NOVEL ANTIBODIES AND COMBINED USE OF Treg-DEPLETING ANTIBODY AND IMMUNOSTIMULATORY ANTIBODY

Abstract

To provide novel aspects of using a Treg-depleting anti-4-1 BB antibody for treatment of cancer.SOLUTION: Described is sequential administration for use in treatment of cancer, comprising first administering Treg-depleting antibody molecules selected from antibody molecules, such as antibody molecules binding specifically to a target belonging to the tumor necrosis factor receptor superfamily (TNFRSF), such as a Treg-depleting anti-4-1 BB antibody or a Treg-depleting OX-40 antibody, and then administering immunostimulatory antibody molecules, such as immunostimulatory anti-4-1 BB antibody or an immunostimulatory OX-40 antibody. Also described are novel anti-4-1 BB antibodies and novel OX-40 antibodies that may be used in such sequential administration.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] The present invention relates to the sequential administration of first a Treg-depleting antibody molecule and then an immunostimulatory antibody molecule for use in the treatment of cancer. The present invention also relates to novel antibodies for use in such treatment, including a novel anti-4-1BB antibody and a novel anti-OX40 antibody. [Background technology]

[0002] Promising clinical results using immunomodulatory mAbs have revived the idea that the immune system holds the key to cancer control. Recent examples have demonstrated that both types of checkpoint blockers (antagonists) and costimulatory molecules (agonists) can combat tumors through activating FcγR binding and depletion of inhibitory regulatory T cells (Tregs), calling into question the classification of these mAbs as checkpoint blockers or activators of costimulatory molecules. In contrast to these findings, anti-CD40 mAbs rely on inhibitory FcγR crosslinking to stimulate agonistic immunity. Thus, immunomodulatory mAbs offer considerable promise for cancer immunotherapy, but the effector mechanisms employed by various mAbs and, consequently, their optimal applications remain unclear.

[0003] Immunomodulatory mAbs, such as ipilimumab (anti-CTLA4), anti-PD-1 / PD-L1, and anti-CD40, have shown positive results when tested in difficult-to-treat malignancies, albeit in small numbers of patients (1-4). These promising results have reinvigorated the idea that the immune system may hold the key to cancer control. These mAbs target key molecular regulators of T cells or APCs and have been generated to enhance anticancer immunity through blocking inhibitory signals (checkpoint blockers) or delivering costimulatory signals (agonists). This dualistic classification has recently been called into question when it was found that the therapeutic activity of anti-CTLA4, anti-GITR, and anti-OX40 (all of which target T cells) is dependent on co-engagement of activating FcγRs and contributes to the deletion of inhibitory CD4+ T regulatory cells (5-7). In contrast, the activity of agonistic APC-targeted anti-CD40 mAbs requires co-engagement of inhibitory FcγRs, which promotes effective mAb cross-linking, necessary for CD40 signaling and immune stimulation (8-10). Thus, immunomodulatory mAbs offer considerable promise for cancer immunotherapy, but the mechanisms employed may depend on both the Fab and Fc regions of the mAb in a disease-defined manner and on the type of cell targeted. T versus direct immune stimulation reg Understanding the relative importance of depletion will be essential for the development of immunomodulatory mAbs and their successful translation to patients. Summary of the Invention

[0004] The present invention demonstrates that anti-4-1BB mAb exerts direct immune stimulation or T cell proliferation in solid tumors, with the primary mechanism dependent on antibody isotype and FcγR availability. regThis work is based on studies demonstrating that either depletion or stimulation can be used. Importantly, depletion and immune stimulation appear to be competing mechanisms, likely limited by FcγR binding limitations. Research leading to the present invention further demonstrates that first depleting Tregs and then stimulating CD8 T cells by sequentially administering an isotype-distinct or isotype-optimal anti-4-1BB mAb followed by an anti-PD-1 mAb leads to an enhanced response, resulting in improved therapy and outcomes. Furthermore, the inventors engineered a depleting anti-4-1BB mIgG2a with a human IgG2 hinge region "B" (mIgG2a / h2B) to provide FcγR-independent agonism, demonstrating the potential for this single mAb to utilize both mechanisms to deliver improved therapy.

[0005] This possibility is then expanded by the demonstration that this can be achieved by Treg depletion followed by immune stimulation using an additional antibody in addition to the anti-4-1BB mAb, i.e., an antibody molecule that specifically binds to a target belonging to the tumor necrosis factor receptor superfamily (TNFRSF), for example, a Treg-depleting antibody molecule such as a Treg-depleting anti-4-1BB antibody or a Treg-depleting anti-OX40 antibody, in combination with an immunostimulatory antibody molecule such as an immunostimulatory anti-4-1BB antibody, an immunostimulatory anti-OX40 antibody, or an immunoactivating PD-1-blocking antibody, with the order of administration being important to achieve the desired effect. This research also led to the development of novel anti-4-1BB antibodies and novel anti-OX40 antibodies.

[0006] Thus, the present invention relates to Treg-depleting antibody molecules for use in the treatment of cancer, wherein the Treg-depleting antibody molecules are administered sequentially with immunostimulatory antibody molecules, the Treg-depleting antibody molecules being administered prior to administration of the immunostimulatory antibody molecules.

[0007] The present invention further relates to a method of treating cancer in a subject, the treatment comprising the administration of a Treg-depleting antibody molecule followed by the sequential administration of an immunostimulatory antibody molecule.

[0008] The present invention further relates to an anti-4-1BB antibody molecule selected from the group consisting of antibody molecules comprising one or more CDRs selected from SEQ ID NOs: 1 to 6, 9 to 14, 17 to 22, 25 to 30, 33 to 38, 41 to 46, 49 to 54, 57 to 62, 65 to 70, 153 to 158, and 163 to 168. "One or more" in this context means that the antibody molecule comprises 1, 2, 3, 4, 5, or 6 of the indicated sequences, i.e., 1 to 6 of the indicated sequences. Thus, an anti-4-1BB antibody molecule may comprise one to six CDRs selected from SEQ ID NOs: 1 to 6, one to six CDRs selected from SEQ ID NOs: 9 to 14, one to six CDRs selected from SEQ ID NOs: 17 to 22, one to six CDRs selected from SEQ ID NOs: 25 to 30, one to six CDRs selected from SEQ ID NOs: 33 to 38, one to six CDRs selected from SEQ ID NOs: 41 to 46, one to six CDRs selected from SEQ ID NOs: 49 to 54, one to six CDRs selected from SEQ ID NOs: 57 to 62, one to six CDRs selected from SEQ ID NOs: 65 to 70, one to six CDRs selected from SEQ ID NOs: 153 to 158, or one to six CDRs selected from SEQ ID NOs: 163 to 168.

[0009] The present invention further relates to nucleic acids encoding the above-described anti-4-1BB antibody molecules.

[0010] The present invention further relates to an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising one or more 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. Again, "one or more" in this context means that the antibody molecule comprises 1, 2, 3, 4, 5, or 6 of the indicated sequences, i.e., 1 to 6 of the indicated sequences. Thus, an anti-OX40 antibody molecule can comprise one to six CDRs selected from SEQ ID NOs: 73-78, one to six CDRs selected from SEQ ID NOs: 81-86, one to six CDRs selected from SEQ ID NOs: 89-94, one to six CDRs selected from SEQ ID NOs: 97-102, one to six CDRs selected from SEQ ID NOs: 105-110, one to six CDRs selected from SEQ ID NOs: 113-118, one to six CDRs selected from SEQ ID NOs: 121-126, one to six CDRs selected from SEQ ID NOs: 129-134, one to six CDRs selected from SEQ ID NOs: 137-142, one to six CDRs selected from SEQ ID NOs: 145-150, or one to six CDRs selected from SEQ ID NOs: 171-176.

[0011] The present invention further relates to a vector comprising the above-described nucleic acid.

[0012] The present invention further relates to a host cell comprising the above-mentioned nucleic acid and / or the above-mentioned vector. DETAILED DESCRIPTION OF THE INVENTION

[0013] Regulatory T cells, T cells, Treg cells, Treg or T reg (formerly known as suppressor T cells and sometimes referred to as suppressive regulatory T cells) are a subpopulation of T cells that are able to suppress other immune cells in normal and pathological immune environments.

[0014] As used herein, Treg depletion, or Treg depletion, refers to the depletion, deletion, or elimination of Tregs through physical clearance of cells, specifically, the depletion of intratumoral Tregs.

[0015] Effector T cells are T cells or T lymphocytes that respond to stimuli and activate, attack, or destroy antigen-expressing cells in an antigen:MHC:TCR-restricted manner. Effector T cells can control cancer and eradicate tumor cells directly (cytotoxic T cells) or indirectly through activation of other immune cells (T helper cells).

[0016] Antibodies are well known to those skilled in the art of immunology and molecular biology. Typically, an antibody comprises two heavy chains (H) and two light chains (L). This complete antibody molecule is sometimes referred to herein as a full-size antibody or full-length antibody. The heavy chain of an antibody comprises one variable domain (VH) and three constant domains (CH1, CH2, CH3), and the light chain of an antibody molecule comprises one variable domain (VL) and one constant domain (CL). The variable domains (sometimes referred to as F V The variable domains (collectively referred to as "variable domains") bind to the antibody's target, i.e., antigen. Each variable domain is composed of three loops called complementarity-determining regions (CDRs), which are responsible for target binding. The constant domains are not directly involved in binding the antibody to the antigen but exhibit various effector functions. Antibodies or immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant regions of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. In humans, some of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4; IgA1, and IgA2. Another part of the antibody is the Fc domain (also known as the fragment crystallizable domain), which contains two constant domains from each of the antibody's heavy chains. The Fc domain is responsible for the interaction between the antibody and the Fc receptor.

[0017] Fc receptors are membrane proteins that are often found on the cell surface of cells of the immune system (i.e., Fc receptors are found on target cell membranes - also known as plasma membranes or cytoplasmic membranes). The role of Fc receptors is to bind antibodies via the Fc domain and internalize the antibody. In the immune system, this can lead to antibody-mediated phagocytosis and antibody-dependent cell-mediated cytotoxicity.

[0018] As used herein, the term antibody molecule encompasses full-length or full-size antibodies, as well as functional fragments of full-length antibodies and derivatives of such antibody molecules.

[0019] A functional fragment of a full-sized antibody has the same antigen-binding characteristics as the corresponding full-sized antibody, and contains either the same variable domains (i.e., VH and VL sequences) and / or the same CDR sequences as the corresponding full-sized antibody. Having the same antigen-binding characteristics as the corresponding full-sized antibody means that the functional fragment binds to the same epitope on the target as the full-sized antibody. Such a functional fragment may correspond to the Fv portion of a full-sized antibody. Alternatively, such a fragment may be a Fab, also referred to as F(ab), which is a monovalent antigen-binding fragment that does not contain the Fc portion, or a bivalent antigen-binding fragment containing two antigen-binding Fab portions linked together by disulfide bonds, or a monovalent variant of F(ab'), i.e., F(ab')2. Such a fragment may also be a single-chain variable fragment (scFv).

[0020] Functional fragments do not always contain all six CDRs of the corresponding full-size antibody.It is understood that molecules containing three or fewer CDR regions (sometimes only a single CDR or a portion thereof) can retain the antigen binding activity of the antibody derived from that CDR(s).For example, Gao et al., 1994, J.Biol.Chem.,269:32389-93, has described that the entire VL chain (including all three CDRs) has high affinity for its substrate.

[0021] Molecules containing two CDR regions are described, for example, in Vaughan & Sollazzo 2001, Combinatorial Chemistry & High Throughput Screening, 4:417-430. On page 418 (right column -3 (Our Strategy for Design)), a minibody containing only H1 and H2 CDR hypervariable regions interspersed within framework regions is described. Minibodies are described as being capable of binding to targets. Pessi et al., 1993, Nature, 362:367-9, and Bianchi et al., 1994, J. Mol. Biol., 236:649-59, referenced by Vaughan & Sollazzo, provide more detailed descriptions of H1 and H2 minibodies and their properties. Qiu et al., 2007, Nature Biotechnology, 25:921-9, demonstrate that a molecule consisting of two linked CDRs can bind to an antigen. Quiocho 1993, Nature, 362:293-4 provides an overview of "minibody" technology. Ladner 2007, Nature Biotechnology, 25:875-7 states that molecules containing two CDRs can retain antigen-binding activity.

[0022] Antibody molecules containing a single CDR region are described, for example, in Laune et al., 1997, JBC, 272:30937-44, which demonstrated that a series of hexapeptides derived from the CDR exhibited antigen-binding activity and noted that synthetic peptides of a complete single CDR exhibited strong binding activity. Monnet et al., 1999, JBC, 274:3789-96, demonstrated that a series of 12-mer peptides and related framework regions had antigen-binding activity, and stated that a CDR3-like peptide alone could bind to the antigen. Heap et al., 2005, J. Gen. Virol., 86:1791-1800, reported that "microantibodies" (molecules containing a single CDR) were capable of antigen binding, and demonstrated that a cyclic peptide of an anti-HIV antibody had antigen-binding activity and function. Nicaise et al., 2004, Protein Science, 13:1882-91, show that a single CDR can confer antigen binding activity and affinity for its lysozyme antigen.

[0023] Thus, antibody molecules having five, four, three or fewer CDRs can retain the antigen-binding properties of the full-length antibody from which they are derived.

[0024] An antibody molecule may be a derivative of a full-length antibody or a fragment of such an antibody. A derivative has the same antigen binding characteristics as the corresponding full-size antibody, meaning that it binds to the same epitope on the target as the full-size antibody.

[0025] Thus, as used herein, the term "antibody molecule" includes all types of antibody molecules, including monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, single-chain Fvs (scFvs), Fab fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), antibody heavy chains, antibody light chains, antibody heavy chain homodimers, antibody light chain homodimers, antibody heavy chain heterodimers, antibody light chain heterodimers, antigen-binding functional fragments of such homo- and heterodimers, as well as functional fragments thereof and derivatives thereof.

[0026] Furthermore, as used herein, the term "antibody molecule" includes all classes of antibody molecules and functional fragments, including IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, and IgE.

[0027] In some embodiments, the antibody is human IgG1. Those skilled in the art will recognize that mouse IgG2a and human IgG1 share the ability to productively bind to activating Fc gamma receptors and activate target cell deletion through activation of activating Fc gamma receptor-bearing immune cells (e.g., macrophages and NK cells), for example, by ADCP and ADCC. Thus, mouse IgG2a is the preferred isotype for deletion in mice, while human IgG1 is the preferred isotype for deletion in humans. Conversely, optimal costimulation of TNFR superfamily agonist receptors, such as 4-1BB, ox40, TNFRII, and CD40, is known to depend on antibody binding of inhibitory FcγRII. In mice, the IgG1 isotype, which preferentially binds to inhibitory Fc gamma receptors (FcγRIIB) and only weakly binds to activating Fc gamma receptors, is known to be optimal for the costimulatory activity of TNFR superfamily targeting mAbs. Although a direct human equivalent of the mouse IgG1 isotype has not been described, antibodies can be engineered to similarly exhibit improved binding to inhibitory human Fc gamma receptors over activating human Fc gamma receptors. Such engineered TNFR superfamily-targeting antibodies also have improved in vivo costimulatory activity in transgenic mice engineered to express human activating and inhibitory Fc gamma receptors (49).

[0028] As outlined above, different types and forms of antibody molecules are encompassed by the present invention and will be known to those skilled in the art of immunology. It is well known that antibodies used for therapeutic purposes are often modified with additional moieties that alter the properties of the antibody molecule.

[0029] Thus, antibody molecules of the invention or used in accordance with the invention (e.g., monoclonal and / or polyclonal and / or bispecific antibody molecules) include those that comprise a detectable moiety and / or a cytotoxic moiety.

[0030] "Detectable moieties" include one or more from the group consisting of enzymes, radioactive atoms, fluorescent moieties, chemiluminescent moieties, and bioluminescent moieties. Detectable moieties allow for the visualization of antibody molecules in vitro, and / or in vivo, and / or ex vivo.

[0031] "Cytotoxic moieties" include radioactive moieties and / or enzymes, where the enzymes are caspases and / or toxins, where the toxins are bacterial toxins or venoms, and where the cytotoxic moiety is capable of inducing cell lysis.

[0032] Furthermore, the antibody molecules may be in isolated and / or purified form and / or may be PEGylated.

[0033] As described above, the CDRs of an antibody bind to the antibody target. The amino acid assignments for each CDR described herein are as defined by Kabat EA et al., 1991, "Sequences of Proteins of Immulogical Interest," Fifth Edition, NIH Publication No. 91-3242, pp. xv-xvii.

[0034] As one skilled in the art will recognize, there are other methods for assigning amino acids to each CDR, such as the International ImMunoGeneTics information system (IMGT®) (http: / / www.imgt.org / and Lefranc and Lefranc "The Immunoglobulin Facts Book" published by Academic Press, 2001).

[0035] In a further embodiment, an antibody molecule of or for use in accordance with the present invention is an antibody molecule that can compete with the specific antibodies provided herein, e.g., an antibody molecule comprising any of the amino acid sequences set forth in SEQ ID NOs: 1-152, for binding to a specific target.

[0036] By "competitive" it is meant that a competing antibody is capable of at least partially inhibiting or interfering with the binding of an antibody molecule as defined herein to a particular target.

[0037] For example, such a competing antibody molecule may be capable of inhibiting binding of an antibody molecule described herein by at least about 10%, e.g., 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 may be capable of inhibiting the ability of an antibody described herein to bind to a particular target, preventing or reducing by at least about 10%, e.g., 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%.

[0038] Competitive binding can be determined by methods well known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0039] ELISA assay can be used to evaluate epitope-modified or blocking antibodies. Additional suitable methods for identifying competitive antibodies are disclosed in Antibodies: A Laboratory Manual, Harlow & Lane (see, for example, pages 567-569, 574-576, 583, and 590-612, 1988, CSHL, NY, ISBN 0-87969-314-2), which is incorporated herein by reference.

[0040] It is well known that antibodies specifically bind to defined target molecules or antigens, i.e., they bind preferentially and selectively to their target and not to molecules that are not targets.

[0041] Targets of antibodies according to the invention, or targets of antibodies used according to the invention, are cell surface antigens that are expressed on the surface of cells, i.e., they will contain epitopes (alternatively known in this context as cell surface epitopes) for the antibodies. Cell surface antigens and epitopes are terms that will be readily understood by those skilled in the art of immunology or cell biology.

[0042] The term "cell surface antigen" includes cases where the cell surface antigen is exposed on the extracellular side of the cell membrane, but is only transiently exposed on the extracellular side of the cell membrane. "Transiently exposed" includes cases where the cell surface antigen is either internalized within the cell or released from the extracellular side of the cell membrane into the extracellular space. Cell surface antigens can be released from the extracellular side of the cell membrane by cleavage, which can be mediated by proteases.

[0043] Also, cell surface antigens may be connected to the cell membrane, but may only be transiently associated with the cell membrane. "Transiently associated" includes cases where the cell surface antigen is released from the extracellular side of the cell membrane into the extracellular space. Cell surface antigens may be released from the extracellular side of the cell membrane by cleavage, which may be mediated by proteases.

[0044] Furthermore, the cell surface antigen may be an epitope present on a peptide, or a polypeptide, or a carbohydrate, or an oligosaccharide chain, or a lipid, and / or a protein, or a glycoprotein, or a lipoprotein.

[0045] Methods for assessing protein binding are known to those skilled in the art of biochemistry and immunology.Those skilled in the art will understand that these methods can be used to assess the binding of an antibody to a target and / or the binding of an Fc domain of an antibody to an Fc receptor, and the relative strength, specificity, inhibition, prevention, or reduction of these interactions.Examples of methods that can be used to assess protein binding include, for example, immunoassay, BIAcore, Western blot, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) (for a discussion of antibody specificity, see Fundamental Immunology Second Edition, Raven Press, New York, pp. 332-336 (1989)).

[0046] Thus, a "specifically binding antibody molecule" or a "target-specific antibody molecule" includes an antibody molecule that specifically binds to a target but does not bind to a non-target, or that binds to the non-target weaker than it does to the target (e.g., with lower affinity).

[0047] It also includes the meaning that the antibody specifically binds to a target at least 2 times stronger, or at least 5 times stronger, or at least 10 times stronger, or at least 20 times stronger, or at least 50 times stronger, or at least 100 times stronger, or at least 200 times stronger, or at least 500 times stronger, or at least about 1000 times stronger to a target than to a non-target.

[0048] In addition, the antibody targets at least about 10 -1 K d , or at least about 10 -2 K d , or at least about 10 -3 K d , or at least about 10 -4 K d , or at least about 10 -5 K d , or at least about 10 -6 K d , or at least about 10 -7K d , or at least about 10 -8 K d , or at least about 10 -9 K d , or at least about 10 -10 K d , or at least about 10 -11 K d , or at least about 10 -12 K d , or at least about 10 -13 K d , or at least about 10 -14 K d , or at least about 10 -15 K d K d By binding, it is meant that the antibody specifically binds to the target.

[0049] As used herein, the term Treg-depleting antibody refers to an antibody that, when administered to a subject, such as a human, specifically binds to a target expressed on the surface of Tregs, resulting in the depletion of Tregs. Thus, a Treg-depleting antibody selected from antibodies that specifically bind to a target belonging to the tumor necrosis factor receptor superfamily (TNFRSF) is an antibody that, when administered to a subject, such as a human, specifically binds to a target belonging to the tumor necrosis factor receptor superfamily expressed on the surface of Tregs, resulting in the depletion of Tregs. In some embodiments, the target belonging to the tumor necrosis factor receptor superfamily is a target that is preferentially expressed on tumors or within the tumor microenvironment.

[0050] In some embodiments, the Treg-depleting antibody has no immunostimulatory effect in addition to its Treg-depleting effect. In some embodiments, the Treg-depleting antibody also has an immunostimulatory effect in addition to its Treg-depleting effect, and in such embodiments, the Treg-depleting antibody has sufficiently poor immunostimulatory activity to allow for improved therapeutic activity following sequential administration of a second immunostimulatory antibody.

[0051] To determine whether an antibody is a Treg-depleting antibody within the meaning of the present invention, in vitro antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) assays can be used.

[0052] ADCC assays can be performed by labeling target cells with calcein AM followed by the addition of diluted concentrations of Ab. The target cells are then co-cultured with human PBMCs at a 50:1 E:T ratio for 4 hours at 37°C. The plate is centrifuged at 400 x g for 5 minutes 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 wavelength of 485 nm and an emission wavelength of 530 nm. The percentage of maximum release is calculated as follows: % Maximum Release = (Sample / Triton-treated) * 100. The ADCP assay can be performed by labeling target cells with 5 mM CFSE for 10 minutes at room temperature, followed by washing in complete medium. The CFSE-labeled targets are then opsonized with diluted Abs and then co-cultured with BMDMs at a 1:5 E:T ratio in a 96-well plate for 1 hour at 37°C. BMDMs are then labeled with anti-F4 / 80-allophycocyanin for 15 minutes at room temperature and washed twice with PBS. The plate is kept on ice, and the wells are scraped to collect the BMDMs. 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.

[0053] To determine whether an antibody has an immunostimulatory effect, an in vitro agonism assay can be used. For such an assay, the following general method can be used: Cell cultures were grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum, glutamine (2 mM), pyruvate (1 mM), penicillin, and streptomycin (100 IU / mL) at 37°C in 5% CO. (商標)Fresh PBMCs are labeled with 2 mM carboxyfluorescein succinimidyl ester (CFSE). PBMCs are then plated at 1 × 10 in 24-well plates 48 h prior to the mAb stimulation assay, as described by Romer et al. (51). 7 For PBMC stimulation, wet coat round-bottom 96-well plates with 0.01 μg / mL OKT3 antibody (in-house) in PBS for 4 hours, then discard excess antibody and wash the plate with PBS. 5 PBMCs / well are transferred to plates and stimulated with 5 μg / mL of test mAb. On day 4 or 5 post-stimulation, cells are labeled with anti-CD8-APC (BioLegend) and anti-CD4-PE (in-house), and proliferation is assessed by CFSE dilution on a FACSCalibur (BD Biosciences).

[0054] To determine whether sequential treatment with Treg depletion and immunostimulatory antibodies results in improved therapeutic activity, in vivo assays using tumor-bearing, immunocompetent animals expressing activating and inhibitory Fc gamma receptors can be used, such as those illustrated in Figures 1a and 4c and described in the Examples below.

[0055] As used herein, the term immunostimulatory antibody or immunostimulatory antibody refers to an antibody that, when administered to a subject, such as a human, specifically binds to a target present on the surface of effector T cells. Binding of the immunostimulatory antibody to its target results in stimulation of an immune response, either directly through agonism (e.g., of antibodies against TNF superfamily agonist receptors, such as anti-4-1BB, OX40 antibodies) or indirectly through stimulation of effector T cells through blockade of inhibitory signals (e.g., through antibody blockade of the PD1 / PDL1 axis). Such effector T cells are CD8 + In such embodiments, the immunostimulatory antibody is a CD8 activating and / or CD8 enhancing antibody. Alternatively, or in addition, such effector T cells may be CD4+ In such embodiments, the immunostimulatory antibody is a CD4-activating and / or CD4-enhancing antibody.

[0056] It is shown herein that different antibodies directed against different targets with immunostimulatory or co-inhibitory properties, acting in an Fc:FcγR-dependent or -independent manner, can be used for immune stimulation. Immunostimulatory antibodies can be antibodies such as 4-1BB, which act on immunostimulatory receptors expressed on effector T cells in an Fc:FcγR-dependent manner, or antibodies such as PD-1, which antagonize immune checkpoint receptors expressed on effector T cells in an Fc:FcγR-independent manner.

[0057] To determine whether an antibody is an immunostimulatory antibody within the meaning of the present invention, an in vitro assay demonstrating T cell proliferation in response to the mAb can be used. The assays described above can be used for this purpose.

[0058] To determine whether an antibody lacks or only poorly depletes Tregs, an in vitro phagocytosis assay or an in vivo depletion test can be used. The assays described in the Examples can be used for this purpose. For example, a group of test mice receives tumors on day 0. When the tumors are palpable (or at a stage appropriate for the tumor model), the mice receive intravenous administration of mAb, followed by three additional intraperitoneal administrations every other day (final dose or established dose of mAb, 200 μg). The mice are then sacrificed one or two days after the final mAb administration, and the spleens and tumors are analyzed by flow cytometry for TIL content and the frequency of Foxp3+ cells within the CD4+ population of the tumors and spleens (control tissues) plotted.

[0059] In some embodiments, the Treg-depleting antibody is a human antibody.

[0060] In some embodiments, the Treg-depleting antibody is a humanized antibody.

[0061] In some embodiments, the immunostimulatory antibody is a human antibody.

[0062] In some embodiments, the immunostimulatory antibody is a humanized antibody.

[0063] The Treg-depleting and immunostimulatory antibodies used in combination according to the present invention may both comprise the same CDRs, since the depleting / immunostimulatory effect can be modulated by modifying other parts of the antibody, as described above.

[0064] For example, a Treg-depleting antibody can be obtained using an antibody in the form of a human IgG1 antibody, and thus in some embodiments, the Treg-depleting antibody is a human IgG1 antibody. A Treg-depleting antibody can also be obtained by using an antibody in the form of a human IgG1 antibody that exhibits improved binding to one or several activating Fc receptors and / or has been engineered for improved binding to one or several activating Fc receptors, and thus in some embodiments, the Treg-depleting antibody is an Fc-engineered human IgG1 antibody. A Treg-depleting antibody can also be obtained by using a mouse or humanized mouse IgG2a antibody, and thus in some embodiments, the Treg-depleting antibody is a humanized mouse IgG2a antibody.

[0065] Furthermore, immunostimulatory antibodies can be obtained by using antibodies in the form of human IgG2 antibodies, such as human IgG2b antibodies, or in the form of human IgG4 antibodies. Thus, in some embodiments, the immunostimulatory antibody is a human IgG2 antibody. In some embodiments, the immunostimulatory antibody is a human IgG2b antibody. In some embodiments, the immunostimulatory antibody is a human IgG4 antibody. Immunostimulatory antibodies can also be obtained by using mouse or humanized mouse IgG1 antibodies, and in some embodiments, the immunostimulatory antibody is a humanized mouse IgG1 antibody.

[0066] In some embodiments, the immunostimulatory antibody is an antibody that exhibits improved binding to inhibitory Fcγ receptors over activating Fcγ receptors, hi some embodiments, the immunostimulatory antibody is an antibody that exhibits improved binding to human FcγRIIB over activating Fcγ receptors.

[0067] In some embodiments, the immunostimulatory antibody is engineered for improved binding to inhibitory Fcγ receptors over activating Fcγ receptors, hi some embodiments, the immunostimulatory antibody is engineered for improved binding to human FcγRIIB over activating Fcγ receptors.

[0068] The target to which the Treg-depleting antibody of the present invention or used in accordance with the present invention binds may be selected from the group consisting of targets belonging to TNFRS. The target belonging to TNFRS may be selected from the group consisting of 4-1BB, OX40, and TNFR2.

[0069] Alternatively, the target to which the Treg-depleting antibody of the invention or used in accordance with the invention binds may be selected from the group consisting of ICOS, GITR, CTLA-4, CD25, and neuropilin-1. In some embodiments, the target is not CD25.

[0070] The target to which the immunostimulatory antibody of the invention or immunostimulatory agent used in accordance with the invention binds may be selected from the group consisting of 4-1BB and OX40.

[0071] Alternatively, the target to which the immunostimulatory antibody of the invention or Treg-depleting antibody used in accordance with the invention binds may be selected from the group consisting of ICOS, GITR, CTLA-4, TNFR2, CD25, and PD-1, hi some embodiments, the target is not CD25.

[0072] In some embodiments of the present invention, at least one target is 4-1BB, also known as CD137 and tumor necrosis factor receptor superfamily member 9 (TNFRSF9). 4-1BB is expressed on Tregs and subsequently activates CD4+ and CD8+ T cells, and its ligation is required for optimal protective CD8+ T cell responses against viruses and B-cell lymphomas in mice (11, 12). Anti-4-1BB-specific antibodies enhance the proliferation and survival of antigen-stimulated T cells in vitro, and, like anti-CD40, anti-4-1BB mAbs promote antitumor immunity in preclinical cancer models that primarily depend on CD8+ T cells (12, 13). 4-1BB is a downstream target of the Treg lineage-defining transcription factor Foxp3, is expressed on resting Treg cells, and is upregulated upon Treg activation (14, 15). Therefore, it is possible that anti-4-1BB may act in part through Treg cell depletion. Whether an anti-4-1BB antibody is a depleting or stimulatory antibody likely depends on its FcγR usage, and in the studies leading to this invention, the inventors performed in vitro and in vivo experiments to explore the optimal isotype for therapeutic anti-4-1BB mAbs in the tumor environment.

[0073] Although mIgG1 isotype mAbs exerted superior agonistic activity and direct immune stimulation of CD8+ T cells compared with the mIgG2a version of the same specificity, mIgG2a mAbs were found to provide optimal therapeutic activity in established solid tumor settings. The efficacy of mIgG2a mAbs was found to be due to the depletion of intratumoral Tregs. Even when depletion was prevented, the therapeutic potential of mIgG2a was retained in mice lacking activating FcγRs. Under these conditions, mIgG2a was converted into an agonist by binding to the inhibitory FcγRIIB. In addition, it was confirmed that depletion and agonism are competing mechanisms, and simultaneous binding of both reduces efficacy. This blunted activity could be overcome through sequential administration of Treg depletion followed by an immunostimulatory isotype or through Fc engineering to generate a dual-active anti-4-1BB mAb with optimal FcγR depletion capacity along with FcγR-independent agonism. Together, these results further demonstrate that immunomodulatory mAbs with the same target specificity can utilize different mechanisms to mediate therapy, and that their optimal use depends on the isotype, local FcγR repertoire, abundance, and function of both immunosuppressive and effector cells, as well as their relative and absolute target expression in the tumor microenvironment. Importantly, the results demonstrate that the temporal administration of immunomodulatory mAbs with complementary but competing mechanisms of action can be used to optimize outcomes, and that through mAb engineering, it is possible to generate a single agent capable of utilizing multiple mechanisms to deliver improved therapeutic efficacy. These results have implications for the administration of existing and developmental immunomodulatory mAbs, as well as for the design of next-generation immunomodulatory antibodies.

[0074] In some embodiments, the Treg-depleting antibody molecule is an anti-4-1BB antibody molecule selected from the group presented in Table 1 below.

[0075] In some embodiments, the Treg-depleting antibody molecule is an anti-4-1BB antibody molecule selected from the group consisting of antibody molecules comprising 1 to 6 of the CDRs from each group selected from SEQ ID NOs: 1 to 6, SEQ ID NOs: 9 to 14, SEQ ID NOs: 17 to 22, SEQ ID NOs: 25 to 30, SEQ ID NOs: 33 to 38, SEQ ID NOs: 41 to 46, SEQ ID NOs: 49 to 54, SEQ ID NOs: 57 to 62, SEQ ID NOs: 65 to 70, SEQ ID NOs: 153 to 158, and SEQ ID NOs: 163 to 168.

[0076] In some embodiments, the Treg-depleting antibody molecule is an anti-4-1BB antibody molecule selected from the group consisting of antibody molecules comprising six 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.

[0077] In some embodiments, the Treg-depleting antibody molecule is an anti-4-1BB antibody 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.

[0078] In some embodiments, the Treg-depleting antibody molecule is an anti-4-1BB antibody 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.

[0079] In some embodiments, the Treg-depleting antibody molecule is an anti-4-1BB 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: 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.

[0080] In some embodiments, the immunostimulatory antibody molecule is an anti-4-1BB antibody molecule selected from the group presented in Table 1 below.

[0081] In some embodiments, the immunostimulatory antibody molecule is an anti-4-1BB antibody molecule selected from the group consisting of antibody molecules comprising 1 to 6 of the CDRs from each of the groups set forth in SEQ ID NOs: 1 to 6, 9 to 14, 17 to 22, 25 to 30, 33 to 38, 41 to 46, 49 to 54, 57 to 62, 65 to 70, 153 to 158, and 163 to 168.

[0082] In some embodiments, the immunostimulatory antibody molecule is an anti-4-1BB antibody molecule selected from the group consisting of antibody molecules comprising six 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.

[0083] In some embodiments, the immunostimulatory antibody molecule is an anti-4-1BB antibody 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.

[0084] In some embodiments, the immunostimulatory antibody molecule is an anti-4-1BB antibody 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.

[0085] In some embodiments, the immunostimulatory antibody molecule is an anti-4-1BB antibody 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. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0086] In some embodiments, the Treg-depleting antibody molecule is an anti-OX40 antibody molecule selected from the group presented in Table 2 below.

[0087] In some embodiments, the Treg-depleting antibody molecule is an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising 1 to 6 CDRs from each of the groups 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.

[0088] In some embodiments, the Treg-depleting antibody molecule is an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising six 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.

[0089] In some embodiments, the Treg-depleting antibody molecule is an anti-OX40 antibody molecule selected from the group comprising a VH selected from the group consisting of SEQ ID NOs: 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, and 177.

[0090] In some embodiments, the Treg-depleting 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, 112, 120, 128, 136, 144, 152, and 178.

[0091] In some embodiments, the Treg-depleting antibody molecule is an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising a VH and VL selected from the group consisting of sequences 79-80, 87-88, 95-96, 103-104, 111-112, 119-120, 127-128, 135-136, 143-144, 151-152, and 177-178.

[0092] In some embodiments, the immunostimulatory antibody molecule is an anti-OX40 antibody molecule selected from the group presented in Table 2 below.

[0093] In some embodiments, the immunostimulatory antibody molecule is an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising 1 to 6 CDRs from each of the groups 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.

[0094] In some embodiments, the immunostimulatory antibody molecule is an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising six 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.

[0095] In some embodiments, the immunostimulatory antibody molecule is an anti-OX40 antibody molecule selected from the group consisting of antibody binding molecules comprising a VH selected from the group consisting of SEQ ID NOs: 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, and 177.

[0096] 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, 112, 120, 128, 136, 144, 152, and 178.

[0097] 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 sequences 79-80, 87-88, 95-96, 103-104, 111-112, 119-120, 127-128, 135-136, 143-144, 151-152, and 177-178. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0098] 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.

[0099] 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.

[0100] In some embodiments, the immunostimulatory antibody is an anti-PDL1 antibody, preferably a human anti-PDL1 antibody. The anti-PDL1 antibody can be atezolizumab.

[0101] In some embodiments, the immunostimulatory antibody is an anti-CTLA-4 antibody, preferably a human anti-CTLA-4 antibody. The anti-CTLA-4 antibody may be selected from the group consisting of ipilimumab and tremilimumab.

[0102] The Treg-depleting antibody is administered to a subject, such as a human, before the administration of the immunostimulatory antibody. This means that the Treg-depleting antibody is first administered to the tumor to achieve the Treg-depleting effect. Once the Treg-depleting effect has appeared, the immunostimulatory antibody is administered. This sequential administration can be achieved by temporally separating the two antibodies. Alternatively, or in combination with the first option, the sequential administration can also be achieved by spatially separating the two antibodies, such as by administering the Treg-depleting antibody intratumorally or in other ways to reach the tumor before the immunostimulatory antibody, and then administering the immunostimulatory antibody systemically or in other ways to reach the tumor after the Treg-depleting antibody.

[0103] Those skilled in the art of medicine will know that drugs can be modified with different additives, for example to change the rate at which the drug is absorbed by the body, or can be modified in different formats to allow for specific routes of administration to the body.

[0104] Thus, the compositions, and / or antibodies, and / or agents, and / or drugs of the present invention include those in combination with excipients, and / or pharmaceutically acceptable carriers, and / or pharmaceutically acceptable diluents and / or adjuvants.

[0105] The compositions, and / or antibodies, and / or agents, and / or drugs of the present invention also include those suitable for parenteral administration, including aqueous and / or non-aqueous sterile injection solutions which may contain antioxidants, and / or buffers which render the formulation isotonic with the blood of the intended recipient, and / or bacteriostats, and / or solutes; and / or suspending agents and / or thickening agents. The compositions, and / or antibodies, and / or agents, and / or drugs of the present invention may be contained in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water for injections, immediately prior to use.

[0106] Extemporaneous injection solutions and suspensions may be prepared from sterile powders and / or granules and / or tablets of the type previously described.

[0107] For parenteral administration to human patients, daily dosage levels of Treg-depleting antibodies and / or immunostimulatory antibodies will typically be 1 mg to 20 mg / kg of patient body weight, or in some cases up to 100 mg / kg, administered in single or divided doses. Lower doses may be used in special circumstances, such as in combination with chronic administration. In any event, the physician will determine the actual dosage most appropriate for an individual patient, which will vary with the age, weight, and response of the particular patient. The dosages described above are exemplary of the average case. Of course, there may be individual cases in which higher or lower dosage ranges are appropriate, and these are within the scope of the present invention.

[0108] Typically, compositions and / or medicaments of the invention will contain Treg-depleting antibodies and / or immunostimulatory antibodies at a concentration of approximately 2 mg / ml to 150 mg / ml, or approximately 2 mg / ml to 200 mg / ml. In a preferred embodiment, medicaments and / or compositions of the invention will contain Treg-depleting antibodies and / or immunostimulatory antibodies at a concentration of 10 mg / ml.

[0109] Generally, in humans, oral or parenteral administration of the compositions, and / or antibodies, and / or agents, and / or drugs of the present invention is the preferred route and is most convenient. For veterinary use, the agents, and / or antibodies, and / or agents, and / or drugs of the present invention will be administered in an acceptable formulation as appropriate in accordance with normal veterinary practice, and the veterinarian will determine the dosage regimen and route of administration that will be most appropriate for a particular animal. Accordingly, the present invention provides pharmaceutical formulations comprising antibodies and / or drugs of the present invention in an amount effective to treat various conditions (described above and further below). Preferably, the compositions, and / or antibodies, and / or agents, and / or drugs are adapted for delivery by a route selected from the group including intravenous, intramuscular, and subcutaneous.

[0110] The present invention also includes compositions comprising pharmaceutically acceptable acid or base addition salts of the polypeptide binding moieties of the present invention, and / or antibodies, and / or pharmaceutical agents, and / or drugs. The acids used to prepare pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful in the present invention are, among others, those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate [i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)] salts. Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the agents of the present invention. Chemical bases that can be used as reagents to prepare pharmaceutically acceptable base salts of the present agents, which 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 pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), water-soluble amine addition salts such as ammonium or N-methylglucamine (meglumine), and lower alkanolammonium, as well as other pharmaceutically acceptable organic amine base salts. The agents and / or polypeptide binding moieties of the present invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilization method (e.g., spray drying, cake drying) and / or reconstitution technique may be used. Those skilled in the art will understand that lyophilization and reconstitution may lead to varying degrees of loss of antibody activity (e.g., in conventional immunoglobulins, IgM antibodies tend to have greater loss of activity than IgG antibodies), and that usage levels may need to be adjusted upward to compensate.In one embodiment, a lyophilized (freeze-dried) polypeptide binding moiety loses about 20% or less, or about 25% or less, or about 30% or less, or about 35% or less, or about 40%, or about 45% or less, or about 50% or less of its activity (before lyophilization) when rehydrated.

[0111] A combination of a Treg-depleting antibody molecule and an immunostimulatory antibody molecule, where the Treg-depleting antibody molecule is administered to a subject prior to administration of the immunostimulatory antibody molecule to the subject, can be used to treat cancer.

[0112] The subject may be a mammal or a non-mammal. Preferably, the mammalian subject is a human or non-mammal, such as a horse, cow, sheep, pig, camel, dog, or cat. Most preferably, the mammalian subject is a human.

[0113] "Exhibiting" includes when a subject exhibits cancer symptoms and / or cancer diagnostic markers and / or when cancer symptoms and / or cancer diagnostic markers are measured and / or assessed and / or quantified.

[0114] It will be readily apparent to one skilled in the art of medicine what cancer symptoms and cancer diagnostic markers are, and how to measure and / or assess and / or quantify whether there is a reduction or increase in the severity of cancer symptoms, or a reduction or increase in cancer diagnostic markers, and how cancer symptoms and / or cancer diagnostic markers can be used to form a prognosis for cancer.

[0115] Cancer treatments are often administered as a course of treatment, i.e., the therapeutic agent is administered over a period of time. The length of the course of treatment depends on many factors, including, among other things, the type of therapeutic agent administered, the type of cancer being treated, the severity of the cancer being treated, and the age and health of the subject.

[0116] "Currently undergoing treatment" includes a subject currently undergoing a course of treatment and / or receiving a therapeutic agent and / or a series of therapeutic agents.

[0117] In some embodiments, the cancer treated according to the present invention is a solid tumor.

[0118] In some embodiments, the cancer is selected from the group consisting of sarcoma, carcinoma, and lymphoma.

[0119] In some embodiments, the cancer is selected from the group consisting of squamous cell carcinoma (SCC), thymoma, neuroblastoma, or ovarian cancer.

[0120] Any of the above-mentioned cancers are well known, and the symptoms and cancer diagnostic markers are well described, as are the therapeutic agents used to treat those cancers. Thus, the symptoms, cancer diagnostic markers, and therapeutic agents used to treat the above-mentioned types of cancer will be known to those skilled in the art of medicine.

[0121] The clinical definition of the diagnosis, prognosis, and progression of most cancers is based on a specific classification known as staging. These staging systems collate many different cancer diagnostic markers and cancer symptoms to provide an overview of the diagnosis, prognosis, and / or progression of cancer. Those skilled in the art of oncology will know how to use staging systems to evaluate the diagnosis, prognosis, and / or progression of cancer, and which cancer diagnostic markers and cancer symptoms should be used for this purpose.

[0122] "Cancer staging" includes the Rai staging system, including stage 0, stage I, stage II, stage III, and stage IV, and / or the Binet staging system, including stage A, stage B, and stage C, and / or the Ann Arbour staging system, including stage I, stage II, stage III, and stage IV.

[0123] It is known that cancer can cause abnormalities in cell shape. These abnormalities often occur reproducibly in a particular cancer, which means that examining these changes in shape (also known as histological examination) can be used for cancer diagnosis or prognosis. Techniques for visualizing and preparing samples for examining cell shape are well known in the art, such as light microscopy or confocal microscopy.

[0124] "Histological examination" includes the presence of small mature lymphocytes, and / or the presence of small mature lymphocytes with narrow cytoplasmic borders, the presence of small mature lymphocytes with dense nuclei lacking discernible nucleoli, and / or the presence of small mature lymphocytes with narrow cytoplasmic borders and dense nuclei lacking discernible nucleoli, and / or the presence of atypical cells, and / or cleaved cells, and / or prolymphocytes.

[0125] It is well known that cancer is the result of the mutation of cellular DNA, which can lead to cell death avoidance or uncontrollable proliferation.Therefore, the examination of these mutations (also known as cytogenetic examination) can be a useful tool for assessing the diagnosis and / or prognosis of cancer.An example of this is the deletion of chromosome position 13q14.1, which is characteristic of chronic lymphocytic leukemia.The technique for examining mutations in cells is well known in the art, for example, fluorescence in situ hybridization (FISH).

[0126] "Cytogenetic testing" refers to the testing of DNA in cells, specifically chromosomes. Cytogenetic testing can be used to identify DNA alterations that may be associated with the presence of refractory cancer and / or recurrent cancer. Such alterations include deletion of the long arm of chromosome 13, and / or deletion of chromosome location 13q14.1, and / or trisomy of chromosome 12, and / or deletion of the long arm of chromosome 12, and / or deletion of the long arm of chromosome 11, and / or deletion of 11q, and / or deletion of the long arm of chromosome 6, and / or deletion of 6q, and / or deletion of the short arm of chromosome 17, and / or deletion of 17p, and / or t(11:14) translocation. and / or (q13:q32) translocations, 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) translocations, and / or (q13:q32) translocations, and / or (3:v) translocations, and / or (8:14) translocations, and / or (8:v) translocations, and / or t(11:14) and (q13:q32) translocations.

[0127] It is known that subjects with cancer exhibit certain physical symptoms, which are often the result of cancer's physical burden.These symptoms often recur with the same cancer, and can be diagnostic and / or prognostic and / or progression characteristics of the disease.Those skilled in the art of medicine will understand which physical symptoms are related to which cancer, and how the evaluation of these body systems can be correlated with the diagnosis and / or prognosis and / or progression of the disease."Physical symptoms" include hepatomegaly and / or splenomegaly. [Brief explanation of the drawings]

[0128] The examples below refer to the following diagram: [Figure 1-1]Anti-4-1BB mIgG2a mAb, but not mIgG1, confers a survival benefit in multiple cancer models. Figure 1A: Groups of BALB / c mice were challenged with 5 x 105 subcutaneous doses of CT26 on day 0. When tumors were palpable, mice were administered anti-4-1BB (LOB12.0) mIgG1, mIgG2a, or PBS control intravenously, followed by three additional intraperitoneal doses every other day (final dose of 200 μg). [Figure 1-2] Figure 1B: Groups of A / J mice were challenged with 2 x 10 NXS2 cells subcutaneously. When tumors were palpable, 200 µg of anti-4-1BB mAb or isotype control mAb was administered intraperitoneally. A second 200 µg dose was administered 3 days later. In both experimental models, tumor growth was monitored, and mice were culled when the mean tumor area exceeded 225 mm. Data are expressed as tumor area (mm) several days after tumor challenge, as indicated. Each line represents an individual mouse. The right panel shows the survival rate to the humane endpoint. Data represent examples from at least two independent experiments, with n = 5 mice per group. [Figure 2-1]Anti-4-1BB mIgG1 exerts agonist activity in vitro and in vivo. Figure 2A: Splenocytes from Foxp3-GFP mice, sorted for either GFP+ cells (-Tregs) or non-GFP+ cells (+Tregs), were cultured with 0.1 μg / ml anti-CD3 and the indicated concentrations of either anti-4-1BB (LOB12.0) mIgG1 or mIgG2a, as indicated. [3H]-thymidine incorporation was measured during the final 16 hours of a 72-hour culture. Figure 2B: Splenocytes from C57BL / 6 mice were similarly cultured with 0.1 μg / ml anti-CD3 and the indicated concentrations of anti-4-1BB mIgG1, mIgG2a, or a 1:1 mixture of the two, after which [3H]-thymidine incorporation was assessed. Data in Figures 2A and 2B represent the mean (+ / - SEM) counts per minute from triplicate wells. Figure 2C: Groups of mice were administered 5 mg of OVA and 200 μg of anti-4-1BB mIgG1 or mIgG2a intraperitoneally on day 0. SIINFEKL-specific T cell responses in peripheral blood were quantified by flow cytometry and expressed as % of total CD8+ cells. Data are from three separate experiments and show the time course of the response (mean ± SEM, 6 mice per group) and the peak of the response (mean and individual responses, 9 mice per group, *p=0.023). [Figure 2-2] Figure 2D: Groups of A / J mice were challenged with 2 x 10 NXS2 cells subcutaneously on day 0 and received 200 μg of anti-4-1BB mAb or isotype control mAb intraperitoneally on day 3, along with control (SIINFEKL, left panel) or TH (FETFEAKI, right panel) peptides. A second dose of mAb (200 μg) was administered 6 days later. Data in Figures 2B and 2C represent examples from at least two experiments with n = 5 mice per group. [Figure 3-1]Mouse and human tumor-resident Treg cells preferentially express 4-1BB. Figure 3A: Immunofluorescence microscopy image (left panel) showing 4-1BB expressing intratumoral Foxp3+ Tregs in CT26 tumors. Scale bar = 50 μM. Flow cytometry histogram (right panel) demonstrating 4-1BB expression on T cell subsets from TILs and splenocytes. 4-1BB expression (black line) relative to isotype control (gray line) on CD4+Foxp3+ (inset top panel), CD4+Foxp3- (inset middle panel), and CD8+ T cells (inset bottom panel). Cells were isolated from CT26 tumor-bearing mice. [Figure 3-2]Figure 3B: Freshly surgically resected ovarian tumor, ascites, and blood samples were obtained from patients and compared with healthy PBMCs. Tumor samples were minced and gradient-digested prior to isolation. Matched peripheral blood was obtained, and peripheral blood mononuclear cells were isolated by centrifugation. 4-1BB expression on CD4+CD25+CD127- Treg cells, CD4+ non-Treg cells, and CD8+ effector T cells was assessed by flow cytometry. The top panel shows representative histograms of 4-1BB expression in tumor tissue (solid black line), blood (dashed gray line), and ascites (dashed black line) from the same patient. The isotype control is indicated by a solid gray line. The bottom panel shows 4-1BB expression on T cell subsets from different tissue samples. Data points represent individual patients / donors: n = 11 for healthy PBMCs, n = 20 for ascites, n = 9 for tumor, and n = 5 for patient blood. Figure 3C: Freshly surgically excised squamous cell carcinoma (SCC) and normal skin samples were obtained from patients. Samples were minced and gradient-density digested before isolation. Matched peripheral blood was obtained, and peripheral blood mononuclear cells were isolated by centrifugation. Cells were stained for 4-1BB, and staining was detected by flow cytometry. The top panel shows representative histograms in which 4-1BB staining is shown as open histograms for tumor tissue (solid black line), blood (dashed gray line), normal skin (dashed black line), and isotype control (solid gray line). The bottom panel shows 4-1BB expression on T cell subsets in different tissue samples. Data points represent 10 individual patients. [Figure 4-1]The primary mechanism of anti-4-1BB mAb therapy in solid tumors depends on the antibody isotype and FcγR availability. Figure 4A: On day 0, groups of 3–4 WT, FcγRIIB KO, or γ-chain KO BALB / c mice were challenged with 5 × 10 CT26 cells subcutaneously. When tumors were palpable, mice received anti-4-1BB mIgG1, mIgG2a, or PBS control intravenously, followed by three additional intraperitoneal doses every other day (final dose of 200 μg). Mice were sacrificed on day 13, and spleens and tumors were analyzed by flow cytometry. Data show the frequency of Foxp3+ cells within the CD4+ population in tumors (left panel) or matched spleens (right panel). Data are representative of two independent experiments. Figure 4B: Mice were treated as in (A), and CD8+, Ki67+ T cells were enumerated and plotted as fold change compared to controls. [Figure 4-2] Figure 4C: Groups of WT, γ-chain KO, or FcγR null mice (γ-chain KO x FcγRIIB KO), γ-chain KO, or FcγRIIB KO BALB / c mice were challenged with 5 x 10 CT26 cells and treated with anti-4-1BB mAb as in (A). Tumor growth was monitored, and mice were culled when the mean tumor area exceeded 225 mm. Data are expressed as tumor area (mm) several days after tumor challenge, as indicated, and each line represents an individual mouse. The right panel shows survival to the humane endpoint. Data represent examples from at least two experiments, with n = 5 mice per group. [Figure 4-3]Figure 4D: CFSE-labeled target mouse splenic T cells opsonized with anti-4-1BB mIgG1, mIgG2a, or control mAbs were cocultured with wild-type (solid bars) or FcgRIIB KO (open bars) mBMDMs and then assessed for phagocytosis. (E) (Left panel) CFSE-labeled target human T cells opsonized with anti-human 4-1BB hIgG1 mAb clone SAP3-6, BI5-B02 (also designated 005-BI02), or control were cocultured with hMDMs and then assessed for ADCP. (Right panel) The level of phagocytosis is plotted relative to the level of 4-1BB expression determined by flow cytometry. In all cases, phagocytosis is plotted as the % of double-positive macrophages. [Figure 5-1] The planned administration of heterologous anti-4-1BB or planned combination with anti-PD-1 mAb enhances antitumor activity. Figure 5A: Groups of age- and sex-matched BALB / c mice were challenged subcutaneously with 5 x 104 CT26 on day 0. When tumors were palpable, mice were intravenously administered anti-4-1BB (LOB12.0) mIgG1, mIgG2a, mIgG1 and mIG2a simultaneously, or PBS control, followed by three additional intraperitoneal doses every other day (final dose of 200 μg). For planned administration, mIgG2a was administered intravenously, followed by mIgG1 intraperitoneally 4 days later. Tumor growth was monitored, and mice were culled when the mean tumor area exceeded 225 mm2. Data are expressed as the mean tumor area (mm2) for several days after tumor challenge, as indicated. Data presented are combined from two independent experiments with n = 10 mice per group. Figure 5B: Mice were challenged with CT26 tumors and then administered monotherapy as in Figure 5A, or a planned combination of anti-4-1BB mIgG1 or mIgG2a and / or anti-PD-1 rIgG1(WT), or their deglycosylated forms. For combinations, anti-4-1BB mAb was administered intravenously when tumors first became palpable, followed by anti-PD-1 intraperitoneally 4 days later. Tumor growth was monitored, and data were plotted as in Figure 5A. Data represent examples from at least two independent experiments, with n = 4 or 5 mice per group. [Figure 5-2] The planned administration of heterologous anti-4-1BB or planned combination with anti-PD-1 mAb enhances antitumor activity. Figure 5A: Groups of age- and sex-matched BALB / c mice were challenged subcutaneously with 5 x 104 CT26 on day 0. When tumors were palpable, mice were intravenously administered anti-4-1BB (LOB12.0) mIgG1, mIgG2a, mIgG1 and mIG2a simultaneously, or PBS control, followed by three additional intraperitoneal doses every other day (final dose of 200 μg). For planned administration, mIgG2a was administered intravenously, followed by mIgG1 intraperitoneally 4 days later. Tumor growth was monitored, and mice were culled when the mean tumor area exceeded 225 mm2. Data are expressed as the mean tumor area (mm2) for several days after tumor challenge, as indicated. Data presented are combined from two independent experiments with n = 10 mice per group. Figure 5B: Mice were challenged with CT26 tumors and then administered monotherapy as in Figure 5A, or a planned combination of anti-4-1BB mIgG1 or mIgG2a and / or anti-PD-1 rIgG1(WT), or their deglycosylated forms. For combinations, anti-4-1BB mAb was administered intravenously when tumors first became palpable, followed by anti-PD-1 intraperitoneally 4 days later. Tumor growth was monitored, and data were plotted as in Figure 5A. Data represent examples from at least two independent experiments, with n = 4 or 5 mice per group. [Figure 6]Fc-engineered anti-4-1BB mIgG2a / h2B possesses dual activity and delivers enhanced cancer therapy. Figure 6A: nrCE-SDS profiles of anti-4-1BB (LOB12.0) mIgG2a, mIgG2a / h2, and "biased" mIgG2a / h2B. Figure 6B: C57Bl / 6 mouse splenocytes were cultured with 0.01 μg / ml anti-CD3 and the indicated concentrations of either anti-4-1BB mIgG1, mIgG2a, or mIgG2a / h2B, as indicated. [H]-thymidine incorporation was measured during the final 16 hours of a 72-hour culture. Figure 6C: CFSE-labeled target mouse splenic T cells opsonized with anti-4-1BB mIgG2a, mIgG2a / h2B, or control mAb were cocultured with wild-type mBMDMs and then assessed for phagocytosis. Phagocytosis is plotted as the % of double-positive macrophages. Figure 6D: On day 0, groups of age- and sex-matched C57Bl / 6 mice were challenged subcutaneously with 5 x 10 EG7. On days 3, 5, and 7, mice were intraperitoneally injected with 200 μg of mAb or PBS control, as indicated. On day 20, tumors were harvested and TILs were enumerated by flow cytometry (n = 4 mice per group). (E) Mice were set up as in (D), monitored for tumor growth, and culled when the mean tumor area exceeded 400 mm. Data represent examples from at least two independent experiments, with n = 5 mice per group. [Figure 7-1] Characterization of anti-4-1BB mAb. Figure 7A: Surface plasmon resonance analysis of binding of anti-4-1BB (clone LOB12.0) mIgG1, mIgG2a, and parental rIgG2a to mouse FcγRI, IIB, III, and IV. Immobilized 4-1-BB mAb at 5000 RU outperformed recombinant soluble FcγR protein (0, 6, 23, 94, 375, 1500 nM). Sensorgrams are shown. [Figure 7-2]Figure 7B: A human cell line stably transfected with a construct encoding the extracellular and transmembrane domains of mouse 4-1BB was cultured with a range of concentrations of anti-4-1BB of the mIgG1 or mIgG2a isotype, or the parental rIgG2a mAb, followed by staining with a PE-labeled secondary antibody. Data show the mean fluorescence intensity as a percentage of maximum at each concentration. Figure 7C: Rat anti-4-1BB was mixed with mouse mIgG1 or mIgG2a anti-4-1BB mAb at the indicated concentrations and then cultured with the mouse 4-1BB-transfected cell line. Rat mAb binding was detected with an anti-rat secondary antibody, and data are presented as the mean fluorescence intensity of the rat anti-4-1BB antibody relative to the concentration of competitor mouse anti-4-1BB. [Figure 8] The antitumor efficacy of anti-4-1BB mIgG2a mAb is dependent on CD8+ T cells. Groups of BALB / c mice were treated or not with 500 μg of CD8-depleting antibody on days -1, 1, and 4 in conjunction with a subcutaneous challenge of 5 × 10 CT26 cells on day 0. Anti-41BB mIgG2a mAb was administered intravenously on day 6 and intraperitoneally on days 8, 10, and 12 to a final total dose of 200 μg. Tumor size was recorded, and mice were culled when the mean tumor diameter reached 15 mm. Data show tumor area (mm2) on the indicated days after tumor challenge, with each line representing an individual mouse. (n = 5 mice / group) [Figure 9-1] Primary in vivo proliferation of OT-I cells in response to OVA and anti-4-1BB mAb. Figure 9A: Groups of three wild-type or FcγRIIB- / - mice were administered 2 x 105 OT-I cells intravenously and 24 hours later (day 0), were injected intraperitoneally with 0.5 mg of OVA and 200 μg of mIgG1 or mIgG2a anti-4-1BB. Control mice received OVA alone. Blood samples were collected to measure circulating SIINFEKL tetramer+ CD8+ cells in response and expressed as % of total CD8+ cells (mean ± SEM). Data are representative of two experiments. [Figure 9-2]Figure 9B: Groups of five C57BL / 6 mice were injected intradermally with 2.5 x 10 B16 / BL6 cells on day 0, followed by 1 x 10 irradiated FVAX cells intradermally in the contralateral 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-4-1BB antibody intraperitoneally, as indicated. Survival rates to the humane endpoint are shown. [Figure 10] Characterization of anti-PD-1 mAbs. Figure 10A: Surface plasmon resonance analysis of binding of anti-PD-1 (clone EW1-9) rIgG1 (black solid line) and rIgG1 deglycosylated (gray solid line) to mouse FcγRI, IIB, III, and IV. PD-1 mAb (500 nM) outcompeted recombinant FcγR-his protein (1000 RU) (R&D Systems) captured on a CM5 chip with anti-histidine mAb (GE Healthcare). Sensorgrams are shown with the 0 nM curve subtracted. Figure 10B: Analysis demonstrating that anti-PD-1 mAb binds to PD-1 and blocks PD-L1 binding. Recombinant PD-1-his (R&D Systems) (2000 RU) captured on a CM5 chip with anti-histidine mAb was out-competed by anti-PD-1 rIgG1 (solid black line), rIgG1 deglycosylated (solid gray line), or buffer (dashed black line). At the time points indicated by the arrows, it out-competed recombinant PD-L1-Fc (R&D Systems), demonstrating binding to PD-1 and blocking by anti-PD-1. [Figure 11] Anti-4-1BB mIgG2a / h2B FcγR binding. Surface plasmon resonance analysis of binding of anti-4-1BB (clone LOB12.0) mIgG2a and mIgG2a / h2B to mouse FcγRI, IIB, III, and IV. Immobilized 4-1BB mAb at 5000 RU outperformed recombinant soluble FcγR protein (0, 6, 23, 94, 375, 1500 nM). Sensorgrams are shown. [Figure 12] Binding titration curves for HDLM2 cells are shown. [Figure 13] Ligand blockade is shown. [Figure 14] The left panel shows the results of an in vitro assay using ADCC of 4-1BB+IVA CD4s, and the right panel shows the results of an in vitro assay using CD8 T cell proliferation. [Figure 15] Binding to in vitro activated human CD4+ cells is shown. [Figure 16] Demonstrating cyon cross-reactivity with activated CD4+ T cells. [Figure 17] Ligand blockade is shown. [Figure 18] ADCC in T cells is shown, demonstrating that several mAbs induce significant ADCC in OX40-expressing CD4+ T cells. The figure shows the average of five experiments. Campath was used as a positive control, and Yervoy was used as a comparator. [Figure 19] 1 shows the results of an in vitro proliferation assay. [Figure 20] The agonist activity of different antibodies is demonstrated in vivo using the h OX40 KI / OT1 transfer model. [Figure 21] A table outlining the characteristics of some of the antibodies described herein is provided. [Figure 22] Treg cells and effector cells from different compartments in humans are shown, clearly demonstrating that Tregs in and / or near tumor tissues have distinct potential target expression profiles compared to peripheral Tregs. [Figure 23] Figure 1 shows receptor expression on Treg cells in different organs of mice. [Figure 24] Receptor expression on Treg cells compared to other cell types in mice is shown. [Example]

[0129] Specific non-limiting examples embodying certain aspects of the present invention are described below.

[0130] Examples of sequential administration result The therapeutic activity of anti-4-1BB mAbs is determined by their isotype It has previously been established that immunostimulatory mAb activity targeting TNFR superfamily members is dependent on cross-linking provided by the inhibitory FcγRIIB. To determine whether this requirement similarly applies to anti-4-1BB, we generated mIgG1 and mIgG2a chimeric versions of the rIgG2a anti-4-1BB mAb (in-house generated LOB12.0 (16)), as previously described for other mAb specificities (17-19). The nucleotide sequence encoding the LOB12.0 mIgG1 heavy chain is shown in SEQ ID NO: 179, and the corresponding amino acid sequence is shown in SEQ ID NO: 180. The nucleotide sequence encoding the LOB12.0 mIgG2a heavy chain is shown in SEQ ID NO: 181, and the corresponding amino acid sequence is shown in SEQ ID NO: 182. Surface plasmon resonance and flow cytometry analyses confirmed that these mAbs had the expected mFcγR binding profiles, with mIgG2a having a high activating to inhibitory FcγR ratio (A:I) and, conversely, mIgG1 having a low A:I (Figure 7A, (19, 20)). Both mAbs retained comparable 4-1BB specificity and binding (Figures 7B and 7C). We then evaluated the therapeutic potential of these murine anti-4-1BB mAbs in three different solid tumor models established using CT26 colon carcinoma (Figure 1A) and NXS2 neuroblastoma (Figure 1B). In marked contrast to anti-CD40 studies (18, 19, 21) and published reports on other agonistic anti-TNFR superfamily mAbs targeting DR5 (22, 23), our study showed that anti-4-1BB and mIgG2a mAbs at high A:I ratios provided substantial therapeutic efficacy (80% long-term survival in all models), whereas the mIgG1 version at low A:I ratios was largely ineffective (0–20% survival). Notably, although it was the mIgG2a mAb that was protective in these settings, its therapeutic effect remained dependent on CD8+ T cells (Figure 8) and resulted in long-term productive antitumor immunity, as determined by tumor rechallenge experiments.These results suggest that the protective effect of mIgG2a anti-4-1BB is mediated through the adaptive antitumor immune response, but that this mAb utilizes a molecular mechanism that, in contrast to anti-CD40 mAbs, occurs in an FcγRIIB-independent manner.

[0131] The immunostimulatory activity of anti-4-1BB is optimal with the mouse IgG1 isotype Given the results obtained in tumor models and previous studies demonstrating the critical role of CD8+ T cells in mediating the effects of anti-4-1BB mAbs, we sought to confirm isotype-dependent anti-4-1BB mAb activity in T cell populations in vitro and in vivo. Using an in vitro T cell costimulation assay (Figure 2A), only mIgG1, but not mIgG2a (the same antibody used above), demonstrated agonistic activity. This is consistent with other published results (18, 19). The costimulatory activity of mIgG1 was independent of Treg cells in T cell culture assays, suggesting that this anti-4-1BB mAb mediates its effects through targeting of 4-1BB on effector T cells. Finally, the costimulatory activity of mIgG1 was abolished by the addition of mIgG2a, demonstrating that both mAb variants bind with similar avidity and compete for binding to 4-1BB on effector T cells (Figure 2B). These in vitro results were confirmed using an in vivo immunization model with the model antigen OVA, both in endogenous (Figure 2C) and OT1 T cell-transfer environments (Figure 8A). In this context, the superior agonistic activity of mIgG1 depended on inhibitory FcγRIIB (Figure 2C, Figure 9A), as previously shown for anti-CD40 mAbs (18, 19, 24, 25). Finally, in two immunization models (NXS2 peptide and B16-sFlt3L-Ig, Figures 2D and 9B, respectively), mIgG1 and mIgG2a isotype antibodies were equally therapeutically effective (Figures 2D and 9B). Notably, the efficacy of mIgG1, but not IgG2a, was abolished in the absence of vaccination, confirming that mIgG1, but not mIgG2a, operates via an immune activation-dependent mechanism, likely FcγRIIB cross-linking (Figure 2D).

[0132] 4-1BB is expressed on intratumoral Treg cells in mouse tumor models and human cancer patients. Anti-4-1BB mIgG2a was found to be more active than mIgG1 in treating established tumors in mice. However, these murine mIgG2a lack the ability to deliver costimulatory activity, leading us to explore alternative mechanisms that could explain its immunomodulatory effects. 4-1BB mRNA and protein are preferentially expressed on Treg cells compared with resting effector T cells (14, 26, 27), and its expression is further upregulated following Treg cell activation (27, 28). It has recently been shown to be upregulated, at least at the transcriptional level, in intratumoral Tregs of human solid tumors (29, 30). Therefore, we investigated the possibility that anti-4-1BB mIgG2a, with its high A:I FcγR binding profile, could enhance antitumor responses via Treg cell depletion. We began by confirming the presence of 4-1BB on Treg cells in two mouse tumor models, CT26 (Figure 3A) and NXS2. We found that 4-1BB was expressed on a significant proportion of tumor-infiltrating Tregs and only a minority of effector T cells. Furthermore, only a small proportion of splenic Treg cells expressed 4-1BB. To confirm that these observations could be translated to humans at the protein level, we determined by flow cytometry whether 4-1BB was present on intratumoral Tregs from patients with ovarian cancer and squamous cell carcinoma. 4-1BB was found on CD4+Foxp3+ Treg cells but not on tumor effector CD4+ or CD8+ T cells. 4-1BB was expressed at lower levels in Tregs isolated from healthy PBMCs, matched blood, ascites, or normal skin, as can be seen in Figures 3B and 3C.

[0133] Role of FcγR in mediating the antitumor activity of anti-4-1BB mAb Because intratumoral Tregs were confirmed to express 4-1BB, we used the CT26 tumor model to determine the potential role and relative depletion potency of anti-4-1BB mAbs. The data demonstrate that in wild-type mice, the mIgG2a mAb efficiently depleted intratumoral Tregs, whereas the mIgG1 mutant (the same antibody used above (Figure 4A)) was ineffective. This depletion effect was restricted to the tumor and dependent on expression of the common γ chain, a critical component of the activating FcγR complex. Furthermore, in FcγRIIB knockout mice, the depletion activity of the mIgG1 mAb was enhanced to a level similar to that of mIgG2a, demonstrating that the depletion efficiency of these mAbs is closely related to the FcγR A:I ratio and that depletion potency can be manipulated through alterations in FcγR expression. We next sought to determine whether we could observe activation of tumor-infiltrating CD8 T cells in these mice. We observed that administration of the anti-4-1BB mIgG1 mAb resulted in a clear and significant increase in CD8 T cell proliferation, as monitored by Ki-67 positivity, despite the lack of efficacy of the mIgG1 mAb in mice bearing wild-type tumors (Figure 4B). The mIgG2a isotype also induced an increase in CD8 activation, although this was significantly less than that of mIgG1. These data likely demonstrate the dominant role of Tregs in the CT26 tumor model and suggest that inducing CD8 responses with mIgG1 in wild-type mice without ablation of Treg suppression is insufficient to induce a productive antitumor response.

[0134] Because anti-4-1BB mIgG2a was efficient at mediating intratumoral Treg cell depletion in a manner dependent on the expression of activating FcγRs, we reasoned that the absence of activating FcγRs would be detrimental to the therapeutic efficacy of this mAb. Surprisingly, however, in the CT26 tumor model (Figure 4C), anti-4-1BB mIgG2a retained antitumor activity in the absence of activating FcγRs, suggesting that Treg cell depletion may not be sufficient to support its therapeutic activity. In contrast to the minimal effect on the efficacy of anti-4-1BB mIgG2a, there was a substantial improvement in the ability of anti-4-1BB mIgG1 to promote antitumor immunity in the absence of activating FcγRs (2 / 5 in CT26). Considering the FcγR binding profiles demonstrated by mIgG1 and mIgG2a mAbs (Figure 7), these findings suggest that in the absence of competitive binding of mAbs to activating FcγRs, there is productive binding of FcγRIIB by both mIgG1 and mIgG2a, and thus optimal cross-linking of mAbs allows for the delivery of costimulatory activity. This concept is further supported by the T cell activation observed with mIgG2a in FcRg KO animals in the OVA model (Figure 2C). Consistent with the improved Treg-depleting activity of mIgG1 in FcγRIIB KO mice (Figure 4A), upon treatment with FcγRIIB KO mice, mIgG1 isotype mAbs demonstrated improved activity equivalent to that of mIgG2a. These results, coupled with the observation that neither mAb produced therapeutic activity in the absence of all FcγRs (Figure 4C), support the importance of efficient, noncompetitive FcγR binding for optimal in vivo activity.

[0135] Next, we sought to formally demonstrate the depletion potential of anti-4-1BB mAbs using both mouse and human targets and effectors in vitro. Using WT mouse bone marrow-derived macrophages and 4-1BB-expressing T cell targets, we observed that mIgG2a induced effective phagocytosis of target cells, whereas mIgG1 mAb was ineffective (Figure 4D). Consistent with the in vivo depletion results (Figure 4B) and therapeutic response (Figure 4C), when FcγRIIB KO macrophages were used as effectors, a significant increase in mIgG1-mediated phagocytosis was observed, consistent with the levels obtained with mIgG2a 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, two distinct huIgG1 anti-human 4-1BB clones (SAP3-6 and 005-B02) were found to be capable of mediating effective phagocytic clearance (Figure 4E). Finally, we sought to confirm that it was the level of 4-1BB expression, rather than the cell type itself, that determined the efficacy of depletion. We did this using both human (Figure 4E) and mouse (data not shown) macrophages and target cells expressing 4-1BB at various levels in vitro, and found a direct correlation between 4-1BB expression and the efficiency of target cell depletion. This supports the notion that high levels of 4-1BB expression on Tregs in the tumor microenvironment make them good targets, while low-expressing CD8+ cells are likely not used.

[0136] Treg depletion and scheduled administration of immunostimulatory mAbs leads to improved anti-cancer therapy Our results demonstrating that the therapeutic activity of the anti-4-1BB mAb isotype variants occurs via different mechanisms suggested the possibility of combining them for improved therapeutic efficacy. However, because depletion of Treg cells (mIgG2a) and delivery of costimulatory (mIgG1) both rely on FcγR binding and appear to be dependent in a competitive manner, we speculated that sequential administration, rather than simultaneous administration, might be optimal. Therefore, we next compared the therapeutic efficacy following simultaneous and sequential administration of anti-4-1BB mIgG2a and mIgG1 mAbs (the same antibodies used above). As previously observed, the mIgG2a variant, but not mIgG1, was active as a single agent. Coadministration of mIgG2a and mIgG1 anti-4-1BB mAb resulted in reduced therapeutic efficacy, as indicated by increased tumor size (Figure 5A) and a reduced number of tumor-free mice (Figure 5B), compared with mIgG2a monotherapy. In striking contrast, sequential delivery of first mIgG2a to deplete Treg cells, followed by the agonist mIgG1 to provide costimulation, improved both tumor growth inhibition and the number of tumor-free mice compared to monotherapy with either antibody variant alone (Figures 5A and 5B). These findings demonstrated that the therapeutic efficacy of FcγR-dependent immunomodulatory mAbs can be optimized by sequential administration. Importantly, this finding also demonstrated that Treg depletion, particularly when used in an FcγR-noncompetitive manner, may have broad utility for improving immunostimulatory antibodies beyond anti-4-1BB.

[0137] We next investigated the therapeutic potential of combining Treg-depleting anti-4-1BB with the clinically validated immune agonist anti-PD-1. We reduced the dose of mAb to obtain a suboptimal monotherapy, then sequentially combined the isotype-optimal anti-4-1BB mIgG2a with an FcγR-null-binding deglycosylated (31) mutant anti-PD-1 blocking antibody (Figures 10A and 10B) to mimic the lack / poor FcγR binding of the clinically validated anti-PD-1 antibodies nivolumab and pembrolizumab. This combination resulted in a significant increase in response, yielding 80% long-term responders compared to 20–25% with the monotherapy (Figure 5C). Notably, suboptimal isotype combinations of mAbs did not result in improved responses, demonstrating that understanding the isotype and programmatic requirements of each component of any combination is essential for optimal combination therapy.

[0138] Dual-active engineered anti-4-1BB mIgG2a / h2B delivers enhanced cancer therapy Having demonstrated that better responses can be achieved through an optimal combination of Treg depletion and agonism / immunosuppression release than either mechanism alone, we sought to demonstrate the feasibility of delivering these multiple mechanisms through the engineering of a single mAb. Given the observation that mAb-mediated Treg depletion and immunostimulatory agonism have distinct and competing FcγR requirements, we sought to exploit previous findings that the human IgG2 hinge region can provide FcγR-independent agonistic properties to anti-TNFR superfamily member mAbs (25). Here, we cloned the human IgG2 region into the murine mIgG2a constant region of anti-4-1BB, as previously detailed (25), and then biased the hinge toward the agonism-enhancing "B" form to generate anti-4-1BB mIgG2a / h2B (Figure 6A). The nucleotide sequence 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 sequence encoding LOB12.0 HuIgGhinge2.mIgG2aFc (mIgG2a / h2B) is set forth in SEQ ID NO: 185, and the corresponding amino acid sequence is set forth in SEQ ID NO: 186. The nucleotide sequence encoding LOB12 human kappa is set forth in SEQ ID NO: 187, and the corresponding amino acid sequence is set forth in SEQ ID NO: 188. When tested in vitro for T cell proliferation, mIgG2a / h2B had significantly improved agonistic activity compared to the mIgG2a parent (FIG. 6B), despite an unchanged FcγR binding profile (FIG. 11). Despite this improved agonistic activity, the engineered mAb also retained potent phagocytic capacity in vitro using BMDM and 4-1BB-expressing target cells (FIG. 6C), demonstrating the generation of a reagent with both agonistic and depleting potential without competing FcγR requirements. Finally, we compared this mAb with the parental mIgG2a in an EG7 tumor model and found that the dual-active mIgG2a / h2B possessed a potent Treg-depleting capacity comparable to that of the parental mIgG2a, but also possessed a significant CD8-stimulating capacity resulting in an improved CD8 / Treg ratio (Figure 6D).This improved dual activity mAb also showed greater therapeutic potential, curing 100% of treated mice compared to 60% for standard mIgG2a (Figure 6E). These data demonstrate for the first time that a single mAb can be engineered to optimally mediate depletion and agonism and deliver better therapy through this improved dual activity.

[0139] Consideration A variety of in vitro and in vivo models have confirmed that anti-TNFR superfamily mAbs require efficient cross-linking to elicit agonistic effects, which for most mAbs is best provided by inhibitory FcγR binding (8, 9, 18, 19, 23, 32, 33). Despite these findings, it is unclear whether such agonistic binding is the primary mechanism of action contributing to the therapeutic activity of these mAbs in the solid tumor environment. We investigated this question using mIgG2a and mIgG1 isotype anti-4-1BB mAbs, which have high and low activating:inhibitory FcγR ratios, respectively, and consequently good depleting and agonistic potential (10).

[0140] Using two different solid tumor models in different wild-type strains of mice, we found that mIgG2a mAb produced substantial therapeutic effects, whereas mIgG1 had little effect (Figure 1A, Figure 1B). These results contrast sharply with the agonistic activity of these mAbs on CD8+ T cells, where mIgG1 was more effective (Figure 2A, Figure 2B, and Figure 9A). Notably, in contrast to previous publications on anti-CD40 mAbs, anti-4-1BB mIgG2a was not devoid of T cell agonistic activity in vivo, suggesting that 4-1BB may have a lower crosslinking threshold for signaling than CD40 (18, 19). The mIgG2a-dependent therapeutic activity displayed in these different tumor models likely suggested that therapy was mediated by an effector cell-dependent depletion effect. None of the tumors used were 4-1BB-positive, implying that this was not due to direct tumor-targeting, as seen with anti-CD20 mAbs (34).

[0141] mAbs targeting CTLA-4, OX40, and GITR inhibit intratumoral T reg Given recent findings that 4-1BB can mediate therapy through depletion (5-7), we examined 4-1BB expression in these models and found that 4-1BB was specifically upregulated on intratumoral Treg cells (Figure 3A). Importantly, regarding the potential translation of these findings to humans, we demonstrated that 4-1BB is expressed restrictively on intratumoral Treg cells in both ovarian cancer and squamous cell carcinoma patients (Figure 3B and Figure 3C, respectively), supporting the therapeutic potential of this mechanistic approach in patients. Furthermore, mIgG2a depleted this suppressor population in an activating FcγR-dependent manner, whereas mIgG1 had little effect (Figure 4A). Consistent with the requirement for high activation relative to inhibitory FcR binding for productive depletion, when these experiments were performed in FcγRIIB KO mice, mIgG1 became comparable to mIgG2a in its depletion capacity both in vivo (Figure 4A) and in vitro (Figure 4D).

[0142] Although Treg depletion was the most effective mechanistic effect of 4-1BB Ab in these models, we hypothesized that in the absence of competition for binding to activating FcγRs, these mAbs might exert their therapeutic effect through their agonist function. We tested this possibility using the CT26 model and found that in the absence of activating FcγRs, mIgG1 mAb was indeed therapeutic (Figure 4C). Also noteworthy, consistent with the assertion that 4-1BB has a relatively low in vivo cross-linking threshold, activity was maintained even in the absence of activating FcγR mIgG2a. When treated in FcγRIIB KO mice, mIgG1 isotype mAb demonstrated improved activity comparable to mIgG2a, consistent with the improved Treg depletion activity of mIgG1 in FcγRIIB KO mice (Figures 4A and 4C). Furthermore, neither mAb was able to protect mice in the absence of FcγRs (Fig. 4C), demonstrating that both agonist and depletion mechanisms are FcγR dependent.

[0143] The fact that anti-4-1BB mAb can be a therapeutic agent that utilizes two distinct mechanisms, given the appropriate FcγR delivery, suggests that both mechanisms may be involved when the mAbs are administered sequentially. Indeed, we found this to be the case when mIgG2a was given first to deplete Treg cells, followed by mIgG1 to deliver an agonistic signal (Figures 5A and 5B). Importantly, when the mAbs were administered simultaneously, there was little therapeutic effect. These observations support the hypothesis that although simultaneous engagement of these two FcγR-dependent mechanisms through single antigen binding may be impossible, as shown herein, temporal or even spatial (intratumoral vs. systemic) separation of these mAbs may facilitate their combined efficacy.

[0144] To further substantiate the likely isotype and programmatic requirements for anti-4-1BB mAbs in patients whose clinical results suggest a combination approach is likely necessary, we investigated combinations of different mAbs with anti-PD-1. In this setting, we found that isotype-optimal versions of both anti-4-1BB and anti-PD-1 produced significant combination efficacy, resulting in cures in 80% of treated mice, in stark contrast to the 20-25% cures of the monotherapy drugs and subisotype-optimal combinations.

[0145] There has been considerable clinical interest in targeting 4-1BB using agonistic antibodies. However, data indicate that only approximately 1% of CD8+ or CD4+ T cells at tumor sites express 4-1BB. Furthermore, recent findings indicate that only approximately 10% of CD3+CD8+ cells infiltrating tumor sites in melanoma patients express 4-1BB, yet these are enriched for tumor-reactive clones (35). Therefore, the current finding that anti-4-1BB can be used to deplete Tregs to unleash an immunotherapeutic response suggests that this strategy may be particularly attractive to patients. Clinical studies using other putative Treg-depleting immunotherapeutic agents (e.g., anti-OX40 and anti-CTLA-4) appear promising (5, 36, 37), further confirming the potential of Treg-depleting anti-4-1BB mAbs in patients.

[0146] Two fully humanized anti-4-1BB mAbs are currently in development: urelumab (BMS-663513), an IgG4 antibody manufactured by Bristol-Myers Squibb, and PF-05082566, a fully humanized IgG2 antibody manufactured by Pfizer. To date, PF-05082566 has proven safe, causing only grade 1 toxicity in patients (38). However, urelumab caused adverse effects in 15% of patients, including elevated liver enzymes, pruritis, and diarrhea (39). Despite the promising safety profile, neither urelumab nor PF-05082566 are predicted to bind strongly to FcγRIIB, raising questions about whether either antibody will provide efficacy in patients (40). Recent data from our group indicate that human IgG2 antibodies targeting 4-1BB can act as FcγR-independent superagonists, and it remains possible that PF-05082566 may act in a similar manner (25). The data presented herein demonstrate improved efficacy of Treg depletion compared with immunoagonistic variant anti-4-1BB antibodies and selective intratumoral 4-1BB expression on Tregs compared with CD8 effector cells, supporting the development of human therapeutic anti-4-1BB IgG1 isotype antibodies selected for their Treg-depleting capabilities (40). Recently, it has been demonstrated that such Treg-depleting antibodies synergize to enhance responses and help overcome resistance to checkpoint blockade (50).

[0147] These findings support the assertion that immunomodulatory mAbs can utilize multiple mechanisms of action for therapy, and we explored the possibility of engineering a single antibody to optimally perform both depletion and agonism. Given data demonstrating competing FcγR requirements for these mechanisms in vitro and in vivo, engineering a mAb with improved activating and inhibitory FcγR binding would likely not work if any one mAb could only bind to a single FcγR at a time. Given these limitations, we generated an mIgG2a mAb with optimal depletion capabilities that incorporates a hIgG2 hinge region biased toward the optimal "B" form of agonism. We hypothesized that this mAb would be capable of performing both functions and found this to be the case both in vitro (Figures 6B and C) and in vivo (Figure 6D), resulting in improved therapy in solid tumor models (Figure 6E). These results have direct implications for the administration of existing and developmental immunomodulatory mAbs, as well as for the design and development of future reagents and strategies for their use.

[0148] array SEQ ID NO:179 - Nucleotide sequence encoding LOB12.0 mIgG1 heavy chain. [Table 3] SEQ ID NO:180 - Amino acid sequence of LOB12.0 mIgG1 heavy chain. The underlined sequence indicates the leader sequence. [Table 4] SEQ ID NO:181 - Nucleotide sequence encoding LOB12.0 mIgG2a heavy chain. [Table 5] SEQ ID NO:182 - Amino acid sequence of LOB12.0 mIgG2a heavy chain. The underlined sequence indicates the leader sequence. [Table 6] SEQ ID NO:183 - Nucleotide sequence encoding LOB12.0 mkappa. [Table 7] SEQ ID NO:184 - Amino acid sequence of LOB12.0 mKappa. The underlined sequence indicates the leader sequence. [Table 8] SEQ ID NO:185 - Nucleotide sequence encoding LOB12.0 HuIgGhinge2.mIgG2aFc (mIgG2a / h2B). [Table 9] SEQ ID NO: 186—Amino acid sequence of LOB12.0 HuIgGhinge2.mIgG2aFc (mIgG2a / h2B). The underlined sequence indicates the leader sequence. [Table 10] SEQ ID NO:187 - Nucleotide sequence encoding LOB12 human kappa. [Table 11] SEQ ID NO:188 - Amino acid sequence of LOB12 human kappa. The underlined sequence indicates the leader sequence. [Table 12]

[0149] method Animals and Cells. Mice were bred and maintained in local facilities. The genetically modified strains used were OT1 TCR transgenic C57BL / 6 mice (Dr. Matthias Merkenschlager, Imperial College, London, UK), Foxp3-GFP, γ-chain KO, FcγRIIB KO, and FcγR null (γ-chain KO x FcγRIIB KO). Mice were obtained by breeding with genotypes confirmed by polymerase chain reaction (PCR) and / or flow cytometry. CT26 colon carcinoma (16), NXS2 neuroblastoma (41), B16 Flt3vax melanoma (42), and EG7 thymoma (43) models have all been previously described.

[0150] Immunotherapy. On day 0, groups of CT26-age- and sex-matched WT, γ-chain KO, FcγRIIB KO, or FcγR null (γ-chain KO x FcγRIIB KO) BALB / c mice were treated with 5 × 10 4 Mice were challenged with subcutaneous administration of CT26. When tumors were palpable, mice were administered mAb or PBS control intravenously, followed by three additional intraperitoneal doses every other day (final dose of 200 μg unless otherwise indicated). Upon CD8+ T cell depletion, 0.5 mg of anti-CD8 (YTS169) was administered intraperitoneally on days -1, +1, and +4, as previously described (44), prior to tumor and mAb administration. On day 0, age- and sex-matched A / J mice were injected with 2 × 10 6 Mice were challenged with subcutaneous administration of NXS2 cells and given the antibody / peptide vaccine designated in each experiment. All antibodies were administered intraperitoneally in PBS. Prior to intradermal injection, tyrosine hydroxylase (FETFEAKI) and control (SIINFEKL or FEANGNLI) peptides in PBS were emulsified with an equal volume of incomplete Freund's adjuvant (IFA). Tumor size in all models was monitored regularly with calipers and reached a cross-sectional area of ​​225 mm. 2 On day EG7-0, mice were divided into groups of age- and sex-matched C57BL / 6 mice, with 5 × 10 5Mice were challenged with subcutaneous administration of EG7 cells. On days 3, 5, and 7, mice were intraperitoneally administered 200 μg of mAb or PBS control, as indicated. Survival times to the humane endpoint were plotted using the Kaplan-Meier method and analyzed for significance by the log-rank test using GraphPad Prism 6.0 for Windows (GraphPad Software Inc, La Jolla, CA).

[0151] Antibodies and Reagents. Anti-41BB (clone LOB12.0) mAbs mIgG1, mIgG2a, and mIgG2a huIgG2 hinge (mIgG2a / h2B) isotypes were constructed as previously described (antibodies 18, 25). Anti-CD8 (YTS169) was generated in-house. Anti-mouse PD-1 (EW1-9) mAb rIgG1 was produced using conventional hybridoma technology after immunizing Wistar rats with recombinant mouse PD-1 (Leu25-Gln167) Fc fusion protein (RnD Systems). Spleens from immunized rats or mice were fused with NS-1 melanoma cells, and plates were screened by ELISA and flow cytometry. mAbs were initially screened, and cells from positive wells were cloned twice and expanded in culture for IgG production. Antibodies were produced from hybridomas or CHOK1 cells, purified with Protein A, and purity was assessed by electrophoresis (Beckman EP system, Beckman Coulter, Buckinghamshire, UK) and the absence of aggregation by SEC HPLC. All preparations were low in endotoxin (<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 0.05 U PNGase F / µg antibody. N-glycosidase F (PNGase F) was obtained from Promega (V483A). Samples were stored overnight at 37°C. Deglycosylation was confirmed by either EP or SPR analysis. Purification of the antibody from the enzyme was achieved through size exclusion chromatography using Sephadex™ 200. Peptides (SIINFEKL, FETFEAKI, and FEANGNLI) were obtained from Peptide Protein Research Ltd.

[0152] In vitro T cell expansion. Spleens from Foxp3-GFP mice were sorted to eliminate GFP+ cells (-Treg cells, 99% of Treg cells are depleted) or null sorted and treated with 1 × 10 spleens containing 0.1 μg / ml of anti-CD3 and a range of concentrations of anti-4-1BB mAb as indicated. 5 After 56 hours, 1 μCi / well of [ 3 H]-thymidine was added and plates were harvested after an additional 16 hours of incubation.

[0153] Endogenous OVA-specific immune responses. Mice were immunized with 5 mg of OVA (Sigma) and 200 μg of mAb on day 0, as specified in the figure legends. The proliferation of endogenous OVA-specific CD8+ T cells in peripheral blood was monitored over time and analyzed by flow cytometry as previously described (18).

[0154] Isolation of lymphocytes. Tumors from mice challenged with CT26 or EG7 were excised and analyzed. oThe cells were digested with 0.5 Wu / ml Liberase DL (Roche) and 50 μg / ml DNase I (Roche) at 37°C for 20 minutes. The cells were then passed through a 100 μm cell strainer and either used directly in the assay or tumor-infiltrating lymphocytes were isolated using a 40% and 70% Percoll gradient. Human ascites was evaluated as an isolated single-cell suspension. Ovarian tumor samples were obtained from patients undergoing surgery at the Department of Obstetrics and Gynecology, Skane University Hospital. The material was cut into small pieces and incubated in R10 containing DNase I (Sigma) and Liberase™ (Roche Diagnostics) at 37°C for 20 minutes. The remaining tissue was mechanically dissociated and passed through a 70 μm cell strainer along with the cell suspension. Freshly excised squamous cell carcinoma (cSCC) and normal skin samples were obtained from patients undergoing surgery at the Dermatology Department of 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 / ml DNAse I (Sigma) in RPMI medium (Gibco) for 1.5 hours at 37°C, then filtered through a 70 μm cell filter (BD) and centrifuged (600 x g, 20 minutes) on an Optiprep (Axis-Shield) density gradient. Matched peripheral blood samples were obtained, and peripheral blood mononuclear cells were isolated by centrifugation on Lymphoprep (Axis-Shield) at 600 x g for 30 minutes.

[0155] Flow cytometry. Mouse cell surface staining: Isolated lymphocytes were washed and incubated with antibodies in PBS + 1% BSA (Sigma) on ice for 30 minutes in the dark. Cells were then 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. Data were analyzed using FCS Express. Intracellular staining: After surface staining, cells were fixed and stained intracellularly using an anti-mouse / rat Foxp3 staining set (BD Biosciences). Antibodies used 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 control. Ovarian cancer patient cells were incubated with 10 mg / ml KIOVIG (Baxalta) for 10 minutes before staining with human-relevant antibodies. Cell viability: Cells were stained with either eFluor 780 Live / Dead Cell Stain (eBioscience) or aqua Live / Dead Cell Stain (Invitrogen), fixable in PBS at 4°C. Cell surface staining: Antibodies were incubated with cells in PBS + 1% BSA (Sigma) + 10% FCS (Gibco) for 30 minutes at 4°C in the dark. Intracellular staining was performed using Foxp3 staining buffer set (eBioscience). Cells were analyzed 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), 41BB-PE (4B4-1), mouse IgG2a isotype, kappa control-PE (G155-178, all from BD Biosciences), SCC-CD3-APC-Cy7, CD4-FITC, or PerCP Cy5.5, CD8-PE Cy7 (all Biolegend), 4-1BB-PE, and Foxp3-APC (both eBioscience).

[0156] Antibody-dependent cellular phagocytosis. ADCP assays were performed as previously described using mouse (17, 45) or human macrophages (18, 46). Briefly, bone marrow-derived macrophages (BMDMs) were generated from 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 stained with CFSE (5 μM) and then opsonized with antibodies before co-culture with macrophages for approximately 1 h. Macrophages were stained with CD16-APC or F4 / 80-APC, and samples were assessed for the percentage of double-positive (CFSE / APC) macrophages by flow cytometry.

[0157] Statistical analysis. Data were analyzed using an unpaired Student's t-test, or for tumor treatment experiments, survival times to the humane endpoint were plotted using the Kaplan-Meier method with analysis of significance by the log-rank test. All statistical analyses were performed using GraphPad Prism 6.0 for Windows (GraphPad Software Inc, La Jolla, CA). Significance was accepted when p<0.05.

[0158] Surface plasmon resonance analysis. Analysis of the interaction between anti-41BB mAb and soluble FcγR was performed using a Biacore T100 (GE Healthcare Life Sciences, Buckinghamshire, UK). Antibodies or BSA as a control were immobilized to the flow cell of a CM5 sensor chip (GE Healthcare Life Sciences, Buckinghamshire, UK) at 5000 resonance units (RU) by standard amine coupling according to the manufacturer's instructions. Soluble FcγR (R&D Systems, Abingdon, UK) was injected through the flow cell 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 μl / min. Soluble Fc receptor was injected for 2 min, and dissociation was monitored for 5 min. Background binding to the control flow cell was automatically subtracted. Affinity constants were derived from data by equilibrium binding analysis as indicated using Biacore Bioevaluation software (GE Healthcare Life Sciences, Buckinghamshire, UK).

[0159] In vitro binding assay. Karpas-299 cells stably transduced with the tailless morphology of murine 4-1BB(pTL) (47) were incubated for 4 oCells were incubated with anti-4-1BB mAb at the concentrations indicated in C for 20 minutes, then washed and stained with PE-labeled anti-mouse or PE-labeled anti-rat secondary antibodies (both Jackson Labs). No staining was observed in Karpas-299 cells stably expressing the empty vector control (data not shown). In competitive binding assays, 0.1 μg / ml of the parental rat anti-4-1BB mAb was mixed with either the mIgG1 or mIgG2a version of anti-4-1BB at graded concentrations as indicated and then incubated with Karpas-299 pTL cells. Cells were washed and stained with APC-conjugated and mouse-adsorbed donkey anti-rat secondary antibody; the secondary antibody did not bind to either mIgG1 or mIgG2a. Flow cytometry analysis was performed using a BD FACS Canto II and FACS Diva software.

[0160] OVA-specific immune response. Splenocytes from OTI transgenic mice were harvested and washed. Approximately 2 × 10 5 OVA-specific CD8 T cells were transferred into recipient mice. The following day, mice were immunized with OVA (Sigma) as described in the individual experiments. OTI proliferation in peripheral blood was analyzed by flow cytometry as previously described (18). Results are shown at the peak of the response (4–5 days post-immunization).

[0161] Tumor challenge. B16-sFlt3L-Ig(FVAX)-On day 0, groups of C57BL / 6 mice were injected with 2.5 × 10 4 On days 3, 6, and 9, mice were challenged with 1 × 10 B16 / BL6 cells intradermally in the contralateral flank as indicated. 6 of irradiated FVAX cells, as well as either PBS, 100 μg of anti-CTLA-4 (clone 9D9) or anti-CTLA-4, and 300 μg of anti-4-1BB, were administered intradermally and also intraperitoneally on days 3, 6, and 9, based on a previously published protocol ( 48 ).

[0162] Examples of specific 4-1BB and OX40 antibodies material and method Animals and cells Mice were bred and maintained in a local facility in accordance with Home Office guidelines. Female BALB / c and C57 BL6 mice, 10–12 weeks old, were supplied by Taconic (Bomholt, Denmark) and maintained in a local animal facility. For xenograft studies using primary tumor cells, 6–8 week old female BALB / c and C57 BL6 mice were implanted with the syngeneic tumor cell lines CT26 and TH03, respectively.

[0163] Clinical samples Ethical approval for the use of clinical samples was obtained from the ethics committee of Skane University Hospital. Informed consent was provided in accordance with the Declaration of Helsinki. Samples were obtained through the Department of Gynecology and Oncology at Skane University Hospital, Lund. Ascites fluid was evaluated as an isolated single-cell suspension. Tumor material was cut into small pieces and incubated in R10 containing DNase I (Sigma-Aldrich) and Liberase™ (Roche Diagnostics) for 20 minutes at 37°C. The remaining tissue was mechanically disrupted and passed through a 70 μm cell strainer along with the cell suspension. Cells isolated from ascites and tumor were stained. Data collection was performed using FACSVerse and analyzed using FlowJo.

[0164] cell culture Cell culture was performed in supplemented RPMI (GIBCO, Life Technologies) containing 2 mM glutamine, 1 mM pyruvate, 100 IU / ml penicillin and streptomycin, and 10% FBS. Human peripheral CD4 + T cells were purified by negative selection using the MACS CD4 T cell isolation kit (Miltenyi Biotec, UK).

[0165] Antibodies and reagents The following antibodies and reagents were used: purified anti-CD3 (UCHT1, R&D Systems), purified anti-CD28 (CD28.2, BioLegend), KIOVIG (Baxalta, Lessines, Belgium), Fixable Viability Dye eFluor780 (eBioscience, San Diego, CA). Cell Trace CFSE (dissolved in DMSO) and propidium iodide were obtained from Life Technologies (Carlsbad, CA). Human lymphocytes were stained using the following reagents: CD4-BV510 (RPA-T4), CD25-BV421 (M-A251), anti-CD127-FITC (HIL-7R-M21), Ox40-PE (ACT35), 41BB-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 IgG1, kappa isotype control-PE (MOPC-21), mouse IgG2a isotype, kappa control-PE (G155-178), mouse IgG2b isotype, kappa control-PE (27-53, all from BD Biosciences), and TNFRII-PE (FAB226P, R&D Systems). The following reagents were used to stain mouse lymphocytes: CD4-BV510 (RM4-5), CD25-BV421 (7D4), CD8-Alexa 488 (53-6.7, BD), Ox40, 41BB, TNFRII, ICOS, GITR, PD-1, CTLA-4, and FITC negative control (scFv, BioInvent in-house generated).

[0166] Flow cytometry Flow cytometry was performed according to standard procedures. Dead cells (propidium iodide) were counted. +Cells were identified as chromatin-dependent markers (as chromatin-dependent markers or using Fixable Viability Dye eFluor 780) and cell aggregates were excluded from all analyses. Fluorescently conjugated mAbs were purchased from BD Biosciences, eBiosciences, BioLegend, or generated in-house. Data collection was performed with FACSVerse (BD Biosciences, Franklin Lakes, NJ) and analyzed with FlowJo software (Tree Star, Ashland, OR). For gene expression analysis, cells were sorted using a FACSAria (BD Biosciences). Staining with in-house generated scFvs was detected with an in-house Alexa 647-labeled deglycosylated anti-His tag antibody (AD1.1.10, R&D Systems). CFSE labeling of T cells was performed according to the manufacturer's instructions.

[0167] Antibody-dependent cytotoxicity (ADCC) ADCC assays were performed in two ways: a) using the NK-92 cell line stably transfected to express the CD16-158V allele along with GFP (purchased from Conkwest, San Diego, CA). CD4+ target T cells were isolated from peripheral blood of healthy donors using a CD4+ T cell isolation kit (Miltenyi Biotec). Cells were stimulated with CD3 / CD28 Dynabeads (Life Technologies, Thermo Fisher Scientific) and 50 ng / ml rhIL-2 (R&D Systems) at 37°C for 2 days. Target cells were preincubated with 0.1–10 μg / ml mAb for 30 min at 4°C and then mixed with NK cells. Cells were cultured at a 2:1 effector:target cell ratio for 4 hours in RPMI1640 + GlutaMAX medium (Invitrogen) containing 10 mM HEPES buffer, 1 mM sodium pyruvate, and 10% FBS-low IgG. Lysis was determined by flow cytometry. Briefly, at the end of the culture, cell suspensions were stained with BV510-conjugated anti-CD4 along with 10 nM SYTOX Red dead cell stain (Invitrogen) or Fixable Viability Dye eFluor780 (eBioscience) for 20 minutes in the dark at 4°C, and then the cells were analyzed using a FACSVerse (BD Biosciences). Target cells were labeled with calcein AM, followed by the addition of diluted concentrations of Ab. Target cells were co-cultured with human PBMCs at a 50:1 E:T ratio for 4 hours at 37°C. The plates were centrifuged at 400 x g for 5 minutes to pellet the cells, and the supernatant 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 an emission wavelength of 530 nm. The percentage of maximum release was calculated as follows: % Maximum Release = (Sample / Triton-treated) * 100.

[0168] Antibody-dependent cellular phagocytosis (ADCP) Target cells were labeled with 5 mM CFSE for 10 minutes at room temperature before washing with complete medium. CFSE-labeled targets were then opsonized with diluted Abs and then co-cultured with BMDMs at a 1:5 E:T ratio in a 96-well plate for 1 hour at 37°C. BMDMs were then labeled with anti-F4 / 80-allophycocyanin for 15 minutes at room temperature and washed twice with PBS. Plates were kept on ice, and BMDMs were collected by scraping the wells. Phagocytosis was assessed by flow cytometry using a FACSCalibur (BD) to determine the percentage of F4 / 80+CFSE+ cells within the F4 / 80+ cell population.

[0169] T cell proliferation assay The agonist activity of antibodies was tested using two protocols: a) Antibodies were crosslinked with F(ab')2 goat anti-human IgG, Fcg fragment specific, or F(ab')2 goat anti-mouse IgG, Fcg fragment specific, at a molar ratio of 1.5:1 IgG:F(ab')2 for 1 hour at room temperature. 1 x 105 MACS-purified human CD4+ T cells were stimulated with CFSE-labeled, plate-bound anti-CD3 (0.5 μg / ml) and 4 μg / ml soluble crosslinking IgG at 37°C for 3 days and then analyzed. b) Cell culture was performed in RPMI 1640 medium (Gibco™) supplemented with 10% fetal bovine serum, glutamine (2 mM), pyruvate (1 mM), penicillin, and streptomycin (100 IU / ml) at 37°C in 5% CO2. Fresh PBMCs were labeled with 2 mM carboxyfluorescein succinimidyl ester (CFSE). PBMCs were then cultured at 1 x 107 cells / mL in 24-well plates 48 h prior to mAb stimulation assays, as described by Romer et al. (51). For PBMC stimulation, round-bottom 96-well plates were wet-coated with 0.01 µg / mL OKT3 antibody (in-house) in PBS for 4 h, after which excess antibody was discarded and the plates were washed with PBS. 1 x 105 PBMCs / well were transferred to the plates and stimulated with 5 µg / mL test mAbs (anti-4-1BB, anti-OX40 mAb). On day 4 or 5 post-stimulation, cells were labeled with anti-CD8-APC (BioLegend) and anti-CD4-PE (in-house), and proliferation was assessed by CFSE dilution on a FACSCalibur (BD Biosciences).

[0170] Ligand blocking ELISA Human receptors (hox40, R&D Systems; h41BB, in-house production) were coated onto 96-well plates (Lumitrac 600 LIA plate, Greiner) at 1 pmole / well. After washing, mAbs (10 μg / ml to 0.01 μg / ml) were allowed to bind for 1 hour. Ligands were added at 5 nM (hox40-L, h41BB-L, R&D Systems), and the plates were incubated for an additional 15 minutes. After washing, bound ligands were detected with biotinylated antibodies (anti-hox40-L, anti-h41BB-L, R&D Systems), followed by streptavidin-HRP (Jackson ImmunoResearch) with intermediate washes. Plates were analyzed using a Tecan Ultra Microplate reader with Super Signal ELISA Pico (Thermo Scientific) as substrate.

[0171] Microarray 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 the spleens of healthy C57 / Bl6 and Balb / c mice. CD8+ T cells were isolated from the spleens of healthy Balb / c mice. RNA from all samples was prepared with the RNA Isolation Midi Kit from Macherey-Nagel (Dueren, Germany) according to the manufacturer's instructions. Isolated RNA was amplified and prepared for hybridization to Affymetrix Mouse Gene 2.0ST arrays at the Swegene Center for Integrative Biology, Lund University (SCIBLU), Sweden. Data analysis was performed at SCIBLU according to standard methods.

[0172] The results of the above assays and the characteristics of the antibodies studied are shown in Figures 12-24.

[0173] Embodiment The following provides an itemized list of different embodiments of the present invention. 1. 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, the Treg-depleting antibody molecule being administered prior to administration of the immunostimulatory antibody molecule. 2. The Treg-depleting antibody for use according to embodiment 1, wherein said immunostimulatory antibody molecule is a CD8-activating and / or CD8-enhancing antibody molecule. 3. The Treg-depleting antibody molecule for use according to embodiment 1 or 2, wherein the cancer is a solid tumor. 4. The Treg-depleting antibody molecule for use according to embodiment 3, wherein the solid tumor is selected from the group consisting of sarcoma, carcinoma, lymphoma, and ovarian cancer. 5. The Treg-depleting antibody molecule for use according to embodiment 3, wherein the solid tumor is squamous cell carcinoma (SCC), thymoma, neuroblastoma, or ovarian cancer. 6. The Treg-depleting antibody molecule for use according to any one of Examples 1 to 5, wherein said Treg-depleting antibody molecule and / or said immunostimulatory antibody molecule is selected from the group consisting of a full-size antibody, Fab, Fv, scFv, Fab', and (Fab')2. 7. The Treg-depleting antibody molecule for use according to any one of embodiments 1 to 6, wherein said Treg-depleting antibody molecule and / or said immunostimulatory antibody molecule is a human or humanized antibody. 8. The Treg-depleting antibody molecule for use according to any one of embodiments 1 to 7, wherein said Treg-depleting antibody molecule is a human IgG1 antibody. 9. The Treg-depleting antibody molecule for use according to any one of embodiments 1 to 8, wherein said Treg-depleting antibody molecule is a human IgG1 antibody molecule engineered for improved binding to at least one activating FcγR. 10. The Treg-depleting antibody molecule for use according to any one of embodiments 1 to 9, wherein said Treg-depleting antibody molecule is selected from antibody molecules that specifically bind to targets belonging to the tumor necrosis factor receptor superfamily (TNFRSF). 11. The Treg-depleting antibody molecule for use according to embodiment 10, wherein said Treg-depleting antibody molecule is an antibody molecule that specifically binds to a target selected from the group consisting of 4-1BB, OX40, and TNFR2. 12. The Treg-depleting antibody molecule for use according to any one of embodiments 1 to 9, wherein said Treg-depleting antibody molecule is an antibody molecule that specifically binds to a target selected from GITR, ICOS, CTLA-4, and CD25. 13. The Treg-depleting antibody molecule for use according to embodiment 11, wherein said Treg-depleting antibody molecule is an anti-4-1BB 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 molecules comprising 1 to 6 CDRs selected from SEQ ID NOs: 1-6, 1 to 6 CDRs selected from SEQ ID NOs: 9-14, 1 to 6 CDRs selected from SEQ ID NOs: 17-22, 1 to 6 CDRs selected from SEQ ID NOs: 25-30, 1 to 6 CDRs selected from SEQ ID NOs: 33-38, 1 to 6 CDRs selected from SEQ ID NOs: 41-46, 1 to 6 CDRs selected from SEQ ID NOs: 49-54, 1 to 6 CDRs selected from SEQ ID NOs: 57-62, 1 to 6 CDRs selected from SEQ ID NOs: 65-70, 1 to 6 CDRs selected from SEQ ID NOs: 153-158, and 1 to 6 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 molecules comprising SEQ ID NOs: 1 to 6, SEQ ID NOs: 9 to 14, SEQ ID NOs: 17 to 22, SEQ ID NOs: 25 to 30, SEQ ID NOs: 33 to 38, SEQ ID NOs: 41 to 46, SEQ ID NOs: 49 to 54, SEQ ID NOs: 57 to 62, SEQ ID NOs: 65 to 70, SEQ ID NOs: 153 to 158, and SEQ ID NOs: 163 to 168. 16. The 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. The Treg-depleting antibody for use according to any one of embodiments 14 to 16, wherein the Treg-depleting 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: 8, 16, 24, 32, 40, 48, 56, 64, 72, 160, and 170. 18. The Treg-depleting antibody for use according to any one of embodiments 14 to 17, wherein the Treg-depleting antibody molecule is selected from the group consisting of antibody molecules 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. 19. The Treg-depleting antibody molecule for use according to embodiment 11, wherein said Treg-depleting antibody is a human anti-OX40 monoclonal antibody molecule. 20. The Treg-depleting antibody for use according to embodiment 19, wherein the Treg-depleting antibody molecule is selected from the group consisting of antibody molecules comprising one or more 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 molecules comprising SEQ ID NOs: 73-78, 81-86, 89-94, 97-102, 105-110, 113-118, 121-126, 129-134, 137-142, 145-150, and 177-178. 22. The 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, 111, 119, 127, 135, 143, 151, and 177. 23. The Treg-depleting antibody for use according to any one of embodiments 20 to 22, wherein the Treg-depleting 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. 24. The Treg-depleting antibody for use according to any one of embodiments 20 to 23, wherein the Treg-depleting 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. 25. The Treg-depleting antibody for use according to any one of embodiments 1 to 9, wherein the Treg-depleting antibody molecule is selected from antibody molecules that specifically bind to a target selected from the group consisting of ICOS, GITR, CTLA-4, CD25, and Neuropilin-1. 26. The Treg-depleting antibody for use according to any one of embodiments 1 to 25, wherein the immunostimulatory antibody molecule is a human IgG2 antibody or a human IgG4 antibody molecule. 27. The Treg-depleting antibody for use according to embodiment 26, wherein the immunostimulatory antibody molecule is a human IgG2b antibody molecule. 28. The Treg-depleting antibody molecule for use according to any one of embodiments 1 to 27, wherein the immunostimulatory antibody molecule is engineered for improved binding to human FcγRIIB over activating Fc gamma receptors. 29. The Treg-depleting antibody molecule for use according to any one of embodiments 1 to 28, wherein the immunostimulatory antibody molecule is an antibody that specifically binds to a target selected from the group consisting of 4-1BB, OX40, ICOS, GITR, CTLA-4, CD25, PD-1, and PDL1. 30. The Treg-depleting antibody molecule for use according to embodiment 29, wherein the immunostimulatory antibody molecule is an anti-4-1BB antibody molecule. 31. The 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 CDRs selected from sequences 1-6, 9-14, 17-22, 25-30, 33-38, 41-46, 49-54, 57-62, 65-70, 153-158, and 163-168. 32. The Treg-depleting antibody for use according to embodiment 31, wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules 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. The Treg-depleting antibody for use according to embodiment 31 or 32, wherein the immunostimulatory 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. 34. The Treg-depleting antibody for use according to any one of embodiments 31 to 33, 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: 8, 16, 24, 32, 40, 48, 56, 64, 72, 160, and 170. 35. The Treg-depleting antibody for use according to any one of embodiments 31 to 34, wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules 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. The Treg-depleting antibody molecule for use according to embodiment 29, wherein the immunostimulatory antibody molecule is an anti-OX40 antibody molecule. 37. The Treg-depleting antibody molecule for use according to embodiment 36, wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising one or more 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. 38. The 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: 73-78, 81-86, 89-94, 97-102, 105-110, 113-118, 121-126, 129-134, 137-142, 145-150, and 171-176. 39. The Treg-depleting antibody for use according to embodiment 37 or 38, wherein the immunostimulatory 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, 111, 119, 127, 135, 143, 151, and 177. 40. The Treg-depleting antibody for use according to any one of 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. The Treg-depleting antibody for use according to any one of 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: 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: 177-178. 42. The Treg-depleting antibody molecule for use according to embodiment 29, wherein the immunostimulatory antibody molecule is a human anti-PD1 monoclonal antibody molecule, a human anti-PDL1 monoclonal antibody molecule, or a human anti-CTLA-4 monoclonal antibody molecule. 43. The Treg-depleting antibody molecule for use according to embodiment 42, 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 selected from the group consisting of ipilimumab and tremilimumab. 44. An anti-4-1BB antibody molecule selected from the group consisting of antibody molecules comprising one or more CDRs selected from sequences 1-6, 9-14, 17-22, 25-30, 33-38, 41-46, 49-54, 57-62, 65-70, 153-158, and 163-168. 45. The anti-4-1BB antibody molecule according to embodiment 44, selected from the group consisting of antibody molecules comprising SEQ ID NOs: 1 to 6, 9 to 14, 17 to 22, 25 to 30, 33 to 38, 41 to 46, 49 to 54, 57 to 62, 65 to 70, 153 to 158, and 163 to 168. 46. ​​The anti-4-1BB antibody molecule of embodiment 44 or 45, 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. 47. The anti-4-1BB antibody molecule of any one of embodiments 44 to 46, selected from the group consisting of antibody molecules 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. The anti-4-1BB antibody molecule according to any one of embodiments 44 to 47, selected from the group consisting of antibody molecules 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 to 170. 49. The anti-4-1BB antibody molecule of any one of embodiments 44 to 48, which is selected from the group consisting of a full-length IgG antibody, a Fab, an Fv, an scFv, an Fab', and an (Fab')2. 50. The anti-4-1BB antibody molecule of embodiment 49, wherein the full-length IgG antibody is selected from the group consisting of IgG1, IgG2, IgG4, and Fc-engineered variants thereof. 51. The anti-4-1BB antibody molecule of any one of embodiments 44 to 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 antibody molecules comprising one or more 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. 53. The anti-OX40 antibody molecule of embodiment 52, selected from the group consisting of antibody molecules comprising SEQ ID NOs: 73 to 78, 81 to 86, 89 to 94, 97 to 102, 105 to 110, 113 to 118, 121 to 126, 129 to 134, 137 to 142, 145 to 150, and 171 to 176. 54. The anti-OX40 antibody molecule of embodiment 52 or 53, 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, 111, 119, 127, 135, 143, 151, and 177. 55. The anti-OX40 antibody molecule of any one of 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. The anti-OX40 antibody molecule of any one of embodiments 52 to 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. The anti-OX40 antibody molecule of any one of embodiments 52 to 56, wherein the Treg-depleting antibody molecule and / or the immunostimulatory antibody molecule is selected from the group consisting of a full-length IgG antibody, Fab, Fv, scFv, Fab', and (Fab')2. 58. The anti-OX40 antibody molecule of embodiment 57, wherein the full-length IgG antibody is selected from the group consisting of IgG1, IgG2, IgG4, and Fc-engineered variants thereof. 59. The anti-OX40 antibody molecule of any one of 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 embodiments 44 to 59. 61. A vector comprising the nucleic acid of embodiment 60. 62. A host cell comprising the vector of embodiment 61. 63. The antibody according to any one of embodiments 44 to 59 for use in medicine. 64. A pharmaceutical composition comprising an antibody according to any one of embodiments 44 to 59. 65. The antibody of embodiment 63 or the pharmaceutical composition of embodiment 64 for use in treating cancer. 66. The antibody or pharmaceutical composition of embodiment 65, wherein the cancer is a solid tumor. 67. The antibody or pharmaceutical composition of embodiment 66, wherein the solid tumor is selected from the group consisting of sarcoma, carcinoma, and lymphoma. 68. The antibody or pharmaceutical composition of embodiment 67, wherein the solid tumor is squamous cell carcinoma (SCC), thymoma, neuroblastoma, or ovarian cancer. 69. A pharmaceutical composition comprising an antibody according to any one of embodiments 44 to 59, or an antibody according to any one of embodiments 44 to 51 and an antibody according to any one of embodiments 52 to 59. 70. The antibody according to any one of embodiments 44 to 59 or the pharmaceutical composition according to embodiment 69, wherein the pharmaceutical composition is for the treatment of cancer. 71. The antibody of any one of embodiments 44 to 59 or the pharmaceutical composition of embodiment 70, wherein the cancer is a solid tumor. 72. The antibody of any one of embodiments 44 to 59 or the pharmaceutical composition of embodiment 71, wherein the solid tumor is selected from the group consisting of sarcoma, carcinoma, and lymphoma. 73. The antibody of any one of embodiments 44 to 59 or the pharmaceutical composition of embodiment 72, wherein the solid tumor is squamous cell carcinoma (SCC), thymoma, neuroblastoma, or ovarian cancer. 74. Use of an antibody according to any one of embodiments 44 to 59 for the manufacture of a pharmaceutical composition for use in the treatment of cancer. 75. The use according to embodiment 74, wherein the cancer is a solid tumor. 76. The use according to embodiment 75, wherein the solid tumor is selected from the group consisting of sarcoma, carcinoma, and lymphoma. 77. The use according to embodiment 76, wherein the solid tumor is squamous cell carcinoma (SCC), thymoma, neuroblastoma, or ovarian cancer. 78. A method for treating cancer in a subject, comprising administering a Treg-depleting antibody molecule to the subject, and administering an immunostimulatory antibody molecule sequentially to the administration of the Treg-depleting antibody molecule. 79. The method of embodiment 78, wherein the immunostimulatory antibody molecule is a CD8-activating and / or CD8-enhancing antibody molecule. 80. The method of embodiment 78 or 79, wherein the cancer is a solid tumor. 81. The method of embodiment 80, wherein the solid tumor is selected from the group consisting of sarcoma, carcinoma, and lymphoma. 82. The method of embodiment 81, wherein the solid tumor is squamous cell carcinoma (SCC), thymoma, neuroblastoma, or ovarian cancer. 83. The method of any one of embodiments 78-82, wherein the Treg-depleting antibody molecule and / or the immunostimulatory antibody molecule is selected from the group consisting of full-length IgG antibodies, Fab, Fv, scFv, Fab', and (Fab')2. 84. The method of embodiment 83, wherein the full-length IgG antibody is selected from the group consisting of IgG1, IgG2, IgG4, and Fc-engineered variants thereof. 85. The method of any one of embodiments 78-84, wherein said Treg-depleting antibody molecule and / or said immunostimulatory antibody molecule is a human or humanized antibody. 86. The method of any one of embodiments 78-85, wherein the Treg-depleting antibody molecule is a human anti-IgG1 antibody. 87. The method of any one of embodiments 78-86, wherein said Treg-depleting antibody molecule is a human IgG1 antibody molecule engineered for improved binding to at least one activating FcγR. 88. The method of any one of embodiments 78 to 87, wherein the Treg-depleting antibody molecule is selected from antibody molecules that specifically bind to targets belonging to the tumor necrosis factor receptor superfamily (TNFRSF). 89. The method of embodiment 88, wherein said Treg-depleting antibody molecule is an antibody molecule that specifically binds to a target selected from the group consisting of 4-1BB, OX40, and TNFR2. 90. The method of embodiment 87, wherein said Treg-depleting antibody molecule is an anti-4-1BB 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 sequences 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 molecules comprising 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. 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 variable 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 embodiments 91-93, wherein the Treg-depleting 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: 8, 16, 24, 32, 40, 48, 56, 64, 72, 160, and 170. 95. The method of any one of embodiments 91-94, wherein the Treg-depleting antibody molecule is selected from the group consisting of antibody molecules 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: 169-170. 96. The method of embodiment 89, wherein the 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 molecules comprising one or more 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: 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 embodiments 96 to 98, 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, 111, 119, 127, 135, 143, 151, and 177. 100. The method of any one of embodiments 96-99, wherein the Treg-depleting 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. 101. The method of any one of embodiments 96-100, wherein the Treg-depleting 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 embodiments 78-87, wherein the Treg-depleting antibody molecule is selected from antibody molecules that specifically bind to a target selected from the group consisting of ICOS, GITR, CTLA-4, CD25, and neuropilin-1. 103. The method of any one of embodiments 78-102, wherein the immunostimulatory antibody molecule is a human IgG2 antibody or a human IgG4 antibody molecule. 104. The method of embodiment 103, wherein the immunostimulatory antibody molecule is a human IgG2b antibody molecule. 105. The method of any one of embodiments 78-104, wherein the immunostimulatory antibody molecule is engineered for improved binding to human FcγRIIB over activating Fc gamma receptors. 106. The method of any one of embodiments 76 to 105, wherein the immunostimulatory antibody molecule is an antibody that specifically binds to a target selected from the group consisting of 4-1BB, 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 sequences 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 claim 108, wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules 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 embodiments 107-109, wherein the immunostimulatory 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 embodiments 107-110, 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: 8, 16, 24, 32, 40, 48, 56, 64, 72, 160, and 170. 112. The method of any one of embodiments 107-111, wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules 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 molecules comprising one or more 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: 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 embodiments 113-115, wherein the immunostimulatory 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, 111, 119, 127, 135, 143, 151, and 177. 117. The method of any one of embodiments 113-116, 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. 118. The method of any one of embodiments 113-117, 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: 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. 119. The method of embodiment 106, wherein the immunostimulatory antibody molecule is a human anti-PD1 monoclonal antibody molecule, a human anti-PDL1 monoclonal antibody molecule, or a human anti-CTLA-4 monoclonal antibody molecule. 120. The method of embodiment 119, 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 selected from the group consisting of ipilimumab and tremilimumab. 121. Any use, method, antibody, nucleic acid, vector, host cell, or pharmaceutical composition described in the description, examples and / or figures herein.

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Claims

1. 1. 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, the Treg-depleting antibody molecule being administered prior to administration of the immunostimulatory antibody molecule.

2. The Treg-depleting antibody for use according to claim 1 , wherein the immunostimulatory antibody molecule is a CD8-activating and / or CD8-enhancing antibody molecule.

3. 3. The Treg-depleting antibody molecule for use according to claim 1 or 2, wherein the cancer is a solid tumor selected from the group consisting of sarcoma, carcinoma, lymphoma, and ovarian cancer, and / or a solid tumor such as a solid tumor selected from the group consisting of squamous cell carcinoma (SCC), thymoma, neuroblastoma, or ovarian cancer.

4. The Treg-depleting antibody molecule and / or the immunostimulatory antibody molecule may be a full-size antibody, Fab, Fv, scFv, Fab', and (Fab') 2 The Treg-depleting antibody molecule for use according to any one of claims 1 to 3, selected from the group consisting of:

5. 8. The Treg-depleting antibody molecule for use according to any one of claims 1 to 7, wherein said Treg-depleting antibody molecule is a human IgG1 antibody which may optionally be engineered for improved binding to at least one activating FcγR.

6. 6. The Treg-depleting antibody molecule for use according to any one of claims 1 to 5, wherein the Treg-depleting antibody molecule is selected from antibody molecules that specifically bind to a target belonging to the tumor necrosis factor receptor superfamily (TNFRSF).

7. The Treg-depleting antibody molecule for use according to claim 6, wherein the Treg-depleting antibody molecule is an antibody molecule that specifically binds to a target selected from the group consisting of 4-1BB, OX40, and TNFR2.

8. The Treg-depleting antibody molecule for use according to any one of claims 1 to 5, wherein the Treg-depleting antibody molecule is an antibody molecule that specifically binds to a target selected from GITR, ICOS, CTLA-4, CD25, and neuropilin-1.

9. The Treg-depleting antibody molecule for use according to claim 7, wherein said Treg-depleting antibody molecule is an anti-4-1BB monoclonal antibody molecule.

10. 10. The Treg-depleting antibody for use according to claim 9, wherein said Treg-depleting antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 CDRs selected from SEQ ID NOs: 1-6, 1 to 6 CDRs selected from SEQ ID NOs: 9-14, 1 to 6 CDRs selected from SEQ ID NOs: 17-22, 1 to 6 CDRs selected from SEQ ID NOs: 25-30, 1 to 6 CDRs selected from SEQ ID NOs: 33-38, 1 to 6 CDRs selected from SEQ ID NOs: 41-46, 1 to 6 CDRs selected from SEQ ID NOs: 49-54, 1 to 6 CDRs selected from SEQ ID NOs: 57-62, 1 to 6 CDRs selected from SEQ ID NOs: 65-70, 1 to 6 CDRs selected from SEQ ID NOs: 153-158, and 1 to 6 CDRs selected from SEQ ID NOs: 163-168.

11. 11. The Treg-depleting antibody for use according to claim 10, 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, and / or 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.

12. 12. The Treg-depleting antibody for use according to claim 10 or 11, wherein the Treg-depleting antibody molecule is selected from the group consisting of antibody molecules 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: 159-160, and SEQ ID NOs: 169-170.

13. The Treg-depleting antibody molecule for use according to claim 7, wherein the Treg-depleting antibody is a human anti-OX40 monoclonal antibody molecule.

14. 14. The Treg-depleting antibody for use according to claim 13, wherein said Treg-depleting antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 CDRs selected from SEQ ID NOs: 73-78, 1 to 6 CDRs selected from SEQ ID NOs: 81-86, 1 to 6 CDRs selected from SEQ ID NOs: 89-94, 1 to 6 CDRs selected from SEQ ID NOs: 97-102, 1 to 6 CDRs selected from SEQ ID NOs: 105-110, 1 to 6 CDRs selected from SEQ ID NOs: 113-118, 1 to 6 CDRs selected from SEQ ID NOs: 121-126, 1 to 6 CDRs selected from SEQ ID NOs: 129-134, 1 to 6 CDRs selected from SEQ ID NOs: 137-142, 1 to 6 CDRs selected from SEQ ID NOs: 145-150, and 1 to 6 CDRs selected from SEQ ID NOs: 171-176.

15. 15. The Treg-depleting antibody for use according to claim 14, wherein said 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, 111, 119, 127, 135, 143, 151, and 177, and / or 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.

16. 16. The Treg-depleting antibody for use according to any one of claims 1 to 15, wherein the immunostimulatory antibody molecule is a human IgG2 or IgG4 antibody molecule that may optionally be engineered for improved binding to human FcγRIIB over activating Fc gamma receptors.

17. 17. The Treg-depleting antibody for use according to claim 16, wherein the immunostimulatory antibody molecule is a human IgG2b antibody molecule that can optionally be engineered for improved binding to human FcγRIIB over activating Fc gamma receptors.

18. 18. The Treg-depleting antibody molecule for use according to any one of claims 1 to 17, wherein the immunostimulatory antibody molecule is an antibody that specifically binds to a target selected from the group consisting of 4-1BB, OX40, ICOS, GITR, CTLA-4, CD25, PD-1, and PDL1.

19. The Treg-depleting antibody molecule for use according to claim 18, wherein the immunostimulatory antibody molecule is an anti-4-1BB antibody molecule.

20. 20. The Treg-depleting antibody molecule for use according to claim 19, wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 CDRs selected from SEQ ID NOs: 1-6, 1 to 6 CDRs selected from SEQ ID NOs: 9-14, 1 to 6 CDRs selected from SEQ ID NOs: 17-22, 1 to 6 CDRs selected from SEQ ID NOs: 25-30, 1 to 6 CDRs selected from SEQ ID NOs: 33-38, 1 to 6 CDRs selected from SEQ ID NOs: 41-46, 1 to 6 CDRs selected from SEQ ID NOs: 49-54, 1 to 6 CDRs selected from SEQ ID NOs: 57-62, 1 to 6 CDRs selected from SEQ ID NOs: 65-70, 1 to 6 CDRs selected from SEQ ID NOs: 153-158, and 1 to 6 CDRs selected from SEQ ID NOs: 163-168.

21. 21. The Treg-depleting antibody for use according to claim 20, wherein the immunostimulatory 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, and / or 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.

22. 22. A Treg-depleting antibody for use according to claim 20 or 21, wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules 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.

23. 19. The Treg-depleting antibody molecule for use according to claim 18, wherein the immunostimulatory antibody molecule is an anti-OX40 antibody molecule.

24. 24. The Treg-depleting antibody molecule for use according to claim 23, wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 CDRs selected from SEQ ID NOs: 73-78, 1 to 6 CDRs selected from SEQ ID NOs: 81-86, 1 to 6 CDRs selected from SEQ ID NOs: 89-94, 1 to 6 CDRs selected from SEQ ID NOs: 97-102, 1 to 6 CDRs selected from SEQ ID NOs: 105-110, 1 to 6 CDRs selected from SEQ ID NOs: 113-118, 1 to 6 CDRs selected from SEQ ID NOs: 121-126, 1 to 6 CDRs selected from SEQ ID NOs: 129-134, 1 to 6 CDRs selected from SEQ ID NOs: 137-142, 1 to 6 CDRs selected from SEQ ID NOs: 145-150, and 1 to 6 CDRs selected from SEQ ID NOs: 171-176.

25. 25. The Treg-depleting antibody for use according to claim 24, wherein the immunostimulatory 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, 111, 119, 127, 135, 143, 151, and 177, and / or 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.

26. 26. A Treg-depleting antibody for use according to claim 24 or 25, 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: 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: 177-178.

27. 19. The Treg-depleting antibody molecule for use according to claim 18, wherein the immunostimulatory antibody molecule is a human anti-PD1 monoclonal antibody molecule, a human anti-PDL1 monoclonal antibody molecule, or a human anti-CTLA-4 monoclonal antibody molecule.

28. 28. The Treg-depleting antibody molecule for use according to claim 27, 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 selected from the group consisting of ipilimumab and tremilimumab.

29. an anti-4-1BB antibody molecule selected from the group consisting of antibody molecules comprising 1 to 6 CDRs selected from SEQ ID NOs: 1-6, 1 to 6 CDRs selected from SEQ ID NOs: 9-14, 1 to 6 CDRs selected from SEQ ID NOs: 17-22, 1 to 6 CDRs selected from SEQ ID NOs: 25-30, 1 to 6 CDRs selected from SEQ ID NOs: 33-38, 1 to 6 CDRs selected from SEQ ID NOs: 41-46, 1 to 6 CDRs selected from SEQ ID NOs: 49-54, 1 to 6 CDRs selected from SEQ ID NOs: 57-62, 1 to 6 CDRs selected from SEQ ID NOs: 65-70, 1 to 6 CDRs selected from SEQ ID NOs: 153-158, and 1 to 6 CDRs selected from SEQ ID NOs: 163-168.

30. 30. The anti-4-1BB antibody molecule of claim 29, 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, and / or 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.

31. The anti-4-1BB antibody molecule according to claim 29 or 30, selected from the group consisting of antibody molecules 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 to 170.

32. Full-length IgG antibodies, Fab, Fv, scFv, Fab', and (Fab') 2 The anti-4-1BB antibody molecule of any one of claims 44 to 48, selected from the group consisting of:

33. 50. The anti-4-1BB antibody molecule of claim 49, wherein the full-length IgG antibody is selected from the group consisting of IgG1, IgG2, IgG4, and Fc-engineered variants thereof.

34. The anti-4-1BB antibody molecule of any one of claims 29 to 33, wherein the Treg-depleting antibody molecule and / or the immunostimulatory antibody molecule is a human or humanized antibody.

35. an anti-OX40 antibody molecule selected from the group consisting of antibody molecules comprising one to six CDRs selected from SEQ ID NOs:73-78, one to six CDRs selected from SEQ ID NOs:81-86, one to six CDRs selected from SEQ ID NOs:89-94, one to six CDRs selected from SEQ ID NOs:97-102, one to six CDRs selected from SEQ ID NOs:105-110, one to six CDRs selected from SEQ ID NOs:113-118, one to six CDRs selected from SEQ ID NOs:121-126, one to six CDRs selected from SEQ ID NOs:129-134, one to six CDRs selected from SEQ ID NOs:137-142, one to six CDRs selected from SEQ ID NOs:145-150, and one to six CDRs selected from SEQ ID NOs:171-176.

36. 36. The anti-OX40 antibody molecule of claim 35, 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, 111, 119, 127, 135, 143, 151, and 177, and / or 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.

37. The Treg-depleting antibody molecule and / or the immunostimulatory antibody molecule may be a full-length IgG antibody, Fab, Fv, scFv, Fab', and (Fab') 2 37. The anti-OX40 antibody molecule of claim 35 or 36, selected from the group consisting of:

38. 38. The anti-OX40 antibody molecule of claim 37, wherein the full-length IgG antibody is selected from the group consisting of IgG1, IgG2, IgG4, and Fc-engineered variants thereof.

39. The anti-OX40 antibody molecule of any one of claims 35 to 38, wherein the Treg-depleting antibody molecule and / or the immunostimulatory antibody molecule is a human or humanized antibody.

40. An isolated nucleic acid encoding the antibody of any one of claims 29 to 39.

41. A vector comprising the nucleic acid of claim 40.

42. A host cell comprising the vector of claim 41.

43. An antibody according to any one of claims 29 to 39 for use in medicine.

44. A pharmaceutical composition comprising the antibody of any one of claims 29 to 39.

45. 45. The antibody of claim 43 or the pharmaceutical composition of claim 44 for use in the treatment of cancer.

46. 46. ​​The antibody or pharmaceutical composition of claim 45, wherein the cancer is a solid tumor selected from the group consisting of sarcoma, carcinoma, and lymphoma, and / or a solid tumor such as a solid tumor selected from the group consisting of squamous cell carcinoma (SCC), thymoma, neuroblastoma, or ovarian cancer.

47. A pharmaceutical composition comprising an antibody according to any one of claims 29 to 39, or an antibody according to any one of claims 29 to 34 and an antibody according to any one of claims 35 to 39.

48. The antibody of any one of claims 29 to 39 or the pharmaceutical composition of claim 47, wherein the pharmaceutical composition is for the treatment of cancer.

49. The antibody of any one of claims 29 to 39 or the pharmaceutical composition of claim 48, wherein the cancer is a solid tumor such as a solid tumor selected from the group consisting of sarcoma, carcinoma, and lymphoma, and / or a solid tumor selected from the group consisting of squamous cell carcinoma (SCC), thymoma, neuroblastoma, or ovarian cancer.

50. Use of an antibody according to any one of claims 29 to 39 for the manufacture of a pharmaceutical composition for use in the treatment of cancer.

51. 51. The use of claim 50, wherein the cancer is a solid tumor selected from the group consisting of sarcoma, carcinoma, and lymphoma, and / or a solid tumor selected from the group consisting of squamous cell carcinoma (SCC), thymoma, neuroblastoma, or ovarian cancer.

52. 1. A method for treating cancer in a subject, comprising administering to said subject a Treg-depleting antibody molecule, and sequentially with said administration of said Treg-depleting antibody molecule, an immunostimulatory antibody molecule.

53. 53. The method of claim 52, wherein the immunostimulatory antibody molecule is a CD8-activating and / or CD8-enhancing antibody molecule.

54. 54. The method of claim 52 or 53, wherein the cancer is a solid tumor selected from the group consisting of sarcoma, carcinoma, and lymphoma, and / or a solid tumor selected from the group consisting of squamous cell carcinoma (SCC), thymoma, neuroblastoma, or ovarian cancer.

55. The Treg-depleting antibody molecule and / or the immunostimulatory antibody molecule may be a full-length IgG antibody, Fab, Fv, scFv, Fab', and (Fab') 2 55. The method of any one of claims 52 to 54, selected from the group consisting of:

56. 56. The method of claim 55, wherein the full-length IgG antibody is selected from the group consisting of IgG1, IgG2, IgG4, and Fc-engineered variants thereof.

57. 85. The method of any one of claims 52-84, wherein the Treg-depleting antibody molecule is a human IgG1 antibody that can optionally be engineered for improved binding to at least one activating FcγR.

58. 58. The method of any one of claims 52 to 57, wherein the Treg-depleting antibody molecule is selected from antibody molecules that specifically bind to targets belonging to the tumor necrosis factor receptor superfamily (TNFRSF).

59. 59. The method of claim 58, wherein the Treg-depleting antibody molecule is an antibody molecule that specifically binds to a target selected from the group consisting of 4-1BB, OX40, and TNFR2.

60. 58. The method of any one of claims 52 to 57, wherein the Treg-depleting antibody molecule is selected from antibody molecules that specifically bind to a target selected from the group consisting of ICOS, GITR, CTLA-4, CD25, and neuropilin-1.

61. 60. The method of claim 59, wherein the Treg-depleting antibody molecule is an anti-4-1BB monoclonal antibody molecule.

62. 62. The method of claim 61, wherein the Treg-depleting antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 CDRs selected from SEQ ID NOs: 1-6, 1 to 6 CDRs selected from SEQ ID NOs: 9-14, 1 to 6 CDRs selected from SEQ ID NOs: 17-22, 1 to 6 CDRs selected from SEQ ID NOs: 25-30, 1 to 6 CDRs selected from SEQ ID NOs: 33-38, 1 to 6 CDRs selected from SEQ ID NOs: 41-46, 1 to 6 CDRs selected from SEQ ID NOs: 49-54, 1 to 6 CDRs selected from SEQ ID NOs: 57-62, 1 to 6 CDRs selected from SEQ ID NOs: 65-70, 1 to 6 CDRs selected from SEQ ID NOs: 153-158, and 1 to 6 CDRs selected from SEQ ID NOs: 163-168.

63. 63. The method of claim 62, 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, and / or 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.

64. 65. The method of claim 63 or 64, wherein the Treg-depleting antibody molecule is selected from the group consisting of antibody molecules 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: 169-170.

65. 60. The method of claim 59, wherein the Treg-depleting antibody is a human anti-OX40 monoclonal antibody molecule.

66. 66. The method of claim 65, wherein the Treg-depleting antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 CDRs selected from SEQ ID NOs: 73-78, 1 to 6 CDRs selected from SEQ ID NOs: 81-86, 1 to 6 CDRs selected from SEQ ID NOs: 89-94, 1 to 6 CDRs selected from SEQ ID NOs: 97-102, 1 to 6 CDRs selected from SEQ ID NOs: 105-110, 1 to 6 CDRs selected from SEQ ID NOs: 113-118, 1 to 6 CDRs selected from SEQ ID NOs: 121-126, 1 to 6 CDRs selected from SEQ ID NOs: 129-134, 1 to 6 CDRs selected from SEQ ID NOs: 137-142, 1 to 6 CDRs selected from SEQ ID NOs: 145-150, and 1 to 6 CDRs selected from SEQ ID NOs: 171-176.

67. 67. The method of claim 65 or 66, 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, 111, 119, 127, 135, 143, 151, and 177, and / or 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.

68. 103. The method of any one of claims 78-102, wherein the immunostimulatory antibody molecule is a human IgG2 or human IgG4 antibody molecule that can optionally be engineered for improved binding to human FcγRIIB over activating Fc gamma receptors.

69. 69. The method of claim 68, wherein the immunostimulatory antibody molecule is a human IgG2b antibody molecule that can optionally be engineered for improved binding to human FcγRIIB over activating Fc gamma receptors.

70. 70. The method of any one of claims 52-69, wherein the immunostimulatory antibody molecule is an antibody that specifically binds to a target selected from the group consisting of 4-1BB, OX40, ICOS, GITR, CTLA-4, CD25, PD-1, and PDL1.

71. 71. The method of claim 70, wherein the immunostimulatory antibody molecule is an anti-4-1BB antibody molecule.

72. 72. The method of claim 71, wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 CDRs selected from SEQ ID NOs: 1-6, 1 to 6 CDRs selected from SEQ ID NOs: 9-14, 1 to 6 CDRs selected from SEQ ID NOs: 17-22, 1 to 6 CDRs selected from SEQ ID NOs: 25-30, 1 to 6 CDRs selected from SEQ ID NOs: 33-38, 1 to 6 CDRs selected from SEQ ID NOs: 41-46, 1 to 6 CDRs selected from SEQ ID NOs: 49-54, 1 to 6 CDRs selected from SEQ ID NOs: 57-62, 1 to 6 CDRs selected from SEQ ID NOs: 65-70, 1 to 6 CDRs selected from SEQ ID NOs: 153-158, and 1 to 6 CDRs selected from SEQ ID NOs: 163-168.

73. 72. The method of claim 71, wherein the immunostimulatory 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, and / or 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.

74. 73. The method of claim 71 or 72, wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules 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.

75. 71. The method of claim 70, wherein the immunostimulatory antibody molecule is an anti-OX40 antibody molecule.

76. 75. The method of claim 74, wherein the immunostimulatory antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 CDRs selected from SEQ ID NOs: 73-78, 1 to 6 CDRs selected from SEQ ID NOs: 81-86, 1 to 6 CDRs selected from SEQ ID NOs: 89-94, 1 to 6 CDRs selected from SEQ ID NOs: 97-102, 1 to 6 CDRs selected from SEQ ID NOs: 105-110, 1 to 6 CDRs selected from SEQ ID NOs: 113-118, 1 to 6 CDRs selected from SEQ ID NOs: 121-126, 1 to 6 CDRs selected from SEQ ID NOs: 129-134, 1 to 6 CDRs selected from SEQ ID NOs: 137-142, 1 to 6 CDRs selected from SEQ ID NOs: 145-150, and 1 to 6 CDRs selected from SEQ ID NOs: 171-176.

77. 76. The method of claim 74 or 75, wherein the immunostimulatory 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, 111, 119, 127, 135, 143, 151, and 177, and or 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.

78. 77. The method of any one of claims 74-76, 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: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.

79. 71. The method of claim 70, wherein the immunostimulatory antibody molecule is a human anti-PD1 monoclonal antibody molecule, a human anti-PDL1 monoclonal antibody molecule, or a human anti-CTLA-4 monoclonal antibody molecule.

80. 79. The method of claim 78, 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 selected from the group consisting of ipilimumab and tremilimumab.

81. Any use, method, antibody, nucleic acid, vector, host cell or pharmaceutical composition described in the description, examples and / or figures herein.

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