WO2020169698A1 - Sensibilisation de cellules cancéreuses au tnf par inhibition de bet - Google Patents
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Definitions
- the present invention relates to the use of a BET inhibitor for sensitizing a cancer cell to TNF induced cell death.
- CIT cancer immunotherapy
- 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.
- BETi BET inhibitors
- 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.
- 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.
- 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.
- Figure 1 shows that MKN45 and HCT116 cells’ viability is decreased with TNF and increasing amounts of BETi.
- FIGS 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.
- FIG. 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.
- FIG. 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.
- FIG. 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.
- an“immune activating agent” is an agent capable of activating a naive T cell into a cytotoxic T cell against cancer cells.
- 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®
- 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 TNF a
- TNFa 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 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.
- bispecific means that the antibody is able to specifically bind to at least two distinct antigenic determinants.
- a bispecific antibody comprises two antigen binding sites, each of which is specific for a different antigenic determinant.
- the bispecific antibody is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.
- 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.
- 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).
- ECM extracellular matrix
- an antigen binding moiety refers to a polypeptide molecule that specifically binds to an antigenic determinant.
- 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.
- 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.
- 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.
- ELISA enzyme-linked immunosorbent assay
- SPR surface plasmon resonance
- 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 8 M or less, e.g. from 10 8 M to 10 13 M, e.g., from 10 9 M to 10 13 M).
- K D dissociation constant
- 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).
- 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.
- 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.
- CEA Carcinoembryonic antigen
- CEACAM5 Carcinoembryonic antigen-related cell adhesion molecule 5
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- scFv single-chain antibody molecules
- Diabodies are antibody fragments with two antigen binding sites that may be bivalent or bispecific.
- 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.
- 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.
- 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.
- 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).
- 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.
- 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)
- CL light chain constant domain
- Kabat EU index numbering system see pages 661-723
- CHI heavy chain constant domains
- hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence
- antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
- HVRs herein include:
- HVR residues and other residues in the variable domain are numbered herein according to Rabat et al., supra.
- FR 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.
- 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.
- 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).
- the peptide chain comprising the heavy chain constant domain 1 CHI is referred to herein as the“heavy chain” of the (crossover) Fab molecule.
- the peptide chain comprising the heavy chain variable domain VH is referred to herein as the“heavy chain” of the (crossover) Fab molecule.
- 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).
- immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody.
- 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.
- 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.
- VL variable domain
- CL constant light
- 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 (IgA 2 ).
- 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.
- K kappa
- l lambda
- An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.
- 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.
- 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.
- 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.
- 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.
- 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
- 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.
- 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
- 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.
- 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
- the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution.
- 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.
- effector functions refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
- 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.
- % 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.
- 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.
- 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.
- increased binding refers to an increase in binding affinity for the respective interaction.
- 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.
- the immune activating agent is a bispecific CD3 antibody.
- the immune activating agent is a bispecific CD3 antibody capable of specifically binding to CEA (CEA CD3 bispecific antibody).
- CEA CD3 bispecific antibody particularly 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.
- 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:
- 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.
- the CEA CD3 bispecific antibody comprises
- 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
- 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.
- 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.
- 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.
- 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.
- 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.
- the CEA CD3 bispecific antibody comprises
- 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.
- CEA CD3 bispecific antibody comprises
- 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;
- 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.
- CEA CD3 bispecific antibody comprises
- 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
- 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.
- CEA CD3 bispecific antibody comprises
- 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;
- 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.
- the first and/or the second antigen binding moiety is a Fab molecule.
- 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.
- the second antigen binding moiety preferably is a conventional Fab molecule.
- 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
- 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).
- 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).
- 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).
- 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
- E glutamic acid
- D aspartic acid
- 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).
- 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)
- the amino acid at position 124 is substituted by lysine (R)
- the constant domain CL of the second antigen binding moiety is of kappa isotype.
- the first and the second antigen binding moiety are fused to each other, optionally via a peptide linker.
- 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.
- the CEA CD3 bispecific antibody provides monovalent binding to CD3.
- 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.
- the CEA CD3 bispecific antibody comprises a third antigen binding moiety that specifically binds to CEA.
- 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).
- the CEA CD3 bispecific antibody further comprises an Fc domain composed of a first and a second subunit.
- the Fc domain is an IgG Fc domain.
- the Fc domain is an IgGi Fc domain.
- the Fc domain is an IgG 4 Fc domain.
- the Fc domain is an IgG 4 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 IgG 4 antibodies (see
- the Fc domain is a human Fc domain.
- 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.
- the first, the second and, where present, the third antigen binding moiety are each a Fab molecule
- 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.
- said modification is in the CH3 domain of the Fc domain.
- 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).
- 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).
- 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.
- 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).
- 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.
- 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).
- 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).
- the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W
- the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).
- the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.
- 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.
- 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.
- the same one or more amino acid substitution is present in each of the two subunits of the Fc domain.
- the one or more amino acid substitution reduces the binding affinity of the Fc domain to an Fc receptor.
- 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.
- 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.
- the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index).
- 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).
- the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S.
- the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index).
- the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”,“PGLALA” or“LALAPG”).
- each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e.
- 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)
- the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index).
- the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain.
- the CEA CD3 bispecific antibody comprises
- 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;
- 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
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- the CEA CD3 bispecific antibody is CEA- TCB (cibisatamab).
- CEA CD3 antibody is a bispecific antibody comprising
- 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;
- 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;
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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.
- 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).
- BET inhibitor refers to agents that prevent activity of BET proteins with an IC50 of about 0.001 mM to about 2 pM.
- 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 comprising the administration of a BET inhibitor to a patient in need thereof;
- 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 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;
- 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;
- CEA CD3 bispecific antibody comprises
- 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
- 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 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;
- 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 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
- 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 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;
- 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;
- 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;
- 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.
- 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;
- 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);
- T cell bispecific (TCB) antibody T cell bispecific antibody
- 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;
- CEA CD3 bispecific antibody comprises
- 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
- 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 in the manufacture of medicament according to the invention wherein the immune activating agent is CEA-TCB (cibisatamab) or CEA-TCB 2;
- 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;
- BET inhibitor 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;
- BET inhibitor 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);
- TCB antibody is a CEA CD3 antibody
- 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;
- 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
- 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;
- TCB antibody is CEA-TCB (cibisatamab) or CEA- TCB 2;
- 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 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 (RG61
- 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.
- 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.
- 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.
- 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.
- 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.
- Example 1 Increased response to TNF in gastric and colorectal cancer cells when treated with a BET inhibitor
- RG6146, JQ1, OTX015 and ⁇ BET151 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.
- 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
- 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.
- FCS fetal calf serum
- Gibco penicillin/streptomycin
- RG6146, JQ1, IBET151, IBET762, Y803, RVX-208, and dBETl were dissolved in DMSO to generate a lOmM stock solution.
- 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.
- BET inhibitors RG6146, JQ1, IBET151, IBET762, dBET ImM; RVX-208 IOmM
- Example 2 TNF and a BET inhibitor induce cell death as measured by cleaved PARP
- 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, ⁇ BET151 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.
- the lysis buffer contained Cell Lysis Buffer (Cell Signaling Technology #9803), Phosphatase Inhibitor (Cocktail Set P, Calbiochem
- 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.
- FCS fetal calf serum
- Gibco penicillin/streptomycin
- 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 ‘ZIP score’ is the overall average % inhibition beyond the expectation by the ZIP model over the whole dose-response matrix.
- Example 4 The sensitization of cancer cells to TNF by BET inhibition is found across multiple cancer subtypes
- 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.
- TCA trichloracetic acid
- SRB Sulforhodamine B
- 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
- 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.
- 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
- 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).
- 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)].
- 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.
- BET inhibitors RG6146, JQ1, IBET151, IBET762, dBET ImM; RVX-208 IOmM
- 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
- 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).
- 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)].
- 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.
- PI propidium iodide
- 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.
- 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.
- PBMCs Peripheral blood mononuclear cells
- enriched lymphocyte preparations 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 10 6 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.
- 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.
- 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
- 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.
- 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.
- Example 8 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).
- Example 10 Cytokines released by CEA-TCB activated T cells is able to induce cell death in the presence of BET inhibition
- Example 8 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.
- 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
- Example 11 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
- Percentage specific killing was determined using the formula: (Sample 51 Cr release - Spontaneous background 51 Cr release)/ (Total 51 Cr release - Spontaneous Background 51 Cr 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.
- 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.
- 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.
- 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%
- 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.
- 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.
- 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).
- 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.
- 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.
- FCS fetal calf serum
- Gabco penicillin/streptomycin
- 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
- 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.
- 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).
- 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 5xl0 5 together with matrigel.
- mice 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.
- 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).
- anti TNF alfa Mab was injected iv at 2mg/kg twice weekly as single agent and in combination (4x).
- 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.
- 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.
- 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.
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Abstract
L'invention concerne un procédé de sensibilisation d'une cellule cancéreuse à la mort cellulaire induite par TNF comprenant l'administration d'un inhibiteur BET à un patient en ayant besoin, en particulier chez un patient soumis à une thérapie avec un agent d'activation immunitaire.
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WO2022101120A1 (fr) * | 2020-11-10 | 2022-05-19 | F. Hoffmann-La Roche Ag | Prévention ou atténuation d'effets secondaires liés à un agent de mise en contact de lymphocytes t |
WO2022132049A1 (fr) * | 2020-12-17 | 2022-06-23 | National University Of Singapore | Traitement de cancers à l'aide d'inhibiteurs de bet |
WO2022223651A1 (fr) * | 2021-04-23 | 2022-10-27 | F. Hoffmann-La Roche Ag | Prévention ou atténuation d'effets secondaires liés à un agent engageant les cellules nk |
Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0404097A2 (fr) | 1989-06-22 | 1990-12-27 | BEHRINGWERKE Aktiengesellschaft | Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application |
WO1993001161A1 (fr) | 1991-07-11 | 1993-01-21 | Pfizer Limited | Procede de preparation d'intermediaires de sertraline |
WO1993016185A2 (fr) | 1992-02-06 | 1993-08-19 | Creative Biomolecules, Inc. | Proteine de liaison biosynthetique pour marqueur de cancer |
US5571894A (en) | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
US5587458A (en) | 1991-10-07 | 1996-12-24 | Aronex Pharmaceuticals, Inc. | Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof |
US5731168A (en) | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
US5869046A (en) | 1995-04-14 | 1999-02-09 | Genentech, Inc. | Altered polypeptides with increased half-life |
US6248516B1 (en) | 1988-11-11 | 2001-06-19 | Medical Research Council | Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors |
WO2011143669A2 (fr) | 2010-05-14 | 2011-11-17 | Dana-Farber Cancer Institute, Inc | Compositions et méthodes de traitement des néoplasies, des maladies inflammatoires et d'autres affections |
US20140242079A1 (en) * | 2013-02-26 | 2014-08-28 | Roche Glycart Ag | Bispecific t cell activating antigen binding molecules |
WO2017055389A1 (fr) | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Molécules bispécifiques de liaison à l'antigène activant les lymphocytes t anti-ceaxcd3 |
US20170182025A1 (en) * | 2015-12-24 | 2017-06-29 | Celgene Quanticel Research, Inc. | Bromodomain and extra-terminal protein inhibitor combination therapy |
WO2017223268A1 (fr) * | 2016-06-22 | 2017-12-28 | Yale University | Compositions et procédés de resensibilisation de cellules à des inhibiteurs des protéines de bromodomaine et de domaine extraterminal (beti) |
WO2019020610A1 (fr) * | 2017-07-26 | 2019-01-31 | F. Hoffmann-La Roche Ag | Polythérapie faisant appel à un inhibiteur de bet et à un inhibiteur de bcl-2 |
-
2020
- 2020-02-20 WO PCT/EP2020/054413 patent/WO2020169698A1/fr active Application Filing
Patent Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6248516B1 (en) | 1988-11-11 | 2001-06-19 | Medical Research Council | Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors |
EP0404097A2 (fr) | 1989-06-22 | 1990-12-27 | BEHRINGWERKE Aktiengesellschaft | Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application |
US5571894A (en) | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
WO1993001161A1 (fr) | 1991-07-11 | 1993-01-21 | Pfizer Limited | Procede de preparation d'intermediaires de sertraline |
US5587458A (en) | 1991-10-07 | 1996-12-24 | Aronex Pharmaceuticals, Inc. | Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof |
WO1993016185A2 (fr) | 1992-02-06 | 1993-08-19 | Creative Biomolecules, Inc. | Proteine de liaison biosynthetique pour marqueur de cancer |
US7695936B2 (en) | 1995-03-01 | 2010-04-13 | Genentech, Inc. | Knobs and holes heteromeric polypeptides |
US5731168A (en) | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
US5869046A (en) | 1995-04-14 | 1999-02-09 | Genentech, Inc. | Altered polypeptides with increased half-life |
WO2011143669A2 (fr) | 2010-05-14 | 2011-11-17 | Dana-Farber Cancer Institute, Inc | Compositions et méthodes de traitement des néoplasies, des maladies inflammatoires et d'autres affections |
US20140242079A1 (en) * | 2013-02-26 | 2014-08-28 | Roche Glycart Ag | Bispecific t cell activating antigen binding molecules |
WO2014131712A1 (fr) | 2013-02-26 | 2014-09-04 | Roche Glycart Ag | Molécules de liaison à l'antigène bispécifiques activant des lymphocytes t |
WO2017055389A1 (fr) | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Molécules bispécifiques de liaison à l'antigène activant les lymphocytes t anti-ceaxcd3 |
US20170182025A1 (en) * | 2015-12-24 | 2017-06-29 | Celgene Quanticel Research, Inc. | Bromodomain and extra-terminal protein inhibitor combination therapy |
WO2017223268A1 (fr) * | 2016-06-22 | 2017-12-28 | Yale University | Compositions et procédés de resensibilisation de cellules à des inhibiteurs des protéines de bromodomaine et de domaine extraterminal (beti) |
WO2019020610A1 (fr) * | 2017-07-26 | 2019-01-31 | F. Hoffmann-La Roche Ag | Polythérapie faisant appel à un inhibiteur de bet et à un inhibiteur de bcl-2 |
Non-Patent Citations (23)
Title |
---|
"NCBI GenBank", Database accession no. BAB71849.1 |
"UniProt", Database accession no. P06731 |
CANCER IMMUNOL RES., vol. 6, no. 10, October 2018 (2018-10-01), pages 1234 - 1245 |
CARTER, J IMMUNOL METH, vol. 248, 2001, pages 7 - 15 |
CELL REP., vol. 18, no. 9, 28 February 2017 (2017-02-28), pages 2162 - 2174 |
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917 |
CLAUS ET AL., SCIENCE TRANSLATIONAL MEDICINE, 2019 |
HEELEY, ENDOCR RES, vol. 28, 2002, pages 217 - 229 |
HOLLINGER ET AL., PROC NATL ACAD SCI USA, vol. 90, 1993, pages 6444 - 6448 |
HUDSON ET AL., NAT MED, vol. 9, 2003, pages 129 - 134 |
IANEVSKI, ALEKSANDR ET AL.: "SynergyFinder: a web application for analyzing drug combination dose-response matrix data", BIOINFORMATICS, vol. 33.15, 2017, pages 2413 - 2415, XP055615806, DOI: 10.1093/bioinformatics/btx162 |
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH, pages: 647 - 660 |
KEARNEY ET AL., CELL DEATH DIFF., vol. 24, no. 10, 2017 |
KEARNEY ET AL., SCIENCE IMMUNOLOGY, 2018 |
KINDT ET AL.: "Kuby Immunology", 2007, W.H. FREEMAN AND CO., pages: 91 |
LILJEBLAD ET AL., GLYCO J, vol. 17, 2000, pages 323 - 329 |
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745 |
PEARSON, GENOMICS, vol. 46, 1997, pages 24 - 36 |
PLUCKTHUN: "The Pharmacology of Monoclonal Antibodies", vol. 113, 1994, SPRINGER-VERLAG, pages: 269 - 315 |
RIDGWAY ET AL., PROT ENG, vol. 9, 1996, pages 617 - 621 |
STUBENRAUCH ET AL., DRUG METABOLISM AND DISPOSITION, vol. 38, 2010, pages 84 - 91 |
W. R. PEARSON: "Effective protein sequence comparison", METH. ENZYMOL., vol. 266, 1996, pages 227 - 258 |
W. R. PEARSOND. J. LIPMAN: "Improved Tools for Biological Sequence Analysis", PNAS, vol. 85, 1988, pages 2444 - 2448 |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022101120A1 (fr) * | 2020-11-10 | 2022-05-19 | F. Hoffmann-La Roche Ag | Prévention ou atténuation d'effets secondaires liés à un agent de mise en contact de lymphocytes t |
WO2022132049A1 (fr) * | 2020-12-17 | 2022-06-23 | National University Of Singapore | Traitement de cancers à l'aide d'inhibiteurs de bet |
WO2022223651A1 (fr) * | 2021-04-23 | 2022-10-27 | F. Hoffmann-La Roche Ag | Prévention ou atténuation d'effets secondaires liés à un agent engageant les cellules nk |
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