Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2815—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/53—Hinge
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/75—Agonist effect on antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the present invention relates to sequential administration of first a Treg depleting antibody molecule and then an immunostimulatory antibody molecule for use in the treatment of cancer.
• the invention also relates to novel antibodies for use in such treat- ment, including novel anti-4-1 BB antibodies and novel anti-OX40 antibodies.
• Immunomodulatory mAb such as ipilimumab (anti-CTLA4), anti-PD-1/PD-L1 and anti-CD40 have shown positive outcomes when trialled in difficult-to-treat malignancies, albeit in a minority of patients (1-4). These promising results have helped to reinvigorate the belief that the immune system can hold the key to controlling cancer. These mAb were generated to target key molecular regulators on T cells or APC and to boost anti- cancer immunity through blockade of inhibitory signals (checkpoint blockers) or delivery of co-stimulatory signals (agonists).
• inhibitory signals checkpoint blockers
• agonists co-stimulatory signals
• the present invention is based on research demonstrating that anti-4-1 BB mAb can employ either direct immune-stimulation or T reg depletion in solid tumours, with the primary mechanism dependant on antibody isotype and FcyR availability. Importantly, depletion and immunostimulation appear to be competitive mechanisms, likely limited by restrictions on FcyR engagement.
• the research leading to the present invention has fur- ther shown that sequential administration of isotype-disparate anti-4-1 BB mAbs or iso- type-optimal anti-4-1 BB mAb followed by anti-PD-1 mAb to initially delete Treg and then stimulate CD8 T cells leads to augmented responses, resulting in enhanced therapy and improved outcome.
• the inventors engineered a depleting anti-4-1 BB mlgG2a with human lgG2 hinge region 'B' (mlgG2a/h2B) to provide FcyR independent agonism and demonstrate that this single mAb is capable of harnessing both mecha- nisms to deliver enhanced therapy.
• Treg depletion followed by im- munostimulation can be achieved with further antibodies in addition to anti-4-1 BB mAb, namely a Treg depleting antibody molecule, such as an antibody molecules binding spe- cifically to target belonging to the tumour necrosis factor receptor superfamily (TNFRSF), for example a Treg depleting anti-4-1 BB antibody or a Treg depleting anti-OX40 anti- body, in combination with an immunostimulatory antibody molecule, such as an im- munostimulatory anti-4-1 BB antibody, an immunostimulatory anti-OX40 antibody or an immune activatory PD-1 blocking antibody, wherein the order of administration is of im- portance to achieve the desired effect.
• TNFRSF tumour necrosis factor receptor superfamily
• the present invention relates to a Treg depleting antibody molecule for use in the treatment of cancer wherein the Treg depleting antibody molecule is administered sequentially with an immunostimulatory antibody molecule with the Treg depleting anti- body molecule being administered prior to administration of the immunostimulatory anti- body molecule.
• the present invention further relates to a method of treating a cancer in a subject, said treatment comprising administration of a Treg depleting antibody molecule followed sequentially by administration of an immunostimulatory antibody molecule.
• the present invention further relates to an anti-4-1 BB antibody molecule selected from the group consisting of antibody molecules comprising one or more of the CDRs selected from SEQ. ID. NOs: 1-6, 9-14, 17-22, 25-30, 33-38, 41-46, 49-54, 57-62, 65-70, 153-158 and 163-168.
• “One or more” means in this context that the antibody molecule comprises 1 , 2, 3, 4, 5 or 6 of the indicated sequences, i.e. 1-6 of the indicated sequenc- es.
• the anti-4-1 BB antibody molecule may comprise 1-6 of the CDRs selected from SEQ. ID. NOs: 1-6; 1-6 of the CDRs selected from SEQ. ID.
• the present invention further relates to nucleotide acids encoding the above anti- 4- BB antibody molecules.
• the present invention further relates to an anti-OX40 antibody molecule selected from the group consisting of antibody molecule comprising one or more of the CDRs se- lected from SEQ. ID. NOs: 73-78, 81-86, 89-94, 97-102, 105-110, SEQ. ID. NOs: 113- 118, SEQ. ID. NOs: 121-126, SEQ. ID. NOs: 129-134, SEQ. ID. NOs: 137-142, SEQ. ID. NOs: 145-150, and SEQ. ID. NOs: 171-176.
• the antibody molecule comprises 1 , 2, 3, 4, 5 or 6 of the indicated sequences, i.e.
• the anti-OX40 antibody molecule may comprise 1-6 of the CDRs selected from SEQ. ID. NOs: 73-78; 1-6 of the CDRs selected from SEQ. ID. NOs: 81-86; 1-6 of the CDRs selected from SEQ. ID. NOs: 89-94; 1-6 of the CDRs selected from SEQ. ID. NOs: 97-102; 1-6 of the CDRs selected from SEQ. ID. NOs: 105- 110; 1-6 of the CDRs selected from SEQ. ID. NOs: 113-118; 1-6 of the CDRs selected from SEQ. ID. NOs: 121-126; 1-6 of the CDRs selected from SEQ. ID.
• the present invention further relates to vectors comprising the above nucleotide acids.
• the present invention further relates to host cells comprising the above nucleo- tide acids and/or the above vectors.
• Treg cells Tregs or T re gs, are a subpopulation of T cells which are capable of suppressing other immune cells in normal and pathological immune settings.
• Treg depletion By depletion of Tregs, or Treg depletion, we refer herein to depletion, deletion or elimination of Tregs through physical clearance of cells. In particular, we refer to deple- tion of intratumoural Tregs.
• Effector T cells are T cells or T lymphocytes that in response to stimulus activate, attack or destroy antigen-expressing cells in an antigen: HC:TCR-resthcted manner. Effector T cells may control cancer and eradicate tumor cells directly (cytotoxic T cells), or indirectly (T Helper cells) through activation of other immune cells.
• an antibody comprises two heavy (H) chains and two light (L) chains.
• the antibody's heavy chain comprises one variable domain (VH) and three constant domains (CH1 , CH2 and CH3)
• the antibody's molecule light chain comprises one variable domain (VL) and one constant domain (CL).
• the variable do- mains (sometimes collectively referred to as the Fv region) bind to the antibody's target, or antigen.
• Each variable domain comprises three loops, referred to as complementary determining regions (CDRs), which are responsible for target binding.
• the constant do- mains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions.
• antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and in humans several of these are further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, and lgG4; lgA1 and lgA2.
• Another part of an antibody is the Fc domain (otherwise known as the fragment crystallisable domain), which comprises two of the constant domains of each of the antibody's heavy chains. The Fc domain is responsible for interactions be- tween the antibody and Fc receptor.
• Fc receptors are membrane proteins which are often found on the cell surface of cells of the immune system (i.e. Fc receptors are found on the target cell membrane - otherwise known as the plasma membrane or cytoplasmic membrane).
• the role of Fc receptors is to bind antibodies via the Fc domain, and to internalize the antibody into the cell. In the immune system, this can result in antibody-mediated phagocytosis and anti- body-dependent cell-mediated cytotoxicity.
• antibody molecule encompasses full-length or full-size antibodies as well as functional fragments of full length antibodies and derivatives of such antibody molecules.
• Functional fragments of a full-size antibody have the same antigen binding char- acteristics as the corresponding full-size antibody and include either the same variable domains (i.e. the VH and VL sequences) and/or the same CDR sequences as the corre- sponding full-size antibody. That the functional fragment has the same antigen binding characteristics as the corresponding full-size antibody means that it binds to the same epitope on the target as the full-size antibody. Such a functional fragment may corre- spond to the Fv part of a full-size antibody.
• such a fragment may be a Fab, also denoted F(ab), which is a monovalent antigen-binding fragment that does not con- tain a Fc part, or a F(ab') 2 , which is an divalent antigen-binding fragment that contains two antigen-binding Fab parts linked together by disulfide bonds or a F(ab'), i.e. a mono- valent-variant of a F(ab')2.
• F(ab) a monovalent antigen-binding fragment that contains two antigen-binding Fab parts linked together by disulfide bonds or a F(ab'), i.e. a mono- valent-variant of a F(ab')2.
• Such a fragment may also be single chain variable fragment (scFv).
• a functional fragment does not always contain all six CDRs of a corresponding full-size antibody. It is appreciated that molecules containing three or fewer CDR regions (in some cases, even just a single CDR or a part thereof) are capable of retaining the antigen-binding activity of the antibody from which the CDR(s) are derived. For example, in Gao er a/., 1994, J. Biol. Chem., 269: 32389-93 it is described that a whole VL chain (including all three CDRs) has a high affinity for its substrate.
• Molecules containing two CDR regions are described, for example, by Vaughan & Sollazzo 2001 , Combinatorial Chemistry & High Throughput Screening, 4: 417-430.
• a minibody including only the H1 and H2 CDR hypervariable regions interspersed within framework regions is described.
• the minibody is described as being capable of binding to a target.
• Pessi er a/., 1993, Nature, 362: 367-9 and Bianchi er a/., 1994, J. Mol. Biol., 236: 649-59 are referenced by Vaughan & Sollazzo and describe the H1 and H2 minibody and its properties in more detail.
• Antibody molecules containing a single CDR region are described, for example, in Laune er a/., 1997, JBC, 272: 30937-44, in which it is demonstrated that a range of hexapeptides derived from a CDR display antigen-binding activity and it is noted that synthetic peptides of a complete, single, CDR display strong binding activity.
• Monnet er a/., 1999, JBC, 274: 3789-96 it is shown that a range of 12-mer peptides and associ- ated framework regions have antigen-binding activity and it is commented on that a CDR3-like peptide alone is capable of binding antigen.
• micro-antibody a molecule containing a single CDR
• a cyclic peptide from an anti-HIV antibody has antigen-binding activity and function.
• Nicaise ei a/., 2004, Protein Sci- ence, 13:1882-91 it is shown that a single CDR can confer antigen-binding activity and affinity for its lysozyme antigen.
• antibody molecules having five, four, three or fewer CDRs are capable of retaining the antigen binding properties of the full-length antibodies from which they are derived.
• the antibody molecule may also be a derivative of a full-length antibody or a fragment of such an antibody.
• the derivative has the same antigen binding characteris- tics as the corresponding full-size antibody in the sense that it binds to the same epitope on the target as the full-size antibody.
• antibody molecule we include all types of an- tibody molecules and functional fragments thereof and derivatives thereof, including: monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly pro- prised antibodies, multi-specific antibodies, bi-specific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, single-chain Fvs (scFv), Fab fragments, F(ab')2 fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), antibody heavy chains, antibody light chains, homo-dimers of antibody heavy chains, homo-dimers of antibody light chains, heterodimers of antibody heavy chains, heterodi- mers of antibody light chains, antigen binding functional fragments of such homo- and heterodimers.
• antibody molecule includes all classes of an- tibody molecules and functional fragments, including: IgG, lgG1 , lgG2, lgG3, lgG4, IgA, IgM, IgD, and IgE.
• the antibody is a human lgG1.
• the skilled person is aware that the mouse lgG2a and human lgG1 productively engage with activatory Fc gamma receptors, and share the ability to activate deletion of target cells through activation of activatory Fc gamma receptor bearing immune cells (e.g. macrophages and NK cells) by e.g. ADCP and ADCC.
• activatory Fc gamma receptor bearing immune cells e.g. macrophages and NK cells
• ADCP and ADCC activatory Fc gamma receptor bearing immune cells
• the mouse lgG2a is the preferred isotype for deletion in the mouse
• human lgG1 is a preferred isotype for deletion in human.
• optimal co-stimulation of TNFR superfamily agonist receptors e.g.
• BB, ox40, TNFRII, CD40 depends on antibody engagement of the inhibitory FcyRII.
• the lgG1 isotype which binds preferentially to inhibitory Fc gamma receptor (FcyRIIB) and only weakly to activatory Fc gamma receptors, is known to be optimal for costimulatory activity of TNFR-superfamily targeting mAb. While no direct equivalent of the mouse lgG1 isotype has been described in man, antibodies may be en- gineered to show a similarly enhanced binding to inhibitory over activatory human Fc gamma receptors. Such engineered TNFR-superfamily targeting antibodies also have improved co-stimulatory activity in vivo, in transgenic mice engineered to express human activatory and inhibitory Fc gamma receptors (49).
• antibody molecules As outlined above, different types and forms of antibody molecules are included in the invention, and would be known to the person skilled in immunology. It is well known that antibodies used for therapeutic purposes are often modified with additional components which modify the properties of the antibody molecule.
• an antibody molecule of the invention or an antibody molecule used in accordance with the invention comprises a detectable moiety and/or a cytotoxic moiety.
• detectable moiety we include one or more from the group comprising of: an enzyme; a radioactive atom; a fluorescent moiety; a chemiluminescent moiety; a biolu- minescent moiety.
• the detectable moiety allows the antibody molecule to be visualised in vitro, and/or in vivo, and/or ex vivo.
• cytotoxic moiety we include a radioactive moiety, and/or enzyme, wherein the enzyme is a caspase, and/or toxin, wherein the toxin is a bacterial toxin or a venom; wherein the cytotoxic moiety is capable of inducing cell lysis.
• the antibody molecule may be in an isolated form and/or purified form, and/or may be PEGylated.
• the antibody molecule of the present invention or used according to the invention is an antibody molecule that is capable of competing with the specific antibodies provided herein, for example antibody molecules comprising any of the amino acid sequences set out in for example SEQ ID NOs: 1-152 for binding to the specific target.
• capable of competing for we mean that the competing antibody is capable of inhibiting or otherwise interfering, at least in part, with the binding of an antibody mole- cule as defined herein to the specific target.
• such a competing antibody molecule may be capable of inhibiting the binding of an antibody molecule described herein by at least about 10%; for example at least about 20%, or at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100% and/or inhibiting the ability of the antibody described herein to prevent or reduce binding to the specific target by at least about 10%; for ex- ample at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100%).
• ELISA Enzyme-linked immunosorbent assay
• ELISA assays can be used to evaluate epitope-modifying or blocking antibodies.
• an antibody specifically binds a defined target molecule or antigen. That is to say, the antibody preferentially and selectively binds its target and not a molecule which is not a target.
• the targets of the antibodies according to the present invention, or of the antibod- ies used in accordance with the invention, are expressed on the surface of cells, i.e. they are cell surface antigen, which would include an epitope (otherwise known in this context as a cell surface epitope) for the antibody.
• Cell surface antigen and epitope are terms that would be readily understood by one skilled in immunology or cell biology.
• cell surface antigen we include that the cell surface antigen is exposed on the extracellular side of the cell membrane, but may only be transiently exposed on the extracellular side of the cell membrane.
• transiently exposed we include that the cell surface antigen may be internalized into the cell, or released from the extracellular side of the cell membrane into the extracellular space.
• the cell surface antigen may be re- leased from the extracellular side of the cell membrane by cleavage, which may be me- diated by a protease.
• the cell surface antigen may be connected to the cell mem- brane, but may only be transiently associated with the cell membrane.
• transiently associated we include that the cell surface antigen may be released from the extracellu- lar side of the cell membrane into the extracellular space. The cell surface antigen may be released from the extracellular side of the cell membrane by cleavage, which may be mediated by a protease.
• the cell surface antigen may be a peptide, or a polypep- tide, or a carbohydrate, or an oligosaccharide chain, or a lipid; and/or an epitope that is present on a protein, or a glycoprotein, or a lipoprotein.
• antibody molecule the specifically binds or "target specific anti- body molecule” we include that the antibody molecule specifically binds a target but does not bind to non-target, or binds to a non-target more weakly (such as with a lower affinity) than the target.
• the antibody specifically binds to the target at least two-fold more strongly, or at least five-fold more strongly, or at least 10-fold more strongly, or at least 20-fold more strongly, or at least 50-fold more strongly, or at least 100-fold more strongly, or at least 200-fold more strongly, or at least 500-fold more strongly, or at least than about 1000-fold more strongly than to a non-target.
• the antibody specifically binds to the target if it binds to the target with a Kd of at least about 10 "1 Kd, or at least about 10 "2 Kd, or at least about 10 "3 Kd, or at least about 10 ⁇ 4 Kd, or at least about 10 "5 Kd, or at least about 10 "6 Kd, or at least about 10 "7 K d , or at least about 10 "8 Kd, or at least about 10 "9 Kd, or at least about 10 "10 K d , or at least about 10 "11 Kd, or at least about 10 "12 Kd, or at least about 10 ⁇ 13 K d , or at least about 10 "14 K d , or at least about 10 "15 K d .
• Treg depleting antibody refers to an antibody that upon administration to a subject, such as a human, specifically binds to a target expressed on the surface of Tregs, wherein this binding results in depletion of Tregs.
• a Treg de- pleting antibody selected from antibodies binding specifically to target belonging to the tumour necrosis factor receptor superfamily (TNFRSF) is an antibody that upon admin- istration to a subject, such as a human, binds to a target belonging to the tumour necro- sis factor receptor superfamily expressed on the surface of Tregs and wherein the bind- ing results in depletion of Tregs.
• the target belonging to the tu- mour necrosis factor receptor superfamily is a target that is preferentially expressed on a tumour or in the tumour microenvironment.
• the Treg depleting antibody does not have any im- munostimulatory effects in addition to the Treg depleting effects.
• the Treg depleting antibody also has an immunostimulatory effect, in addition to the Treg depleting effects; in such embodiments the Treg depleting antibody has a sufficiently poor immunostimulatory activity to allow for enhanced therapeutic activity following se- quential administration of a second immunostimulatory antibody.
• ADCC antibody-dependent cellular cyto- toxicity
• ADCP antibody-dependent cellular phagocytosis
• An ADCP assay may be done by labelling target cells with 5 mM CFSE for 10 min at room temperature before washing in complete media. CFSE-labeled targets is then op- sonized with diluting concentrations of Ab before coculturing at a 1 :5 E:T ratio with BMDMs in 96-well plates for 1 h at 37°C. BMDMs are then labeled with anti-F4/80- allophycocyanin for 15 min at room temperature and washed with PBS twice.
• Plates are kept on ice, wells are scraped to collect BMDMs, and phagocytosis is assessed by flow cytometry using a FACSCalibur (BD) to determine the percentage of F4/80+CFSE+ cells within the F4/80+ cell population.
• BD FACSCalibur
• an in vitro agonism assay For such an assay the following generic method may be used.
• Cell culture is in RPMI 1640 media (GibcoTM) supplemented with 10% foetal calf serum, glutamine (2 mM), pyruvate (1 mM), penicillin, and streptomycin (100 lU/mL) at 37°C in 5% C0 2 .
• Fresh PBMCs are labelled with 2mM carboxyfluorescein succinimidyl ester (CFSE).
• PBMCs are then cultured in a 24-well plate at 1x10 7 cells/mL as described by Romer et al (51 ) for 48 hours prior to mAb stimulation assays.
• PBMC stimulation round-bottomed 96-well plates are wet-coated with 0.01 ⁇ g/mL of OKT3 antibody (in- house) in PBS for 4 hours after which excess antibody is discarded and the plates are washed with PBS. 1X10 5 PBMCs/well are transferred to the plates and stimulated with 5 ⁇ g/mL of test mAb.
• in vivo assays with immune compe- tent animals bearing tumours and expressing activatory and inhibitory Fc gamma recep- tors can be used.
• an assay one described in the examples below may be used, for example as illustrated in Figures 1 a and 4c.
• the term immunostimulatory antibody refers to an antibody that upon administration to a subject, such as a human, spe- cifically binds to a target present on the surface of an effector T cell.
• the binding of the immunostimulatory antibody to the target results in stimulation of an immune response , either directly through agonism (e.g. antibodies to TNF superfamily agonist receptors such as anti-4-1 BB, OX40 antibodies) or indirectly through blockade of inhibitory signals (e.g. through antibody blockade of the PD1/PDL1 axis) through stimulation of an effector T cell.
• agonism e.g. antibodies to TNF superfamily agonist receptors such as anti-4-1 BB, OX40 antibodies
• blockade of inhibitory signals e.g. through antibody blockade of the PD1/PDL1 axis
• Such an effector T cell can be a CD8 + cell; in such embodiments, the im- munostimulatory antibody is a CD8 activating and/or CD8 boosting antibody.
• the im- munostimulatory antibody is a CD8 activating and/or CD8 boosting antibody.
• such an effector T cell can be a CD4 + cell; in such embodiments, the immunostimulatory antibody is a CD4 activating and/or CD4 boosting antibody.
• the immunostimulatory antibody may be an antibody that agonizes an immune stimulatory receptor, such as 4-1 BB, expressed on effector T cells, in an Fc:FcyR dependent manner, or an antibody that antagonises an immune checkpoint receptor, such as PD-1 , expressed on effector T cells, in an Fc:FcyR- independent manner.
• an antibody is an immunostimulatory antibody in the meaning of the present invention
• an in vitro assay that demonstrates T cell proliferation in response ' to mAb.
• the assays described above may be used for this pur- poses.
• mice receive tumour on day 0.
• mice receive the mAb i.v. followed by 3 further ad- ministrations i.p. every other day (200 ⁇ g final dose, or as established for mAb).
• mice are then sacrificed 1 or 2 day(s) after the final mAb administration and the spleen and tumour are analysed by flow cytometry for TIL content and the frequency of Foxp3+ cells within the CD4+ population in the tumour plotted and spleen (control tissue).
• the Treg depleting antibody is a human antibody.
• the Treg depleting antibody is a humanized antibody.
• the immunostimulatory antibody is a human antibody. In some embodiments, the immunostimulatory antibody is a humanized antibody.
• the Treg depleting antibody and the immunostimulatory antibody used in combi- nation in accordance with the present invention may both comprise the same CDRs since the depleting/immunostimulatory effects may be adjusted by modifications of other parts of the antibody, as also discussed above.
• a Treg depleting antibody may be obtained by using an antibody in the form of a human lgG1 antibody; accordingly, in some embodiments, the Treg deplet- ing antibody is a human lgG1 antibody.
• a Treg depleting antibody may also be obtained by using an antibody in the form of a human lgG1 antibody showing improved binding to one or several activatory Fc receptors and/or being engineered for improved binding to one or several activatory Fc receptors; accordingly, in some embodiments, the Treg de- pleting antibody is an Fc-engineered human lgG1 antibody.
• a Treg depleting antibody may also be obtained by using murine or a humanized murine lgG2a antibody, and ac- cordingly, in some embodiments, the Treg depleting antibody is a humanized murine lgG2a antibody.
• an immunostimulatory antibody may be obtained by using an anti- body in the form of a human lgG2 antibody, such as a human lgG2b antibody, or in the form of a human lgG4 antibody.
• the immunostimulatory an- tibody is a human lgG2 antibody.
• the immunostimulatory antibody is a human lgG2b antibody.
• the immunostimulatory antibody is a human lgG4 antibody.
• An immunostimulatory antibody may also be obtained by using a murine or a humanized murine lgG1 antibody, and in some embodiments the im- munostimulatory antibody is a humanized murine lgG1 antibody.
• the immunostimulatory antibody is and antibody showing enhanced binding to inhibitory over activatory Fey receptors. In some embodiments, the immunostimulatory antibody is an antibody showing enhanced binding to human FcyRIIB over activatory Fey receptors. In some embodiments, the immunostimulatory antibody is engineered for en- hanced binding to inhibitory over activatory Fey receptors. In some embodiments, the immunostimulatory antibody is engineered for enhanced binding to human FcyRIIB over activatory Fey receptors.
• the target that the Treg depleting antibody of the present invention or the Treg depleting antibody used in accordance with the present invention binds to may be se- lected from the group consisting of targets belonging to the TNFRS.
• the target belonging to the TNFRS may be selected from the group consisting of 4-1 BB, OX40, and TNFR2.
• the target that the Treg depleting antibody of the present invention or the Treg depleting antibody used in accordance with the present invention binds to may alterna- tively be selected from the group consisting of ICOS, GITR, CTLA-4, CD25, and neu- roplin-1. In some embodiments, the target is not CD25.
• the target that the immunostimulatory antibody of the present invention or the immunostimulatory used in accordance with the present invention binds to may be se- lected from the group consisting of 4-1 BB and OX40.
• the target that the immunostimulatory antibody of the present invention or the Treg depleting antibody used in accordance with the present invention binds to may al- ternatively be selected from the group consisting of ICOS, GITR, CTLA-4, TNFR2, CD25 and PD-1. In some embodiments, the target is not CD25.
• At least one target is 4-1 BB, which is also denoted CD137 and tumour necrosis factor receptor superfamily member 9 (TNFRSF9).
• 4-1 BB is expressed on Tregs following activation of CD4+ and CD8+ T cells and its ligation is required for optimal protective CD8 T cell responses against viruses and B cell lymphoma in mice (11 , 12).
• Anti-4-1 BB specific antibodies enhance the prolif- eration and survival of antigen-stimulated T cells in vitro and, similar to anti-CD40, anti-4- 1 BB mAb promote anti-tumour immunity in pre-clinical cancer models dependent largely on CD8 T cells (12, 13).
• 4-1 BB is a downstream target of the Treg lineage-defining tran- scription factor Foxp3, is expressed on resting Treg ceils and is upregulated on Treg ac- tivation (14, 15), and it is possible therefore that anti-4-1 BB may act in part through the depletion of Treg cells.
• anti-4-1 BB antibody is a depleting or a stimulating anti- body is likely to depend on its FcyR usage and in the work leading to the present inven- tion, the inventors carried out in vitro and in vivo experiments to explore the optimal iso- type for a therapeutic anti-4-1 BB mAb in a tumour setting.
• mlgG1 isotype mAb exerted superior agonistic activity and direct immune-stimulation of CD8+ T cells compared with a mlgG2a version of the same specificity
• the mlgG2a mAb provided optimal therapeutic activity.
• the potency of the mlgG2a mAb is due to intratumour- al Treg depletion.
• the therapeutic potential of the mlgG2a was retained. Under these conditions mlgG2a was converted to an agonist by engaging the inhibitory FcyRIIB.
• the Treg depleting antibody molecule is an anti-4-1 BB an- tibody molecule selected from the group presented in Table 1 below.
• the Treg depleting antibody molecule is an anti-4-1 BB an- tibody molecule selected from the group consisting of antibody molecules comprising 1-6 of the CDRs from each group selected from of SEQ ID. Nos: 1-6, SEQ ID. Nos: 9-14, SEQ ID. Nos: 17-22, SEQ ID. Nos: 25-30, SEQ ID. Nos: 33-38, SEQ ID. Nos: 41-46, SEQ ID. Nos: 49-54, SEQ ID. Nos: 57-62, SEQ ID. Nos: 65-70, SEQ ID. Nos: 153-158 and SEQ ID. Nos: 163-168.
• the Treg depleting antibody molecule is an anti-4-1 BB an- tibody molecule selected from the group consisting of antibody molecules comprising the 6 CDRs selected from SEQ ID. Nos: 1-6, SEQ ID. Nos: 9-14, SEQ ID. Nos: 17-22, SEQ ID. Nos: 25-30, SEQ ID. Nos: 33-38, SEQ ID. Nos: 41-46, SEQ ID. Nos: 49-54, SEQ ID. Nos: 57-62, SEQ ID. Nos: 65-70, SEQ ID. Nos: 153-158 and SEQ ID. Nos: 163-168.
• the Treg depleting antibody molecule is an anti-4-1 BB an- tibody molecule selected from the group consisting of antibody molecules comprising a VH selected from the group consisting of SEQ ID. Nos: 7, 15, 23, 31 , 39, 47, 55, 63, 71 ,
• the Treg depleting antibody molecule is an anti-4-1 BB an- tibody molecule selected from the group consisting of antibody molecules comprising a VL selected from the group consisting of SEQ ID. Nos: 8, 16, 24, 32, 40, 48, 56, 64, 72,
• the Treg depleting antibody molecule is an anti-4-1 BB an- tibody molecule selected from the group consisting of antibody molecules comprising a VH and a VL selected from the group consisting of SEQ ID. Nos: 7-8, 15-16, 23-24, 31- 32, 39-40, 47-48, 55-56, 63-64, 71-72, 159-160, 161-162 and 169-170.
• the immunostimulatory antibody molecule is an anti-4- 1 BB antibody molecule selected from the group presented in Table 1 below.
• the immunostimulatory antibody molecule is an anti-4- 1 BB antibody molecule selected from the group consisting of antibody molecules com- prising 1-6 of the CDRs from each group SEQ ID. Nos: 1-6, SEQ ID. Nos: 9-14, SEQ ID. Nos: 17-22, SEQ ID. Nos: 25-30, SEQ ID. Nos: 33-38, SEQ ID. Nos: 41-46, SEQ ID. Nos: 49-54, SEQ ID. Nos: 57-62, SEQ ID. Nos: 65-70, SEQ ID. Nos: 153-158 and SEQ ID. Nos: 163-168.
• the immunostimulatory antibody molecule is an anti-4- 1 BB antibody molecule selected from the group consisting of antibody molecules com- prising the 6 CDRs selected from SEQ ID. Nos: 1-6, SEQ ID. Nos: 9-14, SEQ ID. Nos: 17-22, SEQ ID. Nos: 25-30, SEQ ID. Nos: 33-38, SEQ ID. Nos: 41-46, SEQ ID. Nos: 49-
• the immunostimulatory antibody molecule is an anti-4- 1 BB antibody molecule selected from the group consisting of antibody molecules com- prising a VH selected from the group consisting of SEQ ID. Nos: 7, 15, 23, 31 , 39, 47,
• the immunostimulatory antibody molecule is an anti-4- 1 BB antibody molecule selected from the group consisting of antibody molecules com- prising a VL selected from the group consisting of SEQ ID. Nos: 8, 16, 24, 32, 40, 48, 56, 64, 72, 160, 162 and 170.
• the immunostimulatory antibody molecule is an anti-4- 1 BB antibody selected from the group consisting of antibody molecules comprising a VH and VL selected from the group consisting of SEQ ID. Nos: 7-8, 15-16, 23-24, 31-32, 39- 40, 47-48, 55-56, 63-64, 71-72, 159-160, 161-162 and 169-170.
• the Treg depleting antibody molecule is an anti-OX40 an- tibody molecule selected from the group presented in Table 2 below.
• the Treg depleting antibody molecule is an anti-OX40 an- tibody molecule selected from the group consisting of antibody molecules comprising 1-6 of the CDRs from each group selected from SEQ ID. Nos: 73-78, SEQ ID. Nos:81-86, SEQ ID. Nos:89-94, SEQ ID. Nos:97-102, SEQ ID. Nos: 105-110, SEQ ID. Nos:113-118, SEQ ID. Nos-,121-126, SEQ ID. Nos:129-134, SEQ ID. Nos:137-142, SEQ ID. Nos:145- 150 and SEQ ID. Nos: 171 -176.
• the Treg depleting antibody molecule is an anti-OX40 an- tibody molecule selected from the group consisting of antibody molecules comprising the 6 CDRs from each group selected from SEQ ID. Nos: 73-78, SEQ ID. Nos:81-86, SEQ ID. Nos:89-94, SEQ ID. Nos:97-102, SEQ ID. Nos: 105-110, SEQ ID. Nos: 113-118, SEQ ID. Nos:121-126, SEQ ID. Nos:129-134, SEQ ID. Nos: 137-142, SEQ ID. Nos:145-150 and SEQ ID. Nos:171-176.
• the Treg depleting antibody molecule is an anti-OX40 an- tibody molecule selected from the group comprising a VH selected from the group con- sisting of SEQ ID. Nos: 79, 87, 95, 103, 111 , 119, 127, 135, 143, 151 and 177.
• the Treg depleting antibody molecule is an anti-OX40 an- tibody molecule selected from the group consisting of antibody molecules comprising a VL selected from the group consisting of SEQ ID. Nos: 80, 88, 96, 104, 1 12, 120, 128, 136, 144, 152 and 178.
• the Treg depleting antibody molecule is an anti-OX40 an- tibody molecule selected from the group consisting of antibody molecules comprising a VH and a VL selected from the group consisting of SEQ ID. Nos: 79-80, 87-88, 95-96, 103-104, 111-1 12, 1 19-120, 127-128, 135-136, 143-144, 151-152 and 177-178.
• the immunostimulatory antibody molecule is an anti-OX40 antibody molecule selected from the group presented in Table 2 below.
• the immunostimulatory antibody molecule is an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising 1-6 of the CDRs from each group selected from SEQ ID. Nos: 73-78, SEQ ID. Nos: 81- 86, SEQ ID. Nos: 89-94, SEQ ID. Nos: 97-102, SEQ ID. Nos: 105-110, SEQ ID. Nos: 113-118, SEQ ID. Nos: 121-126, SEQ ID. Nos: 129-134, SEQ ID. Nos: 137-142, SEQ ID. Nos: 145-150 and SEQ ID. Nos: 171 -176.
• the immunostimulatory antibody molecule is an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising the 6 CDRs selected from SEQ ID. Nos: 73-78, SEQ ID. Nos: 81-86, SEQ ID. Nos: 89- 94, SEQ ID. Nos: 97-102, SEQ ID. Nos: 105-110, SEQ ID. Nos: 113-1 18, SEQ ID. Nos: 121-126, SEQ ID. Nos: 129-134, SEQ ID. Nos: 137-142, 145-150 and SEQ ID. Nos: 171- 176.
• the immunostimulatory antibody molecule is an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising a VH selected from the group consisting of SEQ ID. Nos: 79, 87, 95, 103, 1 11 , 1 19, 127,
• the immunostimulatory antibody molecule is an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising a VL selected from the group consisting of SEQ ID. Nos: 80, 88, 96, 104, 1 12, 120, 128,
• the immunostimulatory antibody molecule is an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising a VH and a VL selected from the group consisting of SEQ ID. Nos: 79-80, 87-88, 95-96, 103-104, 11 1-112, 119- 120, 127- 128, 135- 136, 143- 144, 151-152 and 177-178.
• the immunostimulatory antibody is an anti-PD1 antibody, preferably a human anti-PD1 antibody.
• the anti-PD1 antibody may be selected from the group consisting of nivolumab and pembrolizumab.
• the immunostimulatory antibody is an anti-PD1 antibody, preferably a human anti-PD1 antibody.
• the anti-PD1 antibody may be selected from the group consisting of nivolumab and pembrolizumab.
• the immunostimulatory antibody is an anti-PDL1 antibody, preferably a human anti-PDL1 antibody.
• the anti-PDL1 antibody may be atezolizumab.
• the immunostimulatory antibody is an anti-CTLA-4 anti- body, preferably a human anti-CTLA-4 antibody.
• the anti-CTLA-4 antibody may be se- lected from the group consisting of ipilimumab and tremilimumab.
• the Treg depleting antibody is administered to the subject, such as a human, pri- or to administration of the immunostimulatory antibody.
• the Treg deplet- ing antibody is administered to the tumour first in order to achieve the Treg depleting ef- fect.
• the immunostimulatory antibody is administered. This sequential administration may be achieved by temporal separation of the two antibodies.
• the sequential administration may also be achieved by spatial separation of the two antibodies, by ad- ministration of the Treg depleting antibody in a way, such as intratumoural, so that it reaches the tumour prior to the immunostimulatory antibody, which is then administerd in a way, such as systemically, so that it reaches the tumour after the Treg depleting anti- body.
• medicines can be modi- fied with different additives, for example to change the rate in which the medicine is ab- sorbed by the body; and can be modified in different forms, for example to allow for a particular administration route to the body.
• composition, and/or antibody, and/or agent, and/or medicament of the invention may be combined with an excipient and/or a phar- maceutically acceptable carrier and/or a pharmaceutically acceptable diluent and/or an adjuvant.
• composition, and/or antibody, and/or agent, and/or me- dicament of the invention may be suitable for parenteral administration including aque- ous and/or non-aqueous sterile injection solutions which may contain anti-oxidants, and/or buffers, and/or bacteriostats, and/or solutes which render the formulation isotonic with the blood of the intended recipient; and/or aqueous and/or non-aqueous sterile sus- pensions which may include suspending agents and/or thickening agents.
• the composi- tion, and/or antibody, and/or agent, and/or medicament of the invention may be present- ed in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (i.e. lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
• a freeze-dried (i.e. lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from ster- ile powders, and/or granules, and/or tablets of the kind previously described.
• the daily dosage level of the Treg depleting antibody and/or the immunostimulatory antibody will usually be from 1 mg/kg bodyweight of the patient to 20 mg/kg, or in some cases even up to 100 mg/kg administered in single or divided doses. Lower doses may be used in special circum- stances, for example in combination with prolonged administration.
• the physician in any event will determine the actual dosage which will be most suitable for any individual pa- tient and it will vary with the age, weight and response of the particular patient.
• the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.
• the composition and/or medicament of the invention will contain the Treg depleting antibody and/or the immunostimulatory antibody at a concentration of be- tween approximately 2 mg/ml and 150 mg/ml or between approximately 2 mg/ml and 200 mg/ml.
• the medicaments and/or compositions of the inven- tion will contain the Treg depleting antibody and/or the immunostimulatory antibody at a concentration of 10 mg/ml.
• compositions, and/or antibody, and/or agent, and/or medicament of the invention are administered as a suitably acceptable formula- tion in accordance with normal veterinary practice and the veterinary surgeon will deter- mine the dosing regimen and route of administration which will be most appropriate for a particular animal.
• the present invention provides a pharmaceutical formulation comprising an amount of an antibody and/or agent of the invention effective to treat vari- ous conditions (as described above and further below).
• the composition, and/or antibody, and/or agent, and/or medicament is adapted for delivery by a route se- lected from the group comprising: intravenous; intramuscular; subcutaneous.
• the present invention also includes composition, and/or antibody, and/or agent, and/or medicament comprising pharmaceutically acceptable acid or base addition salts of the polypeptide binding moieties of the present invention.
• the acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful in this invention are those which form non-toxic acid addition salts, i.e.
• salts containing pharmacologically acceptable anions such as the hydrochloride, hydro- bromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p- toluenesulphonate and pamoate [i.e. 1 ,1 '-methylene-bis-(2-hydroxy-3 naphthoate)] salts, among others.
• pharmacologically acceptable anions such as the hydrochloride, hydro- bromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, male
• Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the agents according to the present invention.
• the chemical bases that may be used as reagents to prepare pharmaceutical- ly acceptable base salts of the present agents that are acidic in nature are those that form non-toxic base salts with such compounds.
• Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g. potassium and sodium) and alkaline earth metal cations ⁇ e.g. calcium and magnesium), ammonium or water-soluble amine addition salts such as N- methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.
• the agents and/or polypep- tide binding moieties of the invention may be lyophilised for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilisation method (e.g. spray drying, cake drying) and/or reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of anti- body activity loss (e.g. with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted upward to compensate.
• lyophilisation and reconstitution can lead to varying degrees of anti- body activity loss (e.g. with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted upward to compensate.
• the lyophilised (freeze dried) polypeptide binding moiety loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (prior to lyophilisation) when re-hyd rated.
• the combination of a Treg depleting antibody molecule and immunostimulatory antibody molecule, wherein the Treg depleting antibody molecule is administered to a subject prior to administration of the immunostimulatory antibody molecule to the subject can be used use in the treatment of cancer.
• the subject could be mammalian or non-mammalian.
• the mammalian subject is a human or is a non-mammalian, such as a horse, or a cow, or a sheep, or a pig, or a camel, or a dog, or a cat.
• the mammalian subject is a human.
• exhibit we include that the subject displays a cancer symptom and/or a can- cer diagnostic marker, and/or the cancer symptom and/or a cancer diagnostic marker can be measured, and/or assessed, and/or quantified.
• cancer symptoms and cancer diagnostic markers would be and how to measure and/or assess and/or quantify whether there is a reduction or increase in the severity of the cancer symptoms, or a reduction or increase in the cancer diagnostic markers; as well as how those cancer symptoms and/or cancer diagnostic markers could be used to form a prog- nosis for the cancer.
• Cancer treatments are often administered as a course of treatment, which is to say that the therapeutic agent is administered over a period of time.
• the length of time of the course of treatment will depend on a number of factors, which could include the type of therapeutic agent being administered, the type of cancer being treated, the severity of the cancer being treated, and the age and health of the subject, amongst others reasons.
• “during the treatment” we include that the subject is currently receiving a course of treatment, and/or receiving a therapeutic agent, and/or receiving a course of a therapeutic agent.
• the cancer to be treated in accordance with the present invention is a solid tumour.
• the cancer is selected from the group consisting of sar- comas, carcinomas and lymphomas.
• the cancer is selected from the group consisting of squa- mous cell carcinoma (SCC), thymoma, neuroblastoma or ovarian cancer.
• SCC squa- mous cell carcinoma
• thymoma thymoma
• neuroblastoma ovarian cancer.
• staging Clinical definitions of the diagnosis, prognosis and progression of a large number of cancers rely on certain classifications known as staging. Those staging systems act to collate a number of different cancer diagnostic markers and cancer symptoms to provide a summary of the diagnosis, and/or prognosis, and/or progression of the cancer. It would be known to the person skilled in oncology how to assess the diagnosis, and/or progno- sis, and/or progression of the cancer using a staging system, and which cancer diagnos- tic markers and cancer symptoms should be used to do so.
• cancer staging we include the Rai staging, which includes stage 0, stage I, stage II, stage III and stage IV, and/or the Binet staging, which includes stage A, stage B and stage C, and/or the Ann Arbour staging, which includes stage I, stage II, stage III and stage IV.
• cancer can cause abnormalities in the morphology of cells. These abnormalities often reproducibly occur in certain cancers, which means that examining these changes in morphology (otherwise known as histological examination) can be used in the diagnosis or prognosis of cancer.
• Techniques for visualizing samples to examine the morphology of cells, and preparing samples for visualization, are well known in the art; for example, light microscopy or confocal microscopy.
• lympho- cyte we include the presence of small, mature lympho- cyte, and/or the presence of small, mature lymphocytes with a narrow border of cyto- plasm, the presence of small, mature lymphocytes with a dense nucleus lacking discern- ible nucleoli, and/or the presence of small, mature lymphocytes with a narrow border of cytoplasm, and with a dense nucleus lacking discernible nucleoli, and/or the presence of atypical cells, and/or cleaved cells, and/or prolymphocytes.
• cancer is a result of mutations in the DNA of the cell, which can lead to the cell avoiding cell death or uncontrollably proliferating. Therefore, examin- ing these mutations (also known as cytogenetic examination) can be a useful tool for as- sessing the diagnosis and/or prognosis of a cancer.
• An example of this is the deletion of the chromosomal location 13q14.1 which is characteristic of chronic lymphocytic leu- kaemia.
• Techniques for examining mutations in cells are well known in the art; for exam- ple, fluorescence in situ hybridization (FISH).
• cytogenetic examination we include the examination of the DNA in a cell, and, in particular the chromosomes. Cytogenetic examination can be used to identify changes in DNA which may be associated with the presence of a refractory cancer and/or relapsed cancer.
• Such may include: deletions in the long arm of chromosome 13, and/or the deletion of chromosomal location 13q14.1 , and/or trisomy of chromosome 12, and/or deletions in the long arm of chromosome 12, and/or deletions in the long arm of chromosome 11 , and/or the deletion of 11q, and/or deletions in the long arm of chromo- some 6, and/or the deletion of 6q, and/or deletions in the short arm of chromosome 17, and/or the deletion of 17p, and/or the t(11 :14) translocation, and/or the (q13:q32) trans- location, and/or antigen gene receptor rearrangements, and/or BCL2 rearrangements, and/or BCL6 rearrangements, and/or t(14:18) translocations, and/or t(11:14) transloca- tions, and/or (q13:q32) translocations, and/or (3:v) translocations,
• FIG. 1 A Groups of BALB/c mice were challenged with 5 x 10 5 CT26 s.c. on day 0. When tumours were palpable mice received anti-4-1 BB (LOB12.0) mlgG1 , mlgG2a or PBS control i.v. followed by 3 further administrations i.p. every other day (200 ⁇ g final dose).
• Fig. 1 B Groups of A/J mice were challenged with 2 x 10 6 NXS2 cells s.c. and received 200 ⁇ g anti-4-1 BB mAb or isotype control mAb i.p.
• tumour growth was monitored and mice culled when mean tumour area exceed- ed 225mm 2 .
• Data are expressed as tumour area (mm 2 ) on the days after tumour chal- lenge as indicated; each line represents an individual mouse. Panels on the right show percentage survival to the humane end-point.
• FIG. 2A Splenocytes from Foxp3-GFP mice either sorted to remove GFP+ cells (-T reg ) or not (+Treg) were incubated with 0.1 ⁇ g/ml anti-CD3 and the indicated concentrations of either anti-4-1 BB (LOB12.0) mlgG1 or mlgG2a as indicated. Incorporation of [ 3 H]- thymidine was measured during the last 16 hours of a 72 hour culture. Fig.
• Fig. 2D Groups of A/J mice were challenged with 2 x 10 6 NXS2 cells s.c. on day 0 and received 200 ⁇ g anti-4-1 BB mAb or isotype control mAb i.p. and control (SIINFEKL; left panel) or TH (FETFEAKI; right panel) pep- tide on day 3. A second dose of mAb (200 ⁇ g) was given on day 6.
• 3B Samples of freshly excised ovarian tumours, ascites and blood were obtained from patients at surgery and compared to healthy PBMC. Tumour samples were minced and digested before separation with a density gradient. Matched peripheral blood was obtained and peripheral blood mononuclear cells were separated by centrifugation. 4- 1 BB expression was assessed on CD4+CD25+CD127- Treg cells, CD4+ non-Treg cells and CD8+ effector T cells by flow cytometry. Top panels show representative histograms with 4-1 BB expression in tumour tissue (solid black), blood (dashed grey) and ascites (dashed black) from the same patient. Isotype control depicted in solid grey.
• FIG. 3C Samples of freshly excised cutaneous squamous cell carcinoma (SCC) and normal skin were obtained from patients at surgery. Samples were minced and digested before separation with a density gradient. Matched peripheral blood was obtained and peripheral blood mononuclear cells were separated by centrifugation. Cells were stained for 4-1 BB and staining detected by flow cytometry.
• SCC cutaneous squamous cell carcinoma
• Top panels show representative histograms with 4-1 BB staining shown as open histo- grams; tumour tissue (solid black), blood (dashed grey), normal skin (dashed black) and isotype control (solid grey).
• Lower panel shows 4-1 BB expression on T cell subsets from different tissue samples. Data points represent 10 individual patients.
• FIG. 4A Groups of 3- 4 WT, FcyRIIB KO or ⁇ chain KO BALB/c mice received were challenged with 5 x 104 CT26 cells s.c. on day 0. When tumours were palpable mice received anti-4-1 BB mlgG1 , mlgG2a or PBS control i.v. followed by 3 further administrations i.p. every other day (200 ⁇ ig final dose). Mice were sacrificed on day 13 and spleen and tumour analysed by flow cytometry.
• Fig. 4B Mice were treated as in (A) and CD8+, Ki67+ T cells enumerated and plotted as fold change compared to control.
• Fig. 4C Groups of WT, ⁇ chain KO, or FcyR null mice ( ⁇ chain KO x FcyRIIB KO), ⁇ chain KO or FCYRIIB KO BALB/c mice were challenged with 5 x 104 CT26 cells and treated with anti-4-1 BB mAb as in (A).
• Fig. 4D CFSE labelled target murine splenic T cells opsonised with anti-4-1 BB mlgG1 , mlgG2a or control mAb were co-cultured with wild type (solid bars) or FcgRIIB KO (open bars) mBMDM and then assessed for phago- cytosis.
• FIG. 5A Groups of age and sex matched BALB/c mice were challenged with 5 x 104 CT26 s.c. on day 0. When tumours were palpable mice received anti-4-1 BB (LOB12.0) mlgGI , mlgG2a, con- current mlgGI and mlG2a or PBS control i.v. followed by 3 further administrations i.p. every other day (200 ⁇ g final dose). For scheduled administration mlgG2a was given i.v. and then mlgGI given i.p. 4 days later.
• Fig. 5B Mice were challenged with CT26 tumour and then given monotherapy as in Fig. 5A or sched- uled combinations of anti-4-1 BB mlgGI or mlgG2a and/or anti-PD-1 rlgG1 (WT) or its deglycosylated form. For combinations anti-4-1 BB mAb were administered i.v. when tu- mours were first palpable and then anti-PD-1 given i.p. 4 days later.
• Figure 6. Fc engineered anti-4- 1BB mlgG2a/h2B possesses dual activity and delivers augmented cancer therapy.
• Fig. 6A nrCE-SDS profiles of anti-4-1 BB (LOB12.0) mlgG2a, mlgG2a/h2 and "skewed" mlgG2a/h2B.
• Fig. 6A nrCE-SDS profiles of anti-4-1 BB (LOB12.0) mlgG2a, mlgG2a/h2 and "skewed" mlgG2a/h2B.
• Fig. 6B Splenocytes from C57BI/6 mice were incubated with 0.01 ⁇ g/ml anti-CD3 and the indicated concentrations of either anti-4-1 BB mlgGI , mlgG2a or mlgG2a/h2B as indicated. Incorporation of [3H]-thymidine was measured during the last 16 hours of a 72 hour culture.
• Fig. 6C CFSE labelled target murine splenic T cells opso- nised with anti-4-1 BB mlgG2a, mlgG2a/h2B or control mAb were co-cultured with wild type mBMDM and then assessed for phagocytosis.
• Phagocytosis is plotted as % of dou- ble positive macrophages.
• (E) Mice were set up as in (D) and tu- mour growth was monitored and mice culled when mean tumour area exceeded 400 mm2. Data represent examples of at least 2 independent experiments where n 5 mice per group.
• Fig. 7A Surface plasmon reso- nance analysis of anti-4-1 BB (clone LOB12.0) mlgG1 , mlgG2a and the parental rlgG2a binding to mouse FcyRI, MB, III and IV. Recombinant, soluble FcyR protein (0, 6, 23, 94, 375, 1500 nM) was passed over 4-1 -BB mAb immobilized at 5000 RU. Sensorgrams are shown. Fig.
• FIG. 7B A human cell line stably transfected with a construct encoding the extra- cellular and transmembrane region of murine 4-1 BB was incubated with anti-4-1 BB of mlgG1 , mlgG2a isotype or with the parental rlgG2a mAb at a range of concentrations prior to staining with a PE-labelled secondary antibody. Data show mean fluorescence intensity at each concentration as a percentage of maximum.
• Fig. 7C Rat anti-4-1 BB was mixed with mouse mlgG1 or mlgG2a anti-4-1 BB mAb at the concentrations indicat- ed, prior to incubation with a murine 4-1 BB transfected cell line. Rat mAb binding was detected with an anti-rat secondary antibody and data are expressed as mean fluores- cence intensity of the rat anti-4-1 BB antibody relative to the concentration of competitive mouse anti-4-1 BB.
• Anti-tumour efficacy of anti-4-1 BB mlgG2a mAb is dependent up- on CD8+ T cells.
• Groups of BALB/c mice were treated, or not, with 500 ⁇ g of a CD8- depleting antibody on days -1 , 1 and 4 relative to challenge with 5 x 10 4 CT26 cells s.c. on day O.
• Anti-41 BB mlgG2a mAb was administered on day 6 i.v., and on days 8, 10 and 12 i.p, to a final total dose of 200 ⁇ g.
• Tumour sizes were recorded and mice culled when mean tumour diameter reached 15mm. Data show tumour area (mm 2 ) on the days indi- cated after tumour challenge with each line representing an individual mouse.
• FIG. 9A Groups of 3 wild type or FcyRIIB-/- mice received 2 x 10 5 OT- I cells i.v., followed 24 hours later (day 0) by i.p. injection of 0.5 mg OVA and 200 ⁇ g mlgG1 or mlgG2a anti-4-1 BB. Control mice received OVA alone. Blood samples were taken to measure circulating SIINFEKL tetramer+ CD8+ cells over the course of the re- sponse, expressed as a % (mean ⁇ SEM) of total CD8+ cells. Data are representative of 2 experiments. Fig.
• FIG. 10A Surface plasmon reso- nance analysis of anti-PD-1 (clone EW1-9) rlgG1 (solid black) and rlgG1 deglycosylated (solid grey) binding to mouse FcyRI, IIB, III and IV.
• PD-1 mAb 500 nM was passed over recombinant FcyR-his protein (1000 RU) (R&D Systems) captured onto a CM5 chip with an anti-histidine mAb (GE Healthcare). Sensorgrams are shown with OnM curve sub- tracted.
• Fig. 10A Surface plasmon reso- nance analysis of anti-PD-1 (clone EW1-9) rlgG1 (solid black) and rlgG1 deglycosylated (solid grey) binding to mouse FcyRI, IIB, III and IV.
• PD-1 mAb 500 nM was passed over recombinant FcyR-his protein (1000 RU
• FIG. 11 Anti-4-1 BB mlgG2a/h2B FcyR binding.
• Recombinant, soluble FcvR protein (0, 6, 23, 94, 375, 1500 nM) was passed over 4-1 BB mAb immobilized at 5000 RU. Sensorgrams are shown.
• Figure 12 shows binding titration cuves for HDLM2 cells.
• Figure 13 shows ligand blocking
• Figure 14 shows the results of in vitro assays with ADCC for 4-1 BB+ IVA CD4s in the left panel and CD8 T cell proliferation in the right panel.
• Figure 15 shows binding to in vitro activated human CD4+ cells.
• Figure 16 shows cyon cross-reactivity on activated CD4+ T cells.
• Figure 17 shows ligand blocking
• Figure 18 shows ADCC on T cells and demonstrates that several mAbs induce significant ADCC on OX40 expressing CD4+ T cells.
• Figure show mean of 5 experi- ments. Campath is used as positive Ctrl and Yervoy as comparator.
• Figure 19 shows results from proliferation assays in vitro.
• Figure 20 demonstrates agonistic activity in vivo using h OX40 KI/OT1 transfer model for different antibodies.
• Figure 21 shows a table summarizing the characteristics of some of the ant ibod- ies described herein.
• Figure 22 shows Treg cells and effector cells from different compartments in hu- man and demonstrates that Tregs in tumor tissue and/or in close vincinity of tumor tissue has clearly disticnt potential target expression profiles compared to peripheral Tregs.
• Figure 23 shows receptor expression on Treg cells from different organs in mouse.
• Figure 24 shows receptor expression on Treg cells compared to other cell types in mouse.
• Therapeutic activity of anti-4-1BB mAb is determined by isotype
• LOB12.0 generated in-house (16) as previously described for other mAb specificities (17-19).
• the nucleotide sequences encoding LOB12.0 mlgG1 heavy chain is shown in SEQ. ID. NO: 179, and the corresponding amino acid sequence is shown in SEQ ID NO: 180.
• the nucleotide sequences encoding LOB 12.0 mlgG2a heavy chain is shown in SEQ. ID. NO: 181 , and the corresponding amino acid sequence is shown in SEQ ID NO: 182.
• 4-1 BB is expressed on intra-tumoural Treg cells in mouse tumour models and human cancer patients Having established that anti-4-1 BB mlgG2a is more active than mlgG1 in treating mice with established tumours, but in these mice mlgG2a lacks the ability to deliver co-stimulatory activity, we looked for alternative mechanisms that could explain its immunomodulatory effects.
• 4-1 BB mRNA and protein are preferentially expressed in Treg cells compared to resting effector T cells (14, 26, 27), its expression is further upregulated following activation of Treg cells (27, 28) and very recently has been shown to be upregulated, at least at the transcriptional level, in intratumoral Treg in hu- man solid cancers (29, 30).
• anti-4-1 BB mlgG2a was efficient in mediating depletion of intra-tumoural Treg cells in a manner dependent on the expression of activatory FcyR, we reasoned that the absence of activatory FcyR would be detrimental for the therapeutic effects of this mAb.
• anti-4-1 BB mlgG2a re- tained anti-tumour activity in the absence of activatory FcyR, suggesting that Treg cell depletion may not fully account for its therapeutic activity.
• Anti-4-1 BB mlgG2a/h2B engineered to possess dual activity delivers augmented cancer therapy
• tumour models sug- gested that therapy was likely mediated by an effector cell dependent depletion effect. None of the tumours used were 4-1 BB positive meaning this could not be a direct tumour targeting effect as seen for anti-CD20 mAb (34).
• Treg depletion was the most effective mechanism of action for 4-1 BB Ab in these models, we postulated that in the absence of competition for binding with ac- tivatory FcyR these mAb may produce a therapeutic effect through their agonistic func- tion.
• anti-4-1 BB mAb could be therapeutic using two separate mecha- nisms, given the provision of the appropriate FcyR, suggests that both mechanisms could be engaged if mAb were administered sequentially. Indeed this was found to be the case when mlgG2a was given first to delete Treg cells and then mlgG1 given to de- liver an agonistic signal (Figure 5A and Figure 5B). Importantly, if the mAb were adminis- tered simultaneously then little therapeutic effect was evident.
• urelumab BMS-663513
• lgG4 antibody manufactured by Bristol-Myers Squibb
• PF- 05082566 a fully humanised lgG2 produced by Pfizer.
• PF-05082566 has prov- en safe causing only grade 1 toxicities in patients (38) whereas urelumab caused ad- verse effects in 15% of patients including increased liver enzymes, pruritis and diarrhea (39).
• neither urelumab or PF-05082566 are pre- dicted to strongly bind FcyRIIB calling into question whether either antibody will prove effective in patients (40).
• mice Animals and cells. Mice were bred and maintained in local facilities. Genetically altered strains used were OT1 TCR transgenic C57BL/6 mice (from Dr. Matthias
• mice were obtained by crossbreeding with genotypes confirmed by polymerase chain reaction (PCR) and/or flow cytometry.
• PCR polymerase chain reaction
• the CT26 colon carcinoma (16), NXS2 neuroblastoma (41 ), B16 Flt3vax melanoma (42) and EG7 thymoma (43) models have all been described previously.
• FETFEAKI Ty- rosine Hydroxylase
• SIINFEKL or FEANGNLI incomplete Freund's adjuvant
• mice received 200 ⁇ g mAb or PBS control i.p. as indicated. Survival period to the hu- mane end point were plotted using the Kaplan-Meier method with analysis for signifi- cance by the log-rank test using GraphPad Prism 6.0 for Windows (GraphPad Software Inc, La Jolla, CA).
• Antibodies and reagents Antibodies and reagents.
• Anti-41 BB (clone LOB12.0) mAb mlgG1 , mlgG2a and mlgG2a hulgG2 hinge (mlgG2a/h2B) isotypes were constructed as previously described (18, 25; antibodies as described above).
• Anti-CD8 (YTS169) was produced in house.
• Anti-mouse PD-1 (EW1-9) mAb rlgG1 was raised using conventional hybridoma technol- ogy after immunisation of Wistar rats with recombinant mouse PD-1 (Leu25 - Gln167) Fc fusion protein (RnD Systems).
• PNGaseF N-Glycosidase F
• PNGaseF N-Glycosidase F
• V483A Promega
• PNGaseF N-Glycosidase F
• Peptides SIINFEKL, FETFEAKI and FEANGNLI
• Lymphocyte isolation Mouse - Mice challenged with CT26 or EG7 had their tumours excised and digested with 0.5 Wu/ml Liberase DL (Roche) and 50 ⁇ g/ml DNasel (Roche) for 20 mins at 37°C. Cells were then passed through a 100 ⁇ m cell strainer and used for assays directly or tumour infiltrating lymphocytes were isolated using percoll gradient of 40% and 70%. Human - Ascitic fluid was assessed as single cell suspension that had been isolated. Ovarian tumour samples were obtained from patients undergoing surgery at the Department of Obstetrics and Gynaecology at Skanes University Hospital.
• DNAse I (Sigma) in RPMI medium (Gibco) at 37°C for 1.5 hours before straining through a 70 ⁇ cell filter (BD) and centrifugation (600 x g, 20 minutes) over an Optiprep (Axis-Shield) density gradient. Matched peripheral blood samples were obtained and peripheral blood mononuclear cells were separated by centrifugation over Lymphoprep (Axis-Shield) at 600 x g for 30 minutes.
• Antibodies were anti-CD4 eF450 (GK1.5), anti-CD8-APC-eF780 (53-6.7), anti- Foxp3 APC (FJK-16), anti-4-1-BB (17-B5) (all eBioscience), anti-Ki67 APC (B56) (BD Biosciences) or isotype controls.
• Cell viability Cells were stained with either fixable eFluor780 Live/Dead stain (eBiosci- ence) or aqua live/dead viability stain (Invitrogen) at 4°C in PBS.
• Fluorophore conjugated antibodies against the following cell markers were used: Ovarian- CD4-BV510 (RPA-T4), CD25-BV421 (M-A251 ), anti-CD127-FITC (HIL- 7R-M21 ), CD8-APC (RPA-T8), 41 BB-PE (4B4-1 ), mouse lgG2a isotype, ⁇ control-PE (G155-178; all from BD Biosciences); SCC- CD3-APC-Cy7, CD4-FITC or PerCP Cy5.5, CD8-PE Cy7 (all Biolegend), 4-1 BB-PE and Foxp3-APC (both eBioscience).
• ADCP assays were performed as described previously with mouse (17, 45) or human macrophages (18, 46). Briefly, bone marrow derived macrophages (BMDM) were generated from the femurs of C57BL/6 mice and cultured in complete RPMI containing 20% L929 supernatant. Alternatively, human monocyte derived macrophages (hMDMs) were generated from PBMCs and cultured in complete RPMI containing M-CSF (in house). Target cells were CFSE-stained (5 ⁇ ) then opsonised with antibody before being co-cultured with macrophages for ⁇ 1 h. Mac- rophages were stained with CD16-APC or F4/80-APC and samples assessed for the percentage of double positive (CFSE/APC) macrophages by flow cytometry.
• BMDM bone marrow derived macrophages
• hMDMs human monocyte derived macrophages
• Soluble FcyR (R&D Systems, Abingdon, U.K.) were injected through the flow cells at 1500, 375, 94, 23, 6, and 0 nM in HBS-EP+ running buffer (GE Healthcare Life Sciences, Buckinghamshire, UK) at a flow rate of 30 ⁇ /min. Soluble Fc receptor was injected for 2 min, and dissociation was monitored for 5 min. Background binding to the control flow cell was subtracted automatically. Affinity constants were derived from the data by equi- librium binding analysis as indicated using Biacore Bioevaluation software (GE
• 0.1 ⁇ g/ml parental rat anti-4-1 BB mAb was mixed with graded concentrations of either mlgG1 or mlgG2a versions of anti-4-1 BB as indicated prior to incubation with Karpas-299 pTL cells.
• Cells were washed and stained with an APC-conjugated and mouse-adsorbed donkey anti-rat secondary antibody; the second- ary antibody did not bind to either mlgG1 or mlgG2a.
• Flow cytometric analysis was per- formed using a BD FACS Canto II and FACS Diva software.
• OVA-specific immune responses Splenocytes from OTI transgenic mice were harvested and washed. Approximately 2 x 10 5 OVA-specific CD8 T cells were then trans- ferred into recipient mice by tail vein injection. The following day mice were immunised with OVA (Sigma) as described for individual experiments. OTI expansion in peripheral blood was analysed by flow cytometry as described previously (18). Results at the peak of the response are shown (4-5 days post immunisation).
• mice were bred and maintained in local facilities in accordance with home office guide- lines. Ten to twelve week-old female BALB/c and C57 bl6 mice were supplied by Taconic (Bomholt, Denmark) and maintained in local animal facilities. For the xenograft studies with primary tumor cells, 6-8 week-old female BALB/c and C57 bl6 mice were grafted with syngeneic tumor cell lines CT26 and TH03, respectively.
• RPMI RPMI containing 2 mM glutamine, 1 mM pyruvate, 100 lU/ml penicillin and streptomycin and 10% FBS (GIBCO by Life Tech- nologies).
• Human peripheral CD4 + T-cells were purified by negative selection using MACS CD4 T-cell isolation kit (Miltenyi Biotec, UK). Antibodies and reagents
• the following antibodies and reagents were used: purified anti-CD3 (UCHT1 ; R&D Sys- tems); purified anti-CD28 (CD28.2; BioLegend); KIOVIG (Baxalta, Lessines, Belgium); Fixable Viability Dye eFluor780 (eBioscience, San Diego, CA).
• Cell Trace CFSE (dis- solved in DMSO) and Propidium Iodide were from Life Technologies (Carlsbad, CA).
• CD4-BV510 RPA-T4
• CD25-BV421 M-A251
• anti-CD127-FITC HIL-7R-M21
• Ox40-PE ACT35
• 41 BB-PE 4B4-1
• ICOS-PE DX29
• GITR-PE 621
• PD-1-PE MIH4
• CTLA-4-PE BNI3
• CD4- APC RPA-T4
• CD8-APC RPA-T8
• mouse lgG1 K isotype control-PE (MOPC-21 )
• mouse lgG2a isotype
• ⁇ control-PE G155-178
• mouse lgG2b isotype
• ⁇ control-PE 27- 53; all from BD Biosciences
• TNFRII-PE FAB226P; R&D Systems
• CD4-BV510 (RM4-5), CD25- BV421 (7D4), CD8-Alexa 488 (53-6.7; BD), Ox40, 41 BB, TNFRII, ICOS, GITR, PD-1 , CTLA-4, FITC negative control (scFv, in-house generated Biolnvent).
• Flow cytometry was performed according to standard procedures. Dead cells (identified as propidium iodide + or using Fixable Viability Dye eFluor780) and cell aggregates were excluded from all analyses. Fluorescently conjugated mAb were purchased from BD Bio- sciences, eBiosciences, BioLegend or made in-house. Data acquisition was performed on a FACSVerse (BD Biosciences, Franklin Lakes, NJ) and analyzed with FlowJo soft- ware (Tree Star, Ashland, OR). For Genexpression analysis cells were sorted using a FACSAria (BD Biosciences).
• ADCC Antibody dependent cellular cytotoxicity
• ADCC assays were performed in two ways: a) ADCC assays were performed using an NK-92 cell line stably transfected to express the CD 16- 158V allele together with GFP (purchased from Conkwest, San Diego, CA) 24.
• CD4+ target T cells were isolated from peripheral blood of healthy donors using CD4+ T cell isolation kit (Miltenyi Biotec). Cells were stimulated for 2d with CD3/CD28 dynabeads (Life Technologies, Thermo Fisher) and 50 ng/ml rh IL-2 (R&D Systems) at 37 °C. Target cells were pre-incubated with mAB at 0.1-10 Mg/ml for 30min at 4°C prior to mixing with NK cells.
• the cells were incubated for 4h in RPMI 1640 + GlutaMAX medium (Invitrogen) containing 10 mM HEPES buffer, 1 mM sodium Pyruvate and 10% FBS low IgG at a 2:1 effector.target cell ratio. Lysis was determined by flow cytometry.
• the cell suspension was stained with BV510-conjugated anti-CD4 together with 10 nM SYTOX Red dead cell stain (Invitrogen) or Fixable Viability Dye eFluor780 (eBioscience) for 20 min in the dark at 4°C and the cells were then analyzed using a FACSVerse (BD Biosciences), b) Target cells were labelled with calcein AM, followed by the addition of diluting concentra- tions of Ab. Target cells were cocultured with human PBMCs at a 50:1 E:T ratio for 4 h at 37°C.
• % max release (sample/triton treated )*100.
• ADCP Antibody dependent cellular phagocytosis
• Target cells were labelled with 5 mM CFSE for 10 min at room temperature before wash- ing in complete media.
• CFSE-labelled targets were then opsonized with diluting concen- trations of Ab before coculturing at a 1 :5 E:T ratio with BMDMs in 96-well plates for 1 h at 37°C.
• BMDMs were then labelled with anti-F4/80-allophycocyanin for 15 min at room temperature and washed with PBS twice.
• PBMCs were then cultured in a 24-well plate at 1x107 cells/mL as described by Romer ef a/ (51 ) for 48 hours prior to mAb stimulation assays.
• PBMC stimulation round-bottomed 96-well plates were wet-coated with 0.01 ⁇ g/mL of OKT3 antibody (in-house) in PBS for 4 hours after which excess antibody was dis- carded and the plates were washed with PBS.
• 1X105 PBMCs/well were transferred to the plates and stimulated with 5 ⁇ g/mL of test mAb (anti-4-1 BB, anti-OX40 mAb).
• Ligands were added at 5 nM (hox40- L, h41 BB-L; R&D Systems) and the plates were further incubated for 15 minutes. After washing, bound ligand was detected with biotinylated antibodies (anti-hox40-L, anti- h41 BB-L; R&D Systems) followed by Streptavidin-HRP (Jackson ImmunoResearch) with intermediate washing. Super Signal ELISA Pico (Thermo Scientific) was used as sub- strate and the plates were analyzed using Tecan Ultra Microplate reader. Microarrav analysis
• CD4+CD25+ target cells and CD4+CD25- non-target cells were sorted from lymph nodes of tumor-bearing mice (CT26 and TH03).
• CD3- non-target cells were sorted from spleens of healthy C57/BI6 and Balb/c mice.
• CD8+ T cells were isolated from spleens of healthy Balb/c mice.
• RNA from all the samples was prepared with RNA isolation Midi kit from Macherey-Nagel (Dueren, Germany) according to manufactures' instructions. Iso- lated RNA was amplified and prepared for hybridization to the Affymetrix Mouse Gene 2.0 ST Array at Sáne Centre for Integrative Biology at Lund University (SCIBLU), Sweden. Data analysis was performed at SCIBLU according to standard methods. The results of the above assays and the characteristics of the antibodies studied are shown in Figures 12-24.
• a Treg depleting antibody molecule for use in the treatment of cancer wherein the Treg depleting antibody molecule is administered sequentially with an immunostimu- latory antibody molecule with the Treg depleting antibody molecule being administered prior to administration of the immunostimulatory antibody molecule.
• a Treg depleting antibody for use according to embodiment 1 wherein said immunostimulatory antibody molecule is a CD8 activating and/or CD8 boosting antibody molecule.
• a Treg depleting antibody molecule for use according to embodiment 3, where- in the solid tumour is squamous cell carcinoma (SCC), thymoma, neuroblastoma or ovar- ian cancer
• a Treg depleting antibody molecule for use according to any one of the embod- iments 1-8, wherein said Treg depleting antibody molecule is a human lgG1 antibody molecule engineered for improved binding to at least one activatory FcyR.
• TNFRSF tumour necrosis factor receptor superfamily
• a Treg depleting antibody molecule for use according to embodiment 11 wherein said Treg depleting antibody molecule is an anti-4-1 BB monoclonal antibody molecule.
• Treg depleting antibody molecule is selected from the group consisting of antibody mole- cule comprising SEQ. ID. NOs: 1-6, SEQ. ID. NOs: 9-14, SEQ. ID. NOs: 17-22, SEQ. ID. NOs: 25-30, SEQ. ID. NOs: 33-38, SEQ. ID. NOs: 41-46, SEQ. ID. NOs: 49-54, SEQ. ID. NOs: 57-62, SEQ. ID. NOs: 65-70, SEQ. ID. NOs: 153-158, and SEQ. ID. NOs: 163-168.
• a Treg depleting antibody for use according to any one of the embodiments 14-17 wherein the Treg depleting antibody molecule is selected from the group consist- ing of antibody molecule comprising SEQ. ID. NOs: 7 and 8; SEQ. ID. NOs: 15 and 16; SEQ. ID. NOs: 23 and 24; SEQ. ID. NOs: 31 and 32; SEQ. ID. NOs: 39 and 40; and SEQ. ID. NOs: 47 and 48; SEQ. ID. NOs: 55 and 56; SEQ. ID. NOs: 63 and 64, SEQ. ID. NOs: 71 and 72; SEQ. ID. NOs: 159 and160; and SEQ. ID. NOs: 169 and 170.
• a Treg depleting antibody molecule for use according to embodiment 11 wherein said Treg depleting antibody is a human anti-OX40 monoclonal antibody mole- cule.
• a Treg depleting antibody for use according to embodiment 20 or 21 wherein the Treg depleting antibody molecule is selected from the group consisting of antibody molecules comprising a variable heavy chain selected from the group consisting of SEQ. ID. NOs: 79, 87, 95, 103, 11 1 , 119, 127, 135, 143, 151 , and 177.
• the Treg depleting antibody molecule is selected from the group consist- ing of antibody molecules comprising a variable light chain selected from the group con- sisting of SEQ. ID. NOs: 80, 88, 96, 104, 1 12, 120, 128, 136, 144, 152 and 178.
• immunostimulatory antibody molecule is a human lgG2 antibody or a human lgG4 antibody molecule.
• a Treg depleting antibody for use according to embodiment 31 wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecule comprising SEQ. ID. NOs: 1-6, SEQ. ID. NOs: 9-14, SEQ. ID. NOs: 17-22,
• a Treg depleting antibody for use according to embodiment 37 or 38 wherein the immunostimulatory antibody molecule is selected from the group consisting of anti- body molecules comprising a variable heavy chain selected from the group consisting of SEQ. ID. NOs: 79, 87, 95, 103, 111 , 119, 127, 135, 143, 151 and 177.
• the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising a variable light chain selected from the group consisting of SEQ. ID. NOs: 80, 88, 96, 104, 112, 120, 128, 136, 144, 152 and 178.
• the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising SEQ. ID. NOs: 79 and 80; SEQ. ID. NOs: 87 and 88; SEQ. ID. NOs: 95 and 96; SEQ. ID. NOs: 103 and 104; SEQ. ID. NOs: 1 11 and 112; SEQ. ID. NOs: 119 and 120; SEQ. ID. NOs: 127 and 128; SEQ. ID. NOs: 135 and 136; SEQ. ID. NOs: 143 and 144; SEQ. ID. NOs: 151 and 152; and SEQ. ID. NOs: 177-178.
• An anti-4-1 BB antibody molecule selected from the group consisting of anti- body molecules comprising one or more of the CDRs selected from SEQ. ID. NOs: 1-6,9- 14, 17-22, 25-30, 33-38, 41-46, 49-54, 57-62, 65-70, 153-158 and 163-168.
• An anti-4-1BB antibody molecule according to embodiment 44 selected from the group consisting of antibody molecule comprising SEQ. ID. NOs: 1-6, SEQ. ID. NOs:
• An anti-4-1BB antibody molecule according to embodiment 44 or 45 selected from the group consisting of antibody molecule comprising a variable heavy chain se- lected from the group consisting of SEQ. ID. NOs: 7, 15, 23, 31 , 39, 47, 55, 63, 71 , 159, and 169 .
• An anti-4-1 BB antibody molecule according to any one of the embodiments 44-46 selected from the group consisting of antibody molecule comprising a variable light chain selected from the group consisting of SEQ. ID. NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 160, and 170.
• An anti-4-1 BB antibody molecule according to any one of the embodiments 44-47 selected from the group consisting of antibody molecule comprising SEQ. ID. NOs: 7 and 8; SEQ. ID. NOs: 15 and 16; SEQ. ID. NOs: 23 and 24; SEQ. ID. NOs: 31 and 32; SEQ. ID. NOs: 39 and 40; SEQ. ID. NOs: 47 and 48; SEQ. ID. NOs: 55 and 56; SEQ. ID. NOs: 63 and 64; SEQ. ID. NOs: 71 and 72; SEQ. ID. NOs: 159 and 160; and SEQ. ID. NOs: 169-170.
• An anti-4-1 BB antibody molecule according to any one of the embodiments 44-48 selected from the group consisting of a full-length IgG antibody, a Fab, a Fv, an scFv, a Fab', and a (Fab')2.
• An anti-4-1 BB antibody molecule according to any one of the embodiments 44-50, wherein said Treg depleting antibody molecule and/or said immunostimulatory antibody molecule is a human or humanized antibody.
• An anti-OX40 antibody molecule selected from the group consisting of anti- body molecule comprising one or more of the CDRs selected from SEQ. ID. NOs: 73-78, 81-86, 89-94, 97-102 105-110, 1 13-1 18, 121-126, 129-134, 137-142, 145-150, and 171 - 176 .
• An anti- OX40 antibody molecule according to embodiment 52 selected from the group consisting of antibody molecule comprising SEQ. ID. NOs: SEQ. ID. NOs: 73- 78, SEQ. ID. NOs: 81 -86, SEQ. ID. NOs: 89-94, SEQ. ID. NOs: 97-102, SEQ. ID. NOs: 105-1 10, SEQ. ID. NOs: 1 13-1 18, SEQ. ID. NOs: 121 -126, SEQ. ID. NOs: 129-134, SEQ. ID. NOs: 137-142, SEQ. ID. NOs: 145-150, and SEQ. ID. NOs: 171-176.
• An anti- OX40 antibody molecule according to embodiment 52 or 53 selected from the group consisting of antibody molecule comprising a variable heavy chain se- lected from the group consisting of SEQ. ID. NOs: 79, 87, 95, 103, 1 11 , 1 19, 127, 135, 143 151 and 177.
• 52-54 selected from the group consisting of antibody molecules comprising a variable light chain selected from the group consisting of SEQ. ID. NOs: 80, 88, 96, 104, 112, 120, 128, 136, 144, 152 and 178.
• An anti- OX40 antibody molecule according to any one of the embodiments 52-55 selected from the group consisting of antibody molecules comprising SEQ. ID. NOs: 79 and 80; SEQ. ID. NOs: 87 and 88; SEQ. ID. NOs: 95 and 96; SEQ. ID. NOs: 103 and 104; SEQ. ID. NOs: 111 and 112; SEQ. ID. NOs: 119 and 120; SEQ. ID. NOs: 127 and 128; SEQ. ID. NOs: 135 and 136; SEQ. ID. NOs: 143 and 144; SEQ. ID. NOs: 151 and 152; and SEQ. ID. NOs: 177 and 178.
• An anti- OX40 antibody molecule according to any one of the embodiments 52-56, wherein said Treg depleting antibody molecule and/or said immunostimulatory antibody molecule is selected from the group consisting of a full-length IgG antibody, a Fab, a Fv, an scFv, a Fab', and a (Fab')2.
• An anti- OX40 antibody molecule according to any one of the embodiments 52-58, wherein said Treg depleting antibody molecule and/or said immunostimulatory antibody molecule is a human or humanized antibody.
• a vector comprising the nucleic acid according to embodiment 60.
• a host cell comprising the vector according to embodiment 61.
• a pharmaceutical composition comprising an antibody according to any one of the embodiments 44-59
• An antibody or a pharmaceutical composition according to embodiment 66, wherein the solid tumour is selected from the group consisting of sarcomas, carcinomas and lymphomas.
• SCC squamous cell carcinoma
• thymoma thymoma
• neuroblastoma ovarian cancer.
• An antibody according to any one of the embodiments 44-59 or a pharmaceu- tical composition comprising an antibody according to any one of the embodiments 44-51 and an antibody according to any one of the embodiments 52-59.
• SCC squamous cell carcinoma
• thymoma thymoma
• neuroblastoma ovarian cancer.
• Solid tumour is selected from the group consisting of sarcomas, carcinomas and lymphomas.
• Solid tumour is squamous cell carcinoma (SCC), thymoma, neuroblastoma or ovarian cancer.
• SCC squamous cell carcinoma
• thymoma thymoma
• neuroblastoma ovarian cancer.
• a method for treatment of cancer in a subject wherein a Treg depleting anti- body molecule is administered to the subject, and wherein the administration of the Treg depleting antibody molecule is sequentially by administration of an immunostimulatory antibody molecule.
• the solid tumour is squamous cell carcinoma (SCC), thymoma, neuroblastoma or ovarian cancer.
• SCC squamous cell carcinoma
• thymoma thymoma
• neuroblastoma ovarian cancer.
• Treg deplet- ing antibody molecule and/or said immunostimulatory antibody molecule is selected from the group consisting of a full-length IgG antibody, a Fab, a Fv, an scFv, a Fab', and a (Fab') 2 .
• the full-length IgG antibody is select- ed from the group consisting of an lgG1 , lgG2, lgG4, and an Fc-engineered variant thereof.
• Treg deplet- ing antibody molecule and/or said immunostimulatory antibody molecule is a human or humanized antibody.
• Treg deplet- ing antibody molecule is a human lgG1 antibody.
• Treg deplet- ing antibody molecule is a human lgG1 antibody molecule engineered for improved bind- ing to at least one activatory FcyR.
• Treg deplet-ing antibody molecule is selected from antibody molecules binding specifically to a target belonging to the tumour necrosis factor receptor superfamily (TNFRSF).
• TNFRSF tumour necrosis factor receptor superfamily
• Treg depleting antibody mole- cule is an antibody molecule that binds specifically to a target selected from the group consisting of 4-1 BB, OX40, and TNFR2.
• Treg depleting antibody mole- cule is an anti-4-1 BB monoclonal antibody molecule.
• Treg depleting antibody molecule is selected from the group consisting of antibody molecules comprising one or more of the CDRs selected from SEQ. ID. NOs: 1-6, 9-14, 17-22, 25-30, 33-38, 41-46, 49-54, 57- 62, 65-70, 153-158 and 163-168.
• Treg depleting antibody molecule is selected from the group consisting of antibody molecules comprising a varia- ble heavy chain selected from the group consisting of SEQ. ID. NOs: 7, 15, 23, 31 , 39, 47, 55, 63, 71 , 159 and 169.
• any one of the embodiments 91-93, wherein the Treg deplet- ing antibody molecule is selected from the group consisting of antibody molecule com- prising a variable light chain selected from the group consisting of SEQ. ID. NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 160 and 170.
• the Treg deplet- ing antibody molecule is selected from the group consisting of antibody molecule com- prising SEQ. ID. NOs: 7 and 8; SEQ. ID. NOs: 15 and 16; SEQ. ID. NOs: 23 and 24; SEQ. ID. NOs: 31 and 32; SEQ. ID.
• Treg depleting antibody is a human anti-OX40 monoclonal antibody molecule.
• CDRs selected from SEQ. ID. NOs: 73-78, 81-86, 89-94, 97-102 105-110, 113-118, 121- 126, 129-134, 137-142, 145-150, and 171-176.
• the Treg depleting antibody molecule is selected from the group consisting of antibody molecules comprising SEQ. ID. NOs: SEQ. ID. NOs: 73-78, SEQ. ID. NOs: 81-86, SEQ. ID. NOs: 89-94, SEQ. ID. NOs: 97- 102, SEQ. ID. NOs: 105-110, SEQ. ID. NOs: 113-118, SEQ. ID. NOs: 121-126, SEQ. ID. NOs: 129-134, SEQ. ID. NOs: 137-142, SEQ. ID. NOs: 145-150, and SEQ. ID. NOs: 171-176.
• Treg deplet- ing antibody molecule is selected from the group consisting of antibody molecules com- prising a variable heavy chain selected from the group consisting of SEQ. ID. NOs: 79, 87, 95, 103, 111, 1 19, 127, 135, 143, 151 and 177.
• Treg deplet- ing antibody molecule is selected from the group consisting of antibody molecules com- prising a variable light chain selected from the group consisting of SEQ. ID. NOs: 80, 88, 96, 104, 112, 120, 128, 136, 144, 152 and 178.
• the Treg de- pleting antibody molecule is selected from the group consisting of antibody molecules comprising SEQ. ID. NOs: 79 and 80; SEQ. ID. NOs: 87 and 88; SEQ. ID. NOs: 95 and 96; SEQ. ID. NOs: 103 and 104; SEQ. ID. NOs: 111 and 112; SEQ. ID. NOs: 119 and 120; SEQ. ID. NOs: 127 and 128; SEQ. ID. NOs: 135 and 136; SEQ. ID. NOs: 143 and 144; SEQ. ID. NOs: 151 and 152; and SEQ. ID. NOs: 177 and 178.
• Treg de- pleting antibody molecule is selected from antibody molecules binding specifically to a target selected from the group consisting of ICOS, GITR, CTLA-4, CD25 and neuropilin- 1.
• the im- munostimulatory antibody molecule is a human lgG2 antibody or a human lgG4 antibody molecule.
• the im- munostimulatory antibody molecule is an antibody that binds specifically to a target se- lected from the group consisting of 4-1 BB, OX40, ICOS, GITR, CTLA-4 CD25, PD-1 and PDL1.
• the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising one or more of the CDRs selected from SEQ. ID. NOs: 1-6, 9-14, 17-22, 25-30, 33-38, 41-46, 49-54, 57-62, 65-70, 153-158 and 163-168.
• the im- munostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising a variable heavy chain selected from the group consisting of SEQ. ID. NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71 , 159 and 169.
• the im- munostimulatory antibody molecule is selected from the group consisting of antibody molecule comprising a variable light chain selected from the group consisting of SEQ. ID. NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 160 and 170.
• immunostimu- latory antibody molecule is selected from the group consisting of antibody molecule comprising SEQ. ID. NOs: 7 and 8; SEQ. ID. NOs: 15 and 16; SEQ. ID. NOs: 23 and 24; SEQ. ID. NOs: 31 and 32; SEQ. ID. NOs: 39 and 40; or SEQ. ID. NOs: 47 and 48; SEQ. ID. NOs: 55 and 56; SEQ. ID. NOs: 63 and 64; SEQ. ID. NOs: 71 and 72; SEQ. ID. NOs: 159 and 160; and SEQ. ID. NOs: 169 and 170.
• the immunostimulatory antibody molecule is selected from the group consisting of antibody molecule comprising one or more of the CDRs selected from SEQ. ID. NOs: 73-78, 81-86, 89-94, 97-102 105-110, 113-118, 121-126, 129-134, 137-142, 145-150, and 171-176.
• the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising SEQ. ID. NOs: SEQ. ID. NOs: 73-78, SEQ. ID. NOs: 81-86, SEQ. ID. NOs: 89-94, SEQ. ID. NOs: 97-102, SEQ. ID. NOs: 105-110, SEQ. ID. NOs: 113-118, SEQ. ID. NOs: 121-126, SEQ. ID. NOs: 129-134, SEQ. ID. NOs: 137-142, SEQ. ID. NOs: 145-150, and SEQ. ID. NOs: 171-176.
• the im- munostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising a variable heavy chain selected from the group consisting of SEQ. ID. NOs: 79, 87, 95, 103, 1 11 , 119, 127, 135, 143, 151 and 177.
• the im- munostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising a variable light chain selected from the group consisting of SEQ. ID. NOs: 80, 88, 96, 104, 112, 120, 128, 136, 144, 152 and 178.
• the im- munostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising SEQ. ID. NOs: 79 and 80; SEQ. ID. NOs: 87 and 88; SEQ. ID. NOs: 95 and 96; SEQ. ID. NOs: 103 and 104; SEQ. ID. NOs: 11 1 and 112; SEQ. ID. NOs: 119 and 120; SEQ. ID. NOs: 127 and 128; SEQ. ID. NOs: 135 and 136; SEQ. ID. NOs: 143 and 144; SEQ. ID. NOs: 151 and 152; and SEQ. ID. NOs: 177 and 178.
• the immunostimulatory antibody molecule is human anti-PD1 monoclonal antibody molecule, a human anti- PDL1 mono- clonal antibody molecule or a human anti-CTLA-4 monoclonal antibody molecule.
• the immunostimulato- ry antibody molecule is a human anti-PD1 monoclonal antibody molecule selected from the group consisting of nivolumab and pembrolizumab or the anti- PDL1 antibody ate- zolizumab or an anti-CTLA-4 antibody selected from the group consisting of ipilimumab and tremilimumab.
• CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans. Science 331 :1612-1616.
• CD4(+)CD25(+) immunoregulatory T cells gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16:31 1-323.
• Type II Beers, S.A., Chan, C.H., James, S., French, R.R., Attfield, K.E., Brennan, CM., Ahuja, A., Shlomchik, M.J., Cragg, M.S., and Glennie, M.J. 2008.
• Anti-mouse FcgammaRIV antibody 9E9 also blocks FcgammaRIII in vivo. Blood 126:2643-2645.
• OX40 is a potent immune-stimulating target in late-stage cancer patients. Cancer Res 73:7189-7198.
• Combination CTLA-4 blockade and 4-1 BB activation enhances tumor rejection by increasing T-cell infiltration, proliferation, and cytokine production.

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