no OT-1 cells (tumour cells only), were also prepared. Small molecules were added to co-cultures and control wells at a final concentration of 1·M. Cultures were subsequently incubated at 37_*C with 10% C02 for 17-18 hours prior to harvest. At harvest, supernatants containing OT-1 T cells and dead cells were collected from each well and surviving, adherent MC38-OVA.GFP+ cells were harvested using trypsin. Total well contents were washed in flow cytometry buffer (PBS + 4% FCS + 0.5M EDTA) and OT-1 cells stained with anti-mouse CD5 APC-conjugated antibody (clone 53-7.3; eBioscience). Samples were washed twice with flow cytometry buffer and propidium iodide (PI; 2_*g/ml) was added immediately prior to analysis. Analysis was performed on an LSR P flow cytometer (BD Biosciences) and data were analysed with FlowJo (Tree Star). CD5+ OT-1 cells were gated out of analysis and dead MC38-OVA cells were recognized as GFP-PI+. The results from Figure 32 were verified in the mouse coculture system. Various BET -inhibitors enhanced OT-1 T cell mediated killing significantly as compared to DMSO control (Figure 33). Data represents mean +/- SEM from three biologically independent experiments (for Figure 32 each individual experiment consisted of three technical replicates). Significance was calculated using a two-way ANOVA with Sidak’s multiple comparison test. For Figure 32 a change in cell viability induced by a small molecule in a coculture with CMV- specific T cells and HCTI 16-NLV cells was compared to treatment of a small molecule in a coculture with HCTI 16-EBV cells. For Figure 33 cell death induced by a small molecule in a coculture with MC38-Ova cells and OT-f cells was compared to the effect of a small molecule in a MC38-Ova monoculture. Significance was defined as *p<0.05, **p<0.01,

***p<0.001, ****p<0.0001.

Claims

1. A BET inhibitor for use in a method of sensitizing a cancer cell to TNF induced cell death.

2. A method of sensitizing a cancer cell to TNF induced cell death comprising the administration of a BET inhibitor to a patient in need thereof.

3. A BET inhibitor for use in a method of enhancing TNF mediated killing of cancer cells in a cancer patient undergoing a therapy with an immune activating agent, wherein the immune activating agent is capable of causing the release of TNF by T cells. 4. A method of enhancing TNF mediated killing of cancer cells in a cancer patient undergoing a therapy with an immune activating agent comprising the administration of a BET inhibitor to a patient in need thereof, wherein the immune activating agent is capable of causing the release of TNF by T cells.

5. A BET inhibitor for use or a method according to claim 1 or 2 in a cancer patient undergoing a therapy with an immune activating agent, wherein the immune activating agent is capable of causing the release of TNF by T cells.

6. A BET inhibitor for use or a method according to any one of claims 1 to 5, wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a- tetraaza-cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]-acetamide (RG6146), INCB-054329, INCB-057643, GSK525762, GS-5829, CPI-0610,

Birabresib, PLX51107, ABBV-075, BI 894999, FT-1101, ZEN-3694, GSK-2820151 or BMS-986158.

7. A BET inhibitor for use or a method according to any one of claims 1 to 6, wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a- tetraaza-cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]-acetamide

(RG6146).

8. A BET inhibitor for use or a method according to any one of claims 3 to 7, wherein the immune activating agent is an antibody.

9. A BET inhibitor for use or a method according to any one of claims 3 to 8, wherein the immune activating agent is a T cell bispecific (TCB) antibody.

10. A BET inhibitor for use or a method according to any one of claims 2 to 7, wherein the immune activating agent is a CEA CD3 bispecific antibody. 11. A BET inhibitor for use or a method according to claim 10, wherein the CEA CD3 bispecific antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the

LCDR3 of SEQ ID NO: 6.

12. A BET inhibitor for use or a method according to claim 10 or 11, wherein the CEA CD3 bispecific antibody comprises

(i) a first antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID

NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the LCDR3 of SEQ ID NO: 6; and

(ii) a second antigen binding moiety that specifically binds to CEA and comprises (i) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ

ID NO: 9, the HCDR2 of SEQ ID NO: 10, and the HCDR3 of SEQ ID NO: 11; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 12, the LCDR2 of SEQ ID NO: 13 and the LCDR3 of SEQ ID NO: 14; or (ii) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 17, the HCDR2 of SEQ ID NO: 18, and the HCDR3 of SEQ ID NO: 19; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 20, the LCDR2 of SEQ ID NO: 21 and the LCDR3 of SEQ ID NO: 22.

13. A BET inhibitor for use or a method according to any one of claims 3 to 12, wherein the immune activating agent is CEA-TCB (cibisatamab) or CEA-TCB 2. 14. A BET inhibitor for use or a method according to any one of claims 3 to 13, wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a- tetraaza-cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]-acetamide (RG6146) and the immune activating agent is CEA-TCB (cibisatamab) or CEA-TCB 2 15. A BET inhibitor and a TCB antibody for use in the treatment of cancer.

16. A method of treating cancer, comprising administering a BET inhibitor and a TCB antibody to a patient in need thereof. 17. A pharmaceutical composition comprising a BET inhibitor, a TCB antibody and one or more pharmaceutically acceptable excipients.

18. A kit comprising a BET inhibitor and a TCB antibody for the simultaneous, separate or sequential administration of said BET inhibitor and TCB antibody to a patient in need thereof.

19. A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical

composition or a kit according to any one of claims 15 to 18, wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a-tetraaza- cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]-acetamide

(RG6146), INCB-054329, INCB-057643, GSK525762, GS-5829, CPI-0610, Birabresib, PLX51107, ABBV-075, BI 894999, FT-1101, ZEN-3694, GSK-2820151 or BMS-986158.

20. A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical

composition or a kit according to any one of claims 15 to 19, wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a-tetraaza- cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]-acetamide

(RG6146).

21. A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical

composition or a kit according to any one of claims 15 to 20, wherein the TCB antibody is a CEA CD3 antibody.

22. A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical

composition or a kit according to claim 21, wherein the CEA CD3 bispecific antibody comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR)

1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the LCDR3 of SEQ ID NO: 6.

23. A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical

composition or a kit according to claim 21 or 22, wherein the CEA CD3 bispecific antibody comprises

(i) a first antigen binding moiety that specifically binds to CD3 and comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5 and the LCDR3 of SEQ ID NO: 6; and (ii) a second antigen binding moiety that specifically binds to CEA and comprises (i) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 9, the HCDR2 of SEQ ID NO: 10, and the HCDR3 of SEQ ID NO: 11; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 12, the LCDR2 of SEQ ID NO: 13 and the LCDR3 of SEQ ID NO: 14; or (ii) a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 17, the HCDR2 of SEQ ID NO: 18, and the HCDR3 of SEQ ID NO: 19; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 20, the LCDR2 of SEQ ID NO: 21 and the LCDR3 of SEQ ID NO: 22. 24. A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical

composition or a kit according to any one of claims 15 to 23, wherein the TCB antibody is CEA-TCB (cibisatamab) or CEA-TCB 2.

25. A BET inhibitor and a TCB antibody for use, a method, a pharmaceutical

composition or a kit according to any one of claims 15 to 24, wherein the BET inhibitor is 2-[(S)-4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-l-thia-5,7,8,9a-tetraaza- cyclopenta[e]azulen-6-yl]-N-[3-(4-methyl-piperazin-l-yl)-propyl]-acetamide (RG6146) and the TCB antibody is CEA-TCB (cibisatamab) or CEA-TCB 2.

26. The invention as hereinbefore described.