NOVEL ANTIBODIES AND COMBINED USE OF A TREG DEPLETING ANTIBODY AND AN IMMUNOSTIMULATORY ANTIBODY

FIELD OF THE INVENTION

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. BACKGROUND OF THE INVENTION

Promising clinical results with immunomodulatory mAb have revived the belief that the immune system holds the key to controlling cancer. The classification of these mAb into checkpoint blockers (antagonists) or activators of co-stimulatory molecules (agonists) has recently come into question with the finding that examples of both types may combat tumours through activatory FcyR engagement and depletion of suppressive regulatory T cells (Treg). In contrast to these findings, anti-CD40 mAb depend on inhibi- tory FcyR cross-linking for agonistic immune stimulation. Therefore whilst immunomodu- latory mAb offer considerable promise for cancer immunotherapy the effector mecha- nisms employed by various mAb, and consequently their optimal application, remain to be defined.

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). Recently this binary classification has come into question when the therapeutic activity of anti-CTLA4, anti-GITR and anti-OX40, which all target T cells, was found to involve deletion of suppressive CD4+ T regulatory cells de- pendent on co-engagement of activatory FcyRs (5-7). The activity of the agonist APC- targeting anti-CD40 mAb, in contrast, requires co-engagement of the inhibitory FcyR to facilitate effective mAb cross-linking, which is necessary for CD40 signalling and immune stimulation (8-10). Therefore, whilst immunomodulatory mAb offer considerable promise for cancer immunotherapy, the mechanisms employed depend on both the Fab and Fc regions of the mAb in ways which are ill-defined and which may depend on the cell type being targeted. Understanding the relative importance of T _reg depletion versus direct im- mune-stimulation will be vital to the development and successful translation of immuno- modulatory mAb to patients.

SUMMARY OF THE INVENTION

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. Furthermore, 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.

This was then broadened by demonstration that 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. The research also lead to the development of novel anti-4-1 BB antibodies and novel anti-OX40 antibodies.-

Thus, 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. Thus, 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. NOs: 9-14; 1-6 of the CDRs selected from SEQ. ID. NOs: 17-22; 1-6 of the CDRs selected from SEQ. ID. NOs: 25- 30; 1-6 of the CDRs selected from SEQ. ID. NOs: 33-38; 1-6 of the CDRs selected from SEQ. ID. NOs: 41-46; 1-6 of the CDRs selected from SEQ. ID. NOs: 49-54; 1-6 of the CDRs selected from SEQ. ID. NOs: 57-62; 1-6 of the CDRs selected from SEQ. ID. NOs: 65-70; 1-6 of the CDRs selected from SEQ. ID. NOs: 153-158; or 1-6 of the CDRs se- lected from SEQ. ID. NOs: 163-168.

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. Again "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 sequences. Thus, 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. NOs: 129-134; 1- 6 of the CDRs selected from SEQ. ID. NOs: 137-142; 1-6 of the CDRs selected from SEQ. ID. NOs: 145-150, or 1-6 of the CDRs selected from SEQ. ID. NOs: 171-176.

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.

DETAILED DESCRIPTION OF THE INVENTION

Regulatory T cells, Treg cells, Tregs or T _re gs, (formerly known as suppressor T cells, sometimes also called suppressive regulatory T cells), are a subpopulation of T cells which are capable of suppressing other immune cells in normal and pathological immune settings.

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.

Antibodies are well known to those skilled in the art of immunology and molecular biology. Typically, an antibody comprises two heavy (H) chains and two light (L) chains. Herein, we sometimes refer to this complete antibody molecule as a full-size or full- length antibody. The antibody's heavy chain comprises one variable domain (VH) and three constant domains (CH1 , CH2 and CH3), and 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. Depending on the amino acid sequence of the constant region of their heavy chains, 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.

The term antibody molecule, as used herein, 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. Alternatively, 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. 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. On page 418 (right column - 3 Our Strategy for Design) 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. In Qiu et al., 2007, Nature Biotechnology, 25:921-9 it is demonstrated that a mole- cule consisting of two linked CDRs are capable of binding antigen. Quiocho 1993, Na- ture, 362: 293-4 provides a summary of "minibody" technology. Ladner 2007, Nature Bio- technology, 25:875-7 comments that molecules containing two CDRs are capable of re- taining antigen-binding activity.

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. In 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. In Heap et al., 2005, J. Gen. Vi- rol., 86: 1791-1800 it is reported that a "micro-antibody" (a molecule containing a single CDR) is capable of binding antigen and it is shown that a cyclic peptide from an anti-HIV antibody has antigen-binding activity and function. In 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.

Thus, 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.

Thus, by the term "antibody molecule", as used herein, we include all types of an- tibody molecules and functional fragments thereof and derivatives thereof, including: monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly pro- duced 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.

Further, the term "antibody molecule", as used herein, includes all classes of an- tibody molecules and functional fragments, including: IgG, lgG1 , lgG2, lgG3, lgG4, IgA, IgM, IgD, and IgE.

In some embodiments, 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. As such, whereas the mouse lgG2a is the preferred isotype for deletion in the mouse, human lgG1 is a preferred isotype for deletion in human. Con- versely, it is known that optimal co-stimulation of TNFR superfamily agonist receptors e.g. 4-1 BB, ox40, TNFRII, CD40 depends on antibody engagement of the inhibitory FcyRII. In the mouse 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).

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.

Accordingly, we include that an antibody molecule of the invention or an antibody molecule used in accordance with the invention (for example, a monoclonal antibody molecule, and/or polyclonal antibody molecule, and/or bi-specific antibody molecule) comprises a detectable moiety and/or a cytotoxic moiety.

By "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.

By "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.

We further include that the antibody molecule may be in an isolated form and/or purified form, and/or may be PEGylated.

As discussed above, the CDRs of an antibody bind to the antibody target. The assignment of amino acids to each CDR described herein is in accordance with the defi- nitions according to Kabat EA et al. 1991 , In "Sequences of Proteins of Immulogical In- terest" Fifth Edition, NIH Publication No. 91-3242, pp xv- xvii.

As the skilled person would be aware, other methods also exist for assigning amino acids to each CDR. For example, the International ImMunoGeneTics information system (IMGT(R)) (http://www.imgt.org/ and Lefranc and Lefranc "The Immunoglobulin FactsBook" published by Academic Press, 2001 ).

In a further embodiment, 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. By "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.

For example, 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%).

Competitive binding may be determined by methods well known to those skilled in the art, such as Enzyme-linked immunosorbent assay (ELISA).

ELISA assays can be used to evaluate epitope-modifying or blocking antibodies.

Additional methods suitable for identifying competing antibodies are disclosed in Antibodies: A Laboratory Manual, Harlow & Lane, which is incorporated herein by reference (for example, see pages 567 to 569, 574 to 576, 583 and 590 to 612, 1988, CSHL, NY, ISBN 0-87969-314-2).

It is well known that 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.

By "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. By "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.

We also include that the cell surface antigen may be connected to the cell mem- brane, but may only be transiently associated with the cell membrane. By "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.

We further include that 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.

Methods of assessing protein binding are known to the person skilled in biochem- istry and immunology. It would be appreciated by the skilled person that those methods could be used to assess binding of an antibody to a target and/or binding of the Fc do- main of an antibody to an Fc receptor; as well as the relative strength, or the specificity, or the inhibition, or prevention, or reduction in those interactions. Examples of methods that may be used to assess protein binding are, for example, immunoassays, BIAcore, western blots, radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISAs) (See Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 (1989) for a discussion regarding antibody specificity).

Accordingly, by "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.

We also include the meaning that 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.

Additionally, we include the meaning that 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 .

As used herein, the term 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. Thus, 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. In some embodiments, 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.

In some embodiments, the Treg depleting antibody does not have any im- munostimulatory effects in addition to the Treg depleting effects. In some embodiments, 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.

To decide whether an antibody is a Treg depleting antibody in the meaning of the present invention or not, it is possible to use an in vitro antibody-dependent cellular cyto- toxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) assay.

An ADCC assay may be done by labelling target cells with calcein AM, followed by the addition of diluting concentrations of Ab. Target cells is then cocultured with hu- man PBMCs at a 50:1 E:T ratio for 4 h at 37°C. The plate is centrifuged at 400 3 g for 5 min to pellet the cells, and the supernatant is transferred to a white 96-well plate. Calcein release is measured using a Varioskan (Thermo Scientific) using an excitation wave- length of 485 nm and emission wavelength, 530 nm. The percentage of maximal release is calculated as follows: % max release = (sample/triton treated) ^* 100.

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.

To decide whether an antibody has immunostimulatory effects, it is possible to use an in vitro agonism assay. For such an assay the following generic method may be used. Cell culture is in RPMI 1640 media (Gibco™) 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. For PBMC stimulation, round-bottomed 96-well plates are wet-coated with 0.01 μg/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 μg/mL of test mAb. On day 4 or day 5 post-stimulation, cells are labelled with anti-CD8- APC (BioLegend), and anti-CD4-PE (in-house) and proliferation is assessed by CFSE dilution on a FACSCalibur (BD Biosciences).

To decide whether sequential treatment of a Treg deleting and immunostimulato- ry antibody results in improved therapeutic activity, in vivo assays with immune compe- tent animals bearing tumours and expressing activatory and inhibitory Fc gamma recep- tors can be used. For such an assay, one described in the examples below may be used, for example as illustrated in Figures 1 a and 4c.

As used herein, the term immunostimulatory antibody, or immunostimulating anti- body, 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. 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. Alternative- ly, or in addition, 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.

We show herein that different antibodies to different targets of immunostimulatory or co-inhibitory nature, which act in an Fc:FcyR dependent or independent manner can be used for immune stimulation. 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.

To decide whether an antibody is an immunostimulatory antibody in the meaning of the present invention, it is possible to use an in vitro assay that demonstrates T cell proliferation in response ^' to mAb. The assays described above may be used for this pur- poses.

To decide whether an antibody lacks Treg depleting effects or has a poor Treg depleting effect, it is possible to use in vitro phagocytosis assays or in vivo depletion studies. The assays described in the examples may be used for this purposes. For ex- ample, groups of test mice receive tumour on day 0. When tumours are palpable (or at appropriate stage for tumour model) mice receive the mAb i.v. followed by 3 further ad- ministrations i.p. every other day (200 μg 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).

In some embodiments, the Treg depleting antibody is a human antibody.

In some embodiments, the Treg depleting antibody is a humanized antibody.

In some embodiments, 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.

For example, 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.

Furthermore, 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. Thus, in some embodiments the immunostimulatory an- tibody is a human lgG2 antibody. In some embodiments the immunostimulatory antibody is a human lgG2b antibody. In some embodiments 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.

In some embodiments, 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.

In some embodiments of the present invention, 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. Whether 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.

We found that although a mlgG1 isotype mAb exerted superior agonistic activity and direct immune-stimulation of CD8+ T cells compared with a mlgG2a version of the same specificity, in established solid tumour settings the mlgG2a mAb provided optimal therapeutic activity. We found that the potency of the mlgG2a mAb is due to intratumour- al Treg depletion. However when depletion was prevented, in mice lacking activatory FcyR, the therapeutic potential of the mlgG2a was retained. Under these conditions mlgG2a was converted to an agonist by engaging the inhibitory FcyRIIB. Further to this we established that depletion and agonism are competing mechanisms and that engag- ing both simultaneously led to reduced efficacy. This blunting of activity could be over- come through sequential administration of Treg depleting and then immunostimulatory isotypes or through Fc engineering to produce a dual-activity anti-4-1 BB mAb posessing optimal FcyR depleting capacity together with FcyR independent agonism. Together, these results demonstrate that immunomodulatory mAb with the same target specificity can utilise different mechanisms to mediate therapy and that their optimal use depends on both isotype, the local FcyR repertoire, abundance and function of immune suppres- sor and effector cells and their relative and absolute expression of target, in the tumor microenvironment. Importantly, our results further demonstrate that temporal administra- tion of immunomodulatory mAb, with complementary, but competing, mechanisms-of- action may be used to optimize outcome and further that it is possible through mAb engi- neering to generate a single agent capable of harnessing multiple mechanisms to deliver enhanced therapeutic efficacy. These results have implications for the administration of existing and in-development immunomodulatory mAb, and for the design of next genera- tion immunomodulatory antibodies.

In some embodiments, the Treg depleting antibody molecule is an anti-4-1 BB an- tibody molecule selected from the group presented in Table 1 below.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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 ,

159, 161 and 169.

In some embodiments, 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,

160, 162 and 170.

In some embodiments, 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.

In some embodiments, the immunostimulatory antibody molecule is an anti-4- 1 BB antibody molecule selected from the group presented in Table 1 below.

In some embodiments, 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.

In some embodiments, 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-

54, SEQ ID. Nos: 57-62, SEQ ID. Nos: 65-70, 153-158 and SEQ ID. Nos: 163-168.

In some embodiments, 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,

55, 63, 71 , 159, 161 and 169.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the Treg depleting antibody molecule is an anti-OX40 an- tibody molecule selected from the group presented in Table 2 below.

In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the immunostimulatory antibody molecule is an anti-OX40 antibody molecule selected from the group presented in Table 2 below.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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,

135, 143, 151 and 177.

In some embodiments, 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,

136, 144, 152 and 178.

In some embodiments, 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.

Table 2: OX40 antibodies

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the immunostimulatory antibody is an anti-PDL1 antibody, preferably a human anti-PDL1 antibody. The anti-PDL1 antibody may be atezolizumab.

In some embodiments, 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. This means that the Treg deplet- ing antibody is administered to the tumour first in order to achieve the Treg depleting ef- fect. Once the Treg depleting effect is manifested, the immunostimulatory antibody is administered. This sequential administration may be achieved by temporal separation of the two antibodies. Alternatively, or in combination with the first option, 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.

It would be known to the person skilled in medicine, that 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.

Accordingly, we include that the 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.

We also include that the 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.

Extemporaneous injection solutions and suspensions may be prepared from ster- ile powders, and/or granules, and/or tablets of the kind previously described.

For parenteral administration to human patients, 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. Typically, 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. In a preferred embodiment, 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.

Generally, in humans, oral or parenteral administration of the composition, and/or antibody, and/or agent, and/or medicament of the invention is the preferred route, being the most convenient. For veterinary use, the composition, 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. Thus, 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). Preferably, 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. 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. In one embodiment, 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.

We include that the subject could be mammalian or non-mammalian. Preferably, 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. Most preferably, the mammalian subject is a human.

By "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.

It would be readily apparent to the person skilled in medicine what the 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. By "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.

In some embodiments the cancer to be treated in accordance with the present invention is a solid tumour.

In some embodiments, the cancer is selected from the group consisting of sar- comas, carcinomas and lymphomas.

In some embodiments, the cancer is selected from the group consisting of squa- mous cell carcinoma (SCC), thymoma, neuroblastoma or ovarian cancer.

Each one of the above described cancers is well-known, and the symptoms and cancer diagnostic markers are well described, as are the therapeutic agents used to treat those cancers. Accordingly, the symptoms, cancer diagnostic markers, and therapeutic agents used to treat the above mentioned cancer types would be known to those skilled in medicine.

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.

By "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.

It is known that 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.

By "histological examination", 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.

It is well known that 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).

By "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, and/or (8:14) translo- cations, and/or (8:v) translocations, and/or t(11 :14) and (q13:q32) translocations.

It is known that subjects with cancer exhibit certain physical symptoms, which are often as a result of the burden of the cancer on the body. Those symptoms often reoccur in the same cancer, and so can be characteristic of the diagnosis, and/or prognosis, and/or progression of the disease. A person skilled in medicine would understand which physical symptoms are associated with which cancers, and how assessing those physi- cal systems can correlate to the diagnosis, and/or prognosis, and/or progression of the disease. By "physical symptoms", we include hepatomegaly, and/or splenomegaly.

BRIEF DESCRIPTION OF THE DRAWINGS

In the examples below, reference is made to the following figures:

Figure 1. Anti-4-1BB mlgG2a mAb, but not mlgG1, confer survival benefit in multiple cancer models. 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 μg final dose). Fig. 1 B: Groups of A/J mice were challenged with 2 x 10 ⁶ NXS2 cells s.c. and received 200 μg anti-4-1 BB mAb or isotype control mAb i.p. when tumour was palpable. A second dose of 200 μg was given 3 days later. In both experimental models 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. Data represent examples of at least 2 in- dependent experiments where n = 5 mice per group.

Figure 2. Anti-4-1BB migG1 exerts agonist activity in vitro and in vivo. Fig. 2A: Splenocytes from Foxp3-GFP mice either sorted to remove GFP+ cells (-T _reg ) or not (+Treg) were incubated with 0.1 μg/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. 2B Spleno- cytes from C57BL/6 mice were similarly incubated with 0.1 μg/ml anti-CD3 and the indi- cated concentrations of anti-4-1 BB mlgG1 , mlgG2a or a 1 :1 mix of the two prior to as- sessment of [ ³ H]-thymidine incorporation. Data in Fig. 2A and Fig. 2B show mean (+/- SEM) counts per minute of triplicate wells. Fig. 2C: Groups of mice received 5 mg OVA and 200 μg anti-4-1 BB mlgG1 or mlgG2a i.p. on day O. SIINFEKL-specific T-cell re- sponses in peripheral blood were quantified by flow cytometry and expressed as a % of total CD8+ cells. Data are from three separate experiments, and show time course of response (mean ± SEM, 6 mice per group) and peak of response (mean and individual responses, 9 mice per group, ^* p=0.023). Fig. 2D: Groups of A/J mice were challenged with 2 x 10 ⁶ NXS2 cells s.c. on day 0 and received 200 μg 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 μg) was given on day 6. Data in Fig. 2B and Fig. 2C represent examples of at least 2 experiments where n = 5 mice per group.

Figure 3. Mouse and human tumour resident Treg ceils preferentially ex- press 4-1BB. Fig. 3A: Immunofluorescence microscopy image showing 4-1 BB express- ing intratumoural Foxp3+ Treg in CT26 tumour (left panel). Scale bar = 50 μΜ. Flow cy- tometry histograms demonstrating 4-1 BB expression on T cell subsets from TILs and splenocytes (right panels). 4-1 BB expression (black line) on CD4+Foxp3+ (inset top panels), CD4+ Foxp3- (inset middle panels) and CD8+ T cells (inset bottom panels), against isotype control (grey line). Cells were isolated from mice bearing CT26 tumour. Fig. 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. Lower panel shows 4-1 BB expression on T cell subsets from different tissue samples. Data points represent individual patients/donors with n=11 for healthy PBMCs, n=20 for ascites, n=9 for tumour and n=5 for patient blood. 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. 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.

Figure 4. The primary mechanism of anti-4-1BB mAb therapy in solid tu- mours is dependent on antibody isotype and FcyR availability. Fig. 4A: Groups of 3- 4 WT, FcyRIIB KO or γ 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. Data show the frequency of Foxp3+ cells within the CD4+ population in the tumour (left panel) or in matched spleens (right panel). Data are representative of two independent experiments. 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, γ chain KO, or FcyR null mice (γ chain KO x FcyRIIB KO), γ 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). Tumour growth was monitored and mice culled when mean tumour area ex- ceeded 225 mm2. Data are expressed as tumour area (mm2) on the days after tumour challenge as indicated, each line represents an individual mouse. Panels on the right show percentage survival to the humane end-point. Data represent examples of at least 2 experiments where n = 5 mice per group. 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. (E) (left panel) CFSE labelled target human T cells opsonised with anti-human 4- 1 BB hlgG1 mAb clones SAP3-6, BI5-B02 (also denoted 005-BI02) or control were co- cultured with hMDM and then assessed for ADCP. (right hand panel) Level of phagocy- tosis plotted in relation to 4-1 BB expression level as determined by flow cytometry. In all cases phagocytosis is plotted as % of double positive macrophages.

Figure 5. Scheduled administration of disparate anti-4-1 BB or scheduled combination with anti-PD-1 mAb enhances anti-tumour activity. 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 μg final dose). For scheduled administration mlgG2a was given i.v. and then mlgGI given i.p. 4 days later. Tumour growth was monitored and mice culled when mean tumour area exceeded 225 mm ² . Data are expressed as the average tumour area (mm2) on the days after tumour challenge as indicated. Data presented is com- bined from two independent experiments where n = 10 mice per group. 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. Tumour growth was monitored and data plotted as in Fig. 5A. Data represent examples of at least 2 inde- pendent experiments where n = 4 or 5 mice per group. 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. 6B: Splenocytes from C57BI/6 mice were incubated with 0.01 μg/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. Fig. 6D: Groups of age and sex matched C57BI/6 mice were challenged with 5 x 105 EG7 s.c. on day 0. On days 3, 5 and 7 mice received 200 μg mAb or PBS control i.p. as indicated. On day 20 tumours were harvested and TIL enu- merated by flow cytometry, n = 4 mice per group. (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. Figure 7. Anti-4-1 BB mAb characterisation. 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. 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.

Figure 8. 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 μg 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 μg. 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.

(n=5/group)

Figure 9. Primary in vivo expansion of OT-I cells in response to OVA and anti-4-1 BB mAb. 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 μg 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. 9B: Groups of 5 C57BL/6 mice were injected with 2.5 x 10 ⁵ B16/BL6 cells i.d. on day 0 prior to receiving 1 x 10 ⁶ irradiated FVAX cells i.d. on the opposite flank on days 3, 6 and 9. Concurrent with FVAX injection mice received either PBS, 100 μg anti-CTLA-4 (clone 9D9) or anti-CTLA-4 and 300 μg anti-4-1 BB antibodies as indicat- ed i.p. Percentage survival to the humane end point is shown.

Figure 10. Anti-PD-1 mAb characterisation. 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. 10B: Analysis demonstrating that anti-PD-1 mAb bind to PD-1 and block PD- L1 binding. Anti-PD-1 rlgG1 (solid black), rlgG1 deglycosylated (solid grey) or buffer (dashed black) was passed over recombinant PD-1 -his (R&D Systems) (2000 RU) cap- tured onto a CM5 chip with an anti-histidine mAb. At the timepoint indicated by the arrow recombinant PD-L1-Fc (R&D Systems) was passed over to demonstrate binding to PD-1 and blockade by anti-PD-1.

Figure 11. Anti-4-1 BB mlgG2a/h2B FcyR binding. Surface plasmon resonance analysis of anti-4-1 BB (clone LOB12.0) mlgG2a and mlgG2a/h2B binding to mouse FcyRI, I IB, III and IV. 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. EXAMPLES

Specific, non-limiting examples which embody certain aspects of the invention will now be described. EXAMPLES RELATING TO SEQUENTIAL ADMINISTRATION

RESULTS

Therapeutic activity of anti-4-1BB mAb is determined by isotype

We and others have previously established the dependence of immunostimulato- ry mAb activity targeting TNFR superfamily members on cross-linking provided by the inhibitory FcyRIIB. To establish if this requirement similarly applied to anti-4-1 BB we generated mlgG1 and mlgG2a chimeric versions of the rlgG2a anti-4-1BB mAb

(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. Analysis by surface plasmon resonance and flow cytometry established that these mAb possessed an expected mFcyR binding profile, with mlgG2a having a high activa- tory to inhibitory FcyR ratio (A:l) and conversely mlgG1 a low A:l (Figure 7A, (19, 20)); both mAb retained equivalent 4-1 BB specificity and binding (Figure 7B and 7C). We then assessed the therapeutic potential of these mouse anti-4-1 BB mAb in three different es- tablished solid tumour models using the CT26 colon carcinoma (Figure 1A) and NXS2 neuroblastoma (Figure 1 B). In marked contrast to our studies with anti-CD40 (18, 19, 21 ) and published reports with other agonistic anti-TNFR superfamily mAb targeting DR5 (22, 23), for anti-4-1 BB, high A:l ratio mlgG2a mAb gave considerable therapeutic benefit (80% long term survival in all models) while the low A:l ratio mlgG1 version was largely ineffective (0-20% survival). Notably, although it was the mlgG2a mAb which was protec- tive in these settings its therapeutic effect was still dependent upon CD8+ T cells (Figure 8) and led to long term productive anti-tumour immunity, as determined by tumour re- challenge experiments. These results suggest that the protective effect of mlgG2a anti-4- 1 BB is mediated through an adaptive anti-tumoural immune response, but that this mAb utilises molecular mechanisms occurring in an FcyRIIB-independent manner, in contrast to anti-CD40 mAb. Immunostimulatory activity of anti-4-1 BB is optimal with mouse lgG1 isotype

Given the results obtained in our tumour models and previous studies demon- strating a critical role for CD8+ T cells in mediating the effects of anti-4-1 BB imAb, we sought to establish the isotype-dependence of anti-4-1 BB mAb activity on T cell popula- tions in vitro and in vivo. Using an in vitro T cell co-stimulation assay (Figure 2A) only mlgG1 , but not mlgG2a, (same antibodies as used above) demonstrated agonistic activi- ty. This is in keeping with other published results (18, 19). The costimulatory activity of mlgG1 was independent of Treg cells in the T cell culture assay, suggesting that this anti- 4-1 BB mAb mediates its effects through targeting 4-1 BB on effector T cells. Finally, the costimulatory activity of mlgG1 was abolished by the addition of mlgG2a, demonstrating that both mAb variants bind with similar avidity and compete for binding to 4-1 BB on ef- fector T cells (Figure 2B). These in vitro results were confirmed using an in vivo immun- isation model with the model antigen OVA in both an endogenous (Figure 2C) and an OT1 T cell transfer setting (Figure 8A).ln this context the superior agonistic activity of mlgG1 was dependent upon the inhibitory FcyRIIB (Figure 2C, Figure 9A) as previously shown for anti-CD40 mAb (18, 19, 24, 25). Finally, in two immunisation models (NXS2 peptide and B16-sFlt3L-lg; Figure 2D and Figure 9B, respectively) the mlgG1 and mlgG2a isotype antibodies were equally therapeutic (Figure 2D and Figure 9B). Of note, the efficacy of mlgG1 , but not lgG2a, was abolished in the absence of vaccination, con- firming that mlgG1 , but not mlgG2a, is operating through a mechanism dependent on immune activation, and likely FcyRIIB cross-linking (Figure 2D).

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). We therefore examined the possibility that anti-4-1 BB mlgG2a, which possesses a high A: I FcyR binding profile could potentiate an anti-tumour response via deletion of Treg cells. We began by confirming the presence of 4-1 BB on Treg cells in two murine tumour models CT26 (Figure 3A) and NXS2 and found that 4- 1 BB was expressed on a substantial proportion of tumour-infiltrating Tregs and only a small minority of effector T cells. Furthermore, only a small fraction of splenic Treg cells expressed 4-1 BB. In order to confirm that these observations were potentially translata- ble to humans at the protein level, we determined by flow cytometry whether 4-1 BB was present on intratumoural Tregs in patients with ovarian cancer and squamous cell carci- noma. It can be seen in Figure 3B and Figure 3C that 4-1 BB was found on CD4+Foxp3+ Treg cells but not on effector CD4+ or CD8+ T cells in tumours and that 4-1 BB was ex- pressed at lower levels on Tregs isolated from healthy PBMC, matched blood, ascites or normal skin. The role of FcyR in mediating the anti-tumour activity of anti-4-1 BB mAb

Having established that intratumoural Treg express 4-1 BB we used the CT26 tu- mour model to determine the potential role for and relative depleting capacity of anti-4- 1 BB mAb. Our data demonstrate that in wild-type mice the mlgG2a mAb efficiently delet- ed intra-tumoural Tregs, whilst the mlgG1 variant was ineffective (same antibodies as used above(Figure 4A). This depletion effect was restricted to the tumour and dependent upon expression of the common γ chain, a crucial component of activatory FcyR com- plexes. Furthermore, in FcyRMB knockout mice the depleting activity of the mlgG1 mAb was enhanced to similar levels as to that of the mlgG2a, demonstrating that the depletion efficiency of these mAb is intimately linked to their FcyR A.I ratio and that depletion po- tency can be manipulated through changes in FcyR expression. We next sought to de- termine if we could observe activation of tumour infiltrating CD8 T cells in these mice and observed that despite the lack of efficacy of the migG1 mAb in WT tumour bearing mice administration of anti-4-1BB mlgG1 mAb led to a clear and significant increase in prolif- eration of CD8 T cells as monitored by Ki-67 positivity (Figure 4B). While the mlgG2a isotype also induced an increase in CD8 activation this was significantly less than the mlgG1. These data likely demonstrate the dominant role of Treg in the CT26 tumour model and suggest that inducing CD8 responses with mlgG1 in wild-type mice without removing Treg suppression is insufficient to induce a productive anti-tumour response.

As 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. Surprisingly, however, in the CT26 tumour model (Figure 4C), 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. In contrast to the minimal ef- fects on anti-4-1 BB mlgG2a efficacy, there was a substantial improvement in the ability of anti-4-1 BB mlgG1 to promote anti-tumour immunity in the absence of activatory FcyR (2/5 in CT26). Given the FcyR binding profiles demonstrated for mlgG1 and mlgG2a mAb (Figure 7), these findings suggest that in the absence of competitive binding of mAb to activatory FcyR, there is productive engagement of FCYRIIB by both mlgG1 and mlgG2a, thus allowing optimal cross-linking of mAb to deliver costimulation. This notion is further supported by the observed T cell activation of mlgG2a in the FcRg KO animals in the OVA model (Fig 2C). In keeping with the enhanced Treg depleting activity of mlgG1 in FcyRIIB KO mice (Figure 4A) when therapies were carried out in FcyRIIB KO mice the mlgG1 isotype mAb demonstrated enhanced and equivalent activity to mlgG2a. These results coupled with the observation that in the absence of all FcyR, neither mAb produced any therapeutic activity (Figure 4C) support the importance of efficient, non- competing FcyR engagement for optimal in vivo activity.

We next sought to formally demonstrate the depleting capacity of anti-4-1 BB mAb using both mouse and human targets and effectors in vitro. Using WT mouse bone mar- row derived macrophages and 4-1 BB expressing T cell targets we observed that mlgG2a induced effective phagocytosis of target cells and the mlgG1 mAb was ineffective (Figure 4D). In agreement with our in vivo depletion results (Figure 4B) and therapeutic respons- es (Figure 4C), when FcyRIIB KO macrophages were used as effectors a significant in- crease in mlgG1 mediated phagocytosis was observed in line with levels obtained with mlgG2a and WT effectors. We then confirmed the translational potential of our findings in a fully human system using human targets and monocyte derived macrophage effectors. In this system we found that two different hulgGI anti-human 4-1 BB clones (SAP3-6 and 005-B02) could mediate effective phagocytic clearance (Figure 4E). Finally, we sought to confirm that it was the level of 4-1 BB expression rather than the cell type per se that dic- tated the efficacy of depletion. We did this using both human (Figure 4E) and mouse (da- ta not shown) in vitro generated macrophages and target cells with varying levels of 4- 1 BB expression and found a direct correlation between 4-1 BB expression and the effi- ciency of target cell depletion. This supports the notion that it is the high level of 4-1 BB expression on Treg in the tumour microenvironment that makes them a good target and that lower expressing CD8 cells are likely spared.

Scheduled administration of Treg depleting and immunomostimulatory mAb leads to enhanced anti-cancer therapy

Our results demonstrating that the therapeutic activity of isotype variants of anti- 4-BB mAb occurs via different mechanisms indicated a potential for combined use to en- hance therapeutic effects. However, since depletion of Treg cells (mlgG2a) and delivery of costimulation (mlgG1) both relied on engagement of FcyRs, and appeared to do so in a competitive manner, we speculated that sequential rather than concurrent administra- tion might be optimal. We therefore compared the therapeutic effect following concurrent and sequential administration of anti-4-1 BB mlgG2a and mlgG1 mAb. (same antibodies as used above) As previously observed the mlgG2a, but not mlgG1 , variant was active as single agent. Concurrent administration of mlgG2a and mlgG1 anti-4-1 BB mAb re- sulted in reduced therapeutic efficacy as indicated by increased tumour size (Figure 5A) and reduced number of tumour free mice (Figure 5B) compared to mlgG2a single agent treatment. In marked contrast, sequential delivery of first mlgG2a, to delete Treg cells, followed by agonistic mlgG1 to provide costimulation, improved both tumour growth inhi- bition and enhance the number of tumour free mice compared with single agent treat- ment with either antibody variant alone (Figure 5A and B). These findings demonstrated that therapeutic efficacy of FcyR-dependent immunomodulatory mAb can be optimised by sequential administration. Importantly, our findings also indicated that Treg depletion may have broad utility to improve on immune-stimulatory antibodies beyond anti-4-1 BB, especially when used in an FcgR-non competing manner.

Next, we therefore investigated the therapeutic potential of combining Treg de- pleting anti-4-1 BB with clinically validated immune agonist anti-PD1. We reduced the dosing of mAb to obtain suboptimal monotherapy and then combined isotype optimal an- ti-4-1 BB mlgG2a sequentially with an FcyR null binding deglycosylated (31) variant anti- PD-1 blocking antibody (Figures 10A and 10B) mimicking the lack of/poor FcyR- engagement of clinically validated anti-PD-1 antibodies nivolumab and pembrolizumab. This combination produced a significant increase in therapy leading to 80% long term responders compared with 20 - 25 % with monotherapies (Figure 5C). Notably, the combination of suboptimal isotypes of mAb did not lead to any enhancement of respons- es demonstrating that for optimal combination therapies it is vital to understand the iso- type and scheduling requirement of each component of any combination.

Anti-4-1 BB mlgG2a/h2B engineered to possess dual activity delivers augmented cancer therapy

Having demonstrated that better responses can be achieved through optimal combination of Treg depletion and agonism /release of immune suppression than through either mechanism alone we sought to demonstrate that it is possible to deliver these multiple mechanisms through the engineering of a single mAb. Given our observa- tions that mAb mediated Treg depletion and immunostimulatory agonism have differen- tial and competing FcyR requirements we sought to capitalise on our previous finding that the human lgG2 hinge region is able to provide anti-TNFR superfamily member mAb with FcyR independent agonistic properties (25). Here, we cloned the human lgG2 re- gion into the murine mlgG2a constant regions of anti-4-1 BB as detailed previously (25) and then skewed the hinge to the agonism enhanced 'B' form to make anti-4-1 BB mlgG2a/h2B (Figure 6A). The nucleotide sequences encoding LOB12.0 mKappa is shown in SEQ. ID. NO: 183, and the corresponding amino acid sequence is shown in SEQ ID NO: 184. The nucleotide sequences encoding LOB12.0 HulgGhinge2.mlgG2aFc (mlgG2a/h2B) is shown in SEQ. ID. NO: 185, and the corresponding amino acid se- quence is shown in SEQ ID NO: 186. The nucleotide sequences encoding LOB12 hu- man kappa is shown in SEQ. ID. NO: 187, and the corresponding amino acid sequence is shown in SEQ ID NO: 188. When tested in vitro for T cell proliferation mlgG2a/h2B had significantly enhanced agonistic activity compared to mlgG2a parent (Figure 6B), despite an unchanged FcyR binding profile (Figure 11 ). Despite this enhanced agonistic activity the engineered mAb also retained strong phagocytic potential in vitro using BMDM and 4-1 BB expressing target cells (Figure 6C) demonstrating that we had gener- ated a reagent with both agonistic and depleting potential without competing FcyR re- quirements. Finally, we compared this mAb with the parental mlgG2a in the EG7 tumour model and found that the dual activity mlgG2a/h2B had equally potent Treg depleting capacity to the parental mlgG2a but now also possessed marked CD8 stimulating capa- bility leading to an enhanced CD8/Treg ratio (Figure 6D). This enhanced dual activity mAb also demonstrated greater therapeutic potential curing 100% of mice treated com- pared to 60% with the standard mlgG2a (Figure 6E). These data demonstrate for the first time that a single mAb can be engineered to optimally mediate depletion and agonism and through this enhanced dual activity deliver better therapy. DISCUSSION

It has been established in a variety of in vitro and in vivo models that anti-TNFR superfamily mAb require efficient cross-linking to induce their agonistic effects and for most mAb this is best provided by inhibitory FcyR engagement (8, 9, 18, 19, 23, , 32, 33). Despite these findings, it is not clear whether such agonistic engagement is the main mechanism of action that contributes to the therapeutic activity of these mAb in a solid tumour setting. We have investigated this question using mlgG2a and mlgG1 isotype anti-4-1 BB mAb, which have a high and low activatory:inhibitory FcyR ratio, and conse- quently good depleting and agonistic potential, respectively (10).

We found using two different solid tumour models in different wild type strains of mice that mlgG2a mAb produced substantial therapeutic effects whereas mlgG1 was largely ineffective (Figure 1A, Figure 1 B). These results are in marked contrast to the ag- onistic activity of these mAb on CD8+ T cells, where mlgG1 was more effective (Figure 2A, Figure 2B and Figure 9A). Notably in contrast to previous publications with anti-CD40 mAb, anti-4-1 BB mlgG2a is not without T cell agonistic activity in vivo, suggesting that 4- 1 BB may possess a lower crosslinking threshold for signalling than CD40 (18, 19). The mlgG2a dependent therapeutic activity displayed in these different 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).

Given the recent findings that mAb targeting CTLA-4, OX40 and GITR are able to mediate therapy through intratumoural T _reg depletion (5-7) we examined 4-1 BB expres- sion in these models and found that 4-1 BB was upregulated specifically on intra-tumoural Treg cells (Figure 3A). Importantly for the potential translation of these findings to hu- mans we found that 4-1 BB demonstrated restricted expression on intratumoural Treg cells in patients with both ovarian cancer and squamous cell carcinoma (Figure 3B and Figure 3C, respectively) supporting the therapeutic potential of this mechanistic approach in patients. Furthermore, mlgG2a depleted this suppressive population in an activatory FcyR dependant manner, whilst mlgG1 had little effect (Figure 4A). In keeping with the requirement for a high activatory to inhibitory FcR engagement for productive depletion, when these experiments were carried out in FcyRIIB KO mice mlgG1 became compara- ble to mlgG2a for its depleting capacity both in vivo (Figure 4A) and in vitro (Figure 4D).

Although 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. We tested this potential using the CT26 model and found that in the absence of ac- tivatory FcyR, mlgG1 mAb did indeed become therapeutic (Figure 4C). It was also nota- ble, and in keeping with our contention that 4-1 BB has a relatively low threshold for cross-linking in vivo, that in the absence of activatory FcyR mlgG2a also retained activity. In agreement with the enhanced Treg depleting activity of mlgG1 in FcyRIIB KO mice (Figure 4A and Figure 4C) when therapies we carried out in FcyRIIB KO mice the mlgG1 isotype mAb demonstrated enhanced and equivalent activity to mlgG2a. Furthermore, neither mAb was able to protect mice in the absence of FcyR (Figure 4C) demonstrating both agonistic and depletion mechanisms to be FcyR dependent.

The fact that 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. These observations sup- port the hypothesis that simultaneous engagement of these two FcyR dependent mech- anisms through the engagement of a single antigen may not be possible but that tem- poral, as shown here, or potentially spatial (intratumour versus systemic) separation of these mAb may facilitate their combined efficacy.

In order to further demonstrate the likely isotype and scheduling requirements for anti-4-1 BB mAb in patients, where clinical results suggest combination approaches are likely to be required, we investigated different mAb combinations with anti-PD-1. In this setting we found that isotype optimal versions of both anti-4-1 BB and anti-PD-1 produced a significant combination effect leading to cures in 80% of mice treated in marked con- trast to monotherapies 20 - 25% cures and isotype suboptimal combination.

In the clinic there has been much interest in targeting 4-1 BB using agonistic anti- bodies. However, our data show that only around 1% of CD8+ or CD4+ T cells at a tu- mour site express 4-1 BB. Furthermore, recent findings indicate that only around 10% of CD3+CD8+ cells infiltrating the tumour site in patients with melanoma express 4-1 BB, although these are enriched for tumour-reactive clones (35). Thus, our current finding that anti-4-1 BB can be used to deplete Tregs to release an immunotherapeutic response suggests that this strategy may be particularly appealing in patients. Clinical studies with other putative Treg-depleting imunotherapeutics (e.g. anti-OX40 and anti-CTLA-4) look promising (5, 36, 37) and further confirm the potential of a Treg depleting anti-4-1 BB mAb in patients.

Currently two fully humanised anti-4-1 BB mAbs are in development; urelumab (BMS-663513), an lgG4 antibody manufactured by Bristol-Myers Squibb, and PF- 05082566, a fully humanised lgG2 produced by Pfizer. Thus far 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). Despite their promising safety profiles, neither urelumab or PF-05082566 are pre- dicted to strongly bind FcyRIIB calling into question whether either antibody will prove effective in patients (40). Recent data from our group show that a human lgG2 antibody targeting 4-1 BB can act as a superagonist independent of FcyRs and it remains possible that PF-05082566 might act in a similar manner (25). Herein presented data, showing improved efficacy of Treg deleting compared with immune agonist variant anti-4-1 BB an- tibodies, and selective intratumoral 4-1 BB expression on Treg compared with CD8 effec- tor cells, support development of human therapeutic anti-4-1 BB lgG1 isotype antibodies selected for capacity for Treg depletion (40). It was recently demonstrated that such Treg deleting antibodies may synergize to boost responses, and help overcome resistance, to checkpoint blockade (50).

Our findings to this point support the contention that immunomodulatory mAb can harness multiple mechanisms of action for therapy and we considered the possibility of whether a single antibody could be engineered to carry out both depletion and agonism optimally. Given our data demonstrating the competing FcyR requirements for these mechanisms in vitro and in vivo it seemed unlikely that engineering a mAb to possess enhanced activatory and inhibitory FcyR engagement would work given that any one mAb can only engage a single FcyR at a time. Given these potential limitations we gen- erated a mlgG2a mAb with optimal depleting capacity to incorporate the hlgG2 hinge re- gion which we skewed to the agonism optimal 'B' form. We hypothesised that this mAb would be able to perform both functions and found this to be the case both in vitro (Fig- ure 6B and C) and in vivo (Figure 6D) and that this led to enhance therapy in a solid tu- mour model (Figure 6E). These results have direct implications for the administration of existing and in-development immunomodulatory mAb and for the design and develop- ment of future reagents and strategies for their use.

SEQUENCES

METHODS

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

Merkenschlager, Imperial College, London, U.K.), Foxp3-GFP, γ chain KO, FcyRIIB KO and FcyR null (γ chain KO x FcyRIIB KO). Mice were obtained by crossbreeding with genotypes confirmed by polymerase chain reaction (PCR) and/or flow cytometry. The CT26 colon carcinoma (16), NXS2 neuroblastoma (41 ), B16 Flt3vax melanoma (42) and EG7 thymoma (43) models have all been described previously.

Immunotherapy. CT26 - Groups of age and sex matched WT, γ chain KO, FcvRIIB KO or FcvR null (γ chain KO x FcyRIIB KO) BALB/c mice were challenged with 5 x 10 ⁴ CT26 s.c. on day 0. When tumours were palpable mice received mAb or PBS control i.v. followed by 3 further administrations i.p. every other day (200 μg final dose unless otherwise indicated). Where CD8+ T cells were depleted, 0.5 mg of anti-CD8 (YTS169) was administered i.p. on days -1 , +1 , and +4 as previously described (44) prior to administration of tumour and mAb. NXS2 - Groups of age and sex matched A/J mice were challenged with 2 * 10 ⁶ NXS2 cells s.c. on day 0 and received antibody/peptide vaccine as specified in individual experiments. All antibodies were given i.p. in PBS. Ty- rosine Hydroxylase (FETFEAKI) and control (SIINFEKL or FEANGNLI) peptides in PBS were emulsified in equal volumes of incomplete Freund's adjuvant (IFA) before intrader- mal injection. Tumor sizes in all models were regularly monitored by caliper and mice culled when cross-sectional area exceeded 225 mm ² . EG7 - Groups of age and sex matched C57BL/6 were challenged with 5 x 10 ⁵ EG7 cells s.c on day 0. On days 3, 5 and 7 mice received 200 μg 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. 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). Spleens from immunised rats or mice were fused with NS- 1 myeloma cells and plates screened by ELISA and flow cytometry. mAbs were initially screened and cells in positive wells were cloned twice and expanded in culture for IgG production. Antibodies were produced from hybridoma or CHOK1 cells and purified on Protein A with purity assessed by electrophoresis (Beckman EP system; Beckman Coul- ter, Buckinghamshire, UK) and lack of aggregation by SEC HPLC. All preparations were endotoxin low (<1 ng endotoxin/mg) as determined using the Endosafe-PTS portable test system (Charles River Laboratories, L'Arbresle, FR). Anti-CTLA-4 (9D9) was purchased from Bio X Cell. Anti-PD-1 de-gly was produced by treating EW1-9 with 0.05U of

PNGaseF / μg of antibody. N-Glycosidase F (PNGaseF) was obtained from Promega (V483A). Samples were kept at 37°C overnight. De-glycosylation was confirmed either by EP or SPR analysis. Purification of antibody from enzyme was achieved through size exclusion chromatography using sephadex™200. Peptides (SIINFEKL, FETFEAKI and FEANGNLI) were obtained from Peptide Protein Research Ltd.

In vitro T cell proliferation. Spleens from Foxp3-GFP mice were sorted to ex- clude GFP+ cells (-Treg cells; 99% of Treg cells removed) or null sorted and plated at 1 x 10 ⁵ cells/well with 0.1 μg/ml anti-CD3 and a range of anti-4-1 BB mAb concentrations as indicated. 1 μCi/well [ ³ H]-thymidine was added 56 hours later and plates harvested after a further 16 hours culture.

Endogenous OVA-specific immune responses. Mice were immunised on Day

0 with 5 mg OVA (Sigma) and 200 μg mAb as specified in the description of the figures. The endogenous OVA specific CD8+ T cell expansion in peripheral blood was monitored over time and analysed by flow cytometry as described previously (18).

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 μg/ml DNasel (Roche) for 20 mins at 37°C. Cells were then passed through a 100 μm 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. The material was cut into small pieces and incubated in R10 with DNase I (Sigma) and Liberase TM (Roche Diagnostics) for 20 min at 37°C. Remaining tissue was mechanical- ly dissociated and, together with the cell suspension, passed through a 70 μm cell strainer. Samples of freshly excised cutaneous squamous cell carcinoma (cSCC) and normal skin were obtained from patients undergoing surgery at the Dermatology De- partment, University Hospital Southampton NHS Foundation Trust, as approved by the South Central Hampshire B National Research Ethics Service Committee (reference number 07/H0504/187). Samples were minced and treated with 1 mg/ml collagenase IA (Sigma) and 10 μg/m! DNAse I (Sigma) in RPMI medium (Gibco) at 37°C for 1.5 hours before straining through a 70 μηι 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.

Flow cytometry. Mouse - Cell surface staining: Isolated lymphocytes were washed and incubated with antibody in the dark for 30 minutes on ice in PBS + 1 % BSA (Sigma) and the cells washed once with PBS/1 %BSA. After staining, samples were fixed using Erythrolyse Red Blood cell lysis buffer (AbD SeroTec). Samples were washed once with PBS/1 % BSA, and run on either a BD FACSCanto II or FACSCalibur and the data analysed using FCS Express. Intracellular staining: After surface staining cells were fixed and stained intracellularly using the anti-Mouse/Rat Foxp3 Staining Set (BD Biosci- ences). 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. Human - Before staining with relevant antibodies, cells from ovarian cancer patients were incubated for 10 min with 10 mg/ml KIOVIG (Baxalta). 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. Cell surface staining: antibodies were incubated with cells in the dark for 30 minutes at 4°C in PBS + 1 % BSA (Sigma) + 10% FCS (Gibco). Intracellular staining was with a Foxp3 staining buffer set (eBioscience). Cells were analysed by flow cytometry using a BD FACSAria or BD FACSVerse. 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, κ 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).

Antibody Dependent Cellular Phagocytosis. 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 μΜ) 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.

Statistical analyses. Unpaired Students t-test analyses of data were performed or for tumour therapy experiments the survival periods to the humane end point were plotted using the Kaplan-Meier method with analysis for significance by the log-rank test. All statistical analyses were carried out using GraphPad Prism 6.0 for Windows

(GraphPad Software Inc, La Jolla, Ca). Significance was accepted when p<0.05.

Surface Plasmon Resonance. Analyses of anti-41 BB mAb and soluble FcyR in- teractions were assayed using a Biacore T100 (GE Healthcare Life Sciences, Bucking- hamshire, UK). Antibodies or BSA as a control were immobilized at 5000 resonance units [RU]) to the flow cells of CM5 sensor chips (GE Healthcare Life Sciences, Bucking- hamshire, UK) by standard amine coupling according to the manufacturer's instructions. 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 μΙ/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

Healthcare Life Sciences, Buckinghamshire, UK).

In vitro binding assays. Karpas-299 cells stably transduced with a tail-less form of murine 4-1 BB (pTL) (47) were incubated with the concentrations of anti-4-1 BB mAb indicated at 4°C for 20 mins prior to washing and staining with a PE-labelled anti-mouse or PE-labelled anti-rat secondary antibody (both Jackson labs). No staining was ob- served to Karpas-299 cells stably expressing an empty vector control (data not shown). For the competitive binding assay, 0.1 μg/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).

Tumour challenge. B16-sFlt3L-lg (FVAX) - Groups of C57BL/6 mice were chal- lenged with 2.5 x 10 ⁴ B16/BL6 cells intra-dermally on day 0. On days 3, 6 and 9 mice re- ceived 1 x 10 ⁶ irradiated FVAX cells i.d. on the opposite flank and either PBS, 100 μg anti-CTLA-4 (clone 9D9) or anti-CTLA-4 and 300 μg anti-4-1 BB antibodies as indicated i.p. also on day 3, 6 and 9 based on our previously published protocol (48). EXAMPLES RELATING TO SPECIFIC 4-1 BB ANTIBODIES

AND SPECIFIC OX40 ANTIBODIES

Material and Methods Animals and cells

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.

Clinical Samples

Ethical approval for the use of clinical samples was obtained by the Ethics Committee of Skane University Hospital. Informed consent was provided in accordance with the Decla- ration of Helsinki. Samples were obtained through the Department of Gynocology and Department of Oncology at Skane University Hospital, Lund. Ascitic fluid was assessed as single cell suspensions that had been isolated. Tumor material was cut into small pieces and incubated in R10 with DNase I (Sigma Aldrich) and Liberase TM (Roche Di- agnostics) for 20 min at 37°C. Remaining tissue was mechanically crashed and, together with the cell suspension, passed through a 70μm cell strainer. Cells isolated from ascitic fluid and tumors were stained. Data acquisition was performed using FACSVerse and data analyzed using FlowJo.

Cell culture

Cell culture was performed in supplemented 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). Following reagents were used to stain human lymphocytes: 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, κ control-PE (G155-178), mouse lgG2b isotype, κ control-PE (27- 53; all from BD Biosciences); TNFRII-PE (FAB226P; R&D Systems).

Following reagents were used to stain mouse lymphocytes: 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

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). Staining with in-house generated scFv was detected with in-house Alexa 647 labeled, deglycosolated anti-His Tag antibody (AD1.1.10, R&D Sys- tems). CFSE-labeling of T cells was performed according to manufacturer's instructions. Antibody dependent cellular cytotoxicity (ADCC)

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. Briefly, at the end of the incubation, 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. The plate was centrifuged at 400 3 g for 5 min to pellet the cells, and the superna- tant was transferred to a white 96-well plate. Calcein release was measured using a Varioskan (Thermo Scientific) using an excitation wavelength of 485 nm and emission wavelength, 530 nm. The percentage of maximal release was calculated as follows: % max release = (sample/triton treated )*100.

Antibody dependent cellular phagocytosis (ADCP)

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. Plates were kept on ice, wells were scraped to collect BMDMs, and phagocytosis was assessed by flow cytometry using a FACSCalibur (BD) to determine the percentage of F4/80+CFSE+ cells within the F4/80+ cell popula- tion.

T-cell proliferation assay

The agonistic activity of antibodies was tested using two protocols: a) Antibodies were cross-linked with F(ab')2 goat anti-human IgG, Fcg fragment specific or F(ab')2 goat anti- mouse IgG, Fcg fragment specific in a molar ratio lgG:F(ab')2 = 1.5:1 for 1 h at RT. 1 x 105 MACS-purified human CD4+ T-cells were CFSE-labelled and stimulated with plate- bound anti-CD3 (0.5 μg/rnl) and 4μg/ml of soluble, cross-linked IgG for 3 days at 37°C before analysis, b) Cell culture was 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% C02. Fresh PBMCs were labelled with 2 mM carboxyfluores- cein succinimidyl ester (CFSE). 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. For PBMC stimulation, round-bottomed 96-well plates were wet-coated with 0.01 μg/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 μg/mL of test mAb (anti-4-1 BB, anti-OX40 mAb). On day 4 or day 5 post-stimulation, cells were labelled with anti-CD8-APC (BioLegend), and anti- CD4-PE (in-house) and proliferation was assessed by CFSE dilution on a FACSCalibur (BD Biosciences). Ligand blocking ELISA Human receptors (hox40, R&D Systems; h41 BB, in-house produced) were coated to 96- well plates (Lumitrac 600 LIA plate, Greiner) at 1 pmole/well. After washing, mAbs (10 μg/ml-0.01 μg/ml) were allowed to bind for 1 hour. 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 Swegene 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.

EMBODIMENTS

In the following an itemized listing of different embodiments of the invention is presented:

1. 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.

2. 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.

3. A Treg depleting antibody molecule for use according to embodiment 1 or 2, wherein the cancer is a solid tumour. 4. A Treg depleting antibody molecule for use according to embodiment 3, where- in the solid tumour is selected from the group consisting of sarcomas, carcinomas, lym- phomas and ovarian cancer.

5. 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

6. A Treg depleting antibody molecule for use according any one of the embodi- ments 1-5, wherein said Treg depleting antibody molecule and/or said immunostimulato- ry antibody molecule is selected from the group consisting of a full-size antibody, a Fab, a Fv, an scFv, a Fab', and a (Fab')2.

7. A Treg depleting antibody molecule for use according any one of the embodi- ments 1-6, wherein said Treg depleting antibody molecule and/or said immunostimulato- ry antibody molecule is a human or humanized antibody.

8. A Treg depleting antibody molecule for use according to any one of the embod- iments 1-7, wherein said Treg depleting antibody molecule is a human lgG1 antibody.

9. 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.

10. A Treg depleting antibody molecule for use according to any one of the em- bodiments 1-9, wherein said Treg depleting antibody molecule is selected from antibody molecules binding specifically to a target belonging to the tumour necrosis factor receptor superfamily (TNFRSF).

11. A Treg depleting antibody molecule for use according to embodiment 10, wherein said Treg depleting antibody molecule is an antibody molecule that binds specif- ically to a target selected from the group consisting of 4-1 BB, OX40, and TNFR2.

12. A Treg depleting antibody molecule for use according to any one of the em- bodiments 1-9, wherein said Treg depleting antibody molecule is an antibody molecule that binds specifically to a target selected from GITR, ICOS, CTLA-4 and CD25.

13. 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.

14. A Treg depleting antibody for use according to embodiment 13, wherein the Treg depleting antibody molecule is selected from the group consisting of antibody mole- cules comprising 1-6 of the CDRs selected from SEQ. ID. NOs: 1-6; 1-6 of the CDRs se- lected from SEQ. ID. NOs: 9-14; 1-6 of the CDRs selected from SEQ. ID. NOs: 17-22; 1- 6 of the CDRs selected from SEQ. ID. NOs: 25-30; 1-6 of the CDRs selected from SEQ. ID. NOs: 33-38; 1-6 of the CDRs selected from SEQ. ID. NOs: 41-46; 1-6 of the CDRs selected from SEQ. ID. NOs: 49-54; 1-6 of the CDRs selected from SEQ. ID. NOs: 57- 62; 1-6 of the CDRs selected from SEQ. ID. NOs: 65-70; 1-6 of the CDRs selected from SEQ. ID. NOs: 153-158; and 1-6 of the CDRs selected from SEQ. ID. NOs: 163-168.

15. A Treg depleting antibody for use according to embodiment 14, wherein the

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.

16. A Treg depleting antibody for use according to embodiment 14 or 15, 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: 7, 15, 23, 31 , 39, 47, 55, 63, 71, 159, and 169.

17. A Treg depleting antibody for use according to any one of the embodiments 14-16, wherein the Treg depleting antibody molecule is selected from the group consist- ing of antibody molecule comprising a variable light chain selected from the group con- sisting of SEQ. ID. NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 160, and 170.

18. 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.

19. 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.

20. A Treg depleting antibody for use according to embodiment 19, wherein the Treg depleting antibody molecule is selected from the group consisting of antibody mole- cule 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.

21. A Treg depleting antibody for use according to embodiment 20, wherein the Treg depleting antibody molecule is selected from the group consisting of antibody mole- cules 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: 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: 177-178. 22. 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.

23. A Treg depleting antibody for use according to any one of the embodiments

20-22, wherein 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.

24. A Treg depleting antibody for use according to any one of the embodiments 20-23, wherein the Treg depleting antibody molecule is selected from the group consist- ing 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.

25. A Treg depleting antibody molecule for use according to any one of the em- bodiments 1-9, wherein said Treg depleting 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.

26. A Treg depleting antibody for use according to any one of the embodiments

1-25, wherein the immunostimulatory antibody molecule is a human lgG2 antibody or a human lgG4 antibody molecule.

27. A Treg depleting antibody for use according to embodiment 26, wherein the immunostimulatory antibody molecule is a human lgG2b antibody molecule.

28. A Treg depleting antibody molecule for use according to any one of the em- bodiments 1-27, wherein the immunostimulatory antibody molecule is engineered for en- hanced binding to human FcyRIIB over activatory Fc gamma receptors.

29. A Treg depleting antibody molecule for use according to any one of the em- bodiments 1-28, wherein the immunostimulatory antibody molecule is an antibody that binds specifically to a target selected from the group consisting of 4-1 BB, OX40, ICOS, GITR, CTLA-4 CD25, PD-1 and PDL1.

30. A Treg depleting antibody molecule for use according to embodiment 29, wherein the immunostimulatory antibody molecule is an anti-4-1 BB antibody molecule.

31. A Treg depleting antibody molecule for use according to embodiment 30, wherein 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.

32. 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,

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.

33. A Treg depleting antibody for use according to embodiment 31 or 32, 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: 7, 15, 23, 31 , 39, 47, 55, 63, 71 , 159 and 169.

34. A Treg depleting antibody for use according to any one of the embodiments 31-33, wherein the immunostimulatory 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.

35. A Treg depleting antibody for use according to any one of the embodiments 31-34, wherein immunostimulatory 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 and 160, and SEQ. ID. NOs: 169 and 170.

36. A Treg depleting antibody molecule for use according to embodiment 29, wherein the immunostimulatory antibody molecule is an anti-OX40 antibody molecule.

37. A Treg depleting antibody molecule for use according to embodiment 36 wherein 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, 1 13-118, 121-126, 129-134, 137-142, 145-150, and 171-176.

38. A Treg depleting antibody for use according to embodiment 37, wherein 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: 105-1 10, SEQ. ID. NOs: 113-1 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. 39. 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.

40. A Treg depleting antibody for use according to any one of the embodiments

37-39, wherein 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.

41. A Treg depleting antibody for use according to any one of the embodiments

37-40, wherein 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.

42. A Treg depleting antibody molecule for use according to embodiment 29, wherein the immunostimulatory antibody molecule is a human anti-PD1 monoclonal anti- body molecule, a human anti- PDL1 monoclonal antibody molecule or a human anti- CTLA-4 monoclonal antibody molecule.

43. A Treg depleting antibody molecule for use according to embodiment 42, wherein the wherein the immunostimulatory antibody molecule is a human anti-PD1 monoclonal antibody molecule selected from the group consisting of nivolumab and pembrolizumab or the anti- PDL1 antibody atezolizumab or an anti-CTLA-4 antibody se- lected from the group consisting of ipilimumab and tremilimumab.

44. 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.

45. 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:

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.

46. 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 .

47. 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.

48. 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.

49. 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.

50. An anti-4-1 BB antibody molecule according to embodiment 49, wherein the full-length IgG antibody is selected from the group consisting of an lgG1 , lgG2, lgG4, and an Fc-engineered variant thereof.

51. 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.

52. 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 .

53. 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.

54. 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.

55. An anti- OX40 antibody molecule according to any one of the embodiments

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.

56. 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.

57. 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.

58. An anti- OX40 antibody molecule according to embodiment 57, wherein the full-length IgG antibody is selected from the group consisting of an lgG1 , lgG2, lgG4, and an Fc-engineered variant thereof.

59. 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.

60. An isolated nucleic acid encoding an antibody according to any one of the embodiments 44-59.

61. A vector comprising the nucleic acid according to embodiment 60.

62. A host cell comprising the vector according to embodiment 61.

63. An antibody according to any one of the embodiments 44-59 for use in medi- cine.

64. A pharmaceutical composition comprising an antibody according to any one of the embodiments 44-59

65. An antibody according to embodiment 63 or a pharmaceutical composition according to embodiment 64 for use in the treatment of cancer.

66. An antibody or a pharmaceutical composition according to embodiment 65, wherein the cancer is a solid tumour.

67. 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.

68. An antibody or a pharmaceutical composition according to embodiment 67, wherein the solid tumour is squamous cell carcinoma (SCC), thymoma, neuroblastoma or ovarian cancer. 69. 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.

70. An antibody according to any one of the embodiments 44-59 or a pharmaceu- tical composition according to embodiment 69, wherein the pharmaceutical composition is for treatment of cancer.

71. An antibody according to any one of the embodiments 44-59 or a pharmaceu- tical composition according to embodiment 70, wherein the cancer is a solid tumour.

72. An antibody according to any one of the embodiments 44-59 or a pharmaceu- tical composition according to embodiment 71 , wherein the solid tumour is selected from the group consisting of sarcomas, carcinomas and lymphomas.

73. An antibody according to any one of the embodiments 44-59 or a pharmaceu- tical composition according to embodiment 72, wherein the solid tumour is squamous cell carcinoma (SCC), thymoma, neuroblastoma or ovarian cancer.

74. Use of an antibody according to any one of the embodiments 44-59 for the manufacture of a pharmaceutical composition for use in treatment of cancer.

75. Use according to embodiment 74, wherein the cancer is a solid tumour.

76. Use according to embodiment 75, wherein the solid tumour is selected from the group consisting of sarcomas, carcinomas and lymphomas.

77 Use according to embodiment 76, wherein the solid tumour is squamous cell carcinoma (SCC), thymoma, neuroblastoma or ovarian cancer.

78. 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.

79. The method of embodiment 78, wherein said immunostimulatory antibody molecule is a CD8 activating and/or CD8 boosting antibody molecule.

80. The method of embodiment 78 or 79, wherein the cancer is a solid tumour.

81. The method of embodiment 80, wherein the solid tumour is selected from the group consisting of sarcomas, carcinomas and lymphomas.

82. The method of embodiment 81 , wherein the solid tumour is squamous cell carcinoma (SCC), thymoma, neuroblastoma or ovarian cancer.

83. The method of any one of the embodiments 78-82, wherein said 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 . 84. The method of embodiment 83, wherein the full-length IgG antibody is select- ed from the group consisting of an lgG1 , lgG2, lgG4, and an Fc-engineered variant thereof.

85. The method of any one of the embodiments 78-84, wherein said Treg deplet- ing antibody molecule and/or said immunostimulatory antibody molecule is a human or humanized antibody.

86. The method of any one of the embodiments 78-85, wherein said Treg deplet- ing antibody molecule is a human lgG1 antibody.

87. The method of any one of the embodiments 78-86, wherein said Treg deplet- ing antibody molecule is a human lgG1 antibody molecule engineered for improved bind- ing to at least one activatory FcyR.

88. The method of any one of the embodiments 78-87, wherein said Treg deplet- ing antibody molecule is selected from antibody molecules binding specifically to a target belonging to the tumour necrosis factor receptor superfamily (TNFRSF).

89. The method of embodiment 88, wherein said 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.

90. The method of embodiment 87, wherein said Treg depleting antibody mole- cule is an anti-4-1 BB monoclonal antibody molecule.

91. The method of embodiment 89, wherein the 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.

92. The method of embodiment 91 , wherein the Treg depleting 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, 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.

93. The method of embodiment 91 or 92, wherein the 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.

94. The method of 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. 95. The method of any one of the embodiments 91-94, wherein 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. 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 and 160; and SEQ. ID. NOs: 169-170.

96. The method of embodiment 89, wherein said Treg depleting antibody is a human anti-OX40 monoclonal antibody molecule.

97. The method of embodiment 96, wherein the Treg depleting 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.

98. The method of embodiment 97, wherein 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.

99. The method of any one of the embodiments 96-98, wherein the 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.

100. The method of any one of the embodiments 96-99, wherein the 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.

101. The method of any one of the embodiments 96-100, wherein 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.

102. The method of any one of the embodiments 78-87, wherein said 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. 103. The method of any one of the embodiments 78-102, wherein the im- munostimulatory antibody molecule is a human lgG2 antibody or a human lgG4 antibody molecule.

104. The method of embodiment 103, wherein the immunostimulatory antibody molecule is a human lgG2b antibody molecule.

105. The method of any one of the embodiments 78-104, wherein the im- munostimulatory antibody molecule is engineered for enhanced binding to human FcyRIIB over activatory Fc gamma receptors.

106. The method of any one of the embodiments 76-105, wherein 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.

107. The method of embodiment 106, wherein the immunostimulatory antibody molecule is an anti-4-1BB antibody molecule.

108. The method of embodiment 107, wherein 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.

109. The method of 108, wherein the immunostimulatory antibody molecule is se- lected from the group consisting of antibody molecule 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.

110. The method of any one of the embodiments 107-109, wherein 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.

111. The method of any one of the embodiments 107-110, wherein 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.

112. The method of any one of the embodiments 107-111 , wherein 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.

113. The method of embodiment 106, wherein the immunostimulatory antibody molecule is an anti-OX40 antibody molecule.

114. The method of embodiment 113, wherein 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.

115. The method of embodiment 114, wherein 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.

116. The method of any one of the embodiments 113-1 15, wherein 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.

117. The method of any one of the embodiments 113-1 16, wherein 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.

118. The method of any one of the embodiments 1 13-117, wherein 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.

119. The method of embodiment 106, wherein 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.

120. The method of embodiment 119, wherein the wherein 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. 121. A use, method, antibody, nucleic acid, vector, host cell or pharmaceutical composi- tion as described herein in the description, examples and/or figures.

REFERENCES

In the text above, reference is made to the following publications, the contents of which are hereby incorporated by reference.

1. Hodi, F.S., O'Day, S.J., McDermott, D.F., Weber, R.W., Sosman, J.A., Haanen, J.B., Gonzalez, R., Robert, C, Schadendorf, D., Hassel, J.C., et al. 2010.

Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363:71 1 -723.

2. Topalian, S.L., Hodi, F.S., Brahmer, J.R., Gettinger, S.N., Smith, D.C.,

McDermott, D.F., Powderly, J.D., Carvajal, R.D., Sosman, J.A., Atkins, M.B., et al. 2012. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366:2443-2454.

3. Brahmer, J.R., Tykodi, S.S., Chow, L.Q., Hwu, W.J., Topalian, S.L., Hwu, P.,

Drake, C.G., Camacho, L.H., Kauh, J., Odunsi, K., et al. 2012. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366:2455- 2465.

4. Beatty, G.L, Chiorean, E.G., Fishman, M.P., Saboury, B., Teitelbaum, U.R., Sun, W., Huhn, R.D., Song, W., Li, D., Sharp, L.L., et al. 201 1. CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans. Science 331 :1612-1616.

5. Simpson, T.R., Li, F., Montalvo-Ortiz, W., Sepulveda, M.A., Bergerhoff, K., Arce, F., Roddie, C, Henry, J.Y., Yagita, H., Wolchok, J.D., et al. 2013. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-

CTLA-4 therapy against melanoma. J Exp Med 210:1695-1710.

6. Bulliard, Y., Jolicoeur, R., Windman, M., Rue, S.M., Ettenberg, S., Knee, D.A., Wilson, N.S., Dranoff, G., and Brogdon, J.L. 2013. Activating Fc gamma receptors contribute to the antitumor activities of immunoregulatory receptor- targeting antibodies. J Exp Med 210:1685-1693.

7. Marabelle, A., Kohrt, H., Sagiv-Barfi, I., Ajami, B., Axtell, R.C., Zhou, G.,

Rajapaksa, R., Green, M.R., Torchia, J., Brody, J., et al. 2013. Depleting tumor- specific Tregs at a single site eradicates disseminated tumors. J Clin Invest 123:2447-2463. White, A.L., Chan, H.T., French, R.R., Beers, S.A., Cragg, M.S., Johnson, P.W., and Glennie, M.J. 2013. FcgammaRlotalotaB controls the potency of agonistic anti-TNFR mAbs. Cancer Immunol Immunother 62:941-948.

Li, F., and Ravetch, J.V. 2013. Antitumor activities of agonistic anti-TNFR antibodies require differential FcgammaRIIB coengagement in vivo. Proc Natl

Acad Sci U S A 1 10:19501 -19506.

White, A.L., Beers, S.A., and Cragg, M.S. 2014. FcgammaRIIB as a key determinant of agonistic antibody efficacy. Curr Top Microbiol Immunol 382:355- 372.

Middendorp, S., Xiao, Y., Song, J.Y., Peperzak, V., Krijger, P.H., Jacobs, H., and Borst, J. 2009. Mice deficient for CD137 ligand are predisposed to develop germinal center-derived B-cell lymphoma. Blood 1 14:2280-2289.

Snell, L.M., Lin, G.H., McPherson, A.J., Moraes, T.J., and Watts, T.H. 201 1. T- cell intrinsic effects of GITR and 4-1 BB during viral infection and cancer immunotherapy. Immunol Rev 244:197-217.

Melero, I., Hirschhorn-Cymerman, D., Morales-Kastresana, A., Sanmamed, M.F., and Wolchok, J.D. 2013. Agonist antibodies to TNFR molecules that costimulate T and NK cells. Clin Cancer Res 19:1044-1053.

McHugh, R.S., Whitters, M.J., Piccirillo, C.A., Young, D.A., Shevach, E.M., Collins, M., and Byrne, M.C. 2002. CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16:31 1-323.

Marson, A., Kretschmer, K., Frampton, G.M., Jacobsen, E.S., Polansky, J.K., Maclsaac, K.D., Levine, S.S., Fraenkel, E., von Boehmer, H., and Young, R.A. 2007. Foxp3 occupancy and regulation of key target genes during T-cell stimulation. Nature 445:931-935.

Taraban, V.Y., Rowley, T.F., O'Brien, L, Chan, H.T., Haswell, L.E., Green, M.H., Tutt, A.L., Glennie, M.J., and Al-Shamkhani, A. 2002. Expression and

costimulatory effects of the TNF receptor superfamily members CD134 (OX40) and CD137 (4-1 BB), and their role in the generation of anti-tumor immune responses. Eur J Immunol 32:3617-3627.

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. Type II

(tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation. Blood

1 12:4170-4177. 18. White, A.L, Chan, H.T., Roghanian, A., French, R.R., Mockridge, C.I., Tutt, A.L., Dixon, S.V., Ajona, D., Verbeek, J.S., Al-Shamkhani, A., et al. 201 1. Interaction with FcgammaRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody. J Immunol 187:1754-1763.

19. White, A.L, Dou, L, Chan, H.T., Field, V.L., Mockridge, C.I., Moss, K., Williams, E.L., Booth, S.G., French, R.R., Potter, E.A., et al. 2014. Fcgamma receptor dependency of agonistic CD40 antibody in lymphoma therapy can be overcome through antibody multimerization. J Imm unol 193:1828-1835.

20. Nimmerjahn, F., and Ravetch, J.V. 2005. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science 310:1510-1512.

21. Wilson, N.S., Yang, B., Yang, A., Loeser, S., Marsters, S., Lawrence, D., Li, Y., Pitti, R., Totpal, K., Yee, S., et al. 201 1. An Fcgamma receptor-dependent mechanism drives antibody-mediated target-receptor signaling in cancer cells. Cancer Cell 19:101-1 13.

22. Haynes, N.M., Hawkins, E.D., Li, M., McLaughlin, N.M., Hammerling, G.J.,

Schwendener, R., Winoto, A., Wensky, A., Yagita, H., Takeda, K., et al. 2010. CD1 1 c+ dendritic cells and B cells contribute to the tumoricidal activity of anti- DR5 antibody therapy in established tumors. J Immunol 185:532-541.

23. Li, F., and Ravetch, J.V. 2012. Apoptotic and antitumor activity of death receptor antibodies require inhibitory Fcgamma receptor engagement. Proc Natl Acad Sci

U S A 109:10966-10971.

24. Mimoto, F., Katada, H., Kadono, S., Igawa, T., Kuramochi, T., Muraoka, M.,

Wada, Y., Haraya, K., Miyazaki, T., and Hattori, K. 2013. Engineered antibody Fc variant with selectively enhanced FcgammaRllb binding over both

FcgammaRlla(R131 ) and FcgammaRlla(H131 ). Protein Eng Des Sel 26:589-598.

25. White, A.L., Chan, H.T., French, R.R., Willoughby, J., Mockridge, C.I.,

Roghanian, A., Penfold, C.A., Booth, S.G., Dodhy, A., Polak, M.E., et al. 2015. Conformation of the human immunoglobulin g2 hinge imparts superagonistic properties to immunostimulatory anticancer antibodies. Cancer Cell 27:138-148. 26. Gavin, M.A., Clarke, S.R., Negrou, E., Gallegos, A., and Rudensky, A. 2002.

Homeostasis and anergy of CD4(+)CD25(+) suppressor T cells in vivo. Nat Immunol 3:33-41.

27. Elpek, K.G., Yolcu, E.S., Franke, D.D., Lacelle, C, Schabowsky, R.H., and

Shirwan, H. 2007. Ex vivo expansion of CD4+CD25+FoxP3+ T regulatory cells based on synergy between IL-2 and 4-1 BB signaling. J Immunol 179:7295-7304. 28. Zheng, G., Wang, B., and Chen, A. 2004. The 4-1 BB costimulation augments the proliferation of CD4+CD25+ regulatory T cells. J Immunol 173:2428-2434.

29. Plitas, G., Konopacki, C, Wu, K„ Bos, P.D., Morrow, M., Putintseva, E.V.,

Chudakov, D.M., and Rudensky, A.Y. 2016. Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer. Immunity 45: 1122-1134.

30. De Simone, M., Arrigoni, A., Rossetti, G., Gruarin, P., Ranzani, V., Politano, C, Bonnal, R.J., Provasi, E., Sarnicola, M.L., Panzeri, I., et al. 2016. Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor- Infiltrating T Regulatory Cells. Immunity 45:1 135-1 147.

31. Tipton, T.R., Mockridge, C.I., French, R.R., Tutt, A.L., Cragg, M.S., and Beers, S.A. 2015. Anti-mouse FcgammaRIV antibody 9E9 also blocks FcgammaRIII in vivo. Blood 126:2643-2645.

32. Li, F., and Ravetch, J.V. 201 1. Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies. Science

333:1030-1034.

33. Li, F., and Ravetch, J.V. 2012. A general requirement for FcgammaRIIB co- engagement of agonistic anti-TNFR antibodies. Cell Cycle 1 1 :3343-3344.

34. Minard-Colin, V., Xiu, Y., Poe, J.C., Horikawa, M., Magro, CM., Hamaguchi, Y., Haas, K.M., and Tedder, T.F. 2008. Lymphoma depletion during CD20 immunotherapy in mice is mediated by macrophage FcgammaRI, FcgammaRIII, and FcgammaRIV. Blood 112:1205-1213.

35. Gros, A., Robbins, P.F., Yao, X., Li, Y.F., Turcotte, S., Tran, E., Wunderlich, J.R., Mixon, A., Farid, S., Dudley, M.E., et al. 2014. PD-1 identifies the patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors. J Clin Invest

124:2246-2259.

36. Curti, B.D., Kovacsovics-Bankowski, M., Morris, N., Walker, E., Chisholm, L., Floyd, K., Walker, J., Gonzalez, I., Meeuwsen, T., Fox, B.A., et al. 2013. OX40 is a potent immune-stimulating target in late-stage cancer patients. Cancer Res 73:7189-7198.

37. Sharma, P., Wagner, K., Wolchok, J.D., and Allison, J. P. 201 1. Novel cancer immunotherapy agents with survival benefit: recent successes and next steps.

Nat Rev Cancer 1 1 : 805-812.

38. Segal, N.H., Gopal, A.K., Bhatia, S,, Kohrt, H., Levy, R., Pishvaian, M.J., Houot,

R., Bartlett, N., Nghiem, N., Kronenberg, S.A., et al. 2014. A phase 1 study of PF- 05082566 (anti-4-1 BB) in patients with advanced cancer. J Clin Oncol 32:suppl; abstr 3007. 39. Molckovsky, A., and Siu, L.L. 2008. First-in-class, first-in-human phase I results of targeted agents: highlights of the 2008 American society of clinical oncology meeting. J Hematol Oncol 1 :20.

40. Furness, A.J., Vargas, F.A., Peggs, K.S., and Quezada, S.A. 2014. Impact of tumour microenvironment and Fc receptors on the activity of immunomodulatory antibodies. Trends Immunol 35:290-298.

41. Lode, H.N., Xiang, R., Varki, N.M., Dolman, C.S., Gillies, S.D., and Reisfeld, R.A.

1997. Targeted interleukin-2 therapy for spontaneous neuroblastoma metastases to bone marrow. J Natl Cancer Inst 89: 1586-1594.

42. Curran, M.A., and Allison, J. P. 2009. Tumor vaccines expressing flt3 ligand

synergize with ctla-4 blockade to reject preimplanted tumors. Cancer Res 69:7747-7755.

43. Moore MW, Carbone FR, and Bevan MJ. Introduction of soluble protein into the class I pathway of antigen processing and presentation. Cell. 1988;54(6):777-85. 44. French, R.R., Chan, H.T., Tutt, A.L., and Glennie, M.J. 1999. CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T- cell help. Nat Med 5:548-553.

45. Beers, S.A., French, R.R., Chan, H.T., Lim, S.H., Jarrett, T.C., Vidal, R.M.,

Wijayaweera, S.S., Dixon, S.V., Kim, H., Cox, K.L., et al. 2010. Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection. Blood 1 15:5191 -5201.

46. Lim, S.H., Vaughan, AT., Ashton-Key, M., Williams, E.L., Dixon, S.V., Chan, H.T., Beers, S.A., French, R.R., Cox, K.L., Davies, A.J., et al. 201 1. Fc gamma receptor Mb on target B cells promotes rituximab internalization and reduces clinical efficacy. Blood 118:2530-2540.

47. Buchan, S.L., and Al-Shamkhani, A. 2012. Distinct motifs in the intracellular

domain of human CD30 differentially activate canonical and alternative transcription factor NF-kappaB signaling. PLoS One 7:e45244.

48. Curran, M.A., Kim, M., Montalvo, W., Al-Shamkhani, A., and Allison, J. P. 2011.

Combination CTLA-4 blockade and 4-1 BB activation enhances tumor rejection by increasing T-cell infiltration, proliferation, and cytokine production. PLoS One 6:e19499.

49. Dahan R, Barnhart BC, Li F, Yamniuk AP, Korman AJ, Ravetch JV. Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires

Selective FcyR Engagement. Cancer Cell. 2016 Jun 13;29(6):820-31. doi:

10.1016/j.ccell.2016.05.001. Epub 2016 Jun 2. PMID: 27265505. 50. Arce Vargas F, Furness AJS, Solomon I, Joshi K, Mekkaoui L, Lesko MH,

Miranda Rota E, Dahan R, Georgiou A, Sledzinska A, Ben Aissa A, Franz D, Werner Sunderland M, Wong YNS, Henry JY, O'Brien T, Nicol D, Challacombe B, Beers SA; Melanoma TRACERx Consortium; Renal TRACERx Consortium; Lung TRACERx Consortium, Turajlic S, Gore M, Larkin J, Swanton C, Chester KA,

Pule M, Ravetch JV, Marafioti T, Peggs KS, Quezada SA. Fc-Optimized Anti- CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors. Immunity. 2017 Apr 18;46(4):577- 586. doi: 10.1016/j.immuni.2017.03.013. Epub 2017 Apr 11. PMID: 28410988 51. Romer PS1 , Berr S, Avota E, Na SY, Battaglia M, ten Berge I, Einsele H, Hunig T.; Preculture of PBMCs at high cell density increases sensitivity of T-cell responses, revealing cytokine release by CD28 superagonist TGN1412. Blood. 2011 Dec 22;118(26):6772-82. doi: 10.1182/blood-2010-12-319780. Epub 2011 Sep 19.