WO2019008375A1

WO2019008375A1 - Method for identifying responders to cancer treatment

Info

Publication number: WO2019008375A1
Application number: PCT/GB2018/051912
Authority: WO
Inventors: Sergio Quezada; Karl PEGGS; Andrew FURNESS; Frederick ARCE VARGAS; Charles Swanton; Rachel ROSENTHAL; Samra TURAJLIC; Kevin LITCHFIELD
Original assignee: Ucl Business Plc
Priority date: 2017-07-06
Filing date: 2018-07-05
Publication date: 2019-01-10

Abstract

The present invention relates to a method for identifying a subject with cancer who is suitable for treatment with an IgG1 antibody or IgG2 antibody targeting an immune checkpoint molecule, and to methods of treatment of such subjects.

Description

METHOD FOR IDENTIFYING RESPONDERS TO CANCER TREATMENT

FIELD OF THE INVENTION The present invention also relates to a method for treating cancer in a human subject, particularly a solid tumour, comprising the step of administering an lgG2 antibody targeted to an immune checkpoint molecule to the subject, wherein the lgG2 antibody is capable of depleting regulatory T cells in a solid tumour. BACKGROUND TO THE INVENTION

The present invention relates to cancer immunotherapy. Cancer immunotherapy involves the use of a subject's own immune system to treat or prevent cancer. Immunotherapies exploit the fact that cancer cells often have subtly different molecules on their surface that can be detected by the immune system. These molecules, or cancer antigens, are most commonly proteins, but also include molecules such as carbohydrates. Immunotherapy thus involves provocation of the immune system into attacking tumour cells via these target antigens.

However, malignant tumors, in particular solid tumors, can escape immune surveillance by means of various mechanisms both intrinsic to the tumor cell and mediated by components of the tumor microenvironment. Amongst the latter, tumor infiltration by regulatory T cells (Tregs) and, more specifically, an unfavorable balance of effector T cells (Teff) versus Treg, have been proposed as critical factors. Since their discovery, Tregs have been found to be critical in mediating immune homeostasis and promoting the establishment and maintenance of peripheral tolerance.

However, in the context of cancer their role is more complex. As cancer cells express both self- and tumour-associated antigens, the presence of Tregs, which seek to dampen effector cell responses, can contribute to tumor progression.

The infiltration of Tregs in established tumors therefore represent one of the main obstacles to effective anti-tumour responses and to treatment of cancers in general. Suppression mechanisms employed by Tregs are thought to contribute significantly to the limitation or even failure of current therapies, in particular immunotherapies that rely on induction or potentiation of anti-tumour responses. Studies have shown the contribution of Tregs to tumor establishment and progression in murine models. Tumor-infiltration by Tregs has also been associated with worse prognosis in several human cancers. Thus, there is a need in the art for ways of treating cancer, in particular solid tumours, where an effective depletion of Tregs is achieved, allowing for the enhancement of antitumor effector functions.

SUMMARY OF THE INVENTION

The inventors have found that, surprisingly, an lgG2 antibody that is capable of binding to an immune checkpoint molecule is capable of Treg depletion in vivo within solid tumours. The lgG2 isotype was thought in the art to be a poor mediator of ADCC (Schneider-Merck et al. 2010). However, the present inventors have shown that an lgG2 isotype antibody is capable of depleting Tregs. This was a surprising finding and goes against the prevailing view in the art.

As such, the present invention provides an lgG2 antibody which is capable of binding to an immune checkpoint molecule and which is capable of depleting Tregs in a tumour for use in the treatment of cancer in a subject, wherein said subject has a solid tumour or haematological tumour.

The invention provides a method for treating cancer in a subject, comprising administering to a subject an lgG2 antibody which is capable of binding to an immune checkpoint molecule and which is capable of depleting Tregs in a tumour, wherein said subject has a solid tumour or haematological tumour.

The invention also provides use of an lgG2 antibody which is capable of binding to an immune checkpoint molecule and which is capable of depleting Tregs in a tumour in the manufacture of a medicament for use in the treatment of cancer in a subject, wherein said subject has a solid tumour or haematological tumour.

In one aspect the tumour is a solid tumour. Preferably, the immune checkpoint molecule as described herein in respect of the invention is one that is expressed at a higher level on Treg cells when compared to Teff cells. Methods for determining expression are known in the art, and are described in the present Examples. In one aspect the antibody is capable of binding to CTLA4.

In a further aspect the present invention provides a method for identifying a subject with cancer who is suitable for treatment with an lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule, preferably one expressed at high levels on Treg cells compared to Teff cells, said method comprising determining the FcyR polymorphism status of the subject, wherein the presence of a FcyR polymorphism which confers an increased affinity to lgG1 is indicative of response to an lgG1 antibody immune checkpoint intervention and wherein the presence of a FcyR polymorphism which confers an increased affinity to lgG2 is indicative of response to an lgG2 antibody targeting an immune checkpoint molecule.

In one embodiment of the method of the invention, determining the FcyR polymorphism status of the patient comprises the steps of:

i) sequencing of the subject's germline DNA;

ii) identifying the presence of one or more single nucleotide polymorphism(s) within an FcyR gene, such that the FcyR gene having the one or more single nucleotide polymorphism(s) encodes a variant FcyR molecule; and optionally a further step of:

iii) determining the affinity of the FcyR for its cognate IgG molecule.

In one aspect the polymorphism is F158 in FcyRIIIA. In another aspect the polymorphism is V158 in FcyRIIIA. The V158 polymorphism may increase the binding affinity to human lgG1 and lgG3, therefore increasing depleting activity.

In one aspect the polymorphism is H 131 in FcyRIIA. In another aspect the polymorphism is R131 in FcyRIIA. The H131 polymorphism may increase the binding affinity to human lgG1 and lgG2, therefore increasing depleting activity (see for example Bruhns P. et al. Blood 2009. 1 13: 3716-3725).

In one aspect, the method for identifying a subject with cancer who is suitable for treatment of the present invention further comprises the step of determining the mutational burden and/or the neo-antigen burden in the subject, wherein the presence of a higher and/or increased and/or high mutational burden and/or neo-antigen burden is indicative of response to an lgG2 or lgG1 antibody targeting an immune checkpoint molecule, preferably one expressed at higher levels on Treg versus Teff cells. In one aspect of the invention as described herein the lgG2 antibody is not tremelimumab..

In another aspect, the present invention provides a method for identifying patients not suitable for treatment with an lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule, said method comprising the steps of:

i) determining the FcyR polymorphism status of the subject; and

ii) determining the mutational burden and/or the neo-antigen burden in the subject; wherein the presence of a FcyR polymorphism which confers an increased affinity to lgG1 or a FcyR polymorphism which confers an increased affinity to lgG2, in combination with a lower and/or decreased and/or low mutational burden and/or neo-antigen burden is indicative of a decreased response to an lgG1 or lgG2 antibody targeting an immune checkpoint molecule respectively. In another aspect, the present invention provides a method of treating or preventing cancer in a subject, comprising the steps of:

i) identifying a subject with cancer who is suitable for treatment with an antibody targeting an immune checkpoint molecule according to a method of the present invention;

ii) treating the cancer in the subject comprising the step of administering an antibody targeted to the immune checkpoint molecule to the subject.

DESCRIPTION OF THE FIGURES

Figure 1. CTLA-4 is highly expressed by tumor-infiltrating Treg cells. (A-C) Mice were injected subcutaneously (s.c.) with B16, MCA205, MC38 or CT26 cells. Ten days later, cell suspensions from peripheral blood mononuclear cells (PBMC), draining lymph nodes (LN) and tumor-infiltrating lymphocytes (TIL) were stained and analyzed by flow cytometry. (A) Representative histograms of CTLA-4 expression detected by intracellular staining of individual T cell subsets from mice with MCA205 tumors. Dotted lines represent the gating, numbers indicate percentage of CTLA-4+ cells in each subset. (B) Percentage and (C) MFI of CTLA-4-expressing cells in murine tumor models. Data shown correspond to one of two separate experiments (n=5). (D-F) Cells suspensions from samples of advanced melanoma (n=8), early-stage non-small cell lung cancer (NSCLC) (n=8), renal cell carcinoma RCC (n=8) and matched PBMCs were prepared and stained for flow cytometry analysis. (D) Representative histograms of CTLA-4 expression detected by intracellular staining of circulating (PBMC) and tumor-infiltrating (TIL) T cell subsets in a patient with advanced melanoma. Dotted lines represent the gating, numbers indicate percentage of CTLA-4+ cells in each subset. (E) Percentage and (F) MFI of CTLA-4 expression on circulating and tumor- infiltrating T cells in patients with the indicated tumor subtypes. Error bars represent standard error of the mean (SEM).

Figure 2. Expression pattern of hFcyRs in human tumors and hFcyR transgenic mice.

Expression of hFcyRs was analyzed by flow cytometry of leukocytes from blood and MCA205 tumours isolated from hFcyR mice and in samples of human melanoma and matching PBMCs. (A) Representative histograms showing FcyR expression on CD3+ T cells, CD19+ B cells, NK1.1⁺ NK cells, CD1 1 b+NK1.1-Ly6G-CD1 1c^{low "} monocyte/macrophages (Μο/ΜΦ) and CD1 1 b+Ly6G+ granulocytes isolated from hFcyR mice. (B) Representative histograms demonstrating FcyR expression on human CD3+CD56- T cells, CD19+CD3- B cells, CD56+CD3- NK cells, CD1 1 b+CD14+HLA-DR+ monocyte/macrophages (mono/Μφ) and CD11 b+CD15+CD14- granulocytes. (C) Percentage expression of individual FcyRs in hFcyR mice (n = 3). Results are representative of three independent experiments. (D) Percentage expression of FcyRs in metastatic deposits of human melanoma (n = 10). (E) Expression of individual FcyRs in human melanoma samples as a percentage of total CD45+ cells. Error bars represent SEM.

Figure 3. Anti-CTLA-4 antibodies of lgG1 and lgG2 isotypes mediate local depletion of intra-tumoral Treg cells in vivo. (A) Predicted ADCC activity of human IgG Fc variants. (B) In vitro killing of CTLA-4-expressing target cells by human macrophage-mediated ADCC using Fc variants of anti-CTLA-4. (C-E) hFcyR mice were treated with 200 μg of anti-CTLA- 4 on days 6 and 9 after challenge with s.c. MCA205 tumor cells (n=9-21). TILs, LNs and PBMCs were processed on day 11 and stained for flow cytometry analysis. (C) Percentage of FoxP3+CD4+ Treg cells from total CD4+ T cells and (D) CD8+/Treg cell ratio in the indicated sites. (E) Percentage of Ki67-expressing CD4+FoxP3- and CD8+ T cells following treatment. (F) Percentage of CD4+FoxP3- and CD8+ T cells expressing IFNy following re- stimulation with PMA and ionomycin. Cumulative data of two separate experiments. Error bars represent SEM.

Figure 4. Intra-tumoral Treg cell depletion is required for the anti-tumor activity of anti-CTLA-4. hFcyR mice were treated with 50 μg of anti-CTLA-4 on days 6, 9 and 12 after s.c. inoculation of MCA205 tumor cells. (A) Diagramatic representation of the experimental protocol. (B) Tumour growth in individual hFcyR mice with each treatment. Tumour volumes were calculated as the product of three orthogonal diameters. Numbers represent the ratio of mice with complete long-term tumour rejection. (C) Kaplan-Meier curve of accumulated data from mice in (B) (log-rank p<0.0001). Cumulative data of two separate experiments for each condition. Figure 5. Human FcyR polymorphisms impact upon response to ipilimumab in patients with advanced melanoma

(A) Survival analysis of patients with advanced melanoma treated with ipilimumab with low mutational burden (below the median) or high mutational burden (above the median) with or without germline polymorphisms in CD32a-H131 R and CD16a-V158F. (B) Survival analysis of patients with advanced melanoma treated with ipilimumab with low predicted neoantigen burden (below the median) or high predicted neoantigen burden (above the median) with or without germline polymorphisms in CD32a-H131 and CD16a-V158F. Key (lower left) in each plot depicts mutational/predicted neoantigen burden and polymorphism status. Log rank p values are displayed in individual plots. HR, hazard ratio; CI, confidence interval.

Figure 6. Expression profile of B7 and TNFR superfamily checkpoint molecules on T cell subsets in murine and human tumors. Expression of the indicated B7 and TNFR superfamily co-inhibitory and co-stimulatory molecules on T cells was quantified by flow cytometry. (A) Heatmap demonstrating the relative expression of immune checkpoint molecules based on percentage of positive cells within the indicated T cell subsets in murine and human tumor subtypes. Data derived from 5 representative mice and human tumors of each subtype. (B) MFI of the indicated co-inhibitory and co-stimulatory immune checkpoint molecules in blood (PBMC) and tumour (TIL) in human tumours. Error bars represent SEM. Figure 7. The expression pattern of FcyRs in hFcyR mice.

(A) Percentage of expression of individual FcyRs in tumor-infiltrating leukocyte subpopulations in each tumor model. (B) Percentage of expression of individual FcyRs in total CD45+ cells from different organs and tissues from tumor-bearing mice or from patients with melanoma. (C-D) Frequency of cell subtypes present in LNs, spleens and TILS of hFcyR mice (C) or matched blood and tumor samples from patients with melanoma (n=10) (D). All mice data obtained from s.c. MCA205 tumors. Error bars represent SEM.

Figure 8. Expression pattern of FcyRs in monocyte-derived human macrophages.

Expression of human FcyRs quantified by flow cytometry on CD14+ bead-sorted monocytes from healthy donor PBMCs (upper panel) and matched macrophages following a 7-day in vitro differentiation with human recombinant M-CSF (lower panel). Figure 9. Expression profile of B7 and TNFR superfamily checkpoints on T cell subsets in murine tumors. MFI of the co-stimulatory and co-inhibitory molecules in different T cell subpopulations in MCA205 tumours. Figure 10. Percentage killing of cells expressing CTLA-4 by different anti-CTLA-4 antibody isotype variants. Human monocyte-derived macrophages from a healthy donor were co-incubated for 20 hours with CFSE-labelled SupT1 cells that constitutively express murine CTLA-4 in the presence of chimeric anti-mouse CTLA-4 antibodies (clone 4F10) with different human IgG variants: lgG1 , lgG2, lgG1-N297A (Fc silent) and endoglycosidase- treated lgG2 (deglycosylated lgG2). Where indicated, cells were pre-incubated with anti- human CD32a (clone IV.3; a known CD32a/FcYRIIa blocking antibody) for 30 min before adding anti-CTLA-4. The number of CTLA-4+ targets was quantified by flow cytometry and the percentage of killing calculated as: (number of targets no mAb - number of targets with mAb)/number of cells no Ab. E:T = effector to target ratio. Error bars represent standard error of the mean.

Figure 11. Anti-CTLA-4 response rates in human melanoma patients, split by mutational load and FcyR polymorphism status. Anti-CTLA-4 (ipilimumab) clinical response rates are shown for patients split into four groups: i) High mutation load (above the median of the cohort) and presence of the high-affinity CD16a-V158 SNP "High load, SNP+"; ii) High mutation load and presence of the low-affinity CD16a-F158 SNP "High load, SNP-", iii) Low mutation load (below the median of the cohort) and presence of the high-affinity CD16a-V158 SNP "Low load, SNP+", ii) Low mutation load and presence of the low-affinity CD16a-F158 "Low load, SNP-". Mutational load was defined using two measures: a) frameshift indel load (left two bar charts), and b) non-synonymous SNV clonal neoantigen load (right two bar charts). Indel load and non-synonymous SNV clonal neoantigen load was calculated following previously described protocols (Turajlic et al. (2017) Lancet Oncol 18: 1009-1021 ; McGranahan et al. (2016) Science 351 : 1463-1469). Data is shown for two anti- CTLA-4 studies: top two bar charts represents Van Allen et al. (2015) Science 350: 207-21 1 ; bottom two bar charts represents Snyder et al. (2015) N Engl J Med 372: 783.

Figure 12. Quantification of the absolute number of tumor-infiltrating leukocyte subpopulations in B16, MC38 and MCA205 tumors in hFcyR mice. C57BL/6 mice were injected s.c. with the indicated cell lines and after 10 days, the tumours were harvested and processed for flow cytometry analysis. The total number of each cell subpopulation per gram of tumour was quantified by adding a known number of reference beads in the sample. Figure 13. Anti-CTLA-4 antibodies of lgG1 and lgG2 isotypes mediate in vitro cell killing. (A) SPR analysis of anti-murine CTLA-4 with human IgG variants. Large graphs show interaction of free monomeric FcyRs at increasing FcyR concentrations with immobilized IgG variants; inset graphs show interaction of immobilized IgG variants with aggregated low-affinity FcyRs at increasing concentrations. RU, response units. (B) In vitro ADCC assay with human monocyte-derived macrophages and mCTLA-4+ target cells in the presence of anti-mCTLA-4 mAbs with different human IgG variants. (C) ADCC assay in the presence of CD32a or CD32b blocking F(ab')₂ antibody fragments and with a deglycosylated lgG2 mAb (lgG2_Endos)- Results are representative of 3 independent experiments. Error bars show SEM of experimental triplicates.

Figure 14. Anti-CTLA-4-lgG2-mediated intratumoral depletion of Treg depends on CD32a. Quantification the percentage of CD4⁺FoxP3⁺ T cells of total CD4⁺ T cells in mice treated with anti-CTLA-4 mAbs with IgG variants in hFcyR and CD32a^"A hFcyR mice.

Figure 15. Intra-tumoral Treg cell depletion is required for the anti-tumor activity of anti-CTLA-4. hFcyR mice were treated with anti-CTLA-4 on days 6, 9 and 12 after s.c. inoculation of MC38 (treated with 100 μg/dose) or B16 (treated with 200 μg/dose) tumor cells. Kaplan-Meier curves showing survival of hFcyR mice for each tumor model. Table shows the total number of mice in each treatment group.

Figure 16. Human FcvR polymorphisms impact upon response to ipilimumab in patients with advanced melanoma. (A) Anti-CTLA-4 response rate in the van Allen et al. and Snyder et al. patient cohorts based on indel mutational load and nsSNV neoantigen load combined with the high-affinity germline SNP CD32a-H131 (SNP+) or the low-affinity germline SNP CD32a-R131 (SNP-). (B) Response rate of patients treated with anti-PD-1 from the Hugo et al. dataset based on indel mutational load with high-affinity CD16-V158 (SNP+) or low affinity CD16-F158 (SNP-). Figure 17. Boxplot showing the expression level of key immune markers from patients with available RNAseq data from the van Allen et al. cohort (n=30): CD8A, ratio of CD8A divided by FOXP3 and cytolytic activity (defined as the log-average of GZMA and PRF expression). Patients are grouped into responders with high mutational load (based on either measure) and SNP+, compared to all other patients.

Figure 18. Extension of the response rate analysis from Figure 1 1 top left, with the following additional two groups: high mutational load (for both measures) plus high CD8A expression (>median) plus SNP+ and high mutational load (for both measures) plus SNP+ (top bar graph). Additionally, high CD8A expression plus SNP+ and high CD8A expression plus SNP+ were compared (bottom bar graph). DETAILED DESCRIPTION OF THE INVENTION

METHOD FOR IDENTIFYING PATIENTS SUITABLE FOR TREATMENT WITH IPILIMUMAB Ipilimumab is a monoclonal antibody that targets CTLA-4. It has been approved by the U.S. FDA for use in the treatment of melanoma. Ipilimumab, a human lgG1 mAb directed against CTLA-4, mediates durable remissions in patients with advanced melanoma, although such responses are limited to a small subset (Hodi et al. (2010) N Engl J Med 363, 71 1-23; Robert et al. (2011) N Engl J Med 364, 2517-26; Schadendorf et al. (2015) J Am Soc Clin Oncol 33, 1889-94). Thus, not all patients with melanoma respond to treatment with ipilimumab, and there is therefore a need in the art for ways of identifying patients who may respond to treatment with ipilimumab.

As demonstrated in the present Examples (see Example 5), the present inventors have surprisingly found that subjects with advanced melanoma with the V158F polymorphism (CD16a-V158F SNP) in FcYRIIIa (i.e. this alloform of FcYRIIIa) show improved outcomes when treated with ipilimumab compared with patients who do not have this polymorphism.

As such, in one aspect is provided a method for identifying patients suitable for treatment with ipilimumab, comprising analysing for the presence of the V158F polymorphism in a sample from said patient. Presence of the polymorphism may be indicative of improved therapeutic outcomes in the patient. For example, in one aspect, presence of the V158F polymorphism may be indicative of long term response to treatment. The inventors found that patients with high mutational burden derived clinical benefit from ipilimumab if they also had the CD16a-V158F polymorphism.

As such, in one aspect the method for identifying patients suitable for treatment with ipilimumab as described herein further comprises the step of analysing the mutational and/or neoantigen burden in the sample from said patient. A higher or increased mutational or neoantigen burden may be indicative of improved therapeutic outcomes in the patient, for example long term response to ipilimumab treatment, when used in conjunction with the identification of a polymorphism(s) which would lead to an increased binding affinity for the treatment antibody, e.g. ipilimumab, to FcyRs.

In one aspect the method for identifying patients suitable for treatment with ipilimumab as described herein comprises the steps of

(i) analysing for the presence of the V158F polymorphism in a sample from said patient; and

(ii) analysing the mutational and/or neoantigen burden in the sample from said patient.

The mutational and/or neoantigen load or burden may be as described herein.

In a further aspect, the method for identifying patients suitable for treatment with an lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule as described herein may comprise the step of analysing the CD8 (CD8A) expression levels in a sample from the patient, wherein a high level of CD8 (CD8A) expression may be indicative of improved therapeutic outcomes in the patient. In one aspect, the level of CD8 expression may be analysed in place of analysing the mutational and/or neoantigen burden in the sample from the patient. As such, in one aspect the method for identifying patients suitable for treatment with lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule comprises the steps of:

(ii) analysing the level of CD8 expression in a sample from said patient.

Presence of the polymorphism and a high CD8 expression level may be indicative of an improved response to an lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule.

In one aspect the lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule is ipilimumab.

CD8 (CD8A) expression can be determined by methods known in the art. For example, using immunohistochemical methods, flow cytometry or quantification of CD8 transcripts by polymerase chain reaction. NEO-ANTIGENS

A "neo-antigen" is a tumour-specific antigen which arises as a consequence of a mutation within a cancer cell. Thus, a neo-antigen is not expressed by healthy cells in a subject.

The neo-antigen described herein may be caused by any non-silent mutation which alters a protein expressed by a cancer cell compared to the non-mutated protein expressed by a wild-type, healthy cell. For example, the mutated protein may be a translocation or fusion. In one aspect the mutation may be a single nucleotide variant (SNV), multiple nucleotide variants, a deletion mutation, an insertion mutation, a translocation, a missense mutation or a splice site mutation resulting in a change in the amino acid sequence (coding mutation).

In one aspect, the neo-antigen can be generated through an "indel" mutation.

An "indel mutation" as referred to herein refers to an insertion and/or deletion of bases in a nucleotide sequence (e.g. DNA or RNA) of an organism. Typically, the indel mutation occurs in the DNA, preferably the genomic DNA, of an organism. Suitably, the indel mutation occurs in the genomic DNA of a tumour cell in the subject. Suitably, the indel may be an insertion mutation. Suitably, the indel may be a deletion mutation. In one aspect the indel mutation is a frameshift indel mutation.

Suitably, the indel may be from 1 to 100 bases, for example 1 to 90, 1 to 50, 1 to 23 or 1 to 10 bases.

In another aspect, the neoantigen is a clonal neoantigen.

A "clonal" neoantigen is a neoantigen which is expressed effectively throughout a tumour and encoded within essentially every tumour cell. A "sub-clonal" neoantigen is a neoantigen which is expressed in a subset or a proportion of cells or regions in a tumour.

Expressed effectively in essentially every tumour cell or essentially all tumour cells means that the mutation is present in all tumour cells analysed in a sample, as determined using appropriate statistical methods.

By way of the example, the cancer cell fraction (CCF), describing the proportion of cancer cells that harbour a mutation may be used to determine whether mutations are clonal or sub- clonal. For example, the cancer cell fraction may be determined by integrating variant allele frequencies with copy numbers and purity estimates as described by Landau et al. (Cell. 2013 Feb 14; 152(4):714-26). As stated, determining a clonal mutation is subject to statistical analysis and threshold. As such, a mutation may be identified as clonal if it is determined to have a CCF 95% confidence interval >= 0.75, for example 0.80, 0.85, 0.90, 0.95, 1.00 or >1.00. Conversely, a mutation may be identified as sub-clonal if it is determined to have a CCF 95% confidence interval <= 0.75, for example 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10, 0.05, 0.01 in any sample analysed.

The clonal neoantigen may be a non-synonymous SNV clonal neoantigen, that is the mutation results from a non-synonymous SNV clonal mutation. A non-synonymous SNV is a nucleotide mutation that alters the amino acid sequence of a protein. In contrast, synonymous mutations do not alter amino acid sequences.

Cancers may be screened to detect mutations and/or neo-antigens (e.g., to detect mutation load/burden and/or neo-antigen load/burden, and/or to detect a particular signature) using techniques known in the art.

The mutations and neo-antigens may be identified by Exome sequencing, RNA-seq, whole genome sequencing and/or targeted gene panel sequencing and or routine Sanger sequencing of single genes. Suitable methods are known in the art. Examples of appropriate strategies for the detection of mutations and neo-antigens are described in McGranahan et al. (2016) Science 351 , 1463-9.

Descriptions of Exome sequencing and RNA-seq are provided by Boa et al. (Cancer Informatics. 2014; 13(Suppl 2):67-82.) and Ares et al. (Cold Spring Harb Protoc. 2014 Nov 3;2014(1 1):1 139-48); respectively. Descriptions of targeted gene panel sequencing can be found in, for example, Kammermeier et al. (J Med Genet. 2014 Nov; 51 (11):748-55) and Yap KL et al. (Clin Cancer Res. 2014. 20:6605). See also Meyerson et al., Nat. Rev. Genetics, 2010 and Mardis, Annu Rev Anal Chem, 2013. Targeted gene sequencing panels are also commercially available (e.g. as summarised by Biocompare ((http://www.biocompare.com/ Editorial-Articles/161 194-Build-Your-Own-Gene-Panels-with-These-Custom-NGS-Targeting- Tools/)). Sequence alignment to identify nucleotide differences (e.g. SNVs) in DNA and/or RNA from a tumour sample compared to DNA and/or RNA from a non-tumour sample may be performed using methods which are known in the art. For example, nucleotide differences compared to a reference sample may be performed using the method described by Koboldt et al. (Genome Res. 2012; 22: 568-576). The reference sample may be the germline DNA and/or RNA sequence.

SUBJECT SUITABLE FOR TREATMENT The invention provides a method for identifying a subject with cancer who is suitable for treatment with an lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule, said method comprising determining the FcyR polymorphism status of the subject. Preferably the presence of a FcyR polymorphism which confers an increased affinity to lgG1 is indicative of response to an lgG1 antibody immune checkpoint intervention and wherein the presence of a FcyR polymorphism which confers an increased affinity to lgG2 is indicative of response to an lgG2 antibody targeting an immune checkpoint molecule.

As used herein, the term "suitable for treatment" may refer to a subject who is more likely to respond to treatment with an immune checkpoint intervention, or who is a candidate for treatment with an immune checkpoint intervention. A subject suitable for treatment may be more likely to respond to said treatment than a subject who is determined not to be suitable using the present invention. A subject who is determined to be suitable for treatment according to the present invention may demonstrate a durable clinical benefit (DCB), which may be defined as a partial response or stable disease lasting for at least 6 months, in response to treatment with an immune checkpoint intervention.

The FcyR polymorphism may be any polymorphism that increases binding to activatory FcyRs, or alternatively that increases the A: I ratio of the antibody. In one aspect the polymorphism decreases binding to inhibitory FcyRs, such as CD32B.

An example of an FcyR polymorphism which confers an increased affinity to lgG1 is the V158F SNP in FcyRllla (CD16a).

An example of an FcyR polymorphism which confers an increased affinity to lgG2 is the H131 R SNP in FcyRlla (CD32a). In one embodiment, a method for identifying a subject with cancer who is suitable for treatment according to the present invention further comprises the step of determining the mutational burden and/or the neo-antigen burden in the subject, wherein the presence of a higher and/or increased and/or high mutational burden and/or neo-antigen burden is indicative of response to an lgG1 or lgG2 antibody targeting an immune checkpoint molecule. Neoantigens and mutations may be as described herein.

The term "burden" or "load" as used herein, for example in reference to mutation burden/load or neoantigen burden/load, refers generally to the number or rate (e.g., of mutations or neoantigens) in a sample or cohort, in some embodiments relative to that observed in an appropriate reference sample or cohort.

By way of example, the number of neo-antigens identified or predicted in the cancer cells obtained from the subject may be compared to one or more pre-determined thresholds. Using such thresholds, subjects may be stratified into categories which are indicative of the degree of response to treatment.

A threshold may be determined in relation to a reference cohort of cancer patients. The cohort may comprise 10, 25, 50, 75, 100, 150, 200, 250, 500 or more cancer patients. The cohort may be any cancer cohort. Alternatively the patients may all have the relevant or specific cancer type of the subject in question.

As described herein, "high" can mean a value above the median of a cohort. Conversely, "low" can mean below the median value of a cohort.

In one embodiment, a "high" mutational and/or neo-antigen burden/load means a number greater than the median number of neo-antigens predicted or mutations found in a reference cohort of cancer patients, such as the minimum number of neo-antigens or mutations predicted to be in the upper quartile of the reference cohort.

Mutational or neo-antigen load can also be reflected in determining specific mutational types.

Thus in one aspect, the mutational or neo-antigen load/burden can be based on the number of insertion-deletion (Indel) mutations induced by DNA frameshifts. In another aspect, the mutational or neo-antigen load/burden can be based on the number of non synonymous Single Nucleotide Variants (nsSNVs), for example nsSNV clonal neoantigen load/burden. In another embodiment, a "high" number of neo-antigens or mutations may be defined as 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190 or 200 or more neo-antigens or mutations. A skilled person will appreciated that references to "high" or "higher" numbers of neo- antigens or mutations may be context specific, and could carry out the appropriate analysis accordingly.

Alternatively, the inflammatory status of a cancer in a subject can also be an indicator of the mutational and/or neo-antigen burden of said cancer in a subject. Inflammatory status can be defined by the presence of inflammatory immune cells such as effector cells, antigen presenting cells, and inflammatory molecules such as granzymes and interferon gamma. In particular, the concentration of inflammatory immune cells or molecules in a tumour can be compared to one or more pre-determined thresholds.

In one embodiment, a "high" inflammatory status means a number greater than the median concentration of one or more inflammatory cells and/or molecules found in a reference cohort of cancer patients. In one embodiment the method for identifying a patient with cancer who is suitable for treatment according to the present invention comprises the step of determining the level of CD8 expression in a sample from said patient in the subject, wherein the presence of a higher and/or increased level of CD8 expression is indicative of response to an lgG1 or lgG2 antibody targeting an immune checkpoint molecule. In one aspect, the patient also has an FcyR polymorphism as described herein.

In one embodiment a "high" level of CD8 expression means a number greater than the median expression level of CD8 found in a reference cohort of cancer patients. In one embodiment according to the present invention, the subject may be a human. As described above, in one aspect, the present invention provides a method for identifying patients not suitable for treatment with an lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule, said method comprising the steps of:

i) determining the FcyR polymorphism status of the subject; and

ii) determining the mutational burden and/or the neo-antigen burden in the subject; wherein the presence of a FcyR polymorphism which confers an increased affinity to lgG1 or a FcyR polymorphism which confers an increased affinity to lgG2, in combination with a lower and/or decreased and/or low mutational burden and/or neo-antigen burden is indicative of a decreased response to an lgG1 or lgG2 antibody targeting an immune checkpoint molecule respectively.

The absence of a FcyR polymorphism which confers an increased affinity to lgG1 or a FcyR polymorphism which confers an increased affinity to lgG2, for example combined with a lower and/or decreased and/or low mutational burden and/or neo-antigen burden, is also indicative of a lower likelihood of response to an lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule.

CANCER As used herein, the terms "cancer", "cancerous", or "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

Examples of cancer include but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More particular examples of such cancers include squamous cell carcinoma, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (HCC), hodgkin's lymphoma, non- hodgkin's lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer.

In one aspect the cancer involves a solid tumour. Examples of solid tumours are sarcomas (including cancers arising from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, vascular, hematopoietic, or fibrous connective tissues), carcinomas (including tumors arising from epithelial cells), mesothelioma, neuroblastoma, retinoblastoma, etc. Cancers involving solid tumours include, without limitations, brain cancer, lung cancer, stomach cancer, duodenal cancer, esophagus cancer, breast cancer, colon and rectal cancer, renal cancer, bladder cancer, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, mouth cancer, sarcoma, eye cancer, thyroid cancer, urethral cancer, vaginal cancer, neck cancer, lymphoma, and the like.

In a one aspect of the invention the cancer is selected from melanoma, renal cancer, lung cancer, colorectal cancer, and sarcoma. In one aspect the cancer is melanoma.

As used herein, the term "tumour" as it applies to a subject diagnosed with, or suspected of having, a cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size, and includes primary tumours and secondary neoplasms. As used herein, "solid tumours" are an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas, in particular, tumours and/or metastasis (wherever located) other than leukaemia or non-solid lymphatic cancers. Solid tumours may be benign or malignant. Different types of solid tumours are named for the type of cells that form them and/or the tissue or organ in which they are located.

Particularly preferred cancers in accordance with the present invention include those characterized by the presence of a solid tumour, that is to say the subject does not have a non-solid tumour. In all aspects of the invention as discussed herein, it is preferred that the cancer is a solid tumour, i.e. that the subject has a solid tumour (and does not have a non- solid tumour).

Reference to "treat" or "treating" a cancer as used herein defines the achievement of at least one positive therapeutic effect, such as for example, reduced number of cancer cells, reduced tumour size, reduced rate of cancer cell infiltration into peripheral organs, or reduced rate of tumour metastasis or tumour growth.

Positive therapeutic effects in cancer can be measured in a number of ways (e.g. Weber (2009) J Nucl Med 50, 1S-10S). By way of example, with respect to tumour growth inhibition, according to National Cancer Institute (NCI) standards, a T/C ≤ 42% is the minimum level of anti-tumour activity. A T/C < 10% is considered a high anti-tumour activity level, with T/C (%) = Median tumour volume of the treated/Median tumour volume of the control x 100. In some embodiments, the treatment achieved by a therapeutically effective amount is any of progression free survival (PFS), disease free survival (DFS) or overall survival (OS). PFS, also referred to as "Time to Tumour Progression" indicates the length of time during and after treatment that the cancer does not grow, and includes the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease. DFS refers to the length of time during and after treatment that the patient remains free of disease. OS refers to a prolongation in life expectancy as compared to naive or untreated individuals or patients.

Reference to "prevention" (or prophylaxis) as used herein refers to delaying or preventing the onset of the symptoms of the cancer. Prevention may be absolute (such that no disease occurs) or may be effective only in some individuals or for a limited amount of time.

In a preferred aspect of the invention the subject has an established tumour, that is the subject already has a tumour, e.g. that is classified as a solid tumour. As such, the invention as described herein can be used when the subject already has a tumour, such as a solid tumour. As such, the invention provides a therapeutic option that can be used to treat an existing tumour. In one aspect of the invention the subject has an existing solid tumour. The invention may be used as a prevention, or preferably as a treatment in subjects who already have a solid tumour. In one aspect the invention is not used as a preventative or prophylaxis.

In one aspect of the invention the method of treating or preventing cancer as described herein further comprises the step of identifying a subject who has cancer.

ANTIBODY

As used herein, the term "antibody" refers to both intact immunoglobulin molecules as well as fragments thereof that include the antigen-binding site, and includes polyclonal, monoclonal, genetically engineered and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanised antibodies, heteroconjugate and/or multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies), and antigen binding fragments of antibodies, including e.g. Fab', F(ab')₂, Fab, Fv, rlgG, polypeptide-Fc fusions, single chain variants (scFv fragments, VHHs, Trans-bodies®, Affibodies®). In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a detectable moiety, a therapeutic moiety, a catalytic moiety, or other chemical group providing improved stability or administration of the antibody, such as poly-ethylene glycol). "Antibody" may also refer to camelid antibodies (heavy-chain only antibodies) and antibody-like molecules such as anticalins (Skerra (2008) FEBS J 275, 2677-83).

In one aspect of the invention the antibody is monoclonal. The antibody may additionally or alternatively be humanised or human. In one aspect the antibody may be chimeric.

Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. Immunoglobulins may be from any class such as IgA, IgD, IgG, IgE or IgM. Immunoglobulins can be of any subclass such as lgG1 , lgG2, lgG3, or lgG4.

The Fc region of IgG antibodies interacts with several cellular Fey receptors (FcyR) to stimulate and regulate downstream effector mechanisms. There are five activating receptors, namely FcyR I (CD64), FcyRlla (CD32a), FcyRllc (CD32c), FcyRllla (CD16a) and FcyRlllb (CD16b), and one inhibitory receptor FcyRllb (CD32b). IgG subclasses vary in their ability to bind to FcyR and this differential binding determines their ability to elicit a range of functional responses.

In one aspect of the invention, the lgG2 antibody binds an activating Fc receptor with high affinity. Preferably the antibody binds FcyRlla with high affinity. In a particular embodiment, the antibody binds to the FcyR with a dissociation constant of less than about 10^"6 M, 10^"7 M, 10^"8 M, 10^"9 M or 10^"10 M.

In one aspect, the antibody binds an inhibitory receptor, FcyRllb, with low affinity. In one aspect, the antibody binds FcyRllb with a dissociation constant higher than about 10^"7 M, higher than about 10^"6 M or higher than about 10^"5 M. In a particular embodiment, the antibody binds FcyRllb with a dissociation constant higher than about 10^"7 M.

In one aspect the antibody has an activatory to inhibitory ratio (A/I) that is at least superior to 1 , that is the ratio of antibody binding to activatory Fc receptors to inhibitory Fc receptors is at least superior to 1.

The present invention relates in particular to the use of antibodies from the human lgG2 subclass, and preferably has ADCC or ADCP activity, as discussed herein. In a preferred embodiment the lgG2 antibody as described herein is targeted to, or in other words binds, an immune checkpoint molecule, preferably with high affinity. In a particularly preferred embodiment, the immune checkpoint molecule to which the lgG2 antibody is targeted to, or binds, is CTLA-4.

As used herein, "immune checkpoint" or "immune checkpoint molecule" refer to proteins or other molecules belonging to inhibitory or activatory pathways in the immune system, in particular for the modulation of T-cell responses. Under normal physiological conditions, immune checkpoints are crucial to regulating the breadth and potency of immunity assuring effective response and preventing autoimmunity, for example during a response to a pathogen. Cancer cells are able to alter the regulation of the expression of immune checkpoint proteins in order to avoid immune surveillance.

In one aspect, the immune checkpoint is any checkpoint molecule expressed at a higher level on Treg versus Teff. Examples of inhibitory immune checkpoint proteins include but are not limited to PD-1 , CTLA-4, BTLA, KIR, LAG3, TIGIT, CD155, B7H3, B7H4, VISTA and TIM3. Examples of activatory immune checkpoint proteins include but are not limited to GITR, OX40, 4-1 BB, ICOS, HVEM. Immune checkpoint molecules may also refer to proteins which bind to other immune checkpoint proteins which modulate the immune response in an inhibitory or activatory manner. Such proteins include but are not limited to PD-L1 , PD-L2, CD80, CD86, HVEM, GAL9, ICOS-Ligand, OX-40 Ligand, GITR-Ligand, 4-1 BB-Ligand.

Inhibitors of immune checkpoint protein, referring to any protein that can interfere with the signalling and/or protein-protein interactions mediated by an immune checkpoint protein, are known in the art.

Activators or agonists of immune checkpoint proteins, referring to any protein that can increase the signalling mediated by an immune checkpoint protein, are known in the art. In one embodiment of the present invention, the immune checkpoint molecule to which an antibody, preferably an lgG2 antibody, is targeted to is CTLA-4.

In another aspect, a preferred anti-CTLA-4 antibody is ipilimumab. In one aspect the antibody is tremelimumab.

Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at the amino terminus a variable domain (V_H) followed by a number of constant domains. Each light chain has a variable domain at the amino terminus (V|_) and a constant domain at the carboxy terminus. The variable regions are capable of interacting with a structurally complementary antigenic target and are characterized by differences in amino acid sequence from antibodies of different antigenic specificity. The variable regions of either H or L chains contain the amino acid sequences capable of specifically binding to antigenic targets. Within these sequences are smaller sequences dubbed "hypervariable" because of their extreme variability between antibodies of differing specificity. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDR" regions.

These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure. The CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains. The variable heavy and light chains of all antibodies each have 3 CDR regions, each non-contiguous with the others (termed L1 , L2, L3, H1 , H2, H3) for the respective light (L) and heavy (H) chains. The accepted CDR regions have been described previously (Kabat et al. (1977) J Biol Chem 252, 6609-6616).

The antibodies of the present invention may function through complement-dependent cytotoxicity (CDC) and/or antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cell-mediated phagocytosis (ADCP).

"Complement- dependent cytotoxicity" (CDC) refers to lysis of antigen-expressing cells by an antibody of the invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and thereby lead to lysis of the target cell. "Antibody-dependent cell-mediated phagocytosis" (ADCP) refers to a cell-mediated reaction in which phagocytes (such as macrophages) that express Fc receptors (FcRs) recognize bound antibody on a target cell and thereby lead to phagocytosis of the target cell. CDC, ADCC and ADCP can be measured using assays that are known and available in the art (Clynes et al. (1998) Proc Natl Acad Sci USA 95, 652-6). The constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell- dependent cytotoxicity and phagocytosis. Thus, as discussed herein, the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxi city/phagocytosis.

As discussed herein, in an embodiment of the invention, the use of an lgG2 antibody preferably leads to the depletion of Treg cells. For example, an antibody that elicits a strong CDC response and/or a strong ADCC and/or a strong ADCP response may be used. Methods to increase CDC, ADCC and/or ADCP are known in the art. For example, CDC response may be increased with mutations in the antibody that increase the affinity of C1q binding (Idusogie et al. (2001) J Immunol 166, 2571-5). ADCC may be increased by methods that eliminate the fucose moiety from the antibody glycan, such as by production of the antibody in a YB2/0 cell line, or though the introduction of specific mutations on the Fc portion of human lgG1 (e.g., S298A/E333A/K334A, S239D/I332E/A330L, G236A/S239D/A330L/I332E) (Lazar et al. (2006) Proc Natl Acad Sci USA 103, 2005-2010; Smith et al. (2012) Proc Natl Acad Sci USA 109, 6181-6). ADCP may also be increased by the introduction of specific mutations on the Fc portion of human lgG1 (Richards et al. (2008) Mol Cancer Ther 7, 2517-27).

In a preferred embodiment of the present invention the antibody is optimised to elicit an ADCC response, that is to say the ADCC response is enhanced, increased or improved relative to other lgG2 antibodies.

In a preferred embodiment of the present invention the antibody is optimised to elicit an ADCP response, that is to say the ADCP response is enhanced, increased or improved relative to other lgG2 antibodies.

Accordingly, in a preferred embodiment of the present invention, the antibody is optimised to engage CD32A. Such an antibody is capable of promoting ADCC/ADCP.

DEPLETION

As discussed herein, the present invention relates to depleting regulatory T cells (Tregs). Thus in one aspect, the lgG2 antibody of the present invention elicits an ADCC or ADCP response.

In accordance with another embodiment of the present invention, the lgG2 antibody is capable of depleting regulatory T cells (Tregs) in the solid tumour.

In one aspect said depletion is via ADCC. In another aspect, said depletion is via ADCP.

As such, the invention provides a method for depleting regulatory T cells in a tumour in a subject, comprising administering to said subject an lgG2 antibody targeted to an immune checkpoint molecule expressed by such regulatory T cells.

In one aspect the invention provides the use of an lgG2 antibody which is capable of binding an immune checkpoint molecule for depleting Tregs in a subject, for example in a solid tumour.

In a preferred embodiment Tregs are depleted in a solid tumour. By "depleted" it is meant that the number, ratio or percentage of Tregs is decreased relative to when an lgG2 antibdoy targeted to an immune checkpoint molecule is not administered.

In particular embodiments of the invention as described herein, over about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% of the tumour-infiltrating regulatory T cells are depleted. As used herein, "regulatory T cells" ("Treg") refer to a lineage of CD4+ T lymphocytes specialized in controlling anti-tumour immunity, autoimmunity, allergy and infection. Typically, they regulate the activities of T cell populations, but they can also influence certain innate immune system cell types. Tregs are usually identified by the expression of the biomarkers CD4, CD25 and Foxp3.

Naturally occurring Treg cells normally constitute about 5-10% of the peripheral CD4+ T lymphocytes. However, within a tumour microenvironment (i.e. tumour-infiltrating Treg cells), they can make up as much as 20-30% of the total CD4+ T lymphocyte population.

Activated human Treg cells may directly kill target cells such as effector T cells and APCs through perforin- or granzyme B-dependent pathways; cytotoxic T-lymphocyte-associated antigen 4 (CTLA4+) Treg cells induce indoleamine 2,3-dioxygenase (I DO) expression by APCs, and these in turn suppress T-cell activation by reducing tryptophan; Treg cells, may release interleukin-10 (IL-10) and transforming growth factor (ΤΘΡβ) in vivo, and thus directly inhibit T-cell activation and suppress APC function by inhibiting expression of MHC molecules, CD80, CD86 and IL-12. Treg cells can also suppress immunity by expressing high levels of CTLA4 which can bind to CD80 and CD86 on antigen presenting cells and prevent proper activation of effector T cells.

In a preferred embodiment of the present invention the ratio of effector T cells to regulatory T cells in a solid tumour is increased. In some embodiments, the ratio of effector T cells to regulatory T cells in a solid tumour is increased to over 5, 10, 15, 20, 40 or 80.

As used herein, the term "effector cell" refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and carry out specific immune functions. An effector cell can induce antibody-dependent cell-mediated cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes which express FcaR are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. An effector cell can also phagocytose a target antigen, target cell, or microorganism.

As discussed herein, antibodies according to the present invention may be optimised for ability to induce ADCC.

ADMINISTRATION

The antibody according to any aspect of the invention as described herein may be in the form of a pharmaceutical composition which additionally comprises a pharmaceutically acceptable carrier, diluent or excipient. These compositions include, for example, liquid, semi-solid and solid dosage formulations, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, or liposomes. In some embodiments, a preferred form may depend on the intended mode of administration and/or therapeutic application. Pharmaceutical compositions containing the antibody can be administered by any appropriate method known in the art, including, without limitation, oral, mucosal, by-inhalation, topical, buccal, nasal, rectal, or parenteral (e.g. intravenous, infusion, intratumoural, intranodal, subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal, or other kinds of administration involving physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue). Such a formulation may, for example, be in a form of an injectable or infusible solution that is suitable for intradermal, intratumoural or subcutaneous administration, or for intravenous infusion.

In some embodiments, the antibody can be prepared with carriers that protect it against rapid release and/or degradation, such as a controlled release formulation, such as implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used.

Those skilled in the art will appreciate, for example, that route of delivery (e.g., oral vs intravenous vs subcutaneous vs intratumoural, etc) may impact dose amount and/or required dose amount may impact route of delivery. For example, where particularly high concentrations of an agent within a particular site or location (e.g., within a tumour) are of interest, focused delivery (e.g., in this example, intratumoural delivery) may be desired and/or useful. Other factors to be considered when optimizing routes and/or dosing schedule for a given therapeutic regimen may include, for example, the particular cancer being treated (e.g., type, stage, location, etc), the clinical condition of a subject (e.g., age, overall health, etc.), the presence or absence of combination therapy, and other factors known to medical practitioners.

The pharmaceutical compositions typically should be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations as discussed herein. Sterile injectable formulations may be prepared using a non-toxic parenterally acceptable diluent or solvent. Each pharmaceutical composition for use in accordance with the present invention may include pharmaceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers are non-toxic to the subjects at the dosages and concentrations employed.

While an embodiment of the treatment method or compositions for use according to the present invention may not be effective in achieving a positive therapeutic effect in every subject, it should do so in a using pharmaceutical compositions and dosing regimens that are consistently with good medical practice and statistically significant number of subjects as determined by any statistical test known in the art such as the Student's t-test, the x²-test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), Jonckheere- Terpstra test and the Wlcoxon-test.

Where hereinbefore and subsequently a tumour, a tumour disease, a carcinoma or a cancer is mentioned, also metastasis in the original organ or tissue and/or in any other location are implied alternatively or in addition, whatever the location of the tumour and/or metastasis is.

ANTIBODIES TO GITR, ICOS and OX40 Treg depletion by ADCC or ADCP relies on higher expression of relevant target molecules on tumour-infiltrating Tregs relative to tumour-infiltrating CD4 and CD8 effector T cells.

The present inventors found that expression of GITR, ICOS and OX40 are consistently expressed by and at the highest levels on Tregs, relative to other tumour-infiltrating T lymphocyte subsets.

As such, in one aspect an anti-GITR, ICOS or OX40 antibody is provided for use in the selective depletion of Tregs in a solid tumour according to any of the methods as described herein, such as with an lgG2 antibody. An anti-41 BB antibody may also be used according to the present invention.

In another aspect the present invention relates to a bispecific antibody.

As used herein, "bispecific antibody" refers to an antibody having the capacity to bind to two distinct epitopes either on a single antigen or polypeptide, or on two different antigens or polypeptides. Bispecific antibodies of the present invention as discussed herein can be produced via biological methods, such as somatic hybridization; or genetic methods, such as the expression of a non-native DNA sequence encoding the desired antibody structure in an organism; chemical methods (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise to one or more molecular entities such as another antibody or antibody fragment); or a combination thereof.

Accordingly, the present invention provides a bispecific antibody comprising:

(a) a first antigen binding moiety that binds to CD32a; and

(b) a second antigen binding moiety that binds to an immune checkpoint protein or a tumour- associated antigen.

In one aspect, the immune checkpoint is any checkpoint molecule expressed at a higher level on Treg versus Teff, as described above.

Examples of inhibitory immune checkpoint proteins include but are not limited to PD-1 , CTLA-4, BTLA, KIR, LAG 3, TIGIT, CD155, CD47, B7H3, B7H4, VISTA and TIM3. Examples of activatory immune checkpoint proteins include but are not limited to GITR, OX40, 4-1 BB, ICOS, HVEM. Immune checkpoint molecules may also refer to proteins which bind to other immune checkpoint proteins which modulate the immune response in an inhibitory or activatory manner. Such proteins include but are not limited to PD-L1 , PD-L2, CD80, CD86, HVEM, GAL9, ICOS-Ligand, OX-40 Ligand, GITR-Ligand, 4-1 BB-Ligand.

In another aspect, the immune checkpoint is any checkpoint molecule that can also be expressed or upregulated on tumour cells.

Examples of inhibitory immune checkpoint proteins expressed or upregulated on tumour cells include but are not limited to PD-L1 , PD-L2 CD155, CD47, B7H3, B7H4, Hvem, Galectins and VISTA.

As used herein, "tumour-associated antigen" refers to antigens expressed on tumour cells, making them distinguishable from non-cancer cells adjacent to them, and include, without limitation, CD20, CD38, EGFR, EGFRV3, CEA and HER2. Various review articles have been published that describe relevant tumour-associated antigens and the corresponding therapeutically useful antitumor antibody agents (see, for example, Sliwkowski & Mellman (2013) Science 341 , 192-8). Such antigens and corresponding antibodies include, without limitation CD22 (Blinatumomab), CD20 (Rituximab, Tositumomab), CD56 (Lorvotuzumab), CD66e/CEA (Labetuzumab), CD221/IGF1 R (MK-0646), CD326/Epcam (Edrecolomab), CD340/HER2 (Trastuzumab, Pertuzumab), and EGFR (Cetuximab, Panitumumab).

ANTIBODIES TO PD-1

The protein Programmed Death 1 (PD-1) is an inhibitory member of the CD28 family of receptors, that also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Okazaki et al (2002) Curr. Opin. Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 170:711-8).

Blockade of the interaction between PD1 and one of its ligands, PD-L1 , has been shown to enhance tumor-specific CD8+ T-cell immunity and may therefore be helpful in clearance of tumor cells by the immune system. Experimental evidence presented herein suggests that in the presence of an FcyR polymorphism capable of binding an anti-PD-1 mAb, response rates to the treatment appeared worse, suggesting that the polymorphism was promoting depletion of effector T cells expressing PD-1. As such, in another aspect, the present invention relates to an Fc-silent antibody targeted to PD-1.

In another aspect, the present invention relates to an Fc-silent antibody targeted to PD-1 for use in the treatment of cancer.

Fc-silent antibodies are antibodies comprising a modified Fc region comprising mutations which silence the ADCC/ADCP activity of the Fc region (also known as Fc silent mutants). For example, the N297A mutation in the constant region of lgG1 is known to silence the Fc region, while a deglycosylated lgG2 is also Fc silent.

One skilled in the art would be aware of how suitable antibodies could be manufactured. For example as described in Tao M. J Immunol 1989. 143:2595-2601 and Lo M. et al. Journal of Biological Chemistry 2017. 292(9) :3900-3908. The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. EXAMPLES

MATERIALS AND METHODS Mice

C57BL/6 and BALB/c mice were purchased from Charles River Laboratories. FcyRa null, human FcyR transgenic of C57BL/6 background (Smith et al. (2012) Proc Natl Acad Sci USA 109, 6181-6) mice were a kind gift from J. V. Ravetch (The Rockefeller University, New York, USA) and housed and bred in Charles River Laboratories, U.K.. Experiments were typically performed with 6-10 week old females. All animal studies were performed under University College London and UK Home Office ethical approval and regulations.

Cell lines and tissue culture

MCA205 cells were cultured in Dulbecco's modified Eagle medium (DMEM, Sigma) supplemented with 10% fetal calf serum (FCS, Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine (all from Gibco). MC38, CT26, B16 and SupT1 cells were cultured in Roswell Park Memorial Institute (RPMI) media supplemented as above. A cell line with stable membrane-bound expression of CTLA-4 was generated by transduction of Sup-T1 cells with SFG plasmids coding for human CTLA-4 with an IRES-linked truncated human CD34 (dCD34) marker gene. CTLA-4 was generated by Phusion PCR and subcloned into SFG.I2.eBFP2 to permit surface expression of the receptor. For generation of human macrophages, monocytes were isolated from healthy donor PBMCs using CD14⁺ microbeads (Miltenyi Biotec) and cultured for 7 days in RPMI supplemented with 10% FCS and recombinant human macrophage colony-stimulating factor (M-CSF) at 40 ng/mL (CELL guidance systems).

Antibody production

Antibody production was outsourced to Evitria AG (Switzerland). The sequence of the variable regions of the heavy and light chains of anti-mouse CTLA-4, clone 4F10, was kindly provided by J. A. Bluestone and used to generate chimeric antibodies with the constant regions of human lgG1 and lgG2 heavy chains and κ light chain, as well as the mutated lgG1 variants N297A and S239D/A330L/I332E (SDALIE) (Lazar et al. (2006) Proc Natl Acad Sci USA 103, 4005-10). In vitro ADCC assay

CTLA-4-expressing SupT1 cells were labelled with 10μΜ carboxyfluorescein succinimidyl ester (CellTrace CFSE Cell Proliferation Kit, Life Technologies) and co-cultured overnight with human macrophages at the indicated ratios in the presence of the indicated mAbs (50 μg/mL). The absolute number of CFSE-labelled cells in each condition was then quantified by flow cytometry using a defined number of fluorescent beads (Cell Sorting Set-up Beads for UV Lasers, ThermoFisher) as reference. The percentage of killing was determined as: 100-(number CFSE⁺ targets treated/number CFSE⁺ targets untreated).

Tumor experiments

Mice were injected subcutaneously in the flank with 5 x 10⁵ MCA205, MC38 or CT26 cells, or, 5 x 10⁴ B16 cells re-suspended in 100 μΙ_ of phosphate buffer solution (PBS).

Therapeutic antibodies were injected intra-peritoneally at the time points and doses detailed in the figure legends. Tumors were measured twice weekly and volumes calculated as the product of three orthogonal diameters. Mice were euthanized when any diameter reached 150 mm. For functional experiments, tissues were harvested and processed as described previously (Simpson et al. (2013) J Exp Med 210, 1695-710).

Flow cytometry

Acquisition was performed with a BD LSR II Fortessa (BD Biosciences). The following antibodies and fluorescent labels were used to stain mouse cells for flow cytometry: CD4- V500 (RM4-5) (BD Biosciences); viability dye eFluor780, CD3-PE.Cy7 (145-2C11), CD5- PerCP.Cy5.5 (53-7.3), FoxP3-FITC (FJK-16s), Foxp3 (FJL-16s), l-Ab-eFluor450 (AF6- 210.1), NK1.1-AlexaFluor700 (PK136), NK1.1-BrilliantViolet650 (PK136), Ki67-eFluor450 (SolA15), IFNY-AlexaFluor488 (XMG1.2), GITR-eFluor450 (DTA-1), 4-1 BB-biotin (17B-5), PD-1-eFluor450 (RMP1-30), TIM-3-PE (8B.2C12) (eBioscience); CD4-BrilliantViolet785 (RM4-5), CD8-BrilliantViolet650 (53-6.7), CD19-BrilliantViolet605 (6D5), CD11 b- BrilliantViolet71 1 (M1/70), CD11 c-PE (N418), Ly6G-PE.Cy7 (1A8), CTLA-4-BrilliantViolet605 (UC10-4B9), ICOS-PE.Cy7 (C398.4A), OX40-biotin (0X86), streptavidin-BrilliantViolet605, streptavidin-BrilliantViolet705 (BioLegend). The following antibodies were used to stain for human FcyRs: CD64-AF700 (10.1), CD16a/b-V500 (3G8) (BD Biosciences), CD32a-FITC (IV.3) (StemCell) and CD32b-APC (6-G11) (Biolnvent). The following antibodies were used to stain human cells: CD1 1 c-V450 (3.9), CD45-Q655 (HI30), CD56-BV711 (HCD56), CD19- BV785 (HIB19), CD1 1 b-PerCP-Cy5.5 (ICRF44), CD15-PE (HI98), PD-1-Q605 (EH12.2H7), CD25-BV711 (BC96), CD3-BV785 (OKT3), BTLA-PerCP-Cy5.5 (MIH26), CTLA-4-APC (L3D10), Streptavidin-Q650, OX40-PE-Cy7 (ACT35), ICOS-APC (C398.4A), 4-1 BB-PE (4B4-1) (Biolegend); CD16-V500 (3G8), Granzyme B-V450 (GB11), CD8-V510 (SK1), TIM- 3-Q650 (7D3), CD14-PE-Cy7 (M5E2) (BD Biosciences); CD3-e605 (OKT3), HLA-DR-PE (L243), TIGIT-FITC (MBSA43), FoxP3-PE (PCM 01), LAG-3-PE-Cy7 (3DS223H), CD4- AF700 (OKT4), Fixable viability dye-e780 (eBioscience) and GITR-Biotinylated (DT5D3) (Miltenyi).

Intranuclear staining of FoxP3 and Ki67 was performed using the FoxP3 Transcription Factor Staining Buffer Set (eBioscience). For intracellular staining of cytokines, cells were re- stimulated with phorbol 12-myristate 13-acetate (PMA, 20 ng/mL) and ionomycin (500 ng/mL) (Sigma Aldrich) for 4 hours at 37°C in the presence of GolgiPlug (BD Biosciences) and stained following the manufacturer's protocol for the Cytofix/Cytoperm buffer set (BD Biosciences). For quantification of absolute number of cells, a defined number of fluorescent beads (Cell Sorting Set-up Beads for UV Lasers, ThermoFisher) was added to each sample before acquisition and used as a counting reference.

Genomic analyses

Variant calling from previously published cohorts and identification of putative neoantigens was performed as described previously (McGranahan et al. (2016) Science 351 , 1463-9).

Germline variant determination

SAMtools mpileup was used to find non-reference positions in tumor and germline samples. VarScan2 somatic used the output to identify somatic and germline variants. Variants were annotated using Annovarl 6.

Human study oversight

Presented human data derives from three translational studies, each approved by local institutional review board and Research Ethics Committee (Melanoma - REC no. 1 1/LO/0003, NSCLC - REC no.13/LO/1546, RCC - REC no. 1 1/LO/1996). All were conducted in accordance with the provisions of the Declaration of Helsinki and with Good Clinical Practice guidelines as defined by the International Conference on Harmonization. All patients (or their legal representatives) provided written informed consent before enrolment. Processing of human tissue

Tumor samples were digested with Liberase TL (0.3 mg/mL, Roche) and DNAse I (0.2 mg/mL, Roche) at 37°C for 30 minutes, homogenized using gentleMACS (Miltenyi Biotech) and filtered through a 0.7 μηι cell mesh. Leukocytes were enriched by gradient centrifugation with Ficoll-paque (GE Healthcare). Isolated live cells were frozen at -80°C and stored in liquid nitrogen until analysis. Data analysis

Flow cytometry data analysis was performed in FlowJo version 10.0.8 (Tree Star Inc.). Statistical analyses were performed in Prism 6 (GraphPad Software, Inc.); p values were calculated using Kruskall-Wallis analysis of variance and Dunn's post-hoc test, unless otherwise indicated in the figure legends (ns = p > 0.05; * = p < 0.05; ** = p < 0.01 ; *** = p < 0.001 ; **** = p < 0.0001). Analysis of Kaplan-Meier survival curves was performed with use of the log-rank test.

EXAMPLE 1 - CTLA-4 is expressed at highest density on tumor-infiltrating Treg cells in mouse and man

It was sought to comprehensively evaluate the relative expression of CTLA-4 on circulating and tumor-infiltrating CD4⁺FoxP3⁺, CD4⁺FoxP3^" and CD8⁺ T lymphocytes across multiple murine models of transplantable tumor cell lines of variable immunogenicity including B16 melanoma, CT26 colorectal carcinoma, MCA205 sarcoma, MC38 colonic adenocarcinoma (Fig. 1A-C) and human solid tumor subtypes including advanced melanoma, early-stage non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) (Fig. 1 D-F). In mice, CTLA-4 expression was evaluated in tumor-infiltrating lymphocytes (TIL), draining lymph nodes (LN) and peripheral blood mononuclear cells (PBMC) by flow cytometry 10 days after tumor challenge. Human tumor digests and PBMCs were isolated from both resection specimens and blood sampled at matched timepoints and were evaluated similarly (details shown in the Table 1 below).

TABLE 1 - Demographics and clinical characteristics of patients with advanced melanoma (MM)

(SC = subcutaneous; LN = lymph node)

MM3 76 F Cutaneous IV- Small BRAF WT; Nil

M1 c bowel NRAS

mutant

MM4 76 M Cutaneous lllc LN BRAF Nil

mutant

MM5 74 F Cutaneous IV- LN BRAF WT Nil

M1 a

MM6 61 M Cutaneous IV- LN BRAF WT Nil Ipilimumab

M1 a

MM7 42 M Cutaneous IMC LN BRAF Nil

mutant

MM8 49 M Cutaneous IV- LN BRAF WT; Nil Paclitaxel+

M1 c NRAS Trametinib mutant Ipilimumab

Pembrolizumab

Across all studied mouse models, CTLA-4 expression was higher in the tumor and largely restricted to CD4⁺FoxP3⁺ Treg cells (mean expression 68.3%), relative to CD4⁺FoxP3^" effector (CD4⁺eff) T cells (10.2%, p<0.0001) and CD8⁺ T cells (5.4%, p<0.0001) (Figure 1A- B). Where CTLA-4 expression was observed on TIL subsets other than Treg cells, this was at significantly lower levels based on mean fluorescent intensity (MFI) of intra-tumoral Treg cells (MFI 2271.8) relative to CD4⁺eff cells (MFI 498.6, pO.0001) and CD8⁺ T cells (MFI 701.0, pO.0001 , Fig. 1C).

In human tumors, CTLA-4 expression was also higher in TILs relative to PBMCs and its expression profile amongst T cell subsets appeared similar to mouse models (mean expression in Treg cells 82.1 %, relative to CD4⁺eff cells 26.5%, p<0.0001 and CD8⁺ T cells 17.4%, p<0.0001 , Fig. 1 D-E). Although CTLA-4 expression was also observed in a proportion of human CD4⁺eff and CD8⁺ TILs, this was again at significantly lower levels based on MFI (mean MFI Treg cells 1349.6 relative to CD4⁺eff cells 385.9, p<0.0001 , and CD8⁺ T cells 239.4, p<0.0001 , Fig. 1 F). CTLA-4 was consistently expressed at low levels on CD8⁺ T cells within tumors, with a MFI lower than both tumor-infiltrating and circulating Treg cells in mouse and man (p<0.0001) (Fig. 1 C and F). EXAMPLE 2 - Transgenic mice bearing human FcyRs display similar expression profiles to human tumors

It was sought to determine the utility of a transgenic mouse model described to recapitulate human FcyR (hFcyR) structural and functional diversity (Smith et al., 2012), comparing FcyR expression profiles within this model to human melanoma.

Analysis of cell subsets in draining LNs (Fig. 2A), spleens (Fig. 7A) and blood (data not shown) 10 days after inoculating MCA205 tumors in hFcyR transgenic mice (hFcyR mice) demonstrated an expression pattern comparable to previous descriptions (Smith et al., 2012) with activatory FcyRI (CD64), lla (CD32a) and llla/b (CD16a/b) expressed on monocytic and granulocytic myeloid cells, CD16a additionally detected in a fraction of natural killer (NK) cells and the inhibitory FcyRllb (CD32b) present on B cells and myeloid cell subpopulations (Fig. 2A, 2C and 7A-B). Although this expression pattern was maintained on tumor- infiltrating leukocytes, the expression levels of all receptors appeared higher in the tumor compared to the LNs or the spleen reflected in a higher MFI, particularly on myeloid cells, which were the most abundant leukocyte subpopulation present in tumors (Fig. 12). This pattern was consistent across all three studied tumor models, although the percentage of expression of CD32a and CD16 appeared lower in innate effector cells in B16 tumors relative to the more immunogenic MC38 and MCA205 models (Fig. 7A). Of relevance, the absolute number of tumor-infiltrating leukocytes varied between models, with B16 tumors bearing the lowest levels of T cells and innate effector cells relative to MCA205 and MC38 (Fig. 12). Analysis of human melanoma metastases derived from varying anatomical sites including subcutaneous, LN and colonic lesions (Table 2 below) demonstrated consistent FcyR expression profiles on individual cell subsets, but with important differences between tumor and blood (Fig. 2B and 2E). FcyR expression on lymphocytes in blood and tumor was confined to CD19⁺CD3^" B cells, which expressed the inhibitory receptor CD32b. Activatory FcyR expression was observed on tumor-infiltrating CD1 1c⁺HLA-DR⁺CD14^" dendritic cells (DC), CD11 b⁺CD15⁺CD14^" granulocytes, CD56⁺CD3^" NK cells and CD1 1 b⁺CD14⁺HLA-DR⁺ macrophages (ΜΦ'ε). In contrast to tumor-infiltrating ΜΦ'ε and granulocytes, NK cells and DCs accounted for a small fraction of CD45⁺ tumor-infiltrating cell subsets (mean % of NK cells=1.64% and DCs=0.95% of total CD45⁺ cells, Fig. 7D). Moreover, where NK cells were identified, expression of CD16a appeared consistently lower on tumor-infiltrating subsets (mean % CD16⁺ in tumor=41.6%, blood=81.1 %, p<0.05). Circulating monocytes and tumor- associated ΜΦ'ε expressed all three activatory FcyRs (CD64, CD32a and CD16a) as well as the inhibitory receptor CD32b. Although FcyR distribution remained similar between circulating monocytes and tumor-infiltrating ΜΦ'ε, all FcyRs, particularly CD32b, were consistently expressed at higher levels on tumor-infiltrating ΜΦ'ε (Fig. 2C-D). In contrast, FcyR expression by circulating and tumor-infiltrating granulocytes appeared similar, with constitutive expression of the activatory receptors CD32a and CD16b (Fig. 2C). Overall, amongst all tumor-infiltrating leukocyte subsets, CD32a was the most abundantly expressed FcyR in both human and murine tumors (Fig. 2E and Fig. 7B).

TABLE 2 - Demographics and clinical characteristics of patients with advanced melanoma (MM); employed for fresh myeloid analyses. M, male; F, female; LN, lymph node; SC, subcutaneous; WT, wild type

Importantly, intra-tumoral FcyR expression profiles were comparable between hFcyR mice and human melanoma, with the exception of CD32b, which in the mouse model was highly expressed on myeloid cells present in LNs and spleen and further upregulated in tumors. In humans, expression on circulating cells was largely confined to B cells, but upregulated on myeloid cells in tumors. This difference could result in a less favorable A: I FcyR ratio in secondary lymphoid organs in the mouse model relative to human blood and tumors.

EXAMPLE 3 - Human lgG1 and lgG2 anti-CTLA-4 antibodies efficiently deplete intra- tumoral Treg cells in vivo

Based on the comparable expression profile of CTLA-4 on T lymphocytes and FcyRs on tumor-infiltrating innate effector cell subsets in humans and hFcyR mice, it was next evaluated whether anti-CTLA-4 antibodies of a human isotype promoted depletion of intra- tumoral Treg cells in vivo in a similar manner to that mediated by murine FcyRs (Selby et al., 2013; Simpson et al., 2013). Chimeric anti-murine CTLA-4 (mCTLA-4) antibodies were constructed (based on clone 4F10) with the human IgG variants employed in ipilimumab (lgG1) and tremelimumab (lgG2) and compared to mutated lgG1 isotypes with either enhanced binding affinity to activatory CD16a (IgG l sDALiE) or no binding to hFcyRs (lgG1 _N297A) (Fig. 3A). Their capacity to deplete CTLA-4-expressing target cells in vitro was assessed in the presence of monocyte-derived human macrophages at varying effector to target (E:T) cell ratios (Fig. 3B, Fig. 8, Fig. 13A and 13B). As predicted, when using monocyte-derived human macrophages and based on affinity for FcyRs expressed on these cells, the lgG1 and lgG2 mAbs demonstrated superior ADCC activity relative to lgG1 _N297A- Furthermore, the lgG1 SDALIE mAb, which has an optimized A:l hFcyR-binding ratio, promoted enhanced ADCC activity relative to all evaluated isoforms at E:T ratios of 5: 1 and above.

It was next sought to determine the impact of chimeric human anti-mCTLA-4 IgG variants in vivo in hFcyR mice. Mice were treated with human anti-mCTLA-4 on days 7 and 9 after inoculation with MCA205 tumors and the frequency of T cell sub-populations analyzed on day 11 in tumors, draining LNs and blood (Fig. 3C). Consistent with the in vitro data (Fig. 3B), there was a reduction in the proportion of tumor-infiltrating Treg cells in mice treated with the lgG1 mAb (mean percentage of Treg cells of total CD4⁺ T cells 24%) compared to those treated with the lgG1 _N297A variant (37%) or to control mice (44%, p<0.001). The depleting activity of the I gG1 SDALIE isotype appeared superior to the wild-type lgG1 mAb (mean percentage of Treg cells of total CD4⁺ T cells 17% vs. 24%, respectively), but this did not meet statistical significance. The lgG2 isotype is often described as a poor mediator of ADCC since it only binds to activatory CD32a (Schneider-Merck et al., 2010). The lgG2 anti-mCTLA-4 mAb, however, efficiently depleted tumor-infiltrating Treg cells in vivo (13% of total CD4⁺ T cells), with comparable activity to that observed in mice treated with the lgG1 and lgG1 sDALiE isotype variants.

As previously described in wild-type mice (Simpson et al., 2013), the depleting activity of all human IgG antibody variants in this new model was restricted to the tumor microenvironment, with no impact on Treg cells in LNs or blood in hFcyR mice (Fig. 3C). As a result, anti-CTLA-4 mAb of human lgG1 , lgG1_SDAUE and lgG2 isotypes led to an increase in the intra-tumoral ratio of CD8⁺ to Treg cells (Fig. 3D). This was only observed within the tumor microenvironment, demonstrating that in the context of human FcyR-lgG interactions in vivo, depletion of tumor-infiltrating Treg cells is a major contributor to the shift in this ratio, which has previously been associated with therapeutic responses in mouse and man (Hodi et al., 2008; Quezada et al., 2006). Treg cell depletion also correlated with a higher proliferation of CD4⁺eff and CD8⁺ T cells independently of the isotype, although only the I gG1 SDALiE mAb induced a significantly higher production of interferon-γ (IFNv) in CD4⁺eff cells (Fig. 3E-F). EXAMPLE 4 - The extent of intra-tumoral Treg depletion determines the anti-tumor activity of human lgG1 and lgG2 anti-CTLA-4 antibodies

In order to determine the impact of intra-tumoral Treg cell depletion on anti-tumor activity and survival, hFcyR mice were challenged with subcutaneous MCA205 tumors on day 0 and subsequently treated with 50 μg of chimeric anti-mCTLA-4 mAb IgG variants on days 6, 9 and 12 (Fig. 4A).

Tumor growth was equivalent in mice left untreated or in those treated with the Fc-silent lgG1 _N297A anti-mCTLA-4 mAb, demonstrating that CTLA-4 blockade alone is insufficient to promote tumor rejection in the context of human FcyR-lgG interactions. In contrast, the majority of mice treated with either lgG1 or lgG2 anti-CTLA-4 mAb rejected tumors completely (66.67% and 80%, respectively). lgG1_SDAUE anti-CTLA-4 mAb, with enhanced affinity for activating FcyRs, resulted in eradication of established tumors in all treated mice, although there was no statistical significance compared to the lgG1 and lgG2 mAbs (Fig. 4B- C). Importantly, responses appeared durable, with responding mice from all treatment groups alive for more than 80 days (Fig. 4C). These data suggest that in a mouse system that models human FcYR-lgG interactions in vivo, anti-CTLA-4 mAbs with enhanced capacity to deplete Treg cells have a favourable impact tumor response and survival.

Similar responses were observed amongst mice bearing MC38 tumors, where the therapeutic effect, although lower than in MCA205 tumors despite higher doses of mAbs, was only observed in the groups treated with depleting isotypes. Although the proportion of complete responses was higher in the lgG1_SDAUE group (75.0%) compared to the lgG1 and lgG2 treatments (66.67% and 62.5%, respectively), these differences were not statistically significant. In contrast, and correlating with the observed paucity of both T and innate effector cell infiltration (Fig. 12), anti-CTLA-4 mAbs lacked efficacy against B16 tumors despite the use of a higher dose of antibody and regardless of antibody isotype (Fig. 15).

EXAMPLE 5 - Human FcyR polymorphisms impact upon response to ipilimumab in patients with advanced melanoma

We sought to determine the impact of the CD16a-V158F and CD32a-H131 R single nucleotide polymorphisms (SNPs), identified through sequencing of germline DNA, on response to ipilimumab in two separate cohorts of patients with advanced melanoma (Allen et al., 2015; Snyder et al., 2014). Overall survival analysis of the Snyder et al. dataset showed that in patients with low mutational burden (Fig. 5A) or low predicted neoantigen burden (Fig. 5B), neither CD32a-H131 R nor CD16a-V158F SNPs impacted on outcome. Amongst patients with high mutational or predicted neoantigen burden, the CD32a-H131 R SNP also had no impact on outcome. However, patients with the CD16a-V158F SNP and high mutational (p=0.028, Fig. 5A, right panel) or high predicted neoantigen burden (p=0.014, Fig. 5B, right panel) derived in improved overall survival after treatment with ipilimumab. Further, combination of these two metrics appeared to better identify long-term responders than considering mutational or predicted neoantigen burden alone. Although the same trend was observed in the survival analysis of the van Allen et al. dataset, the differences were not statistically significant in this dataset (p=0.87) or in the meta-analysis of both datasets (p_meta=0.066, data not shown). Of relevance, the latter cohort is known to include patients more heavily pre-treated with chemotherapy, which might introduce higher variability in the overall survival results.

Analysis of the clinical response rates showed that amongst tumors with low indel burden (≤median), the CD16-V158F polymorphism was not observed to impact upon response. However, amongst those with high indel burden (>median), presence of the CD16-V158F SNP was associated with higher rates of response in both van Allen et al. and Snyder et al. datasets (Fig. 1 1 , left upper and lower panel). Meta-analysis of both datasets demonstrated significantly higher response rates in those with high indel burden and the CD16-V158F SNP, as compared to all other patients (p=0.016). Similar findings were observed when considering nsSNV neontigens and the presence or absence of the CD16-V158F (Fig. 11 , right upper and lower panel, p=0.043). Once again, meta-analysis of both datasets demonstrated significantly higher response rates amongst those with high neoantigen burden (>median) and the CD16- V158F SNP. Such observations were not common to the CD32a-H131 R polymorphism, which is associated with greater affinity for lgG2 rather than lgG1 (Fig. 5, left panels and Fig. 16A) (Parren et al., 1992; Salmon et al., 1992).

The described observations in patients with the CD16-V158F SNP could also relate to enhancement of other immunological processes mediated by FcyRexpressing cell subsets including antigen presentation. However, analysis of a cohort of patients with advanced melanoma treated with pembrolizumab or nivolumab (Hugo et al., 2016), both lgG4 mAbs directed against PD-1 with low predicted binding affinity to FcyRs, showed no association between the presence of the CD16-V158F SNP and improvement in response rates in patients with high indel burden (p=1.0, Fig. 16B). Indeed, response rates appeared lower in this setting. Intriguingly, the CD16-V158F allele is capable of binding to lgG4, raising the possibility that depletion of PD-1 ^h'^9h effector T cells via lgG4-mediated ADCC might underlie inferior response rates in those with high indel burden and CD16-V158F SNP.

EXAMPLE 6 - Immune checkpoint mapping of tumor-infiltrating T lymphocytes in mice and man informs the development of dual activity immune modulatory antibodies Multiple co-stimulatory and co-inhibitory molecules expressed on T cells are potential targets for antibodies that are in development to treat cancer. Here we demonstrated the therapeutic relevance of target molecule distribution and density as determinants of the response to anti- human CTLA-4 mAb in a mouse model of cancer and correlated these findings to the clinical setting, where we also demonstrated preferential expression of CTLA-4 on tumor-infiltrating Treg cells and the impact of FcyR polymorphisms as a co-determinant of the activity of anti- CTLA-4 in patients with melanoma. In order to inform the development of antibodies with dual (immune regulatory and Treg cell depleting) activity targeting other immune modulatory molecules we determined the expression of an extended panel of immune checkpoint molecules of the B7 and tumor necrosis factor receptor (TNFR) superfamilies on TIL subsets in mouse and man (Fig. 6 and 9). Flow cytometry analysis of single cell suspensions generated from murine (subcutaneous B16, CT26, MCA205 and MC38) and human (melanoma, NSCLC and RCC, Table 2) tumors demonstrated significant heterogeneity in expression profiles between different tumor subtypes, particularly in molecules typically described on effector T cells, including 4-1 BB, PD-1 and TIM-3 (Fig. 6A). The percentage of cells expressing these molecules appeared higher amongst CD8⁺ T cells in the more immunogenic MCA205, MC38 and CT26 mouse tumors relative to the poorly immunogenic mouse B16 melanoma and also higher in human melanoma relative to human NSCLC and RCC (Fig. 6A), potentially related to the immunogenic burden of somatic mutations typically associated with these tumor subtypes (Alexandrov et al., 2013).

Despite this, a number of potentially exploitable patterns were observed. Similar to CTLA-4, the co-stimulatory receptors GITR, ICOS and OX40 were consistently expressed on tumor- infiltrating Treg cells in mouse and human tumors. Although expression of these molecules was also observed on CD4⁺FoxP3^" and CD8⁺ T cell subsets, the level of expression, based on MFI, was significantly lower than on the Treg cell compartment (Fig. 6B). This is in contrast to PD-1 , which was expressed by all studied T cell subsets, but at highest MFI on CD8⁺ T cells, and TIM-3, which was also expressed on CD8⁺ T cells. Thus GITR and OX40 appear to be attractive targets in all three human tumor subtypes for dual activity antibodies with capacity for ADCC of intra-tumoral Treg cells.

EXAMPLE 7 - Mechanism of Action of lgG2 mediated cell killing

This experiment studies the fraction crystallisable (Fc)-mediated effector functions of anti- CTLA-4 antibodies by comparing different antibody isotype variants— including clinically relevant isotypes— on the killing of CTLA-4-expressing targets.

The results are shown in Figure 10. It was observed that antibodies of either lgG1 or lgG2 isotypes induced killing of target cells by antibody-dependent cell-mediated cytotoxicity (ADCC). This effect relies on the binding of the Fc to FcyRs expressed on macrophages, since a mutated variant (N297A) that binds to CTLA-4 but not to FcyRs does not mediate killing. lgG1 is a well-characterised ADCC inducer because it has a high binding affinity to activating receptors (FcyRI, FcyRlla, FcyRIII) relative to inhibitory receptors (FcgRllb), i.e. it has a high A:l ratio. lgG2 has classically been regarded as a poor ADCC inducer because it only binds to a single activating FcyR - FcyRlla. However, because of its poor binding to FcgRllb, it also displays a high A:l ratio that may explain its ability to mediate ADCC. This experiment shows that blockage of FcYRIIa (CD32a) binding (using an anti-FcYRIIa antibody) abolishes the ability of lgG2 to induce killing. Instead, blockade of FcYRIIa reduces but not completely eliminate lgG1- mediated ADCC, since this isotype binds to other activating FcyRs (FcyRI and FCYRI I I) This is further supported by the use of a de-glycosylated form of lgG2— which does not bind to FCYRS— which did not mediate ADCC either.

EXAMPLE 8 - Mutational burden and CPI response rates We analysed anti-CTLA4 response rates across two human melanoma cohorts (Snyder et al, 2015; Van Allen et al, 2015), splitting patients into four groups based on low/high mutational load and presence/absence of the CD16a-V158F FCYR polymorphism. Mutational load was defined by two different measures; the first based on the burden of insertion-deletion (Indel Load) mutations induced by DNA frameshifts, and the second defined by the burden of non synonymous Single Nucleotide Variants (nsSNVs) in each patient. Indel mutational load and nsSNV mutational load were calculated following established protocols (Turajlic et al. (2017); McGranahan et al. (2016)). Mutational load above the median of the cohort was considered as 'Hi[gh] mutational load', and mutational load bellow the median of the cohort was considered as 'Low mutational load'.

The results are presented in Figure 1 1.

Highest response rates were consistently observed in patients with both a high mutational load and CD16a-V158F FCYR polymorphism (P<0.05, Figure 1 1). This finding was consistent across both melanoma cohorts and also across two different definitions of mutational load.

The data shows that the activity of an anti CTLA-4 antibody is affected by its ability to interact with activatory Fc Receptors, likely due to its ability to deplete tumour infiltrating Treg cells. This supports a stratification of patients likely to respond to anti-CTLA-4 antibodies based on high mutational burden and presence of a FCYR SNP.

EXAMPLE 9 - Depletion by human lgG2 is via CD32a engagement Owing to the abundance of CD32a in mouse and human tumors, the main receptor to which human lgG2 binds, we also generated a chimeric anti-mCTLA mAb with lgG2, the isotype deployed in tremelimumab. This mAb (along with the lgG1 mCTLA-4 antibody from Example 3) was compared to mutated lgG1 isotypes with either enhanced binding affinity to activatory CD16a (lgG1 SDALIE) (Lazar et al., 2006) or no binding to hFcyRs (lgG1 _N297A)- Consistent with prior publications (Bruhns et al., 2009), surface plasmon resonance (SPR) analysis of the antibodies generated demonstrated binding of lgG1 and lgG1 SDALIE to all four subtypes of hFcyRs, with a modest increase in the binding affinity of cross-linked lgG1 SDALIE relative to wild-type lgG1. lgG2 showed low binding affinity only to activatory CD32a and no binding to inhibitory CD32b, while the mutant lgG1 N297A showed no binding to any of the low affinity hFcyRs (Fig. 13A, 3A). We first assessed their capacity to deplete CTLA-4-expressing target cells in vitro in the presence of monocyte-derived human macrophages at varying effector to target (E:T) cell ratios (Fig. 13B). As predicted, based on the affinity for FcyRs expressed on monocyte-derived human macrophages (Fig. 8), which mirrored the human melanoma, the lgG1 and lgG2 mAbs demonstrated superior ADCC activity relative to lgG1 _N297A- Furthermore, the lgG1 SDALIE mAb, which has an optimized A: I FcyR-binding ratio, promoted enhanced ADCC activity relative to all evaluated isoforms at E:T ratios of 5:1 and above. lgG2-mediated depletion appeared CD32a-dependent, as previously described (Schneider- Merck et al., 2010), with loss of activity upon CD32a blockade or use of a Fc-silent deglycosylated form of lgG2 (lgG2_Endos, Fig. 13C). The depleting activity of the lgG2 isotype, shown to efficiently deplete tumor-infiltrating Treg cells in vivo herein is further shown to be CD32a-dependent and no Treg cell depletion was observed in mice treated with Fc-silent lgG2_Endos mAb or in CD32a^"A mice (Fig. 14). The data stresses the in vivo role of CD32A as a key promoter of ADCC/ADCP. EXAM PLE 10 - RNAseq data

Finally, a clinically relevant potential surrogate of the mutational burden is the magnitude of the immune infiltrate in the tumor. We therefore interrogated RNA sequencing (RNAseq) data derived from the van Allen et al. cohort and compared the expression of key immune markers in responding patients with high mutational load (based on either indel or putative neoantigen burden) and the CD16-V158F SNP with rest of the cohort. Expression levels of CD8A, cytolytic markers (granzyme A and perforin) as well as the CD8A/FoxP3 ratio (based on gene expression) appeared higher in the group with improved response (Fig. 17). Whilst the size of the cohort with RNAseq data available in the van Allen et al. dataset (n=30) was too small to allow adequate statistical analysis, the presence of high indel or putative neoantigen burden, CD8A and the CD16-V158F SNP was associated with higher response rates than any other combination of metrics (Fig. 18), supporting the hypothesis that in inflamed or highly infiltrated tumors anti-CTLA-4 antibodies function, at least in part, via engagement of FcyRs and depletion of Treg cells.

REFERENCES

Alexandrov, LB., Nik-Zainal, S., Wedge, D.C., Aparicio, S.A.J. R., Behjati, S., Biankin, A.V., Bignell, G.R., Bolli, N., Borg, A., B0rresen-Dale, A.-L, et al. (2013). Signatures of mutational processes in human cancer. Nature 500, 415-421. Bruhns, P., lannascoli, B., England, P., Mancardi, D.A., Fernandez, N., Jorieux, S., and Daeron, M. (2009). Specificity and affinity of human Fey receptors and their polymorphic variants for human IgG subclasses. Blood 113, 3716-3725.

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

Bulliard, Y., Jolicoeur, R., Zhang, J., Dranoff, G., Wlson, N.S., and Brogdon, J.L. (2014). OX40 engagement depletes intratumoral Tregs via activating FcyRs, leading to antitumor efficacy. Immunol. Cell Biol. 92, 475^180.

Cartron, G., Dacheux, L., Salles, G., Solal-Celigny, P., Bardos, P., Colombat, P., and Watier, H. (2002). Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRllla gene. Blood 99, 754-758.

Coe, D., Begom, S., Addey, C, White, M., Dyson, J., and Chai, J.-G. (2010). Depletion of regulatory T cells by anti-GITR mAb as a novel mechanism for cancer immunotherapy. Cancer Immunol. Immunother. CM 59, 1367-1377. Comin-Anduix, B., Escuin-Ordinas, H., and Ibarrondo, F.J. (2016). Tremelimumab: research and clinical development. OncoTargets Ther. 9, 1767-1776.

De Simone, M., Arrigoni, A., Rossetti, G., Gruarin, P., Ranzani, V., Politano, C, Bonnal, R.J. P., Provasi, E., Sarnicola, M.L., Panzeri, I., et al. (2016). Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells. Immunity 45, 1 135-1147. Duncan, A.R., Woof, J.M., Partridge, L.J., Burton, D.R., and Winter, G. (1988). Localization of the binding site for the human high-affinity Fc receptor on IgG. Nature 332, 563-564.

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

Hanson, D.C., Canniff, P.C., Primiano, M.J., Donovan, C.B., Gardner, J. P., Natoli, E.J., Morgan, R.W., Mather, R.J., Singleton, D.H., Hermes, P.A., et al. (2004). Preclinical in vitro characterization of anti-CTLA4 therapeutic antibody CP-675,206. Cancer Res. 64, 877-877.

Hodi, F.S., Butler, M., Oble, D.A., Seiden, M.V., Haluska, F.G., Kruse, A., Macrae, S., Nelson, M., Canning, C, Lowy, I., et al. (2008). Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients. Proc. Natl. Acad. Sci. U. S. A. 105, 3005-3010.

Hodi, F.S., O'Day, S.J., McDermott, D.F., Weber, R.W., Sosman, J.A., Haanen, J.B., Gonzalez, R., Robert, C, Schadendorf, D., Hassel, J.C., et al. (2010). Improved Survival with Ipilimumab in Patients with Metastatic Melanoma. N. Engl. J. Med. 363, 711-723.

Ji, R.-R., Chasalow, S.D., Wang, L, Hamid, O., Schmidt, H., Cogswell, J., Alaparthy, S., Berman, D., Jure-Kunkel, M., Siemers, N.O., et al. (2012). An immune-active tumor microenvironment favors clinical response to ipilimumab. Cancer Immunol. Immunother. CM 61 , 1019-1031.

Jie, H.-B., Schuler, P.J., Lee, S.C., Srivastava, R.M., Argiris, A., Ferrone, S., Whiteside, T.L., and Ferris, R.L. (2015). CTLA-4+ Regulatory T Cells Increased in Cetuximab-Treated Head and Neck Cancer Patients Suppress NK Cell Cytotoxicity and Correlate with Poor Prognosis. Cancer Res. 75, 2200-2210.

Kargl, J., Busch, S.E., Yang, G.H.Y., Kim, K.-H., Hanke, M.L., Metz, H.E., Hubbard, J.J., Lee, S.M., Madtes, D.K., Mcintosh, M.W., et al. (2017). Neutrophils dominate the immune cell composition in non-small cell lung cancer. Nat. Commun. 8, 14381.

Koene, H.R., Kleijer, M., Algra, J., Roos, D., von dem Borne, A.E., and de Haas, M. (1997). Fc gammaRllla-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRllla, independently of the Fc gammaRllla-48L/R/H phenotype. Blood 90, 1109— 1 114.

Larkin, J., Chiarion-Sileni, V., Gonzalez, R., Grob, J. J., Cowey, C.L., Lao, CD., Schadendorf, D., Dummer, R., Smylie, M., Rutkowski, P., et al. (2015). Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N. Engl. J. Med.

Lazar, G.A., Dang, W., Karki, S., Vafa, O., Peng, J.S., Hyun, L, Chan, C, Chung, H.S., Eivazi, A., Yoder, S.C., et al. (2006). Engineered antibody Fc variants with enhanced effector function. Proc. Natl. Acad. Sci. U. S. A. 103, 4005-4010.

Leach, D.R., Krummel, M.F., and Allison, J. P. (1996). Enhancement of antitumor immunity by CTLA-4 blockade. Science 271 , 1734-1736. McGranahan, N., Furness, A.J.S., Rosenthal, R., Ramskov, S., Lyngaa, R., Saini, S.K., Jamal-Hanjani, M., Wilson, G.A., Birkbak, N.J., Hiley, C.T., et al. (2016). Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 351 , 1463-1469. Musolino, A., Naldi, N., Bortesi, B., Pezzuolo, D., Capelletti, M., Missale, G., Laccabue, D., Zerbini, A., Camisa, R., Bisagni, G., et al. (2008). Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER- 2/neu-positive metastatic breast cancer. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 26, 1789-1796.

Nathanson, T., Ahuja, A., Rubinsteyn, A., Aksoy, B.A., Hellmann, M.D., Miao, D.D., Allen, E.V., Merghoub, T., Wolchok, J.D., Snyder, A., et al. (2016). Somatic Mutations and Neoepitope Homology in Melanomas Treated with CTLA-4 Blockade. Cancer Immunol. Res. canimm.0019.2016.

Peggs, K.S., Quezada, S.A., Chambers, C.A., Korman, A.J., and Allison, J. P. (2009). Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the antitumor activity of anti-CTLA-4 antibodies. J. Exp. Med. 206, 1717-1725. Plitas, G., Konopacki, C, Wu, K., Bos, P.D., Morrow, M., Putintseva, E.V., Chudakov, D.M., and Rudensky, A.Y. (2016). Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer. Immunity 45, 1 122-1134. Quezada, S.A., Peggs, K.S., Curran, M.A., and Allison, J. P. (2006). CTLA4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells. J. Clin. Invest. 1 16, 1935-1945.

Read, S., Malmstrom, V., and Powrie, F. (2000). Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J. Exp. Med. 192, 295-302. Read, S., Greenwald, R., Izcue, A., Robinson, N., Mandelbrot, D., Francisco, L, Sharpe, A.H., and Powrie, F. (2006). Blockade of CTLA-4 on CD4+CD25+ regulatory T cells abrogates their function in vivo. J. Immunol. Baltim. Md 1950 177, 4376-4383.

Redpath, S., Michaelsen, T.E., Sandlie, I., and Clark, M.R. (1998). The influence of the hinge region length in binding of human IgG to human Fcgamma receptors. Hum. Immunol. 59, 720-727.

Ribas, A., Puzanov, I., Dummer, R., Schadendorf, D., Hamid, O., Robert, C, Hodi, F.S., Schachter, J., Pavlick, A.C., Lewis, K.D., et al. (2015). Pembrolizumab versus investigator- choice chemotherapy for ipilimumab-refractory melanoma (KEYNOTE-002): a randomised, controlled, phase 2 trial. Lancet Oncol. 16, 908-918.

Robert, C, Thomas, L, Bondarenko, I., O'Day, S., Weber, J., Garbe, C, Lebbe, C, Baurain, J.-F., Testori, A., Grob, J. -J., et al. (2011). Ipilimumab plus Dacarbazine for Previously Untreated Metastatic Melanoma. N. Engl. J. Med. 364, 2517-2526.

Robert, C, Ribas, A., Wolchok, J.D., Hodi, F.S., Hamid, O., Kefford, R., Weber, J.S., Joshua, A.M., Hwu, W.-J., Gangadhar, T.C., et al. (2014). Anti-programmed-death-receptor- 1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase 1 trial. Lancet Lond. Engl. 384, 1109-11 17.

Robert, C, Long, G.V., Brady, B., Dutriaux, C, Maio, M., Mortier, L, Hassel, J.C., Rutkowski, P., McNeil, C, Kalinka-Warzocha, E., et al. (2015). Nivolumab in previously untreated melanoma without BRAF mutation. N. Engl. J. Med. 372, 320-330.

Romano, E., Kusio-Kobialka, M., Foukas, P.G., Baumgaertner, P., Meyer, C, Ballabeni, P., Michielin, O., Weide, B., Romero, P., and Speiser, D.E. (2015). Ipilimumab-dependent cell- mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients. Proc. Natl. Acad. Sci. U. S. A. 1 12, 6140-6145.

Sanders, L.A., Feldman, R.G., Voorhorst-Ogink, M.M., de Haas, M., Rijkers, G.T., Capel, P.J., Zegers, B.J., and van de Winkel, J.G. (1995). Human immunoglobulin G (IgG) Fc receptor IIA (CD32) polymorphism and lgG2-mediated bacterial phagocytosis by neutrophils. Infect. Immun. 63, 73-81.

Sarmay, G., Lund, J., Rozsnyay, Z., Gergely, J., and Jefferis, R. (1992). Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fc gamma receptor. Mol. Immunol. 29, 633-639.

Schadendorf, D., Hodi, F.S., Robert, C, Weber, J.S., Margolin, K., Hamid, O., Patt, D., Chen, T.-T., Berman, D.M., and Wolchok, J.D. (2015). Pooled Analysis of Long-Term Survival Data From Phase II and Phase III Trials of Ipilimumab in Unresectable or Metastatic Melanoma. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 33, 1889-1894.

Schneider-Merck, T., Lammerts van Bueren, J. J., Berger, S., Rossen, K., van Berkel, P.H.C., Derer, S., Beyer, T., Lohse, S., Bleeker, W.K., Peipp, M., et al. (2010). Human lgG2 antibodies against epidermal growth factor receptor effectively trigger antibody-dependent cellular cytotoxicity but, in contrast to lgG1 , only by cells of myeloid lineage. J. Immunol. Baltim. Md 1950 184, 512-520. Selby, M.J., Engelhardt, J.J., Quigley, M., Henning, K.A., Chen, T., Srinivasan, M., and Korman, A.J. (2013). Anti-CTLA-4 Antibodies of lgG2a Isotype Enhance Antitumor Activity through Reduction of Intratumoral Regulatory T Cells. Cancer Immunol. Res. 1 , 32-42.

Shields, R.L., Namenuk, A.K., Hong, K., Meng, Y.G., Rae, J., Briggs, J., Xie, D., Lai, J., Stadlen, A., Li, B., et al. (2001). High resolution mapping of the binding site on human lgG1 for Fc gamma Rl, Fc gamma Rll, Fc gamma Rill, and FcRn and design of lgG1 variants with improved binding to the Fc gamma R. J. Biol. Chem. 276, 6591-6604.

Shields, R.L., Lai, J., Keck, R., O'Connell, L.Y., Hong, K. , Meng, Y.G., Weikert, S.H.A., and Presta, L.G. (2002). Lack of fucose on human lgG1 N-linked oligosaccharide improves binding to human Fcgamma Rill and antibody-dependent cellular toxicity. J. Biol. Chem. 277, 26733-26740. Simpson, T.R., Li, F., Montalvo-Ortiz, W., Sepulveda, M.A., Bergerhoff, K., Arce, F., Roddie, C, Henry, J.Y., Yagita, H., Wolchok, J.D., et al. (2013). Fc-dependent depletion of tumor- infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J. Exp. Med. 210, 1695-1710.

Smith, P., DiLillo, D.J., Bournazos, S., Li, F., and Ravetch, J.V. (2012). Mouse model recapitulating human Fey receptor structural and functional diversity. Proc. Natl. Acad. Sci. U. S. A. 109, 6181-6186. Snyder, A., Makarov, V., Merghoub, T., Yuan, J., Zaretsky, J.M., Desrichard, A., Walsh, LA., Postow, M.A., Wong, P., Ho, T.S., et al. (2014). Genetic Basis for Clinical Response to CTLA-4 Blockade in Melanoma. N. Engl. J. Med. 371 , 2189-2199.

Snyder, A., Wolchok, J. D., and Chan, T. A. (2015). Genetic basis for clinical response to CTLA-4 blockade. N Engl J Med 372: 783.

Tarhini, A.A., Edington, H., Butterfield, L.H., Lin, Y., Shuai, Y., Tawbi, H., Sander, C, Yin, Y., Holtzman, M., Johnson, J., et al. (2014). Immune monitoring of the circulation and the tumor microenvironment in patients with regionally advanced melanoma receiving neoadjuvant ipilimumab. PloS One 9, e87705.

Turajlic, S., Litchfield, K., Xu, H., Rosenthal, R., McGranahan, N., Reading, J. L, Wong, Y. N. S., Rowan, A., Kanu, N., Al Bakir, M., et al. (2017). Insertion-and-deletion-derived tumour- specific neoantigens and the immunogenic phenotype: a pan-cancer analysis. Lancet Oncol 18: 1009-1021

Van Allen, E. M., Miao, D., Schilling, B., Shukla, S. A., Blank, C, Zimmer, L, Sucker, A., Hillen, U., Foppen, M. H. G., Goldinger, S. M., et al. (2015). Genomic correlates of response to CTLA-4 blockade in metastatic melanoma. Science 350: 207-21 1.

Warmerdam, P.A., van de Wnkel, J.G., Vlug, A., Westerdaal, N.A., and Capel, P.J. (1991). A single amino acid in the second Ig-like domain of the human Fc gamma receptor II is critical for human lgG2 binding. J. Immunol. Baltim. Md 1950 147, 1338-1343. Weber, J.S., D'Angelo, S.P., Minor, D., Hodi, F.S., Gutzmer, R., Neyns, B., Hoeller, C, Khushalani, N.I., Miller, W.H., Lao, CD., et al. (2015). Nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4 treatment (CheckMate 037): a randomised, controlled, open-label, phase 3 trial. Lancet Oncol. 16, 375-384.

Weng, W.-K., and Levy, R. (2003). Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 21 , 3940-3947.

White, A.L., Chan, H.T.C., French, R.R., Willoughby, J., Mockridge, C.I., Roghanian, A., Penfold, C.A., Booth, S.G., Dodhy, A., Polak, M.E., et al. (2015). Conformation of the human immunoglobulin G2 hinge imparts superagonistic properties to immunostimulatory anticancer antibodies. Cancer Cell 27, 138-148.

Wing, K., Onishi, Y., Prieto- Martin, P., Yamaguchi, T., Miyara, M., Fehervari, Z., Nomura, T., and Sakaguchi, S. (2008). CTLA-4 control over Foxp3+ regulatory T cell function. Science 322, 271-275.

Wolchok, J.D., Kluger, H., Callahan, M.K., Postow, M.A., Rizvi, N.A., Lesokhin, A.M., Segal, N.H., Ariyan, C.E., Gordon, R.-A., Reed, K., et al. (2013). Nivolumab plus ipilimumab in advanced melanoma. N. Engl. J. Med. 369, 122-133.

Wu, J., Edberg, J.C., Redecha, P.B., Bansal, V., Guyre, P.M., Coleman, K., Salmon, J.E., and Kimberly, R.P. (1997). A novel polymorphism of FcgammaRllla (CD16) alters receptor function and predisposes to autoimmune disease. J. Clin. Invest. 100, 1059-1070. Zhang, W., Gordon, M., Schultheis, A.M., Yang, D.Y., Nagashima, F., Azuma, M., Chang, H.-M., Borucka, E., Lurje, G., Sherrod, A.E., et al. (2007). FCGR2A and FCGR3A polymorphisms associated with clinical outcome of epidermal growth factor receptor expressing metastatic colorectal cancer patients treated with single-agent cetuximab. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 25, 3712-3718.

All documents referred to herein are hereby incorporated by reference in their entirety, with special attention to the subject matter for which they are referred Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, cellular immunology or related fields are intended to be within the scope of the following claims.

Claims

1. A method for identifying a subject with cancer who is suitable for treatment with an lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule, said method comprising determining the FcyR polymorphism status of the subject.

2. A method for identifying a subject with cancer who is suitable for treatment with an lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule, said method comprising determining the FcyR polymorphism status of the subject, wherein the presence of a FcyR polymorphism which confers an increased affinity to lgG1 is indicative of response to an lgG1 antibody immune checkpoint intervention and wherein the presence of a FcyR polymorphism which confers an increased affinity to lgG2 is indicative of response to an lgG2 antibody targeting an immune checkpoint molecule.

3. The method according to claim 1 or 2, wherein determining the FcyR polymorphism status of the comprises the steps of:

i) sequencing of the subject's germline DNA;

ii) identifying the presence of one or more single nucleotide polymorphism(s) within an FcyR gene, such that the FcyR gene having the one or more single nucleotide polymorphism(s) encodes a variant FcyR molecule.

4. The method according to claim 3, further comprising the step of:

iii) determining the affinity of the FcyR for its cognate IgG molecule.

5. The method according to any one of claims 2 to 4, wherein the FcyR polymorphism which confers an increased affinity to lgG1 is a V158F SNP in FcyRllla (CD16a).

6. The method according to any one of claims 2 to 4, wherein the FcyR polymorphism which confers an increased affinity to lgG2 is a H131 R SNP in FcyRlla (CD32a).

7. The method according to any one of claims 1 to 4, further comprising the step of determining the mutational burden and/or the neo-antigen burden in the subject, wherein the presence of a higher and/or increased and/or high mutational burden and/or neo-antigen burden is indicative of response to an lgG1 or lgG2 antibody targeting an immune checkpoint molecule.

8. A method for identifying patients not suitable for treatment with an lgG1 antibody or lgG2 antibody targeting an immune checkpoint molecule, said method comprising the steps of:

i) determining the FcyR polymorphism status of the subject; and

ii) determining the mutational burden and/or the neo-antigen burden in the subject; wherein the presence of a FcyR polymorphism which confers an increased affinity to lgG1 or a FcyR polymorphism which confers an increased affinity to lgG2, in combination with a lower and/or decreased and/or low mutational burden and/or neo-antigen burden is indicative of a lack of response or poor response to an lgG1 or lgG2 antibody targeting an immune checkpoint molecule respectively.

9. A method of treating cancer in a human subject comprising the step of administering an lgG2 antibody targeted to an immune checkpoint molecule to the subject, wherein said subject has a solid tumour, wherein preferably the lgG2 antibody is not tremelimumab.

10. The method according to any one of the preceding claims, wherein the immune checkpoint molecule is expressed at higher levels on Treg versus Teff cells.

1 1. The method according to any one of the preceding claims, wherein the immune checkpoint molecule is CTLA-4, ICOS, GITR, OX-40 or 4-1 BB.

12. The method according to any one of claims 9 to 11 wherein the lgG2 antibody binds to FcyRlla (CD32a) with high affinity, preferably with a dissociation constant of less than about 10^"8 M.

13. The method according to any one of claims 9 to 12, wherein the lgG2 antibody elicits an ADCC or ADCP response.

14. The method according to any one of claims 9 to 13, wherein the lgG2 antibody is capable of depleting regulatory T cells (Tregs) in the solid tumour.

15. The method according to any one of claims 9 to 14, wherein the lgG2 antibody is a human antibody.

16. A method of treating or preventing cancer in a subject, comprising the steps of:

i) identifying a subject with cancer who is suitable for treatment with an lgG1 or lgG2 antibody targeting an immune checkpoint molecule according to any one of claims 1 to 7;

ii) treating the cancer in the subject comprising the step of administering an lgG1 or an lgG2 antibody targeted to the immune checkpoint molecule to the subject.

17. The method according to claim 14, wherein treating the cancer comprises a method according to any one of claims 9 to 15.

18. An lgG2 antibody targeted to an immune checkpoint molecule, as defined in any one of claims 9 to 15, for use in a method of treating cancer in a subject.

19. An lgG1 or lgG2 antibody targeted to an immune checkpoint molecule for use in a method of treating cancer in a subject, wherein the method comprises:

20. An lgG1 or lgG2 antibody targeted to an immune checkpoint molecule for use according to claim 19, wherein the subject has a FcyR polymorphism which confers an increased affinity to lgG1 or lgG2 and/or a higher and/or increased and/or high mutational burden and/or neo-antigen burden.