Antibodies against domain 4 of ICAM-1 and uses thereof

Abstract

The present invention relates to antibody molecules, or fragments thereof, that bind specifically to Domain 4 of ICAM-1, and to their use in the treatment of cancer in a patient. The invention also relates to polynucleotide molecules, vectors, viruses, cells, pharmaceutical compositions and kits relating to the antibody molecules, or fragments thereof.

Description

[0001] ANTIBODIES AND USES THEREOF

[0002] The present invention relates to antibody molecules, or fragments thereof, that bind specifically to Domain 4 of ICAM-1, and to their use in the treatment of cancer in a patient. The invention also relates to polynucleotide molecules, vectors, viruses, cells, pharmaceutical compositions, kits, methods and uses relating to the antibody molecules, or fragments thereof.

[0003] Antibody immunotherapy has revolutionised cancer therapy. It encompasses agents that directly target tumor cells (such as rituximab and trastuzumab), or that target cells of the immune system (such as ipilimumab and nivolumab).

[0004] Despite successes, many patients do not respond to antibody immunotherapy, and those that do often relapse or become refractory to further treatment (Weber et al., N Engl J Med. 2017;377(19): 1824-35; Barrueto et al., Transl. Oncol. 2020;13(3): 100738; Torka et al., Curr Hematol. Malig Rep. 2019;14(5):426-38).

[0005] Additionally, antibody efficacy is impacted by the tumor microenvironment (TME). It is often anti-inflammatory, lacking infiltrating lymphocytes and / or containing suppressive cells such as tumor associated macrophages (TAM) and regulatory T cells ("Treg", or "Treg cells”) (Jin and Jin, Signal Transduct. Target Ther. 2020;5(l): 166). Whilst not universal, it is generally established that tumors which have a high proportion of CD4+ FoxP3+ Tregs have a worse prognosis compared to those without (Liu et al., Breast Cancer Res Treat. 2011;130(2):645-55; Li et al., J Cancer. 2016;7(7):784-93; Zhao et al., Am J Pathol. 2020;190(4):886-99). In agreement, systemic removal of Tregs using conditional Treg knock-out (KO) strategies results in tumor regression of subcutaneous tumor models (Teng et al., Cancer Res. 2010;70(20):7800-9.8, Klages et al., Cancer Res. 2010;70(20):7788- 99)

[0006] Whilst Tregs have been proposed as a target in cancer therapy, attempts to actually target them have had varying success in the clinic.

[0007] Metronomic chemotherapy with cyclophosphamide depletes Tregs systemically, therefore driving anti-tumor responses (Scurr et al., Clin Cancer Res. 2017;23(22):6771-80): however, this approach targets regulatory cells throughout the body and is not maintained across prolonged time-courses (Ge et al., Cancer Immunol Immunother. 2012;61(3) :353- 62).

[0008] Using monoclonal antibodies (mAb) to target and delete Tregs is an attractive concept but there is no single specific surface marker for Treg cells: whilst they constitutively express markers such as CD25, GITR, 0X40, 4-1BB, CCR4, CCR8 and CTLA-4, these same receptors are also present on activated CD8+ effector and conventional CD4+ T cells (Buchan et al., Immunity. 2018;49(5) :958-70 e7; Wegrzyn et al., Front Immunol. 2022; 13: 105580). This means that mAbs targeting these antigens will likely impact both Tregs and important effector populations, potentially reducing the impact specific Treg-depletion could evoke. Whilst Treg-depletion through some of these receptors, such as CTLA-4 and CD25 with ipilimumab (Romano et al., Proc Natl Acad. Sci U S A. 2015; 112(19):6140-5) and daclizumab (The L. End of the road for daclizumab in multiple sclerosis. Lancet. 2018;391(10125) : 1000) respectively, is associated with some success in the clinic, there are often issues with prolonged side-effects and development of severe autoimmune complications due to removing systemic Treg activity (Pol et al., Cell Res. 2018;28(5) : 501- 2; Aamdal et al., Int J Cancer. 2022; 150(l) : 100-11). In addition, these approaches can lead to development of autoimmune conditions and toxicities in patients due to systemic targeting (The L. End of the road for daclizumab in multiple sclerosis. Lancet. 2018;391(10125) : 1000).

[0009] Accordingly, more-effective therapeutic antibodies that target tumor cells, or that target cells of the immune system, are needed for use in cancer therapy.

[0010] Against this background, the present inventors have developed therapeutic antibody molecules for treating cancer which are surprisingly advantageous. As discussed herein, those antibody molecules bind specifically to Domain 4 of Intercellular adhesion molecule- 1 ("ICAM-1"), which the inventors have identified as a particularly effective cellular target for cancer therapy. Importantly, the inventors have surprisingly identified that targeting Domain 4 of ICAM-1 provides a very effective approach for depleting Treg cells, and even permits selective depletion of tumour-associated Treg cells.

[0011] The present invention therefore provides particularly advantageous agents, uses and methods for treating cancer, that address shortcomings of the prior art. It permits, among other things, an approach for the specific depletion of tumor-associated Tregs, thereby addressing current limitations of the prior art which are unable to differentiate between the Treg population that resides within a tumor versus Tregs present in other parts of the body.

[0012] In a first aspect, the invention provides an antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1 for use in the treatment of cancer in a patient.

[0013] In another aspect, the invention provides the use of an antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1, in the manufacture of a medicament for the treatment of cancer in a patient.

[0014] In yet another aspect, the invention provides a method for treating cancer in a patient, the method comprising the step of administering an antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1.

[0015] As discussed in detail herein and in the accompanying Examples, the inventors made their invention by utilising a phenotypic signature of tumor-associated Treg cells in conjunction with the n-CoDeR phage library and F.I.R.S.T. platform (Frendeus et al., Oncoimmunology. 2013;2(8); Soderlind et al., Nat. Biotechnol. 2000;18(8):852-6) to generate antibodies that were selective for tumor-associated Tregs. These were tested for their ability to bind and deplete Tregs across multiple tissues in vivo, and among other things the inventors surprisingly demonstrated specific depletion of tumor-associated Tregs, whilst leaving peripheral Tregs untouched, delayed tumor growth and evoked cures in an in vivo tumor model. As the Examples show, the inventors also demonstrated that Tregs from primary human tumor samples displayed a similar tumor-associated Treg selectivity.

[0016] Antibody molecules are well known to those skilled in the art of immunology and molecular biology. Typically, an antibody comprises two heavy (H) chains and two light (L) chains and, herein, we sometimes refer to this complete antibody molecule as a full-size or full- length antibody. The antibody heavy chain comprises one variable domain (VH) and three constant domains (CHI, CH2 and CH3), and the antibody light chain comprises one variable domain (VL) and one constant domain (CL). The variable domains (sometimes collectively referred to as the Fv region) bind to the antibody target, or antigen. Each variable domain comprises three loops, referred to as complementary determining regions (CDRs), which are responsible for target binding. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions.

[0017] Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and in humans several of these are further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, and IgG4; IgAl and IgA2. Another part of an antibody is the Fc domain (otherwise known as the fragment crystallisable domain), which comprises two of the constant domains of each of the antibody heavy chains: the Fc domain is responsible for interactions between the antibody and Fc receptor.

[0018] The term "antibody molecule", as used herein, encompasses full-length or full-size antibodies, and derivatives of such antibody molecules. As discussed herein, the invention also encompasses the use of fragments of such antibody molecules, including functional fragments thereof, and derivatives of such fragments.

[0019] Further, the term "antibody molecule or fragment thereof", as used herein, includes all classes of antibody molecules and functional fragments thereof, including : IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgD, and IgE, and fragments thereof.

[0020] The antibody molecule or fragment thereof of the invention may be in an isolated form and / or a purified form.

[0021] As outlined above, different types and forms of antibody molecules are included in the invention, and would be known to the person skilled in immunology. It is well known that antibodies used for therapeutic purposes are often modified with additional components which modify the properties of the antibody molecule. Accordingly, we include that an antibody molecule or fragment thereof of the invention (for example, a monoclonal antibody molecule, and / or polyclonal antibody molecule, and / or bi-specific antibody molecule) comprises a detectable moiety and / or a cytotoxic moiety.

[0022] By "detectable moiety", we include one or more from the group comprising of: an enzyme; a radioactive atom; a fluorescent moiety; a chemiluminescent moiety; a bioluminescent moiety. The detectable moiety allows the antibody molecule to be visualised in vitro, and / or in vivo, and / or ex vivo.

[0023] By "cytotoxic moiety", we include a radioactive moiety, and / or enzyme, wherein the enzyme is a caspase, and / or toxin, wherein the toxin is a bacterial toxin or a venom; wherein the cytotoxic moiety is capable of inducing cell lysis.

[0024] The antibody molecule or fragment thereof may be PEGylated. PEGylation is a method by which polyethylene glycol polymers are added to a molecule such as an antibody molecule or derivative to modify its behaviour, for example to extend its half-life by increasing its hydrodynamic size, preventing renal clearance.

[0025] It is well known that antibodies "specifically bind" a defined target molecule or antigen. That is to say, the antibody preferentially and selectively binds its target and not a molecule which is not a target. The targets of the antibodies according to the present invention, or of the antibodies used in accordance with the invention, are expressed on the surface of cells, i.e. they are cell surface antigens, which would include an epitope (otherwise known in this context as a cell surface epitope) for the antibody. Cell surface antigen and epitope are terms that would be readily understood by one skilled in immunology or cell biology.

[0026] As discussed above, the CDRs of an antibody bind to the antibody target. The assignment of amino acids to each CDR described herein is in accordance with the definitions according to Kabat EA et al. (1991, In "Sequences of Proteins of Immunological Interest" Fifth Edition, NIH Publication No. 91-3242, pp xv- xvii). As the skilled person would be aware, other methods also exist for assigning amino acids to each CDR. For example, the International ImMunoGeneTics information system (IMGT(R)) (http: / / www.imgt.org / and Lefranc and Lefranc "The Immunoglobulin FactsBook" published by Academic Press, 2001).

[0027] In the context of the present invention, the target of the antibody molecule, or fragment thereof, is Domain 4 of ICAM-1.

[0028] ICAM-1 (also known as CD54) is a 110 kilodalton cell surface glycoprotein, a member of the immunoglobulin superfamily that is involved in cell adhesion, inflammatory and immune responses. The expression pattern of ICAM-1 is wide-ranging, encompassing endothelial cells, epithelial cells and neutrophils; however, ICAM-1 is expressed on only a limited number of cells and at low levels in the absence of stimulation. ICAM-1 is upregulated in inflammatory conditions and has been linked to the migration and interaction of Treg cells with dendritic cells (Bui TM et al., J Leukoc Biol. 2020;108(3):787- 99; 46). Upon treatment with a number of inflammatory mediators (lipopolysaccharide, y-interferon, tumor necrosis factor-o, or interleukin-1), a variety of cell types (endothelial, epithelial, fibroblastic and hematopoietic cells) in a variety of tissues express high levels of ICAM-1 on their surface. Induction occurs via increased transcription of ICAM-1 mRNA.

[0029] Structurally, ICAM-1 is characterized by heavy glycosylation and the dominant secondary structure of the protein is the beta sheet. ICAM-1 is a transmembrane protein, and it spans the entire membrane of the cell.

[0030] As is well known, the term "domain" includes the meaning of a distinct, stable three- dimensional unit of a protein structure, regardless of size. Each protein domain forms a compact folded three-dimensional structure and is often self-stabilizing, folding independently from the rest of the protein chain. The tertiary structure of a typical domain is generally stable in solution and remains the same whether such a member is isolated or covalently fused to other domains. Protein domains are connected to each other through short segments of the polypeptide chain, often referred to as linkers, and disulfide bridges are generally the primary elements that determine tertiary structure.

[0031] The ICAM-1 molecule consists of five Ig-like domains (called Domains 1-5, or "D1"-"D5"), a short transmembrane region, and a small carboxyl-terminal cytoplasmic domain. In ICAM-1, the second, third, and fourth Ig-like domains are heavily N-glycosylated, with four potential sites in D2, two in D3, and two in D4 (Bella et al., Proc Natl Acad. Sci USA. 1998;95(8):4140-5). Two other molecules, ICAM-2 and ICAM-3 have at least 30% sequence identity with ICAM-1 and have similar adhesive properties. ICAM-1 and ICAM-2 normally have low expression levels, whereas ICAM-3 is more abundant in resting monocytes and lymphocytes. Fibrinogen can also bind to DI of ICAM-1, mediating leukocyte adhesion to vascular endothelium. Furthermore, ICAM-1 D4 can bind CD18 / CDllc (Frick et al., Eur. J. Immunol., 2005; 35:3610-3621).

[0032] There are many isoforms of ICAM-1 as reviewed elsewhere (Hu et al., Mol Immunol. 2010;47(9): 1692-700; Hu et al., Mol Immunol. 2010;47(9): 1692-700; Ramos et al., J Immunol. 2014;192(10):4469-74). It is, however, unclear whether there are cell-specific isoforms present, and how the tumor microenvironment (TME) can influence this. TME itself can influence the glycosylation patterns of proteins due to changes in metabolism and expression of glycan-processing enzymes (Peixoto et al., Front Oncol. 2019;9:380). These changes may therefore alter the accessibility of specific epitopes compared to normal tissue.

[0033] In some aspects, the antibody molecule, or fragment thereof, according to the invention binds to a glycosylation dependent epitope in Domain 4 of ICAM-1, while binding to deglycosylated ICAM-1 is substantially diminished. This means that the antibody molecule, or fragment thereof, is more selective to bind to glycosylated epitopes in Domain 4 in the TME compared to epitopes (deglycosylated or alternatively glycosylated) in Domain 4 of ICAM-1 in normal tissue.

[0034] ICAM-1 has been previously investigated as a target for antibody immunotherapy for multiple myeloma (Dahlhoff et al., Leukemia. 2022;36(3):790-800). However, prior art antibodies, whilst tolerated, displayed little therapeutic benefit, which led to a cease in their clinical development (Wichert et al., PLoS One. 2017;12(2):e0171205). Importantly, those prior art antibodies were shown to bind the "top" two domains of ICAM-1 (i.e. Domains 1 and 2 - "DI" and "D2") and were selected primarily for their ability to induce apoptosis of myeloma cells rather than for their impact on tumor infiltrating leukocytes (TILs).

[0035] Unlike known antibodies against ICAM-1, the antibodies of the present invention bind a different domain of ICAM-1, i.e. Domain 4 ("D4") and provide differential selectivity towards tumor-associated Tregs. That was a surprising finding. Without being bound by any theory, the inventors hypothesise that Domain 4 (D4) of ICAM-1 is more frequently exposed and / or is upregulated in tumour-associated Tregs compared to other Tregs, and / or in intratumoural Tregs compared to Tregs that are not intratumoural, explaining the surprising efficacy of the claimed antibodies in the treatment of cancer, and why it is possible to obtain tumor-specific depletion of Tregs and / or depletion of tumor-associated Tregs.

[0036] What is meant by an antibody molecule, or fragment thereof, that "binds specifically" to a defined target molecule or antigen is well known, and includes that the antibody preferentially and selectively binds its target, and not a molecule which is not a target. In this context, the term "binds to" can be used interchangeably with "interacts with". By "an antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1" and "an anti-ICAM-l-Domain 4 antibody molecule, or fragment thereof" we include an antibody molecule or fragment thereof that specifically binds to Domain 4 of ICAM-1, but does not bind to non-target (such as other domains of ICAM-1, or to other ICAM molecules, or to other domains of other ICAM molecules like ICAM-2 or ICAM-3), or binds only minimally to such a non-target (such as with a lower affinity) relative to the target.

[0037] Methods of assessing protein binding are known to those skilled in biochemistry and immunology. It would be appreciated by the skilled person that those methods could be used to assess binding of an antibody to a target and / or binding of the Fc region of an antibody to an Fc receptor; as well as the relative strength, or the specificity, or the inhibition, or prevention, or reduction in those interactions. Examples of methods that may be used to assess protein binding are, for example, immunoassays, BIAcore, western blots, radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISAs) (See Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 (1989) for a discussion regarding antibody specificity).

[0038] We also include the meaning that the antibody molecule, or fragment thereof, specifically binds to Domain 4 of ICAM-1 at least two-fold more strongly, or at least five-fold more strongly, or at least 10-fold more strongly, or at least 20-fold more strongly, or at least 50- fold more strongly, or at least 100-fold more strongly, or at least 200-fold more strongly, or at least 500-fold more strongly, or at least than about 1000-fold more strongly, than it does to a non-target.

[0039] Additionally, we include the meaning that the antibody molecule, or fragment thereof, specifically binds to Domain 4 of ICAM-1 if it binds to the target with a Kd of at least about 101Kd, or at least about IO-2Kd, or at least about IO-3Kd, or at least about 10-4Kd, or at least about IO-5Kd, or at least about IO-6Kd, or at least about IO-7Kd, or at least about IO-8Kd, or at least about IO-9Kd, or at least about 1010Kd, or at least about 1011Kd, or at least about 1012Kd, or at least about 1013Kd, or at least about 1014Kd, or at least about 1015Kd.

[0040] Preferably, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the ICAM-1 is present on the surface of Tregs. Accordingly, it will be appreciated that the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method as disclosed herein, wherein Domain 4 of ICAM-1 is present on the surface of Tregs.

[0041] By "surface", we include that the antigen (i.e. ICAM-1, and specifically Domain 4 of ICAM- 1) to which the antibody binds is present on the extracellular side of the cell membrane, either permanently or transiently. Examples of methods for assessing binding of a proteins would be known to those skilled in the art. For example, it would be appreciated by a skilled person that the methods discussed above can also be used to assess binding of an antibody to a target / antigen present on the surface of effector cell (such as Treg).

[0042] As used herein, the term "Tregs" refers to (and is used interchangeably with) "regulatory T cells", "Treg cells", or "T-regs", and that cell type was formerly known as "suppressor T cells" and sometimes also called "suppressive regulatory T cells". As is well known to those in the art Tregs maintain tolerance to self-antigens, and prevent autoimmune disease (Makoto et al., Autoimmunity Reviews, 10(12) 2011, 744-755). Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells.

[0043] Tregs can express the biomarkers CD4 and / or CD25 and are thought to be derived from the same lineage as naive CD4+ cells. Tregs are involved in shutting down immune responses after they have successfully eliminated invading organisms, and also in preventing autoimmunity. They suppress activation, proliferation, and cytokine production of CD4+ T cells and CD8+ T cells, and are thought to suppress B cells and dendritic cells.

[0044] In the context of cancer, Tregs tend to be upregulated in individuals with cancer, and they seem to be recruited to the site of many tumors. High numbers of Tregs in the tumor microenvironment is indicative of a poor prognosis, and Tregs are thought to suppress tumor immunity, thus hindering the body's innate ability to control the growth of cancerous cells.

[0045] In the context of the present invention, Tregs are ICAM-1 positive, which means they are preferably CD4 positive, FoxP3 expressing and ICAM-1 positive, i.e. expressing CD4, the transcription factor FoxP3, and ICAM-1. Although ICAM-1 is usually expressed on only a limited number of cells and at low levels, Tregs can express significantly higher levels of ICAM-1. As discussed herein, an unexpected finding of the present invention was that ICAM-1 Domain 4 is particularly highly exposed in Treg cells, even compared to other ICAM- 1 domains. As will be appreciated in light of the teachings herein, this makes Tregs more susceptible to depletion compared to cells that express ICAM-1 Domain 4, or ICAM-1, at lower levels.

[0046] In a preferred embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the Tregs are tumor- associated Tregs.

[0047] By "tumor-associated Tregs" (which are also sometimes referred to as "tumour-specific Tregs", a term that can be used interchangeably), we include Tregs that function in favour of cancer and / or tumour in the patient. In other words, tumor-associated Tregs regulate tumor-related immune responses, and serve to promote survival or progression of the cancer and / or tumour, and / or reduce or inhibit the effectiveness of anti-cancer immune responses or cancer immunotherapy. For example, such Tregs may exert immunosuppressive effects, such as reducing or preventing anti-tumor immune responses, and / or inducing or promoting immune evasion of the cancer or tumour.

[0048] Tumor-associated Tregs can be present within the tumour microenvironment (termed "intratumoral Tregs" herein), for example within the tumor tissue (primary and / or secondary) and / or non-malignant part of the tumor (such as stromal cells, extracellular matrix, blood and / or lymphatic vessels). In other embodiments, tumor-associated Tregs can be present outside of the tumour microenvironment, for example in a tumor-draining lymph node.

[0049] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof is a Treg-depleting antibody molecule.

[0050] In another embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein binding of the antibody, or fragment thereof, to ICAM-1 induces the depletion of Tregs in the patient. In another embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein binding of the antibody, or fragment thereof, to ICAM-1 induces the specific depletion of tumor-associated Tregs in the patient.

[0051] By "Treg-depleting" or "depletion of Tregs", or "Treg depletion", we include depletion, deletion and / or elimination of Tregs through the physical clearance of such cells from the patient in general and / or from a particular location within the patient (for example, from one or more tumour in the patient). In a preferred embodiment, the depletion, deletion and / or elimination of Tregs comprises the depletion, deletion and / or elimination of tumor- associated Tregs. In a more preferred embodiment, the depletion, deletion and / or elimination of Tregs comprises the depletion, deletion and / or elimination of intratumoural tumor-associated Tregs.

[0052] Assays for identifying and assessing Treg depletion are known to those skilled in the art and include, for example: ADCC assays; ADCP assays; or in vivo tumor models.

[0053] An ADCC assay may be performed, for example, by labelling target cells with calcein AM (acetyl methyl ester), followed by the addition of diluting concentrations of the antibody, or fragment thereof. Target cells (such as Tregs) are then cocultured with human peripheral blood mononuclear cells (PBMCs) at a 50: 1 effector: target (E:T) ratio for 4 h at 37°C. The plate is centrifuged at 400 x g for 5 min to pellet the cells, and the supernatant is transferred to a white 96-well plate. Calcein release is measured using a Varioskan (Thermo Scientific) using an excitation wavelength of 485 nm and emission wavelength, 530 nm. The percentage of maximal release is calculated as follows: % max release = (sample / triton treated)*100.

[0054] An ADCP assay may be performed, for example, by labelling target cells (such as Tregs) with 5 mM carboxyfluorescein succinimidyl ester (CFSE) for 10 min at room temperature before washing in media containing foetal calf serum. CFSE-labelled targets are then opsonized with diluting concentrations of antibody before coculturing at a 1 :5 E:T ratio with bone marrow derived macrophages (BMDMs) in 96-well plates for 1 h at 37°C. BMDMs are then labelled with anti-F4 / 80-allophycocyanin for 15 min at room temperature and washed with PBS twice. Plates are kept on ice, wells are scraped to collect BMDMs, and phagocytosis is assessed by flow cytometry using a FACSCalibur (BD) to determine the percentage of F4 / 80+CFSE+ cells within the F4 / 80+ cell population. It is also possible to use a method as described by Cleary et al in J Immunol, April 12, 2017, 1601473.

[0055] In addition, it will be appreciated that an in vivo tumor model can be used to identify or assess Treg depletion, for example, a subcutaneous tumor model. Preferably, a model should have a relatively high number of Treg cells present in the tumor microenvironment (infiltrate) and be sensitive to established Treg-depleting therapeutics. In this model, two control animal groups are formed, where one group receives a desired therapeutic antibody regimen (preferably a Treg-depleting antibody, or fragment thereof), and the second group receives a control antibody isotype. After the treatment period has elapsed, tumor and other organs are harvested and processed into a single cell suspension and immune populations assessed by flow cytometry. Flow cytometry is used to analyse the presence and abundance of specific cell populations based on their surface markers or intracellular molecules. Accordingly, after Treg depletion, the cell population can be analysed by flow cytometry using fluorescently labelled antibodies targeting Treg-specific markers (such as CD4, FoxP3 and / or CD25). A decrease in the fluorescence signal corresponding to the depleted cell population (such as Tregs) in the tumor, compared to isotype level, indicates successful depletion. For such an assay, one described in the Examples below may be used. Additionally, flow cytometry can be used to quantify the purity of the remaining cell population and assess any potential contamination by non-target cells.

[0056] In a preferred embodiment, treatment with an antibody molecule, or fragment thereof, results in depletion of tumor-associated Tregs in the patient. That results in a decrease in the number and / or density of Tregs in the patient, for example in the tumor of the cancer of the patient. Preferably, the number and / or density of tumor-associated Tregs decreases by approximately 1.5-fold to 25-fold or more, for example by approximately 1.5-fold, or 2-fold, or 2.5-fold, or 3-fold, or 3.5-fold, or 4-fold, or 4.5-fold, or 5-fold, or 6-fold, or 7- fold, or 8-fold, or 9-fold, or 10-fold, or 11-fold, or 12-fold, or 13-fold, or 14-fold, or 15- fold, or 16-fold, or 17-fold, or 18-fold, or 19-fold, or 20-fold, or 21-fold, or 22-fold, or 23- fold, or 24-fold, or 25-fold, or more.

[0057] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof comprises an Fc region that binds to at least one activating Fey receptor. Fc receptors (FcRs) are membrane proteins which are found on the cell surface of immune effector cells including monocytes, macrophages, dendritic cells, neutrophils, mast cells, basophils, eosinophils, Natural Killer cells and B lymphocytes. The name is derived from their binding specificity for the Fc region of antibodies. Fc receptors are found on the cell membrane - otherwise known as the plasma membrane or cytoplasmic membrane. Fc receptors can be subdivided into activating FcyR and inhibitory FcyR, which are known to regulate cellular activation through binding of aggregated immunoglobulin G Fc's, and transmission of activating or inhibitory signals into the cell through intracellular ITAM or ITIM motifs. FcR binding of aggregated immunoglobulin or immune complexes, can mediate antibody internalization into the cell, and can result in antibody-mediated phagocytosis, antibody-dependent cell-mediated cytotoxicity, or antigen presentation or cross-presentation. FcRs are also known to mediate or enhance cross-linking of antibodybound cell surface receptors. Such cross-linking is known to be required for some (Li et al., 2011, Science, 333: 1030-4.; White et al., 2011, J Immunol, 187: 1754-63) but not all (Richman et al. 2014., Oncoimmunology, 3: e28610) antibodies ability to activate signaling in targeted cells, and may or may not be required to achieve therapeutic effects.

[0058] A subgroup of the Fc receptors is Fey receptors (Fc-gamma receptors, FcgammaR, FcyR), which are specific for IgG antibodies. There are two types of Fey receptors: activating Fey receptors (also denoted activatory Fey receptors), and inhibitory Fey receptors. The activating and the inhibitory receptors transmit their signals via immunoreceptor tyrosinebased activation motifs (ITAM) or immunoreceptor tyrosine-based inhibitory motifs (ITIM), respectively. In humans, FcyRIIb (CD32b) is an inhibitory Fey receptor, while FcyRI (CD64), FcyRIIa (CD32a), FcyRIIc (CD32c) and FcyRIIIa (CD16a) are activating Fey receptors. FcyRIIIb is a GPI-linked receptor expressed on neutrophils that lacks an ITAM motif but through its ability to cross-link lipid rafts and engage with other receptors is also considered activatory. In mice, the activating receptors are FcyRI, FcyRIII and FcyRIV.

[0059] It is well-known that antibodies can modulate immune cell activity through interaction with Fey receptors. Specifically, how antibody immune complexes modulate immune cell activation is determined by their relative engagement of activating and inhibitory Fey receptors. Different antibody isotypes bind with different affinity to activating and inhibitory Fey receptors, resulting in different A: I ratios (activation inhibition ratios) (Nimmerjahn et al; Science. 2005 Dec 2;310(5753): 1510-2). By binding to an activating Fey receptor, an antibody can activate effector cell functions and thereby trigger mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), cytokine release, and / or antibody dependent endocytosis, as well as NETosis (i.e. activation and release of NETs, Neutrophil extracellular traps) in the case of neutrophils. Antibody binding to an activating Fey receptor can also lead to an increase in certain activation markers, such as CD40, MHCII, CD38, CD80 and / or CD86.

[0060] By binding to inhibitory Fey receptors, an antibody can inhibit, block and / or down-modulate effector cell depletion functions like ADCC and ADCP. However, by binding to an inhibitory FcyR, immunomodulatory antibodies can further stimulate cell activation through aggregation of antibody-targeted signaling receptors on a target cell (Li et al., 2011, Science, 333: 1030-4; White et al., 2011, J Immunol, 187: 1754-63; White et al., 2014, J Immunol, 193: 1828-35).

[0061] As explained above, the term "antibody molecule" encompasses full-length or full-size antibody molecules, including a chimeric antibody, and derivatives of such antibody molecules. In a preferred embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody or fragment thereof comprises or consists of a full-length antibody or a chimeric antibody. The invention encompasses "fragments" of such antibody molecules, including functional fragments thereof, and derivatives of such fragments.

[0062] In an embodiment, the invention provides a single chain antibody; a Fab fragment also denoted F(ab) which is a monovalent antigen-binding fragment that does not contain a Fc part; a (Fab')2 fragment which is a divalent antigen-binding fragment that contains two antigen-binding Fab parts linked together by disulfide bond; a Fab' fragment also denoted F(ab') which is a monovalent-variant of a F(ab')2; an Fv fragment; and an scFv fragment.

[0063] Preferably within the context of the present invention, the fragment of a full-size antibody of the invention will have the same antigen binding characteristics as the corresponding full-size antibody, and / or will include either the same variable domains (i.e. the VH and VL sequences) and / or the same CDR sequences as the corresponding full-size antibody. That the fragment of a full-size antibody has the same antigen binding characteristics as the corresponding full-size antibody means that it binds to the same epitope on the target as the full-size antibody. Such a fragment of a full-size antibody may correspond to the Fv part of a full-size antibody.

[0064] A fragment of a full-size antibody may not contain all six CDRs of the corresponding full- size antibody. It will be appreciated that antibody fragments containing less than six CDRs (for example, three or fewer CDR regions, and in some cases, even just a single CDR or a part thereof) are capable of retaining the antigen-binding activity of the antibody from which the CDR(s) are derived.

[0065] For example, in Gao et al., 1994, J. Biol. Chem., 269: 32389-93 it is described that a whole VL chain (including all three CDRs) has a high affinity for its substrate.

[0066] Molecules containing two CDR regions are described, for example, by Vaughan & Sollazzo 2001, Combinatorial Chemistry & High Throughput Screening, 4: 417-430. On page 418 (right column - 3 Our Strategy for Design) a minibody including only the Hl and H2 CDR hypervariable regions interspersed within framework regions is described. The minibody is described as being capable of binding to a target. Pessi et al., 1993, Nature, 362: 367- 9 and Bianchi et al., 1994, J. Mol. Biol., 236: 649-59 are referenced by Vaughan & Sollazzo and describe the Hl and H2 minibody and its properties in more detail. In Qiu et al., 2007, Nature Biotechnology, 25:921-9 it is demonstrated that a molecule consisting of two linked CDRs are capable of binding antigen. Quiocho 1993, Nature, 362: 293-4 provides a summary of "minibody" technology. Ladner 2007, Nature Biotechnology, 25:875-7 comments that molecules containing two CDRs are capable of retaining antigen-binding activity.

[0067] Antibody molecules containing a single CDR region are described, for example, in Laune et al., 1997, JBC, 272: 30937-44, in which it is demonstrated that a range of hexapeptides derived from a CDR display antigen-binding activity and it is noted that synthetic peptides of a complete, single, CDR display strong binding activity. In Monnet et al., 1999, JBC, 274: 3789-96 it is shown that a range of 12-mer peptides and associated framework regions have antigen-binding activity and it is commented on that a CDR3-like peptide alone is capable of binding antigen. In Heap et al., 2005, J. Gen. Virol., 86: 1791-1800 it is reported that a "micro-antibody" (a molecule containing a single CDR) is capable of binding antigen and it is shown that a cyclic peptide from an anti-HIV antibody has antigenbinding activity and function. In Nicaise et al., 2004, Protein Science, 13: 1882-91 it is shown that a single CDR can confer antigen-binding activity and affinity for its lysozyme antigen.

[0068] Thus, antibody molecules, or fragments thereof, having five, four, three, two or one CDRs are capable of retaining the antigen binding properties of the full-length antibodies from which they are derived.

[0069] The term "antibody molecule, or fragment thereof" as used herein includes all types of antibody molecules, fragments thereof and derivatives thereof, including: monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multi-specific antibodies, bi-specific antibodies, human antibodies, antibodies of human origin, humanized antibodies, chimeric antibodies, or modifications of these antibodies.

[0070] The term "antibody molecule, or fragment thereof" also includes: single chain antibodies, single-chain Fvs (scFv), Fab fragments, F(ab')2 fragments, F(ab') fragments, disulfide- linked Fvs (sdFv), antibody heavy chains, antibody light chains, homo-dimers of antibody heavy chains, homo-dimers of antibody light chains, heterodimers of antibody heavy chains, heterodimers of antibody light chains, antigen binding functional fragments of such homo- and heterodimers.

[0071] In some embodiments of the present invention, the antibody molecule that binds specifically to Domain 4 of ICAM-1 completely lacks Fc region or has reduced binding to Fey receptors, which means that the antibody molecule that binds specifically to Domain 4 of ICAM-1 binds poorly to or cannot at all bind to or interact with Fey receptors. This has at least two therapeutically important consequences:

[0072] 1) lack of Fc-mediated binding to activatory FcyRs leaves a greater number of activatory Fc gamma receptors available for binding to Fc's of (other) therapeutic anti-cancer antibodies. This is important since clustering of an increasing number of activatory FcyRs (vs inhibitory FcyRs; Nimmerjahn et al; Science. 2005 Dec 2;310(5753): 1510-2) is known to increase effector cell mediated target cell deletion, a mechanism underlying activity of both checkpoint inhibitor, immune agonist, and other immunomodulatory antibodies, such as anti-IL-2R. 2) lack of, or reduced, Fc-mediated binding to inhibitory FcyR was shown to reduce inhibitory signalling in FcyR-expressing immune effector cells. Thus, lack of or reduced Fc-mediated binding to FcyR of the FcyRIIB targeting antibody likely improves therapeutic efficacy by at least two mechanisms, involving both improved activatory FcyR and reduced inhibitory Fey signalling in immune effector cells in response to a second immunomodulatory anti-cancer antibody.

[0073] "Reduced binding" or "binding with reduced affinity" means in this context that the antibody molecule has reduced Fc-mediated binding to Fey receptors, or in other words that the Fc region of the antibody molecule that specifically binds FcyRIIb binds to an activating Fey receptor with lower affinity than the Fc region of a normal human IgGl. The reduction in binding can be assessed using techniques such as surface plasmon resonance. In this context "normal IgGl" means a conventionally produced IgGl with a non-mutated Fc region that has not been produced so as to alter its glycosylation. As a reference for this "normal IgGl" it is possible to use rituximab produced in CHO cells without any modifications (Tipton et al, Blood 2015 125: 1901-1909; rituximab is described i.a. in EP 0 605 442).

[0074] "Reduced binding" means that binding of the Fc region of the antibody molecule that specifically binds FcyRIIb binds to an activating Fey receptor is at least 10 fold reduced for all Fc receptors compared to the binding of the Fc region of a normal human IgGl to the same receptors. In some embodiments it is at least 20 fold reduced. In some embodiments it is at least 30 fold reduced. In some embodiments it is at least 40 fold reduced. In some embodiments it is at least 50 fold reduced. In some embodiments it is at least 60 fold reduced. In some embodiments it is at least 70 fold reduced.

[0075] In some embodiments reduced binding means that the antibody has a 20 fold reduced affinity with regards to binding to FcyRI.

[0076] In order to obtain reduced binding of an IgGl antibody, such as an IgGl antibody, to an Fc receptor, it is possible to modify the Fc region of the IgG antibody by aglycosylation. Such aglycosylation, for example of an IgGl antibody, may for example be achieved by an amino acid substitution of the asparagine in position 297 (N297X) in the antibody chain. The substation may be with a glutamine (N297Q), or with an alanine (N297A), or with a glycine (N297G), or with an asparagine (N297D), or by a serine (N297S).

[0077] The Fc region may be modified by further substitutions, for example as described by Jacobsen FW et al., JBC 2017, 292, 1865-1875, (see e.g. Table 1). Such additional substitutions include L242C, V259C, A287C, R292C, V302C, L306C, V323C, I332C, and / or K334C. Such modifications also include the following combinations of substitutions in an IgGl:

[0078] L242C, N297G, K334C,

[0079] A287C, N297G, L306C,

[0080] R292C, N297G, V302C,

[0081] N297G, V323C, I332C, and

[0082] V259C, N297G, L306C.

[0083] Alternatively, the carbohydrate in the Fc region can be cleaved enzymatically and / or the cells used for producing the antibody can be grown in media that impairs carbohydrate addition and / or cells engineered to lack the ability to add the sugars can be used for the antibody production, or by production of antibodies in host cells that do not glycosylate or do not functionally glycosylate antibodies e.g. prokaryotes including E.coli, as explained above.

[0084] Reduced affinity for Fc gamma receptors can further be achieved through engineering of amino acids in the antibody Fc region (such modifications have previously been described by e.g. Xencor, Macrogenics, and Genentech), or by production of anti-bodies in host cells that do not glycosylate or does not functionally glycosylate antibodies e.g. prokaryotes including E. coli.

[0085] In addition to having reduced binding to Fey receptors through the Fc region, it is in some embodiments preferred that the antibody molecule that specifically binds FcyRIIb does not give rise to phosphorylation of FcyRIIb when binding the target. Phosphorylation of the ITIM of FcyRIIb is an inhibitory event that blocks the activity in the immune cell.

[0086] In some embodiments of the present invention, the antibody molecule that binds specifically to Domain 4 of ICAM-1 is selected from the group consisting of: a full-length antibody, a chimeric antibody, a single chain antibody, a Fab fragment, a (Fab')2 fragment, a Fab' fragment, a (Fab')2 fragment, a Fv fragment, an scFv fragment, or human IgG antibody molecule having an aglycosylated Fc region or an IgG antibody molecule of human origin having an aglycosylated Fc region.

[0087] In an embodiment, the antibody molecule, or fragment thereof, is a human antibody, i.e. an antibody molecule, or fragment thereof, obtained from a human (for example, obtained from human serum).

[0088] In an embodiment, the antibody molecule, or fragment thereof, is an antibody of human origin, i.e. an originally human antibody that has been modified as described herein.

[0089] In an embodiment, the antibody molecule, or fragment thereof, is a humanized antibody, i.e. an originally non-human antibody that has been modified to increase its similarity to a human antibody. The humanized antibodies may, for example, be of murine antibodies or lama antibodies.

[0090] Preferably, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody or fragment thereof is IgGl or an Fc-engineered variant thereof.

[0091] It is well known that antibodies, or fragments thereof, used for therapeutic purposes are often modified / engineered to enhance the properties of the antibody molecule.

[0092] Antibodies, or fragments thereof, can be Fc-engineered for improved binding to one or several activating Fc receptors, and / or engineered to provide FcyR independent agonism and / or to enhance antibody-mediated Treg depletion and / or T-cell immunostimulation.

[0093] In an embodiment, antibody molecule, or fragment thereof, is engineered to comprise amino acid mutations in the Fc region that enhance binding affinity to activator Fc receptors, such as FcyRI (CD64), FcyRIIa (CD32a), and FcyRIIIa (CD16a). These mutations can increase the strength of the interaction between the Fc region of the antibody and FcyRs, thereby promoting more efficient immune cell activation and effector functions, such as ADCC and phagocytosis. In an embodiment, the antibody molecule, or fragment thereof, is glycoengineered or glycosylated, for example where sugar moieties (glycans) are attached to specific amino acid residues within the Fc region. Glycoengineering approaches involve modifying the composition and structure of Fc glycans to enhance FcyR binding and effector activity. For example, increasing the levels of terminal sialic acid residues on Fc glycans triggers conformational changes of IgGl that enable interactions with type FcyRIIa.

[0094] In an embodiment, this modification involves replacing the amino acids at positions 234, 235, 237, 238, 318, and 319 of the IgGl Fc region with serine (S), aspartic acid (D), alanine (A), leucine (L), isoleucine (I), and glutamic acid (E), respectively. The "SDALIE" modification is a glycoengineering strategy aimed at enhancing the binding affinity of IgGl antibodies for Fey receptors (FcyRs), particularly FcyRIIIa (CD16a). This modification is achieved by introducing specific amino acid substitutions in the Fc region of IgGl antibodies, which optimize the glycosylation pattern of the Fc glycans. "SDALIE" modification was shown to result in enhanced binding to FcyRIIIa and improved ADCC.

[0095] In an embodiment, the Fc region of the antibody, or fragment thereof, is fused with other proteins or domains to create Fc fusion proteins with enhanced effector functions. For example, fusion of the Fc region with cytokines or immune checkpoint inhibitors can augment the antibody's ability to engage immune cells or modulate immune responses, leading to improved therapeutic outcomes.

[0096] Preferably, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof is a human IgGl antibody.

[0097] In an embodiment, the antibody, or fragment thereof, may be a human IgGl antibody having improved binding to one or several activating Fc receptors and / or engineered to improve binding to one or several activating Fc receptors. Accordingly, in an embodiment, the antibody, or fragment thereof, is an Fc-engineered human IgGl antibody.

[0098] In an embodiment, the antibody, or fragment thereof, is a mouse IgG2a antibody or a humanized mouse IgG2a antibody. The mouse IgG2a and human IgGl antibody isotypes productively engage with activating Fc gamma receptors and deplete target cells through activation of activating Fc gamma receptor-bearing immune cells (e.g. macrophages and NK cells), by e.g. ADCP and ADCC. As such, whereas the mouse IgG2a is the preferred isotype to use to achieve target cell depletion in the mouse, human IgGl is a preferred isotype to use to achieve target cell depletion in human.

[0099] Conversely, optimal agonistic co-stimulation of certain receptors (for example, TNFR superfamily receptors such as 4-1BB, 0X40, TNFRII, CD40) depends on antibody engagement of the inhibitory FcyRII. In the mouse the IgGl isotype, which binds preferentially to inhibitory Fc gamma receptor (FcyRIIB) and only weakly to activating Fc gamma receptors, is known to be optimal for co-stimulatory activity of TNFR-superfamily targeting mAb. While no direct equivalent of the mouse IgGl isotype has been described in man, antibodies may be engineered to show a similarly enhanced binding to inhibitory over activating human Fc gamma receptors. Such engineered antibodies also have improved co-stimulatory activity in vivo, in transgenic mice engineered to express human activating and inhibitory Fc gamma receptors (Dahan et al., Cancer Cell. 2016, 13;29(6):820-31).

[0100] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof is a monoclonal antibody.

[0101] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof comprises 1-6 of the CDRs VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, wherein VH-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs: 1, 9 and 17; wherein VH-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs: 2, 10 and 18; wherein VH-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs: 3, 11 and 19; wherein VL-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs: 4, 12 wherein VL-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs: 5, 13 and 21; and wherein VL-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs: 6, 14 and 22.

[0102] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof comprises a variable heavy chain (VH) comprising the following CDRs:

[0103] (i) SEQ. ID. NO: 1, SEQ. ID. NO: 2 and SEQ. ID. NO. : 3; or

[0104] (ii) SEQ. ID. NO: 9, SEQ. ID. NO: 10 and SEQ. ID. NO: 11; or

[0105] (iii) SEQ. ID. NO: 17, SEQ. ID. NO: 18 and SEQ. ID. NO: 19; and / or wherein the antibody molecule comprises a variable light chain (VL) comprising the following CDRs:

[0106] (i) SEQ. ID. NO: 4, SEQ. ID. NO: 5 and SEQ. ID. NO: 6; or

[0107] (ii) SEQ. ID. NO: 12, SEQ. ID. NO: 13 and SEQ. ID. NO: 14; or

[0108] (iii) SEQ. ID. NO: 20, SEQ. ID. NO: 21 and SEQ. ID. NO: 22.

[0109] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof comprises a variable heavy chain (VH) amino acid sequence selected from the group consisting of SEQ. ID. NOs 7, 15 and 23; and / or wherein the antibody molecule comprises a variable light chain (VL) amino acid sequence selected from the group consisting of SEQ. ID. NOs: 8, 16 and 24.

[0110] In an embodiment, the antibody molecule, or fragment thereof, comprises one of the VH- CDR1 sequences listed in Table 1 below. In another embodiment, the antibody molecule, or fragment thereof, comprises one of the VH-CDR2 sequences listed in Table 1 below. In a further embodiment, the antibody molecule, or fragment thereof, comprises one of the VH-CDR3 sequences listed in Table 1 below.

[0111] In an embodiment, the antibody molecule, or fragment thereof, comprises one of the VL- CDR1 sequences listed in Table 1 below. In another embodiments, the antibody molecule, or fragment thereof, comprises one of the VL-CDR2 sequences listed in Table 1 below. In a further embodiments, the antibody molecule, or fragment thereof, comprises one of the VL-CDR3 sequences listed in Table 1 below.

[0112] In an embodiment, the antibody molecule, or fragment thereof, comprises the six CDRs having SEQ. ID. NOs: 1, 2, 3, 4, 5 and 6; or the antibody molecule, or fragment thereof, comprises the six CDRs having SEQ. ID. NOs: 9, 10, 11, 12, 13 and 14; or the antibody molecule, or fragment thereof, comprises the six CDRs having SEQ. ID. NOs: 17, 18, 19, 20, 21 and 22.

[0113] In an embodiment, the antibody molecule, or fragment thereof, comprises a VH region selected from the group consisting of SEQ. ID. NOs: 7, 15 and 23.

[0114] In an embodiment, the antibody molecule, or fragment thereof, comprises a VL region selected from the group consisting of SEQ. ID. NOs: 8, 16 and 24.

[0115] In an embodiment, the antibody molecule, or fragment thereof, comprises a VH region having SEQ. ID. NO: 7. In another embodiment, the antibody molecule, or fragment thereof, comprises a VL having SEQ. ID. NO: 8.

[0116] In an embodiments, the antibody molecule, or fragment thereof, comprises a VH region having SEQ. ID. NO: 7 and a VL having SEQ. ID. NO: 8.

[0117] In an embodiment, the antibody molecule, or fragment thereof, comprises a CH region having SEQ. ID. NO: 25. In another embodiment, the antibody molecule, or fragment thereof, comprises a CL region having SEQ. ID. NO: 26.

[0118] Table 1. Specific sequences of antibody molecules according to the invention (in the VH and VL sequences, the CDR sequences are marked in bold text)

[0119] In an embodiment, the antibody molecule, or fragment thereof, comprises one or both of the constant regions (CH and / or CL) listed in Table 2 below. Table 2. The constant regions of antibodies of the invention

[0120] The CH (SEQ. ID. NO: 25) and the CL (SEQ. ID. NO: 26) sequences in Table 2 above are of human origin. The CH (SEQ. ID. NO 25) in Table 2 above corresponds to human gamma 1 heavy chain constant region; the CL (SEQ. ID. NO: 26) sequences in Table 2 above corresponds to human lambda light chain constant region.

[0121] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof is capable of competing for binding to Domain 4 of ICAM-1 with the an antibody molecule defined herein, for example an antibody molecule, or fragment thereof, comprising any of the amino acid sequences in SEQ. ID. NOs: 1-24 and / or Table 1.

[0122] By "capable of competing for" we mean that the competing antibody is capable of inhibiting or otherwise interfering, at least in part, with the binding of the antibody molecule, or fragment thereof, as defined herein to Domain 4 of ICAM-1.

[0123] For example, such a competing antibody molecule, or fragment thereof, may be capable of inhibiting the binding of the antibody molecule, or fragment thereof as defined herein by at least about 10%; for example at least about 20%, or at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100% and / or inhibiting the ability of the antibody defined herein to prevent or reduce binding to the specific target by at least about 10%; for example at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100%.

[0124] Competitive binding may be determined by methods well known to those skilled in the art, such as Enzyme-Linked Immunosorbent Assay (ELISA).

[0125] ELISA assays can be used to evaluate epitope-modifying or blocking antibodies. Additional methods suitable for identifying competing antibodies are disclosed in Antibodies: A Laboratory Manual, Harlow & Lane, 1988, CSHL, NY, ISBN 0-87969-314-2 which is incorporated herein by reference (for example, see pages 567 to 569, 574 to 576, 583 and 590 to 612).

[0126] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof binds specifically to human ICAM-1, for example human ICAM-1 which comprises or consists of the following sequence (SEQ. ID. NO: 27):

[0127] MAPSSPRPALPALLVLLGALFPGPGNAQTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKEL LLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQVE GGAPRANLTWLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSAPY QLQTFVLPATPPQLVSPRVLEVDTQGTWCSLDGLFPVSEAQVHLALGDQRLNPTVTYGNDSFSAKASV SVTAEDEGTQRLTCAVILGNQSQETLQTVTIYSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGV PAQPLGPRAQLLLKATPEDNGRSFSCSATLEVAGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQ QTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYEIVI ITWAAAVIMGTAGLSTYLYNRQRKIKKYRLQQAQKGTPMKPNTQATPP

[0128] In another embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof binds specifically to mouse ICAM-1, for example mouse ICAM-1 which comprises or consists of the following sequence (SEQ. ID. NO: 28):

[0129] MASTRAKPTLPLLLALVTWI PGPGDAQVSIHPREAFLPQGGSVQVNCSSSCKEDLSLGLETQWLKDEL ESGPNWKLFELSEIGEDSSPLCFENCGTVQSSASATITVYSFPESVELRPLPAWQQVGKDLTLRCHVDG GAPRTQLSAVLLRGEEILSRQPVGGHPKDPKEITFTVLASRGDHGANFSCRTELDLRPQGLALFSNVSE ARSLRTFDLPATI PKLDTPDLLEVGTQQKLFCSLEGLFPASEARIYLELGGQMPTQESTNSSDSVSATA

[0130] LVEVTEEFDRTLPLRCVLELADQILETQRTLTVYNFSAPVLTLSQLEVSEGSQVTVKCEAHSGSKWLL

[0131] SGVEPRPPTPQVQFTLNASSEDHKRSFFCSAALEVAGKFLFKNQTLELHVLYGPRLDETDCLGNWTWQE

[0132] GSQQTLKCQAWGNPSPKMTCRRKADGALLPIGWKSVKQEMNGTYVCHAFSSHGNVTRNVYLTVLYHSQ

[0133] NNWTI I ILVPVLLVIVGLVMAASYVYNRQRKIRIYKLQKAQEEAIKLKGQAPPP

[0134] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein ICAM-1 comprises or consists of a polypeptide sequence with at least 90% homology to SEQ. ID. NO: 27. Preferably, the ICAM-1 comprises or consists of a polypeptide sequence with at least 91% homology to SEQ. ID. NO: 27, at least 92% homology to SEQ. ID. NO: 27, at least 93% homology to

[0135] SEQ. ID. NO: 27, at least 94% homology to SEQ. ID. NO: 27, at least 95% homology to

[0136] SEQ. ID. NO: 27, at least 96% homology to SEQ. ID. NO: 27, at least 97% homology to

[0137] SEQ. ID. NO: 27, at least 98% homology to SEQ. ID. NO: 27, or at least 99% homology to SEQ. ID. NO: 27, or with 100% homology to SEQ. ID. NO: 27.

[0138] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the ICAM-1 comprises or consists of a polypeptide sequence with at least 90% homology to SEQ. ID. NO: 28. Preferably, the, ICAM-1 comprises or consists of a polypeptide sequence with at least 91% homology to SEQ. ID. NO: 28, at least 92% homology to SEQ. ID. NO: 28, at least 93% homology to SEQ. ID. NO: 28, at least 94% homology to SEQ. ID. NO: 28, at least 95% homology to

[0139] SEQ. ID. NO: 28, at least 96% homology to SEQ. ID. NO: 28, at least 97% homology to

[0140] SEQ. ID. NO: 28, at least 98% homology to SEQ. ID. NO: 28, or at least 99% homology to SEQ. ID. NO: 28, or with 100% homology to SEQ. ID. NO: 28.

[0141] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof binds specifically to Domain 4 of human ICAM-1, which preferably comprises or consists of the sequence (SEQ. ID. NO: 29):

[0142] SFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATLEV AGQLIHKNQTRELRVL In another embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof binds specifically to Domain 4 of mouse ICAM-1, which preferably comprises or consists of the sequence (SEQ. ID. NO: 30):

[0143] NFSAPVLTLSQLEVSEGSQVTVKCEAHSGSKWLLSGVEPRPPTPQVQFTLNASSEDHKRSFFCSAALE VAGKFLFKNQTLELHVL

[0144] As discussed in the accompanying Examples, an exemplary antibody that binds specifically to Domain 4 of ICAM-1 binds to Thr289 of the mouse ICAM-1 sequence of SEQ ID NO:30. In the corresponding human ICAM-1 sequence (SEQ ID NO:29), the residue is Ile289. Those residues are emboldened and underlined in SEQ ID NO: 29 and SEQ ID NO: 30 below:

[0145] SFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATLEV AGQLIHKNQTRELRVL ( SEQ ID NO : 29 )

[0146] NFSAPVLTLSQLEVSEGSQVTVKCEAHSGSKWLLSGVEPRPPTPQVQFTLNASSEDHKRSFFCSAALE VAGKFLFKNQTLELHVL ( SEQ ID NO : 30 )

[0147] In a particularly preferred embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof binds specifically to Ile289 of SEQ ID NO: 29. In another particularly preferred embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof binds specifically to Thr289 of SEQ ID NO: 30.

[0148] In a further embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof binds specifically to Domain 4 of rat ICAM-1, which preferably comprises or consists of the sequence (SEQ. ID. NO: 31):

[0149] NFSAPILTLSQPEVSEGDQVTVKCEAHGGAQWLLNSTSPRPPTSQGTSPRPPTSQIQFTLNASPEDHK RRFFCSAALEVDGKSLFKNQTLELHVL In a further embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof binds specifically to Domain 4 of Rhesus macaque ICAM-1, which preferably comprises or consists of the sequence (SEQ. ID. NO: 32):

[0150] SFPAPNVNLTKPEVSEGTEVIVECEAHPRAKVMLNGVPAQPPGPRAQFLLKATPEDNGRSFSCSATLEV AGQLVHKNQTRELRVL

[0151] In a further embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof binds specifically to Thr289 of SEQ ID NO: 31. In another embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule or fragment thereof binds specifically to Asn289 of SEQ ID NO: 32.

[0152] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein Domain 4 of ICAM-1 comprises or consists of a polypeptide sequence with at least 90% homology to SEQ. ID. NO: 29. Preferably, the Domain 4 of ICAM-1 comprises or consists of a polypeptide sequence with at least 91% homology to SEQ. ID. NO: 29, at least 92% homology to SEQ. ID. NO: 29, at least 93% homology to SEQ. ID. NO: 29, at least 94% homology to SEQ. ID. NO: 29, at least 95% homology to SEQ. ID. NO: 29, at least 96% homology to SEQ. ID. NO: 29, at least 97% homology to SEQ. ID. NO: 29, at least 98% homology to SEQ. ID. NO: 29, or at least 99% homology to SEQ. ID. NO: 29, or with 100% homology to SEQ. ID. NO: 29.

[0153] In another embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein Domain 4 of ICAM-1 comprises or consists of a polypeptide sequence with at least 90% homology to SEQ. ID. NO: 30. Preferably, the Domain 4 of ICAM-1 comprises or consists of a polypeptide sequence with at least 91% homology to SEQ. ID. NO: 30, at least 92% homology to SEQ. ID. NO: 30, at least 93% homology to SEQ. ID. NO: 30, at least 94% homology to SEQ. ID. NO: 30, at least 95% homology to SEQ. ID. NO: 30, at least 96% homology to SEQ. ID. NO: 30, at least 97% homology to SEQ. ID. NO: 30, at least 98% homology to SEQ. ID. NO: 30, or at least 99% homology to SEQ. ID. NO: 30, or with 100% homology to SEQ. ID. NO: 30. In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the antibody molecule, or fragment thereof, is administered with an anti-cancer agent, such as a therapeutic anti-cancer antibody molecule.

[0154] By the "therapeutic anti-cancer antibody molecule" we include the meaning of an antibody that specifically targets and / or interacts with immune or cancer cells, leading to various anti-tumor effects. These antibody molecules can be naturally occurring antibodies or engineered antibodies designed for therapeutic purposes. The therapeutic anti-cancer antibody molecule can be any antibody molecule as defined herein, or fragment thereof. In come embodiments, the therapeutic anti-cancer antibody molecule is a monoclonal antibody, a polyclonal antibody, a bispecific antibody, an immune checkpoint inhibitor antibody or an antibody-drug conjugate.

[0155] The antibody molecules, or fragments thereof, combinations of antibodies or fragments thereof with an anti-cancer agent, polynucleotide molecule, vector, viruses, cells and / or pharmaceutical compositions as defined herein, may be formulated for parenteral administration, including: as aqueous and / or non-aqueous sterile injection solutions which may contain anti-oxidants, and / or buffers, and / or bacteriostats, and / or solutes which render the formulation isotonic with the blood of the intended recipient; and / or as aqueous and / or non-aqueous sterile suspensions which may include suspending agents and / or thickening agents. The antibody molecules, or fragments thereof, combinations of antibodies or fragments thereof with an anti-cancer agent, polynucleotide molecule, vector, viruses, cells and / or pharmaceutical compositions defined herein may be presented in unitdose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (i.e. lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

[0156] Extemporaneous injection solutions and suspensions may be prepared from sterile powders, and / or granules, and / or tablets of the kind previously described.

[0157] In an embodiment, the antibody molecules, or fragments thereof, polynucleotide molecule, vector, viruses, cells and / or pharmaceutical compositions as defined herein are packaged in a hermetically sealed container, such as an ampoule or sachet indicating the quantity of antibody. In an embodiment, the antibody molecules, or fragments thereof, polynucleotide molecule, vector, viruses, cells and / or pharmaceutical compositions as defined herein are packaged in the same container as a therapeutic anti-cancer antibody molecule or anticancer agent. In an embodiment, the antibody molecules, or fragments thereof, combinations of antibodies or fragments thereof with an anti-cancer agent, polynucleotide molecule, vector, viruses, cells and / or pharmaceutical compositions as defined herein are supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted to the appropriate concentration for administration to a subject. In an embodiment, the antibody molecules, or fragments thereof, combinations of antibodies or fragments thereof with an anti-cancer agent, polynucleotide molecule, vector, viruses, cells and / or pharmaceutical compositions as defined herein are supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg.

[0158] In an embodiment, the antibody molecule, or fragment thereof, is supplied as a sterile lyophilized powder at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg in the same hermetically sealed container as a therapeutic anti-cancer antibody supplied as a sterile lyophilized powder at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg.

[0159] In an embodiment, antibody molecule, or fragment thereof, is supplied as a sterile lyophilized powder at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg in a different hermetically sealed container as a therapeutic anti-cancer antibody supplied as a sterile lyophilized powder at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized antibody molecules, or fragments thereof, should be stored at between 2°C. and 8°C. in their original container and the antibodies should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted.

[0160] In an embodiment, for parenteral administration to human patients, the daily dosage level of the antibody molecule, or fragment thereof, and / or the further therapeutic anti-cancer antibody molecule as described herein (such as anti-PD-1, anti-PD-Ll or anti-CTLA-4 antibody molecule) can be from 0.0001 mg / kg to 100 mg / kg of the patient's body weight independently for each antibody in the combination. Preferably, the dosage of the antibody molecule, or fragment thereof, and / or the further therapeutic anti-cancer antibody molecule as described herein administered to a patient is between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.10 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.25 mg / kg or 0.01 to 0.10 mg / kg of the patient's body weight. In some cases even up to 100 mg / kg administered in single or divided doses. In an embodiment, the dose is 10 mg / kg. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of the antibody molecule, or fragment thereof, may be reduced by enhancing uptake and tissue penetration of the antibody by modifications such as, for example, lipidation.

[0161] In an embodiment, the antibody molecule, or fragment thereof, is used in combination with a therapeutic anti-cancer antibody and the dosage administered to a patient are lower than when the antibody molecule, or fragment thereof, is used alone. Lower doses may be used in other circumstances, for example in combination with prolonged administration. The physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.

[0162] In an embodiment, a pharmaceutical composition (or medicament), or a kit defined herein will contain the anti-ICAM-l-Domain 4 antibody molecule, or fragment thereof (and optionally a therapeutic anti-cancer antibody molecule, such as anti-PD-1, anti-PD-Ll or anti-CTLA-4 antibody molecule) at a concentration of between approximately 2 mg / ml and 150 mg / ml or between approximately 2 mg / ml and 200 mg / ml. In an embodiment, the pharmaceutical compositions or a kit will contain the anti-ICAM-l-Domain 4 antibody molecule (and optionally the therapeutic anti-cancer antibody molecule) at a concentration of 10 mg / ml or 25 mg / ml. Preferably, the liquid form of the antibody molecules, or fragments thereof (and optionally the therapeutic anti-cancer antibody molecules) is supplied in a hermetically sealed container at least 1 mg / ml, more preferably at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 100 mg / ml, at least 150 mg / ml, at least 200 mg / ml of the antibodies. In an embodiment, the antibodies, or fragments thereof, are supplied as a liquid form at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg in the same hermetically sealed container as the therapeutic anti-cancer antibody supplied as a sterile lyophilized powder at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg.

[0163] In an embodiment, the antibody molecules, or fragments thereof, are supplied as a liquid form at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg in a different hermetically sealed container as the therapeutic anti-cancer antibody molecule supplied as a sterile lyophilized powder at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg.

[0164] The amount of the antibody molecules, or fragments thereof, which will be effective in the treatment, prevention or amelioration of one or more symptoms associated with cancer can be determined by standard clinical techniques. The precise dose to be employed in the pharmaceutical composition (or medicament), or a kit as defined herein will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0165] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the therapeutic anti-cancer antibody molecule is an immunostimulatory antibody molecule.

[0166] As used herein, the term "immunostimulatory antibody", or "immuno-stimulating antibody", refers to an antibody that upon administration to a subject, such as a human, specifically binds to a target present on the surface of an effector T cell. The binding of the immunostimulatory antibody to the target results in stimulation of an immune response, either directly through agonism, and / or indirectly through blockade of inhibitory signals (e.g. through antibody blockade of the PD1 / PDL1 axis), and / or through stimulation of an effector T cell. Such an effector T cell can be a CD8+ cell; in such embodiments, the immunostimulatory antibody is a CD8 activating and / or CD8 boosting antibody. Alternatively, or in addition, such an effector T cell can be a CD4+ cell; in such embodiments, the immunostimulatory antibody is a CD4 activating and / or CD4 boosting antibody.

[0167] The immunostimulatory antibody may be an antibody that agonizes an immune stimulatory receptor expressed on effector T cells, in an Fc:FcyR dependent manner, or an antibody that antagonises an immune checkpoint receptor, such as PD-1, expressed on effector T cells, in an Fc:FcyR-independent manner.

[0168] In an embodiment, the antibody molecule, or fragment thereof, does not have any immunostimulatory effects in addition to the Treg depleting effects. In an embodiment, the antibody molecule, or fragment thereof, also has an immunostimulatory effect, in addition to the Treg depleting effects; in such embodiments the antibody molecule, or fragment thereof, has a sufficiently poor immunostimulatory activity to allow for enhanced therapeutic activity when co-administered with the immunostimulatory antibody molecule.

[0169] In an embodiment, the immunostimulatory antibody molecule is a human antibody.

[0170] In an embodiment, the immunostimulatory antibody molecule is a humanized antibody, i.e. an originally non-human antibody that has been modified to increase its similarity to a human antibody. The humanized antibodies may, for example, be of murine antibodies or lama antibodies.

[0171] Furthermore, the immunostimulatory antibody molecule may be obtained by using an antibody in the form of a human IgG2 antibody, such as a human IgG2b antibody, or in the form of a human IgG4 antibody. Thus, in an embodiment the immunostimulatory antibody molecule is a human IgG2 antibody. In an embodiment the immunostimulatory antibody molecule is a human IgG2b antibody. In an embodiment the immunostimulatory antibody molecule is a human IgG4 antibody. An immunostimulatory antibody molecule may also be obtained by using a mouse or a humanized mouse IgGl antibody, and in an embodiment the immunostimulatory antibody molecule is a humanized mouse IgGl antibody.

[0172] In an embodiment, the immunostimulatory antibody molecule is an antibody showing enhanced binding to inhibitory over activating Fey receptors. In an embodiment, the immunostimulatory antibody molecule is an antibody showing enhanced binding to human FcyRIIB over activating Fey receptors.

[0173] In an embodiment, the immunostimulatory antibody molecule is engineered for enhanced binding to inhibitory over activating Fey receptors. In an embodiment, the immunostimulatory antibody molecule is engineered for enhanced binding to human FcyRIIB over activating Fey receptors.

[0174] To decide whether an antibody molecule has immunostimulatory effects, it is possible to use an in vitro agonism assay. For such an assay the following generic method may be used. Cell culture is in RPMI 1640 media (Gibco™) supplemented with 10% foetal calf serum, glutamine (2 mM), pyruvate (1 mM), penicillin, and streptomycin (100 lU / mL) at 37°C in 5% CO2. Fresh PBMCs are labelled with 2mM carboxyfluorescein succinimidyl ester (CFSE). PBMCs are then cultured in a 24-well plate at IxlO7cells / mL as described by Romer et al (Romer PS, Blood. 2011, 118(26):6772-82) for 48 hours prior to mAb stimulation assays. For PBMC stimulation, round-bottomed 96-well plates are wet- coated with O.Olpg / mL of OKT3 antibody (in-house) in PBS for 4 hours after which excess antibody is discarded and the plates are washed with PBS. 1X105PBMCs / well are transferred to the plates and stimulated with 5 pg / mL of test mAb. On day 4 or day 5 post-stimulation, cells are labelled with anti-CD8-APC (BioLegend), and anti-CD4-PE (inhouse) and proliferation is assessed by CFSE dilution on a FACSCalibur (BD Biosciences).

[0175] As explained above, the therapeutic anti-cancer antibody molecule can be used in conjunction with the antibody of the invention or fragment thereof in the various embodiments of the invention, including pharmaceutical compositions and kits of the invention.

[0176] In preferred embodiments, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the therapeutic anti- cancer antibody molecule is an anti-PD-1 antibody molecule or is an anti-PD-Ll antibody molecule or is an anti-CTLA-4 antibody molecule.

[0177] Preferably, the anti-PD-1 antibody molecule is pembrolizumab. Where the therapeutic anticancer antibody molecule is pembrolizumab, the antibody or fragment of the invention is used at a dose of approximately 25 mg / ml. In an embodiment, pembrolizumab is used at a dose of 200 mg (iv) every 3 weeks or at a dose of 400 mg (iv) every 6 weeks.

[0178] Preferably, the anti-PD-1 antibody is nivolumab. Where the therapeutic anti-cancer antibody molecule is nivolumab, the antibody or fragment of the invention is used at a dose of approximately 10 mg / ml. In an embodiment, nivolumab is used at a dose of 240 mg (iv) every 2 weeks or at a dose of 480 mg (iv) every 4 weeks. In other embodiments, nivolumab may be used in combination with the anti-CTLA-4 antibody ipilimumab, in which case nivolumab is used at a dose of 1 mg / kg every 3 weeks for a maximum of 4 doses or 3 mg / kg every 2 or 3 weeks.

[0179] Preferably, the anti-PD-Ll antibody is atezolizumab. Where the therapeutic anti-cancer antibody molecules is atezolizumab, the antibody or fragment of the invention is used at a dose of approximately 60 mg / ml. In an embodiment, atezolizumab is used at a dose of 840 mg (iv) every 2 weeks or at a dose of 1200 mg (iv) every 3 weeks or at a dose of 1680 mg (iv) every 4 weeks.

[0180] The anti-CTLA-4 antibody can, for example be ipilimumab (such as Yervoy® from Bristol- Myers Squibb) or tremelimumab (such as Imjudo® from AstraZeneca). Preferably, the anti-CTLA-4 is ipilimumab. The skilled person will understand that the standard tolerated therapeutic doses of ipilimumab can be determined from the approved drug labelling.

[0181] In some embodiments, the dose of ipilimumab is 10 mg / kg, such as an initial does of 10 mg / kg IV every 3 weeks for up to 4 doses followed by a maintenance dose of 10 mg / kg IV every 12 weeks for up to 3 years. In some embodiments, the dose of ipilimumab is 3 mg / kg IV every 3 weeks for up to 4 doses.

[0182] Treatment of a patient with the antibody molecules, or fragments thereof, combinations of antibodies, or fragments thereof with an anti-cancer agent, or pharmaceutical compositions as defined herein can include a single treatment or a series of treatments. For example, a subject is treated one time per week for between about 1 to 10 weeks, about 2 to 8 weeks, about 3 to 7 weeks, or about 4, 5, or 6 weeks. In other embodiments, the antibody molecules, or fragments thereof, combinations of antibodies, or fragments thereof with an anti-cancer agent, or pharmaceutical compositions are administered once a day, twice a day, or three times a day. In other embodiments, the antibody molecules, or fragments thereof, combinations of antibodies, or fragments thereof with an anti-cancer agent, or pharmaceutical compositions are administered once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year or once per year. It will also be appreciated that the effective dosage may increase or decrease over the course of a particular treatment.

[0183] Generally, in humans, oral or parenteral administration of the antibody molecules, polynucleotide molecules, vectors, viruses, cells and / or pharmaceutical compositions as defined herein is the preferred route, being the most convenient. For veterinary use, the antibody molecules, or fragments thereof, polynucleotide molecules, vectors, viruses, cells and / or pharmaceutical compositions defined herein are administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal. Thus, the present invention provides a pharmaceutical formulation comprising an amount of an antibody molecule, or fragments thereof, polynucleotide molecules, vectors, viruses and / or cells of the invention effective to treat cancer (as described above and further below).

[0184] Preferably, the antibody molecules, or fragments thereof, polynucleotide molecules, vectors, viruses, cells and / or pharmaceutical compositions as defined herein is adapted for delivery by a route selected from the group comprising: intravenous; intratumoral; intramuscular; subcutaneous. Administration can be in the form of a single injection or several repeated injections (e.g. with the same or different doses, with the same or different routes, at the same or different sites of administration).

[0185] In an embodiment, the antibody molecule or fragment thereof, and the therapeutic anticancer antibody molecule are administered simultaneously.

[0186] In an embodiment, the antibody molecule or fragment thereof is administered prior to administration of the therapeutic anti-cancer antibody molecule. This means that antibody molecule, or fragment thereof, is administered to the tumour first in order to achieve the Treg depleting effect. Once the Treg depleting effect is manifested, the therapeutic anticancer antibody molecule is administered. This sequential administration may be achieved by temporal separation of the two antibodies. Alternatively, or in combination with the first option, the sequential administration may also be achieved by spatial separation of the two antibodies, by administration of the antibody molecule, or fragment thereof, in a way, such as intratumoral, so that it reaches the tumour prior to the therapeutic anti-cancer antibody molecule, which is then administered in a way, such as systemically, so that it reaches the tumour after the antibody molecule, or fragment thereof.

[0187] In other embodiments the antibody molecule, or fragment thereof, and the therapeutic anti-cancer antibody molecule are administered less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, no more than 24 hours apart or no more than 48 hours apart. In preferred embodiments, two or more components are administered within the same patient visit.

[0188] To decide whether sequential treatment of the antibody molecule or fragment thereof, and therapeutic anti-cancer antibody molecule results in improved therapeutic activity, in vivo assays with immune competent animals bearing tumours and expressing activating and inhibitory Fc gamma receptors can be used. For such an assay, one described in the Examples below may be used.

[0189] In an embodiment, the anti-cancer agent is a chemotherapeutic agent.

[0190] There are a variety of chemotherapeutic agents available for treatment of cancer. A significant majority of cancer chemotherapy drugs act by inhibiting DNA synthesis, either directly, or indirectly by inhibiting the biosynthesis of the deoxyribonucleotide triphosphate precursors, to prevent DNA replication and concomitant cell division (See, for example, Gilman et al., Goodman and Gilman's: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Eighth Ed. (Pergamom Press, New York, 1990)). These agents, which include alkylating agents, such as nitrosourea, anti-metabolites, such as methotrexate and hydroxyurea, and other agents, such as etoposides, campathecins, bleomycin, doxorubicin, daunombicin, etc., although not necessarily cell cycle specific, kill cells during S phase because of their effect on DNA replication. Other agents, specifically colchicine and the vinca alkaloids, such as vinblastine and vincristine, interfere with microtubule assembly resulting in mitotic arrest. Taxanes interfere with microtubules that help move chromosomes during mitosis (cell division). Paclitaxel stops the growth of cancer cells and other dividing cells by blocking cell division. Carboplatin is a chemotherapy drug that belongs to the class of platinum-based drugs. Specifically, it is a second-generation platinum-containing compound, derived from cisplatin, another commonly used chemotherapy drug. Carboplatin exerts its anticancer effects by forming platinum-DNA adducts within the DNA of rapidly dividing cancer cells, leading to DNA damage and ultimately triggering apoptosis (programmed cell death). Like cisplatin, carboplatin interferes with DNA replication and transcription processes, inhibiting the proliferation of cancer cells. Chemotherapy protocols often involve administration of a combination of chemotherapeutic agents, or combination of immunotherapy and chemotherapy to increase the efficacy of treatment.

[0191] Anti-cancer agents can be used in conjunction with the antibodies of the invention, or fragments thereof, in the embodiments of the invention, including in pharmaceutical compositions and kits of the invention. Preferably, the chemotherapeutic agent is selected from the group consisting of: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; carboplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa- nl; interferon alfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include, but are not limited to: 20-epi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti- dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL- 2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+ myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N- acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. Preferred additional anti-cancer drugs are 5-fluorouracil and leucovorin. The antibody, or fragment thereof, of the invention may be administered to a subject simultaneously or sequentially with any of the above-referenced anti-cancer agents.

[0192] It would be known to the person skilled in medicine, that medicines can be modified with different additives, for example to change the rate in which the medicine is absorbed by the body; and can be modified in different forms, for example to allow for a particular administration route to the body.

[0193] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the cancer is a solid cancer, such as a sarcoma and / or carcinoma.

[0194] By "tumor" we include a neoplastic mass resulting from abnormal uncontrolled cell growth, which can be benign or malignant. Benign tumors generally remain localized. The term "malignant" generally means that the tumor can invade and destroy neighbouring body structures and spread to distant sites to cause death.

[0195] By "cancer" we include the meaning of any type of cancer. A "cancer" in a subject refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer is a disease involving cells that have the potential to metastasize to distal sites and exhibit phenotypic traits that differ from those of non-cancer cells, for example, formation of colonies in a three-dimensional substrate such as soft agar or the formation of tubular networks or weblike matrices in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancer cells do not form colonies in soft agar and form distinct sphere-like structures in three- dimensional basement membrane or extracellular matrix preparations. Cancer cells acquire a characteristic set of functional capabilities during their development, albeit through various mechanisms. Such capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion / metastasis, limitless explicative potential, and sustained angiogenesis.

[0196] Herein, the term "cancer" includes both pre-malignant and malignant cancer. In an embodiment, cancer refers to a benign tumor, which has remained localized. In other embodiments, cancer refers to a malignant tumor, which has invaded and destroyed neighbouring body structures and spread to distant sites. In yet other embodiments, the cancer is associated with a specific cancer antigen.

[0197] In an embodiment, the cancer is selected from the group consisting of: melanoma; lung cancer (including non-small cell lung cancer (NSCLC)); malignant pleural mesothelioma; head and neck squamous cell cancer (HNSCC);; urothelial cancer; renal cell carcinoma (RCC); microsatellite instability-high (MSI-H) or a mismatch repair deficient (dMMR) solid tumors; colon or rectal cancer (colorectal cancer); gastric (stomach) cancer; gastroesophageal junction (GEJ) adenocarcinoma; esophageal carcinoma; gastroesophageal junction (GEJ) carcinoma; cervical cancer ;ovarian cancer; hepatocellular carcinoma (HOC); biliary tract cancer (BTC); Merkel cell carcinoma (MCC); endometrial carcinoma; kidney cancer; liver cancer; pancreatic cancer; thyroid cancer; brain cancer; central nervous system cancer; cutaneous squamous cell carcinoma (cSCC); bladder cancer; prostate cancer; neuroblastoma; Wilms tumor; rhabdomyosarcoma; retinoblastoma; bone cancer; breast cancer, leukemia (including acute lymphocytic leukemia, Chronic myeloproliferative disease, acute non-lymphocytic leukemia, B cell acute lymphocytic leukemia, chronic lymphocytic leukemia, T cell acute lymphocytic leukemia, and chronic lymphoproliferative diseases), lymphoma (including classical Hodgkin lymphoma (cHL); non-Hodgkin lymphomas and primary mediastinal B-cell lymphoma (PMBCL)) and multiple myeloma.

[0198] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the cancer is a solid cancer. In an embodiment, the solid cancer is selected from the group consisting of: melanoma; lung cancer (including non-small cell lung cancer (NSCLC)); malignant pleural mesothelioma; head and neck squamous cell cancer (HNSCC); urothelial cancer; renal cell carcinoma (RCC); microsatellite instability-high (MSI-H) or a mismatch repair deficient (dMMR) solid tumors; colon or rectal cancer (colorectal cancer); gastric (stomach) cancer; gastroesophageal junction (GEJ) adenocarcinoma; esophageal carcinoma; gastroesophageal junction (GEJ) carcinoma; cervical cancer ;ovarian cancer; hepatocellular carcinoma (HOC); biliary tract cancer (BTC); Merkel cell carcinoma (MCC); endometrial carcinoma; kidney cancer; liver cancer; pancreatic cancer; thyroid cancer; brain cancer; central nervous system cancer; cutaneous squamous cell carcinoma (cSCC); bladder cancer; prostate cancer; neuroblastoma; Wilms tumor; rhabdomyosarcoma; retinoblastoma; bone cancer; and breast cancer.

[0199] In an embodiment, the invention provides an antibody molecule, or fragment thereof, for use, or a use, or a method, as disclosed herein wherein the cancer is a hematologic cancer. In an embodiment, the hematologic cancer is selected from the group consisting of: leukemia; lymphoma and multiple myeloma.

[0200] More specific examples of leukemic cancers are acute lymphocytic leukemia, Chronic myeloproliferative disease, acute non-lymphocytic leukemia, B cell acute lymphocytic leukemia, chronic lymphocytic leukemia, T cell acute lymphocytic leukemia, and chronic lymphoproliferative diseases. More specific examples of lymphoma cancers are classical Hodgkin lymphoma (cHL); non-Hodgkin lymphomas and primary mediastinal B-cell lymphoma (PMBCL).

[0201] The antibody molecules, or fragments thereof, and / or combinations of the antibody molecules or fragments thereof with anti-cancer agents, can be used to prevent, inhibit and / or reduce the growth of primary tumors or metastasis of cancerous cells that express or are related to the expression or over-expression of ICAM-1. In an embodiment, the antibody molecules, or fragments thereof, and / or combinations of antibody molecules or fragments thereof with anti-cancer agents, inhibits and / or reduces the growth of primary tumor and / or metastasis of cancerous cells by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the growth of primary tumor and / or metastasis in the absence of said antibody of the invention and / or a combination.

[0202] Each one of the above described cancers is well-known, and the symptoms and cancer diagnostic markers are well described, as are the therapeutic agents used to treat those cancers. Accordingly, the symptoms, cancer diagnostic markers, and therapeutic agents used to treat the above mentioned cancer types would be known to those skilled in medicine.

[0203] It should be noted that in the present invention, the term "patient" is used interchangeably with the term "subject".

[0204] We include that the subject could be mammalian or non-mammalian. Preferably, the mammalian subject is a human or is a non-mammalian, such as a horse, or a cow, or a sheep, or a pig, or a camel, or a dog, or a cat. Most preferably, the mammalian subject is a human.

[0205] The patient may exhibit signs or symptoms that suggest that they have cancer. By "exhibit", we include that the subject displays a cancer symptom and / or a cancer diagnostic marker, and / or the cancer symptom and / or a cancer diagnostic marker can be measured, and / or assessed, and / or quantified.

[0206] It would be readily apparent to the person skilled in medicine what the cancer symptoms and cancer diagnostic markers would be and how to measure and / or assess and / or quantify whether there is a reduction or increase in the severity of the cancer symptoms, or a reduction or increase in the cancer diagnostic markers; as well as how those cancer symptoms and / or cancer diagnostic markers could be used to form a prognosis for the cancer.

[0207] Cancer treatments are often administered as a course of treatment, which is to say that the therapeutic agent is administered over a period of time. The length of time of the course of treatment will depend on a number of factors, which could include the type of therapeutic agent being administered, the type of cancer being treated, the severity of the cancer being treated, and the age and health of the subject, amongst others reasons.

[0208] Clinical definitions of the diagnosis, prognosis and progression of a large number of cancers rely on certain classifications known as staging. Those staging systems act to collate a number of different cancer diagnostic markers and cancer symptoms to provide a summary of the diagnosis, and / or prognosis, and / or progression of the cancer. It would be known to the person skilled in oncology how to assess the diagnosis, and / or prognosis, and / or progression of the cancer using a staging system, and which cancer diagnostic markers and cancer symptoms should be used to do so.

[0209] By "cancer staging", we include the Rai staging, which includes stage 0, stage I, stage II, stage III and stage IV, and / or the Binet staging, which includes stage A, stage B and stage C, and / or the Ann Arbour staging, which includes stage I, stage II, stage III and stage IV. It is known that cancer can cause abnormalities in the morphology of cells. These abnormalities often reproducibly occur in certain cancers, which means that examining these changes in morphology (otherwise known as histological examination) can be used in the diagnosis or prognosis of cancer. Techniques for visualizing samples to examine the morphology of cells, and preparing samples for visualization, are well known in the art; for example, light microscopy or confocal microscopy.

[0210] By "histological examination", we include the presence of small, mature lymphocyte, and / or the presence of small, mature lymphocytes with a narrow border of cytoplasm, the presence of small, mature lymphocytes with a dense nucleus lacking discernible nucleoli, and / or the presence of small, mature lymphocytes with a narrow border of cytoplasm, and with a dense nucleus lacking discernible nucleoli, and / or the presence of atypical cells, and / or cleaved cells, and / or prolymphocytes.

[0211] It is well known that cancer is a result of mutations in the DNA of the cell, which can lead to the cell avoiding cell death or uncontrollably proliferating. Therefore, examining these mutations (also known as cytogenetic examination) can be a useful tool for assessing the diagnosis and / or prognosis of a cancer. An example of this is the deletion of the chromosomal location 13ql4.1 which is characteristic of chronic lymphocytic leukaemia. Techniques for examining mutations in cells are well known in the art; for example, fluorescence in situ hybridization (FISH).

[0212] By "cytogenetic examination", we include the examination of the DNA in a cell, and, in particular the chromosomes. Cytogenetic examination can be used to identify changes in DNA which may be associated with the presence of a refractory cancer and / or relapsed cancer. Such may include: deletions in the long arm of chromosome 13, and / or the deletion of chromosomal location 13ql4.1, and / or trisomy of chromosome 12, and / or deletions in the long arm of chromosome 12, and / or deletions in the long arm of chromosome 11, and / or the deletion of llq, and / or deletions in the long arm of chromo-some 6, and / or the deletion of 6q, and / or deletions in the short arm of chromosome 17, and / or the deletion of 17p, and / or the t(ll:14) translocation, and / or the (ql3:q32) translocation, and / or antigen gene receptor rearrangements, and / or BCL2 rearrangements, and / or BCL6 rearrangements, and / or t(14: 18) translocations, and / or t(ll: 14) translocations, and / or (ql3:q32) translocations, and / or (3:v) translocations, and / or (8: 14) translocations, and / or (8:v) translocations, and / or t(ll: 14) and (ql3:q32) translocations.

[0213] It is known that subjects with cancer exhibit certain physical symptoms, which are often as a result of the burden of the cancer on the body. Those symptoms often reoccur in the same cancer, and so can be characteristic of the diagnosis, and / or prognosis, and / or progression of the disease. A person skilled in medicine would understand which physical symptoms are associated with which cancers, and how assessing those physical systems can correlate to the diagnosis, and / or prognosis, and / or progression of the dis-ease. By "physical symptoms", we include hepatomegaly, and / or splenomegaly.

[0214] In a further aspect, the invention provides an antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1, as defined herein in the above-described aspects and embodiments of the invention.

[0215] Preferably, the antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1, comprises 1-6 of the CDRs VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, wherein VH-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs: 1, 9 and 17; wherein VH-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs: 2, 10 and 18; wherein VH-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs: 3, 11 and 19; wherein VL-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs: 4, 12 and 20; wherein VL-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs: 5, 13 and 21; and wherein VL-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs: 6, 14 and 22. It is preferred that the an antibody molecule, or fragment thereof, comprises a variable heavy chain (VH) comprising the following CDRs:

[0216] (i) SEQ. ID. NO: 1, SEQ. ID. NO: 2 and SEQ. ID. NO. : 3; or

[0217] (ii) SEQ. ID. NO: 9, SEQ. ID. NO: 10 and SEQ. ID. NO: 11; or

[0218] (iii) SEQ. ID. NO: 17, SEQ. ID. NO: 18 and SEQ. ID. NO: 19; and / or wherein the antibody molecule comprises a variable light chain (VL) comprising the following CDRs:

[0219] (i) SEQ. ID. NO: 4, SEQ. ID. NO: 5 and SEQ. ID. NO: 6; or

[0220] (ii) SEQ. ID. NO: 12, SEQ. ID. NO: 13 and SEQ. ID. NO: 14; or

[0221] (iii) SEQ. ID. NO: 20, SEQ. ID. NO: 21 and SEQ. ID. NO: 22.

[0222] More preferably, the antibody molecule, or fragment thereof, comprises a variable heavy chain (VH) amino acid sequence selected from the group consisting of SEQ. ID. NOs 7, 15 and 23; and / or wherein the antibody molecule comprises a variable light chain (VL) amino acid sequence selected from the group consisting of SEQ. ID. NOs: 8, 16 and 24.

[0223] In an embodiment, the antibody molecule, or fragment thereof, comprises one of the VH- CDR1 sequences listed in Table 1. In another embodiment, the antibody molecule, or fragment thereof, comprises one of the VH-CDR2 sequences listed in Table 1. In a further embodiment, the antibody molecule, or fragment thereof, comprises one of the VH-CDR3 sequences listed in Table 1.

[0224] In an embodiment, the antibody molecule, or fragment thereof, comprises one of the VL- CDR1 sequences listed in Table 1. In another embodiments, the antibody molecule, or fragment thereof, comprises one of the VL-CDR2 sequences listed in Table 1. In a further embodiments, the antibody molecule, or fragment thereof, comprises one of the VL-CDR3 sequences listed in Table 1.

[0225] In an embodiment, the antibody molecule, or fragment thereof, comprises the six CDRs having SEQ. ID. NOs: 1, 2, 3, 4, 5 and 6; or the antibody molecule, or fragment thereof, comprises the six CDRs having SEQ. ID. NOs: 9, 10, 11, 12, 13 and 14; or the antibody molecule, or fragment thereof, comprises the six CDRs having SEQ. ID. NOs: 17, 18, 19, 20, 21 and 22. In an embodiment, the antibody molecule, or fragment thereof, comprises a VH region selected from the group consisting of SEQ. ID. NOs: 7, 15 and 23.

[0226] In an embodiment, the antibody molecule, or fragment thereof, comprises a VL region selected from the group consisting of SEQ. ID. NOs: 8, 16 and 24.

[0227] In an embodiment, the antibody molecule, or fragment thereof, comprises a VH region having SEQ. ID. NO: 7. In another embodiment, the antibody molecule, or fragment thereof, comprises a VL having SEQ. ID. NO: 8.

[0228] In an embodiments, the antibody molecule, or fragment thereof, comprises a VH region having SEQ. ID. NO: 7 and a VL having SEQ. ID. NO: 8.

[0229] In an embodiment, the antibody molecule, or fragment thereof, comprises a CH region having SEQ. ID. NO: 25. In another embodiment, the antibody molecule, or fragment thereof, comprises a CL region having SEQ. ID. NO: 26.

[0230] In an embodiment, the invention provides an antibody molecule, or fragment thereof, is a monoclonal antibody.

[0231] In another aspect, the invention provides a polynucleotide molecule encoding an antibody, or fragment thereof, as defined herein.

[0232] A polynucleotide molecule encoding the antibody, or fragment thereof, may be generated from nucleic acid from a suitable source (e.g. a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express the antibody or fragment thereof) by hybridization with Ig specific probes and / or PCR amplification using synthetic primers hybridisable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody or fragment thereof. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art. Once the polynucleotide molecule of the antibody is determined, the polynucleotide molecule encoding the antibody may be manipulated using methods well known in the art for the manipulation of polynucleotide molecules, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1989, MOLECULAR CLONING, A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y ; see also, Ausubel et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY series of laboratory technique manuals, 1987-1997, Current Protocols, 1994-1997 John Wiley and Sons, Inc.), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and / or insertions

[0233] In addition, polynucleotide molecules to be inserted in a virus can be optimized for providing high level expression in a particular host cell or subject by modifying one or more codon(s). Further to optimization of the codon usage, various modifications may also be envisaged so as to prevent clustering of rare, non-optimal codons being present in concentrated areas and / or to suppress or modify "negative" sequence elements which are expected to negatively influence expression levels. Such negative sequence elements include without limitation the regions having very high (>80%) or very low (<30%) GC content; AT-rich or GC-rich sequence stretches; unstable direct or inverted repeat sequences; R A secondary structures; and / or internal cryptic regulatory elements such as internal TATA-boxes, chi-sites, ribosome entry sites, and / or splicing donor / acceptor sites.

[0234] In an embodiment, the antibody molecule, or fragment thereof, may be administered through the use of vectors (for example through the use of plasmids, bacteriophages, cosmids, artificial chromosomes) or viruses.

[0235] Accordingly, in another aspect, the invention provides a vector comprising a polynucleotide molecule as defined herein. Preferably, the vector comprising a polynucleotide molecule as defined herein is a plasmid. Such vector then comprise a polynucleotide molecule encoding an antibody of the invention, or fragment thereof.

[0236] In an embodiment, a polynucleotide molecule encoding parts of, or the full sequences of the antibody molecule, or fragment thereof, is integrated in a cell or viral genome or in a viriome in a virus; such a cell or virus then act as a delivery vehicle for the antibody molecule, or fragment thereof, (or a delivery vehicle for a polynucleotide molecule encoding the antibody molecule, or fragment thereof). For example, in an embodiment, such a virus may be in the form of a therapeutic oncolytic virus comprising a polynucleotide molecule encoding the antibody molecule, or fragment thereof, as defined herein. Oncolytic viruses are known to those skilled in the arts of medicine and virology.

[0237] Accordingly, in a further aspect, the invention provides a virus comprising a polynucleotide molecule as defined herein, and / or a vector as defined herein.

[0238] In an embodiment, the virus comprises a polynucleotide molecule encoding an amino acid sequence having at least 80% identity with a sequence set out in Table 1 above. In an embodiment, the virus comprises a polynucleotide molecule encoding an amino acid sequence having at least 85% identity with a sequence set out in Table 1 above. In an embodiment, the virus comprises a polynucleotide molecule encoding an amino acid sequence having at least 90% identity with a sequence set out in Table 1 above. In an embodiment, the virus comprises a polynucleotide molecule encoding an amino acid sequence having at least 95% identity with a sequence set out in Table 1 above.

[0239] In a further aspect, the invention provides a cell comprising a polynucleotide molecule as defined herein, and / or a vector as defined herein, and / or a virus as defined herein.

[0240] A vector comprising the polynucleotide molecule encoding the antibody, or fragment thereof can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the antibody or fragment of the invention.

[0241] In an embodiment, polynucleotide molecule(s) are placed under the control of suitable regulatory elements for their proper expression in a host cell or subject. As used herein, the term "regulatory elements" refers to any element that allows, contributes or modulates the expression of the encoding nucleotide sequence(s) in a given host cell or subject, including their replication, duplication, transcription, splicing, translation, stability and / or transport in or outside the expressing cell. It will be appreciated by those skilled in the art that the choice of the regulatory elements can depend on such factors as the polynucleotide molecule (nucleotide sequence) itself, the vector or virus into which it is inserted, the host cell or subject, the level of expression desired, etc. The promoter is of special importance. In the context of the invention, it can be constitutive directing expression of the polynucleotide molecule that it controls in many types of host cells or specific to certain host cells or regulated in response to specific events or exogenous factors (e.g. by temperature, nutrient additive, hormone, etc.) or according to the phase of a viral cycle (e.g. late or early). Promoters adapted to virus-mediated expression are known in the art.

[0242] The host cells used to express the antibody molecules, or fragments thereof, may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant immunoglobulin molecule. In particular, mammalian cells such as Chinese Hamster Ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins.

[0243] In another aspect, the invention provides an antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1 for use in medicine.

[0244] In a further aspect, the invention provides a pharmaceutical composition comprising or consisting of the antibody molecule, or fragment thereof, as defined herein, and optionally a pharmaceutically acceptable diluent, carrier, vehicle and / or excipient.

[0245] In a still further aspect, the invention provides a pharmaceutical composition comprising or consisting of a polynucleotide molecule as defined herein, a vector as defined herein, a virus as defined herein and / or a cell as defined herein, and optionally a pharmaceutically acceptable diluent, carrier, vehicle and / or excipient.

[0246] Pharmaceutically acceptable diluents, carriers, vehicles and / or excipients are known in the art.

[0247] In an embodiment, the pharmaceutical composition comprises other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, or diluents.

[0248] For injection, the carrier will typically be a liquid. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. For other methods of administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and will preferably be in solid or liquid particulate form.

[0249] Pharmaceutical compositions suitable for oral administration can be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the composition of this invention; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Oral delivery can be performed by complexing a composition of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers include plastic capsules or tablets, as known in the art. Such formulations are prepared by any suitable method of pharmacy, which includes the step of bringing into association the composition and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the pharmaceutical composition according to embodiments of the present invention are prepared by uniformly and intimately admixing the composition with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet can be prepared by compressing or moulding a powder or granules containing the composition, optionally with one or more accessory ingredients. Compressed tablets are prepared by compressing, in a suitable machine, the composition in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and / or surface active / dispersing agent(s). Moulded tablets are made by moulding, in a suitable machine, the powdered compound moistened with an inert liquid binder.

[0250] Pharmaceutical compositions suitable for buccal (sub-lingual) administration include lozenges comprising the composition of this invention in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia.

[0251] Pharmaceutical compositions suitable for parenteral administration can comprise sterile aqueous and non-aqueous injection solutions of the composition of this invention, which preparations are optionally isotonic with the blood of the intended recipient. These preparations can contain anti-oxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended recipient. Aqueous and nonaqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0252] The compositions can be presented in unit / dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for- injection immediately prior to use.

[0253] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, an injectable, stable, sterile composition of this invention in a unit dosage form in a sealed container can be provided. The composition can be provided in the form of a lyophilizate, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The unit dosage form can be from about 1 pg to about 10 grams of the composition of this invention. When the composition is substantially water-insoluble, a sufficient amount of emulsifying agent, which is physiologically acceptable, can be included in sufficient quantity to emulsify the composition in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

[0254] Pharmaceutical compositions suitable for rectal administration can be presented as unit dose suppositories. These can be prepared by admixing the composition with one or more conventional solid carriers, such as for example, cocoa butter and then shaping the resulting mixture.

[0255] Pharmaceutical compositions of this invention suitable for topical application to the skin can take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers that can be used include, but are not limited to, petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof. In an embodiment, for example, topical delivery can be performed by mixing a pharmaceutical composition of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.

[0256] Pharmaceutical compositions suitable for transdermal administration can be in the form of discrete patches adapted to remain in intimate contact with the epidermis of the subject for a prolonged period of time. Compositions suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Tyle, Pharm. Res., 1986, 3:318-326) and typically take the form of an optionally buffered aqueous solution of the composition of this invention. Suitable formulations can comprise citrate or bis\tris buffer (pH 6) or ethanol / water and can contain from 0.1 to 0.2M active ingredient.

[0257] The compositions disclosed herein may be administered to the lungs of a subject by any suitable means, for example, by administering an aerosol suspension of respirable particles comprised of the compositions, which the subject inhales. The respirable particles may be liquid or solid. Aerosols of liquid particles comprising the compositions may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles comprising the compositions of the invention may likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.

[0258] In an embodiment, the antibody molecules, or fragments thereof, polynucleotide molecules, vectors, viruses, cells and / or pharmaceutical compositions defined herein comprise pharmaceutically acceptable acid or base addition salts of the polypeptide binding moieties of the present invention. The acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful in this invention are those which form non-toxic acid addition salts, i.e. salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p- toluenesulphonate and pamoate [i.e. 1 ,l'-methylene-bis-(2-hydroxy-3 naphthoate)] salts, among others. Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the agents according to the present invention. The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of the present agents that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g. potassium and sodium) and alkaline earth metal cations (e.g. calcium and magnesium), ammonium or water-soluble amine addition salts such as N- methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others. The antibody molecules, or fragments thereof, polynucleotide molecules, vectors, viruses and / or cells defined herein may be lyophilised for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilisation method (e.g. spray drying, cake drying) and / or reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of antibody activity loss (e.g. with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted upward to compensate. In one embodiment, the lyophilised (freeze dried) polypeptide binding moiety loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (prior to lyophilisation) when re-hydrated.

[0259] In an embodiment, the viral composition is suitably buffered at a physiological or slightly basic pH (e.g. from approximately pH 7 to approximately pH 9 with a specific preference for a pH comprised between 7 and 8.5 and more particularly close to 8). It might be beneficial to also include in the viral composition a monovalent salt so as to ensure an appropriate osmotic pressure. Said monovalent salt may notably be selected from NaCI and KCI, preferably said monovalent salt is NaCI, preferably in a concentration of 10 to 500 mM (e.g. 50 mM). A suitable viral composition comprises saccharose 50 g / L, NaCI 50 mM, Tris-HCI 10 mM and Sodium glutamate 10 mM, pH8. The composition may also be formulated so as to include a cryoprotectant for protecting the oncolytic virus at low storage temperature. Suitable cryoprotectants include without limitation sucrose (or saccharose), trehalose, maltose, lactose, mannitol, sorbitol and glycerol, preferably in a concentration of 0.5 to 20% (weight in g / volume in L, referred to as w / v) as well as high molecular weight polymers such as dextran or polyvinylpyrrolidone (PVP). In a further aspect, the invention provides a pharmaceutical composition as defined herein for use in the treatment of cancer.

[0260] In a further aspect, the invention provides a pharmaceutical composition as defined herein for use in the manufacture of a medicament for the treatment of cancer in a patient.

[0261] In a further aspect, the invention provides a method for treating cancer in a patient, the method comprising the step of administering the pharmaceutical composition as defined herein to the patient.

[0262] In a further aspect, the invention provides a kit for performing the methods and / or uses of the invention, for example for use in the treatment of cancer in a patient. Preferably, the kit comprises or consists of an antibody molecule, or fragment thereof, as defined herein, and / or a polynucleotide molecule as defined herein, and / or a vector as defined herein, and / or a virus as defined herein, and / or a cell as defined herein and / or a pharmaceutical composition as defined herein.

[0263] In a preferred embodiment, the invention provides a kit comprising an antibody molecule, or fragment thereof, which further comprises an anti-cancer agent as defined herein, such as a therapeutic anti-cancer antibody molecule, and / or a chemotherapeutic agent as defined herein.

[0264] In a preferred embodiment, the kit comprises or consists of an antibody molecule or fragment thereof of the invention, and further comprises an immunostimulatory antibody molecule, as defined herein. It is particularly preferred that the immunostimulatory antibody molecule is an anti-PD-1, anti-PD-Ll or an anti-CTLA-4 antibody molecule.

[0265] In a particularly preferred embodiment, the kit comprises or consists of an antibody molecule or fragment thereof of the invention, and the anti-PD-1 antibody molecule, pembrolizumab.

[0266] In a particularly preferred embodiment, the kit comprises or consists of an antibody molecule or fragment thereof of the invention, and the anti-PD-1 antibody, nivolumab. In a particularly preferred embodiment, the kit comprises or consists of an antibody molecule or fragment thereof of the invention, and the anti-PD-Ll antibody, atezolizumab.

[0267] In a particularly preferred embodiment, the kit comprises or consists of an antibody molecule or fragment thereof of the invention, and the anti-CTLA-4 antibody molecule, ipilimumab.

[0268] In a particularly preferred embodiment, the kit comprises or consists of an antibody molecule or fragment thereof of the invention, and the anti-CTLA-4 antibody molecule, tremelimumab.

[0269] In a preferred embodiment, the kit comprises or consists of an antibody molecule or fragment thereof of the invention, and further comprises a chemotherapeutic agent (such as carboplatin and / or paclitaxel) instead or in addition to the immunostimulatory antibody molecule.

[0270] In another embodiment, a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of cancer in one or more containers.

[0271] Kits may comprise one or more containers (such as vials, tubes, and the like) configured to contain the reagents used in the methods defined herein, and optionally may contain instructions or protocols for using such reagents.

[0272] In another aspect, the invention provides an antibody molecule, or fragment thereof, for use; or a use; or a method; or an antibody; or a polynucleotide molecule; or a vector; or a cell; or a pharmaceutical composition; or a kit; as defined herein with reference to the accompanying description, examples and / or figures.

[0273] Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following drawings and examples:

[0274] FIGURES Figure 1 shows generation of tumor-Treg specific mAbs. (A) Schematic of phage panning strategy employed to isolate Treg-associated scFv. (B) 320 scFv-phages from the Treg phage pool were screened for binding to primary tissue and cell lines by flow cytometry. The MFI of binding was then plotted and clustered to reveal a grouping of scFv which demonstrated similar Treg-enriched specificity for further evaluation. (C) scFv from the Treg cluster (outlined in the box labelled Treg Binders) in (B) were produced as bivalent full length IgG and specificity towards T cells confirmed by flow cytometry on immune cells isolated from CT26 tumors and spleen. Shown are representative examples demonstrating tumor T cell enriched, tumor CD4 binders and tumor Treg specific binding. (D) The mean MFI values from (C) were plotted for all mAbs screened in this manner and are presented as a heatmap to show relative binding levels to different T cells populations from different locations.

[0275] Figure 2 shows that tumor specific depletion of Tregs confers therapeutic responses in the CT26 model. (A) CT26 tumor bearing mice were treated with three doses of mAb over 7 days, before blood, spleen, td-LN (tumor draining inguinal lymph node) and tumor were collected 3 days after the last dose. The tissue was processed into a single cell suspension and the proportion of immune cell populations were assessed by flow cytometry. The foldchange in proportion of Tregs (based on percentage of total CD45+) when compared to isotype treated mice was plotted, where a decrease below 1 indicated depletion and an increase indicated expansion of the Treg population. The number of mice for each group is provided on the x-axis. (B) The CD8:Treg ratio for the same individuals in A was also plotted. Statistically significant changes across the tissues for each individual mAb was determined by one-way ANOVA. (C) The mean fold-change in Treg population was plotted for each mAb across each tissue and arranged based on the euclidean clustering in R to determine similar activities between mAbs. This revealed four distinct groups (emphasised by bold border). Where a statistically significant change from the isotype control was found the corresponding cell within the heatmap was marked based on the p-value obtained. (D) to (E) The MFI of mAb binding (from Figure ID) was plotted against the average Treg foldchange (from Figure 2A) for the Spleen (D) and Tumor (E). Linear regression was assessed in prism and the R2 value for each tissue provided in the plot. (F) CT26 tumor bearing mice were treated with three doses of mAb and tumor growth tracked for survival. The plots are grouped based on the clusters of Treg-depleting activity identified in 2C. Data from survival data is compiled across multiple independent experiments - each one controlled for with an isotype-control treated group. P-values are represented as follows * = 0.05, ** = 0.005, *** = 0.0005, **** < 0.0001. p-values > 0.05 were left blank.

[0276] Figure 3 shows benchmarking of newly identified mAbs against anti-CD25 mAb in CT26 tumors. Mice with CT26 tumors s.c. were treated with three doses of 200 pg of mAb on days 0, 4 and 7 (where day 0 is once tumor becomes palpable). (A) Tumor growth was measured three times a week and curves are presented for each mouse. The mean of the tumor growth for the isotype control group is plotted in the black dashed line for all plots.

[0277] (B) Overall survival between the groups is presented. Number of mice for each group is present on the plots. Responses representative of at least two independent experiments.

[0278] (C) Schematic of in vivo tolerability study; 28-day old mice were treated with lOmg / kg of either anti-CTLA-4 or 12-D10 (alone or combined with anti-PD-1) twice weekly for three weeks. Serum was collected 24 hours after last dose, and spleen was collected on day 65.

[0279] (D) Serum ELISA measuring cardiac troponin I (TNNI3), spleen mass and the mean corpuscular volume (MCV) are presented. One-way ANOVA was performed. P-values are represented as follows * = 0.05, ** = 0.005, *** = 0.0005, **** < 0.0001. p-values > 0.05 were left blank.

[0280] Figure 4 shows that 12-D10 binds ICAM-1 and is able to maintain specificity and depletion activity in different tumor models. (A-B) Flow cytometry data confirming that 12-D10 binds Treg cells from the tumor of CT26 bearing mice. N = 3 from one independent experiment. (C-D) Flow cytometry data confirming that 12-D10 also binds Treg cells from the tumor of MC38 bearing mice. Representative data from one independent experiment, N = 3. (E) Compiled CT26 survival data demonstrating that treating with three doses of 12-D10 cures approximately 50% of mice. N = 17, pooled from three independent experiments. (F) 12-D10-mIgG2a results in a clear depletion of Treg cells from the tumor, whilst leaving Tregs in the spleen and td-LN untouched. (G) 12-D10+ and 12-D10- Tregs from MC38 tumors were co-stained for additional T cell markers. 12-D10 binds mouse ICAM-1 as demonstrated by (H) ELISA, (I) flow cytometry of m-ICAM-1 transfected (but not control non -transfected) CHO-S cells and (J) can be blocked by pre-incubating the antibody with recombinant ICAM-1 protein.

[0281] Figure 5 shows that 12-D10 binds the fourth domain of mICAM-1. (A) MFI of 12-D10-PE and a commercial anti-mICAM-1 PE mAb binding to Treg, CD8+ T cells and NK cells isolated from CT26 tumor bearing mice. (B) Model of extracellular mICAM-1 structure (using alphafold (Varadi et al., Nucleic Acids Res. 2022;50(Dl):D439-D44)) with each Ig-like domain coloured and denoted. (C) Domain truncated variants of mICAMI were cloned (represented by the schematics) and tagged with a rituximab-specific peptide, Rp3. These were transiently transfected into CHO-S cells and binding of 12-D10, and the commercial clone, were assessed. Binding pattern indicates that 12-D10 binds the fourth domain of mICAMI whilst the commercial clone binds the first domain. (D) Cross-blocking assay performed where either unlabelled 12-D10 or ligand were incubated with mICAM-1 expressing cells before adding a fluorescently conjugated variant of the mAb or recombinant ligand-His (pl50, 95) detected with anti-His-APC. No blocking was seen indicating 12-D10 bound a different region to the ligand. Mean and SD of three independent transfections presented. (E) Amino acid sequence of domain four from human and mouse ICAM-1. Species different residues are italicised, whilst those highlighted in grey are predicted to be exposed on the surface of the molecule using pymol sasa_relative command (Shrbdinger L. The PyMOL Molecular Graphics System. 1.2r3pre ed). Location of T289I has been emphasised by dark grey shading. (F) Representative example of 12-D10 losing binding to mutant T289I. (G) Ratio of 12-D10 and RTX binding to CHO-S cells transfected with point mutations of the fourth domain ICAM-1. (H) Visualization of binding data highlighting species differences that do not affect binding, location of T289, proposed dimerization site and the binding site of ligand (pl50, 95). Image used the resolved crystal structure of hICAMI, pdb 2OZ4.

[0282] Figure 6 shows that 12-D10-mIgG2a activity is dependent on FcyR-interaction and depletion of Tregs. (A) Survival graph of WT BALB / c CT26 tumor bearing mice treated with either 12-D10-mIgG2a or 12-D10-mIgGl-NA. (B) Survival data from WT or FcR-y chain KO mice bearing CT26 tumors treated with 12-D10-mIgG2a or an isotype control. 6 mice per group from a single experiment. (C-D) Fold-change in Tregs (C) and the CD8:Treg ratio (D) compared to isotype treated mice in the Spleen, td-LN and tumor assessed as presented in figure 2A-B. N = 5 of a single experiment. (E) Schematic showing the treatment regimen for PD-1 and 12-D10 combination in MC38 tumors in WT C57BL / 6 mice. (F) Individual tumor growth and (G) survival of mice from this experiment. Numbers of mice per group are presented on the plots in (F) and are combined from three independent experiments, except for the CTLA-4 only group which was pooled from a single experiments. Figure 7 shows identification of similar anti-human ICAM-1 clones. (A) Schematic and representative data demonstrating that clones 5-A11 and 3-E11 bind domain four of human ICAM-1 in transfected cells whilst enlimomab binds domain 1. (B) 5-A11, 3-E11 and enlimomab are used to stain Tregs, CD4+, CD8+ and non-CD3- immune cells isolated from healthy blood (n=3), and tumor samples (n = 5) by flow cytometry.

[0283] Figure 8. (A) Gating strategy used to identify and quantify Treg and lymphocyte population changes in primary tissue. Raw data from splenocytes of control treated mouse presented. (B) The mean fold-change in the number of CD8+, CD4+FoxP3- and NK cell populations (based on percentage of total CD45+ cells compared to isotype treated mice was plotted for each mAb across each tissue and presented as a heatmap. A decrease below 1 indicated cell depletion and an increase above 1 indicated expansion of the cell population. The antibodies are ranked based on their Treg depleting characterisation in Figure 2C. Where a statistically significant change from the isotype control was found the corresponding cell within the heatmap was marked based on the p-value obtained. P-values are represented as follows * = 0.05, ** = 0.005, *** = 0.0005, **** < 0.0001. p-values > 0.05 were left blank.

[0284] Figure 9. (A) 0X40 (left) and ICOS (right) were coated onto a 96 well ELISA plate. 17- F04 and 6-B10 were added in serially diluted concentrations and detected using luminescence reader, confirming their specificity. (B) HEK cells transfected with mouse FolR4 were stained with 1-C11 and 15-E09 scFv and detected with anti-His-APC and binding confirmed by flow cytometry. (C) Data analysis demonstrating that 12-D10 bound ICAM- 1 associated peptides provided the LRC-TriCEPS methodology at Dualsystems Biotech according to their standard procedures.

[0285] Figure 10. (A) 12-D10+ and 12-D10- Tregs from CT26 tumors were co-stained for additional T cell markers. (B) Gating of tumour to look at either 12-D10 or commercial ICAM-1 binding on non-immune CD45- cells compared to Treg cells. (C) Agarose gel showing PCR amplification of mouse ICAM-1 transcripts from cDNA isolated from Tregs (CD4+ CD25+) isolated from the spleen or tumour of CT26 tumor bearing mice. NTC = no transcript control. (D) ELISA measuring 12-D10 or commercial ICAM-1 mAb binding recombinant mouse ICAM-1 with or without deglycosylation with PNGase. (E) CD8+ cells isolated from MC38 tumors following treatment with the therapeutic antibodies were stained for the presence of intracellular IFN-y or TNF-o by flow cytometry and enumerated as the proportion of CD8+ T cells.

[0286] EXAMPLES

[0287] Example 1 - Experimental data

[0288] Background

[0289] Antibody immunotherapy has revolutionised cancer therapy. It encompasses agents that directly target tumor cells, such as rituximab and trastuzumab, or that target cells of the immune system, such as ipilimumab and nivolumab. Despite these successes, treatment response is variable; in those whose disease responds to therapy, it often relapses or becomes treatment (Weber et al, N Engl J Med. 2017;377(19): 1824-35; Barrueto et al., Transl Oncol. 2020;13(3): 100738; Torka et al., Curr Hematol Malig Rep. 2019;14(5):426- 38).

[0290] One component impacting antibody efficacy is the tumor microenvironment (TME). It is often anti-inflammatory, lacking infiltrating lymphocytes and / or containing suppressive cells such as tumor associated macrophages (TAM) and regulatory T (Treg) cells (Jin and Jin WL. Signal Transduct Target Then 2020;5(l): 166). Whilst not universal, it is generally established that tumors which have a high proportion of CD4+FoxP3+Tregs have a worse prognosis compared to those without (Liu et al., Breast Cancer Res Treat. 2011;130(2):645-55; Li et al., J Cancer. 2016;7(7):784-93; Zhao et al., Am J Pathol. 2020;190(4):886-99). In agreement, systemic removal of Tregs using conditional Treg knock-out (KO) strategies results in tumor regression of subcutaneous tumor models (Teng et al., Cancer Res. 2010;70(20):7800-9; Klages et al., Cancer Res. 2010;70(20):7788- 99).

[0291] Attempts to target Tregs have had varying success in the clinic. Metronomic chemotherapy with cyclophosphamide depletes Tregs systemically, therefore driving anti-tumor responses (Scurr et al., Clin Cancer Res. 2017;23(22):6771-80). However, this approach targets regulatory cells throughout the body and is not maintained across prolonged time-courses (Ge et al., Cancer Immunol Immunother. 2012;61(3):353-62). Using monoclonal antibodies (mAb) to target and delete Tregs is attractive but there is no single specific surface marker for Treg cells. Whilst they constitutively express markers such as CD25, GITR, 0X40, 4- IBB, CCR.8 and CTLA-4, these same receptors are also present on activated CD8+effector and conventional CD4+T cells (Buchan et al., Immunity. 2018;49(5):958-70 e7; Wegrzyn et al., Front Immunol. 2022;13: 1055805). This means that mAbs targeting these antigens will likely impact both Tregs and important effector populations, potentially reducing the impact specific Treg depletion could evoke. Whilst Treg depletion through some of these receptors, such as CTLA-4 and CD25 with ipilimumab (Romano et al., Proc Natl Acad Sci U S A. 2015;112(19):6140-5) and daclizumab (The L. End of the road for daclizumab in multiple sclerosis. Lancet. 2018;391(10125): 1000), respectively, is associated with impressive efficacy in pre-clinical models and some success in the clinic, direct correlation of efficacy and Treg deletion in humans has not always been demonstrable (Sobhani et al., Cancers (Basel). 2021;13(6)) and there are often issues with prolonged side-effects and development of severe autoimmune complications due to removing systemic Treg activity (Pol et al., Cell Res. 2018;28(5):501-2; Aamdal et al., Int J Cancer. 2022;150(l): 100-11).

[0292] These results demonstrate that more specific Treg deletion is desirable. Transcriptomic and proteomic data reveal that Tregs have unique signatures compared to effector CD8+and CD4+T cells, and that Tregs present within the TME are distinct from Tregs in neighbouring healthy tissues (De Simone et al., Immunity. 2016;45(5): 1135-47; Magnuson et al., Proc Natl Acad Sci U S A. 2018;115(45):E10672-E81). This suggests that there may be tumor-specific Treg markers that can be leveraged for more specific depletion of tumor Tregs allowing for tumor regression with minimal systemic toxicities.

[0293] To identify such markers, we utilised the phenotypic signature of tumor-associated Treg cells in conjunction with the nCoDeR phage library and F.I.R.S.T platform (Frendeus et al., Oncoimmunology. 2013;2(8); Soderlind et al., Nat. Biotechnol. 2000;18(8):852-6) to generate antibodies that were selective for murine Tregs. These were tested for their ability to bind and deplete Tregs across multiple tissues in vivo and demonstrated how tumor specific deletion of Tregs, whilst leaving the peripheral Tregs untouched, delayed tumor growth and evoked cures in the murine CT26 tumor model. Of the antibodies isolated, we identified a distinct epitope on mouse ICAM-1 which had tumor Treg specificity, with Tregs from primary human tumor samples displaying a similar selectivity. Results

[0294] Generatinq Treq specific antibodies with tumour T req selectivity

[0295] To generate antibodies to antigens selectively expressed on tumor-localised Treg cells the inventors took a target agnostic approach, performing live cell panning against tumor Treg versus non Treg cells with the n-CoDeR phage display library (Soderlind et al., Nat. Biotechnol. 2000;18(8):852-6). Treg cells (defined as CD4+ CD25+) were isolated from the spleen, tumor draining lymph node (td-LN) and tumor of tumor bearing mice (harbouring a number of different tumors; see methods for details) as the positive selection population. CD3-depleted splenocytes from naive mice were used as the negative target population to remove any non-T cell binders (figure 1A).

[0296] After 3 rounds of panning, over 400 individual scFv-containing phages were isolated and their specificity evaluated by flow cytometry. Many clones showed pan-T cell specificity (figure IB) - which was anticipated as the non-target panning did not contain CD3+ T cells. Similarly, other clones bound an antigen expressed on CD8+ or CD4+ T cells. However, there was a cluster of scFv which bound Tregs isolated from different tumor models and also demonstrated minimal binding to CD4+ effector cells and CD8+ T cells (figure IB).

[0297] This group of scFvs were converted into full length m!gG2a mAb and screened for binding by flow cytometry against a wider range of immune cell populations isolated from tissue collected from CT26 tumor bearing mice. This analysis confirmed that the majority of clones tested from this group bound Tregs, with minimal binding to the effector (non-Treg) CD4+ and CD8+ T cells (inferred by low MFI / staining on these populations as indicated in the representative histograms in Figure 1C). It was also evident that there was a clear increase in binding to cells isolated from the tumor compared to those from the spleen (Figure 1C / D). This indicated that there was an upregulation of their antigenic target in the tumor microenvironment which further indicated that tumor-Treg targeting by antibodies was possible. Of particular note, clone 12-D10 bound Treg cells isolated from the tumor with high selectivity, reflective of a tumor-associated Treg specific antibody.

[0298] Treq depletion efficacy varies amonqst Treq tarqetinq antibodies Having identified a panel of mAbs with varying degrees of Treg specificity, binding a range of differentially expressed targets (inferred from the relative MFI, following detection with the same polyclonal secondary-PE labelled antibody), the capacity of these mAbs to deplete Treg cells in vivo was assessed. Treg depletion was explored in the CT26 subcutaneous tumor model as it is known to have a relatively high Treg infiltrate and is sensitive to established Treg-depleting therapeutics. (Buchan et al., Immunity. 2018;49(5):958-70 e7; Heckel et al., Commun Biol. 2022;5(l); Semmrich et al., J Immunother Cancer. 2022;10(l). Three days following the third dose of mAb, blood, spleen, tumor-draining inguinal lymph nodes (td-LN) and tumors were collected, processed into a single cell suspension and immune populations assessed by flow cytometry (Figure 8A).

[0299] Varying levels of Treg depletion were achieved with the different mAb, with the impact dependent upon the tissue compartment examined (Figure 2A). This variation was not due to differences in the antibody isotype, as all antibodies assessed had the same mIgG2a format known to engage favourably with activating FcyR to elicit target cell deletion, predominantly through antibody dependent cellular phagocytosis (ADCP) (Wang et al., Eur J Immunol. 2022;52(5):753-9).

[0300] Decreases in the Treg populations also resulted in an increase in the CD8:Treg ratio in different tissues (Figure 2B), indicative of the potential to elicit an anti-tumor immune response. Whilst there were trends that the CD8+ populations were also increasing in the tumor after treatment with some mAb, e.g. 49-E11 and 37-D08, these were generally not significant due to the variance of the data (Figure 8B). Importantly, most of the mAb had their greatest effects on the Treg populations with only minor changes across the other T and NK cell populations (Figure 8B). In contrast, clones 49-G08 and 49-F06 caused a reduction of all detectable CD4+ T cells across the tissue compartments, in keeping with their pan-CD4 binding profile and lack of Treg specificity (Figure ID and Figure 8B).

[0301] Cluster analysis of the antibody clones revealed four distinct groups of activity (Figure 2C) : Non-Treg depleting mAbs (e.g. 18-A07); Non-tumor Treg depleting mAbs - i.e. depletion only from blood, spleen and td-LN but not the tumor (e.g. 15-H03); Systemic Treg depleting mAbs - depletion from all tissue compartments examined (e.g. 17-F04) and Tumor Treg depleting mAbs - depletion from the tumor with no depletion from non-tumor tissue (e.g. 12-D10). To evaluate the basis for mAb deletion efficacy we assessed the relative expression level for each mAb (as measured using the anti-mouse IgG secondary antibody staining in figure ID) versus the level of Treg depletion in the spleen and tumor compartment (Figure 2A). Within the spleen, there was a linear correlation between expression and depletion - with higher mAb binding associated with greater levels of depletion (Figure 2D). This relationship has been noted previously (Buchan et al., Immunity. 2018;49(5):958-70 e7; Derer et al., J Immunol. 2012;189(ll):5230-9) and highlights the importance of antigen expression levels for the engagement of direct-targeting antibody effector mechanisms. However, this association was not maintained in the tumor compartment with no correlation between mAb binding level and depletion efficacy (Figure 2E). This perhaps indicates that either the mAbs do not enter the tumor compartment equivalently to decorate the tumor Tregs, or that the TME impacts the relationship between antigen expression and deletion due to suppressive factors not seen in peripheral locations like the spleen. (Dahal et al., Cancer Res. 2017;77(13):3619-31., Hussain et al., J Exp Clin Cancer Res. 2022;41(l): 131) It further infers that using expression levels alone is insufficient to predict effective targetable antigens within the TME.

[0302] Depletion of Tregs from the tumor compartment is required for anti-tumor activity

[0303] Having observed differences in the Treg depleting activities of the antibodies it was important to establish if they evoked tumor control. To assess this, CT26 tumor-bearing mice were treated using the same strategy as the in vivo depletion experiment before (Figure 2A-C), with the area of the tumors measured until they reached the humane endpoint (tumor area of 300 mm2).

[0304] There were clear differences in the tumor outcome, largely correlated with the prior mAb groupings. Antibodies which did not have Treg depleting activity (such as 18-A07) or were unable to deplete Tregs from the tumor compartment (such as 15-H03) did not result in curative responses and displayed only a reduction in tumor growth rate, with modest increases in the median time to survival when compared to the isotype control treated mice (Figure 2F). mAb which evoked systemic depletion resulted in a range of therapeutic outcomes from delayed tumor growth to complete cures. This wide difference in effect most likely reflects the range of antigens bound by these antibodies. There were no obvious signs of toxicity observed in the mice for the duration of the experiment (defined as weight loss and / or hunched / slow behaviour). Of most interest was the group of antibodies which demonstrated tumor-specific Treg depletion and displayed minimal effects on other immune populations (such as 12-D10); these mAb had curative anti-tumor effects resulting in 50-100% survival. Cured mice from these experiments were rechallenged with CT26 cells between 70-80 days after the initial tumor challenge, with 16 out of 18 remaining tumor free, with a single mouse from the 49-E11 (1 / 4) and 37-B08 (1 / 2) groups succumbing. Without being bound to any theory, this may mean that a sufficiently robust anti-tumor memory response is established after Treg deletion to elicit this protective effect.

[0305] Target deconvolution revealed expected and novel targets

[0306] Having demonstrated tumor resolution with several tumor Treg specific mAb, identification of their target antigens followed. This process involved a combination of targeted screening of known Treg targets (for example members of the TNFRSF; like 4-1BB and 0X40, known to be upregulated on Tregs (Buchan et al., Immunity. 2018;49(5):958-70 e7, Griffiths et al., Journal for Immunotherapy of Cancer. 2020;8(2); Piconese et al., Hepatology. 2014;60(5): 1494-507)); those identified from differential gene expression (DGE) analysis of bulk RNA-seq datasets (upregulated in Treg compared to non-T cell populations) and immunoprecipitation / mass spectrometry of mAb-bound antigen isolated from the surface of the target cells (Figure 9).

[0307] From these approaches the inventors identified mAbs which bound expected targets (Table 1), such as CTLA-4 (clone 5-B07, published in Semmrich et al., Immunother. Cancer. 2022;10(l)), ICOS (clone 6-B01), 0X40 (clone 17-F04), and GITR (clone 49-E11). Other identified targets included FolR4 (which accounted for 7 unique mAb clones, including 1- Cll and 16-A03), and ICAM-1 bound by 12-D10.

[0308] Table 3. Summary of Identified Targets from Treg Panning

[0309] Summary of key targets identified for the Treg depleting antibodies identified in Figure 2.

[0310] Of note, CD25 was not identified as a target for any of the functional clones, potentially due to being masked by antibodies during the isolation and purification of Treg cells for the panning.

[0311] The antibodies which had their antigenic target deconvoluted, were then benchmarked against an isotype matched anti-CD25 mAb (PC61-mIgG2a) and anti-CTLA-4 (9H10- m!gG2a) in the CT26 tumor model (Figure 3A). One mouse was cured with anti-CD25 therapy, in keeping with our experience in our animal facility, however except for anti- FolR.4 (clone 15-E09), all other mAbs were able to induce cures (figure 3A-B). Whilst CTLA- 4 targeting cured the majority of mice, approximately 50% developed dermatitis (exhibited by frequent scratching and the appearance of bald patches on the face) approximately 1 month after treatment had stopped. This development of dermatitis was not seen in mice treated with clone 12-D10 - which binds the target ICAM-1 - which also was able to cure ~50% of mice.

[0312] Knowing that ipilimumab frequently elicits autoimmune side-effects in the clinic (Karimi et al. Front Oncol. 2021;ll :624780), and the observation that dermatitis development in our mouse experiments was specific to the anti-CTLA-4 treatment, further investigations were performed. To assess tolerability, 28-day old mice were treated with lOmg / kg of either anti-CTLA-4, 12-D10 (alone or combined with anti-PD-1) twice weekly for three weeks (figure 3C). Serum samples were collected and assessed for measures of inflammation: cardiac troponin I elevation (Du et al. Cell Res. 2018;28(4):433-4738, Delombaerde et al. ESMO Open. 2021;6(4): 100216), increased spleen weight and reduction in the mean corpuscular volume (MCV) (Du et al. Cell Res. 2018;28(4):433-47). As shown in figure 3D, mice treated with 12-D10, compared to anti-CTLA-4 had lower levels of cardiac troponin I in the serum, smaller spleens and no change in MCV, when combined with anti- PD-1. Together, these data suggest that 12-D10 may be less toxic compared to anti-CTLA- 4; presumably due to the localized tumor Treg depletion which minimizes the broader inflammatory effects associated with systemic Treg depletion (Cohan et al. Biomedicines. 2019;7(l)).

[0313] The 12-D10 antibody recognises an epitope on ICAM-1

[0314] 12-D10 stood out as an interesting antibody to pursue. ICAM-1 had not previously been suggested as enriched on tumor Tregs, whilst 12-D10 demonstrated tumor specific binding of Tregs, exhibited localised depletion of Tregs within the tumor compartment (Figures 1D,2C) and cured approximately 50% of CT26 tumor bearing mice as a monotherapy (Figures 2G, 3A), establishing protection against further tumor rechallenge. To assess if its epitope / expression was mouse strain or tumor model specific the inventors evaluated its binding in different tumor models (CT26 and MC38) derived from different strains of mice (BALB / c and C57BL / 6, respectively), confirming its generality (Figure 4A-D). Of note, no binding of 12-D10 was detected by flow cytometry on the non-immune (CD45-) cells within the tumor (Figure 10B). Compiled CT26 survival data across three independent experiments showed that 12-D10 reproducibly cured approximately 50% of CT26 challenged mice, correlating with a clear depletion of Treg cells from the tumor, whilst leaving Tregs in the spleen and td-LN untouched (Figure 4E-F).

[0315] The 12-D10+ Tregs from control tumors were further phenotyped for additional markers known to be expressed on Treg cells with differing functional states (Van Damme et al. J Immunother Cancer. 2021;9(2)) (Figure 4G and Figure 10A). Tregs which bound 12-D10 had significantly higher levels of expression of many markers associated with more suppressive Tregs (Van Damme et al. J Immunother Cancer. 2021;9(2)), including CTLA- 4, CCR.8, GITR, 4-1BB and PD-1 compared to Tregs not expressing the 12-D10 target. This was true in both the MC38 and CT26 tumor models.

[0316] 12-D10 targeting of ICAM-1 was initially revealed from immunoprecipitation and peptide analysis by mass spectrometry (Figure 9) and subsequently confirmed by ELISA, flow cytometry of transfected CHO-S cells and impaired binding in the presence of soluble recombinant ICAM-1 extracellular domain (Figure 4H-J).

[0317] ICAM-1 is expressed by multiple cell populations outside of the tumor and can be upregulated in the presence of proinflammatory cytokines (Bui TM et al., J Leukoc Biol. 2020;108(3):787-99). Since 12-D10 binding was specific to tumor associated Tregs, primary tissue was collected from MC38-tumor bearing mice and stained with commercially available anti-ICAM-1 flow antibodies and 12-D10 to compare binding patterns. It was clear that the commercially available ICAM-1 antibody displayed a wider binding pattern than 12-D10 (figure 5A), also staining much more highly on CD8+ cells and NK cells. This differential staining pattern suggests that the two mAb bind different epitopes of ICAM-1 which may correspond to the different isoforms or accessible forms of ICAM-1 on the surface of different cells (Hu et al., Mol Immunol. 2010;47(9): 1692-700; Ramos et al., J Immunol. 2014;192(10):4469-74).

[0318] To assess whether Tregs expressed a different isoform of ICAM-1 compared to non-Tregs, CD25+ versus CD25- CD4 T cells were isolated from the spleens and tumors of tumor bearing mice and the cDNA generated. ICAM-1 has five extracellular Ig-like domains and a small intracellular tail (Figure 5B). Using primers designed to amplify the complete extracellular domain of ICAM-1 (Figure 10C), it was seen that there was no difference in transcript length from the spleen or tumor of CD25+ or CD25- CD4 T cells. This was confirmed by sequencing of the isolated ICAM-1 transcript, with all five extracellular domains found to be present. This suggests that alternative splicing of ICAM-1 does not explain the difference in cellular binding observed with 12-D10 and commercial ICAM-1 antibodies and infers a difference in epitope bound. Using a combination of published X- ray structures (PDB: 2OZ4 and HAM) and sequence interrogation, a series of domain truncation mutants of mouse ICAM-1 were generated with the addition of an N-terminal peptide tag that can be recognised by rituximab, to independently evaluate expression and define the domain bound by 12-D10 (Cleary et al., 2017; Varadi et al., 2022; Chen et al., Proc Natl Acad Sci U S A. 2007; 104(39): 15358-63). Constructs were validated by sequencing, then expressed and binding determined by flow cytometry to map 12-D10 binding (figure 5C).

[0319] 12-D10 bound the fourth domain of ICAM-1, whilst the commercial ICAM-1 clone bound the top domain (figure 5C). The fourth domain of ICAM-1 is involved in the homodimerization of the receptor and is also where the ligand pl50,95 (CDllc / CD18) binds (Chen et al. 2007; 14; Frick et al., 2005). However, the epitope bound by 12-D10 differed from that of the ligand as it bound cell surface expressed ICAM-1 in the presence of recombinant ligand and pre-binding of 12-D10 to ICAM-1 expressing cells did not prevent the recombinant ligand from binding (Figure 5D). To further interrogate where 12-D10 bound, point mutations within domain 4 of ICAM-1 were introduced. 12-D10 is specific to mouse ICAM-1 and unable to bind the human homolog, therefore a pairwise alignment was performed comparing the amino acid sequences of the corresponding mouse and human sequence to identify species specific residues (Figure 5E). Subsequently, residues predicted to be exposed on the surface of the molecule, based on resolved X-ray structures, were mutated from the mouse to the equivalent human residue. These mutant chimeric molecules were then transfected into HEK293F cells and the ability of 12-D10 to bind was assessed. Mutation of Thr289 to He was able to diminish binding of 12-D10 as determined by flow cytometry (Figure 5F / G). This residue sits on the opposite surface of Domain 4 to both the receptor dimerization site and the proposed binding site of its ligand pl50,95 (Figure 5H), in agreement with expectations from the previously described competition data (Figure 5D).

[0320] Lastly, to better understand how 12-D10 was able to selectively bind tumor Treg but not Treg elsewhere, we evaluated the potential impact of glycosylation. It was found that 12- D10 binding was substantially diminished when recombinant ICAM-1 was deglycosylated whilst the domain 1 binding commercial mAb was not affected (Figure 10D). Together, this data indicates that glycosylation plays a role in the selective tumor Treg nature of 12-D10 binding.

[0321] 12-D10 therapeutic activity is Fc dependent and different to anti-PD-1

[0322] ICAM-1 can interact with CDllc found on DCs and other immune cells within the TME (Frick et al., Eur. J. Immunol., 2005; 35:3610-3621). To test whether the therapy seen with 12- D10 was solely due to Treg depletion, or whether its activity was related to a functional impact on ICAM-1 itself, CT26 tumor bearing WT mice were treated with either the parental mouse IgG2a isotype, or an Fc-silenced version (mouse IgGl-N297A (Lux et al., J Immunol. 2013;190(8):4315-23). Whilst the mIgG2a isotype was able to cure CT26 tumors, the mlgGl-NA variant was unable to elicit anti-tumor activity and no depletion of Tregs was observed (figure 6A / B). This was also seen in a second model where 12-D10- mIgG2a antibody was administered to either WT BALB / c mice, or Fc receptor gamma chain (FCERG1) (Brandsma et al., Immunity. 2016;45(2):225-6) KO BALB / c mice bearing CT26 tumors (Figure 6C). These mice lack activating FcyRs and are therefore unable to mediate antibody-dependent killing via ADCP or ADCC (Bruhns et al., Immunol Rev. 2015;268(l):25-51). In both models, no Treg depletion or improvement in CD8:Treg ratio was observed (Figure 6B / 6D), with effects only seen in the tumor. Together these models highlight that FcyR:Fc interactions are required for the activity of 12-D10 and support that depletion of Tregs is the main mechanism through which it evokes its anti-tumor responses. Next, the Inventors evaluated whether combining 12-D10 with an immunomodulatory mAb (targeting PD-1) could enhance responses in tumor models where Treg depletion alone was insufficient to drive robust anti-tumor control. The Inventors chose the MC38 model to assess combinatorial therapy responses, as it is partially responsive to anti-PD-1 (Figure 6F), having previously determined that the binding of 12-D10 was similar in WT C57BL / 6 mice to that seen in WT BALB / c mice (figure 5A-D). The Inventors' rationale was to first deplete the tumor Tregs using 12-D10 and then treat with anti-PD-1 mAb 48 hours later (Figure 6E). As expected, 12-D10 monotherapy had little activity in this model, but there was a modest improvement in the percentage survival when combined with anti-PDl mAb, with those mice cured in the primary tumor challenge resistant to tumor rechallenge 75 days later (Figure 6F-G). Mice treated with the combination of 12-D10 and PD-1 displayed an increase in the percentage of TNF-o producing CD8 cells, and an elevated CD8:Treg ratio compared to isotype or anti-PDl treatment alone (Figure 10E). No overt toxicity was observed. This indicates that combining tumor-specific Treg depletion with established immunomodulatory strategies (anti-PDl) is a promising combinatorial approach for improved cancer immunotherapy.

[0323] Generating anti-human ICAM-1 candidate antibodies

[0324] To assess the feasibility of targeting Tregs via ICAM-1 in human tumors we sought to identify anti-human antibodies that bound a similar epitope within the fourth domain of human ICAM-1. To do this, anti-human ICAM-1 scFv were selected from the n-CoDeR phage library and screened for domain specificity using domain truncated mutants of human ICAM-1 in the same manner as above (Figure 5C). Of the 90 clones screened, two were identified as domain 4 binders, whilst the majority of scFv mapped to the top two domains (similar to the commercial reagents), including the previously explored clinical candidate enlimomab (figure 7A), (Veitonmaki et al., Clin Lymphoma Myelom. 2009;9:S157-S; Enlimomab Acute Stroke Trial. Neurology. 2001;57(8): 1428-34) .

[0325] To evaluate the binding profile of these domain 4 binders, T cells from healthy PBMCs and dissociated tumor cells from 4 different tumor indications (liver carcinoma, melanoma, breast cancer and ovarian cancer were screened (figure 7B). Enlimomab (a domain 1 binder) bound across all T cell populations with little differentiation between tumor and blood. However, the domain 4 clones displayed a higher level of selective binding to Tregs from the tumor microenvironment compared to Tregs from healthy peripheral blood, with Treg binding higher than both CD4 effectors and CD8 cells, especially for 5-Al 1, in keeping with the data for 12-D10.

[0326] Materials & Methods scFv isolation and screening on mouse tissue

[0327] Antibodies specific for tumor Treg cell-associated receptors were isolated by subjecting the in vitro CDR shuffled n-CoDeR® antibody library to differential biopanning of tumor- associated CD4+ CD25+ Treg cells (isolated from CT26, 4T1, B16 and Lewis lung tumorbearing mice) versus CD3+ T cell-depleted splenocytes from tumor-bearing mice essentially as described previously (Veitonmaki et al., Cancer Cell. 2013;23(4):502-15). After three consecutive rounds of panning, a negative depletion step against CD3- cells was included to enrich for T cell binders. Phagemid-DNA was then purified and the genes encoding scFv fragment were digested from the vector and ligated into a protein expression vector. Following conversion, more than 1500 individual antibody clones were expressed and purified from E. coli supernatants, sequenced (GATC Biotech, Germany) and screened for binding to primary CD4+ CD25+ Treg cells or CD3- splenocytes in an Operetta instrument (Perkin-Elmer).

[0328] 320 unique scFv antibody clones were then evaluated in a heat map binding pattern analysis. Here, in vitro cultured cell lines (EL4, WEHI, J774A1, 4T1, CT26), total splenocytes from naive BALB / c mice, and CD4+ CD25+ Treg cells isolated from the spleen, td-LN (of 4T1 and CT26 tumor-bearing mice) and tumor (of CT26 tumor-bearing mice only) were stained with purified scFv (10 pg / ml) and binding was detected by flow cytometry (FACSVerse, BD Biosciences). Bound scFv were detected using anti-His-AF647 (R&D Systems, MAB050, labelled in-house). A hierarchical clustering of scFv based on binding pattern was done using Qlucore Omics Explorer (Qlucore, plot type heat and normalization Mean = 0, Var = 0). scFv were selected based on binding properties and converted into full-length m!gG2a mAbs.

[0329] To enable the discovery of low-abundance clones, additional selection rounds were performed using converted m!gG2a antibodies and the phage stock from above. In these deep mining strategies (Ljungars et al., Front Pharmacol. 2019;10:847), CD25+ target cells were blocked with dominating purified mIgG2a antibodies (75 clones) from the input pool before the phage stock was added. Alternatively, a non-target cell depletion step was performed aiming to reduce the fraction of antibodies binding to non-target cells. To this end, the input phage stock was incubated with CD8+ T cells, CD4+CD25- T cells and CD25- cells for 4 hours on ice. The non-target cells were then pelleted and the unbound phage were converted to scFv. Clones resulting from these additional steps were screened, sequenced and mapped as described for the initial pool above.

[0330] Target deconvolution

[0331] Different techniques were used for target deconvolution of antibodies:

[0332] Bulk RNA-seq analysis was performed on sorted CD4+ CD25+ Tregs from tumor-draining lymph nodes (td-LN) of CT26 tumor bearing mice and compared to non-target cells such as CD4+ CD25- cells from the same source, CD3- cells sorted from spleens of naive mice and in vitro activated CD8+ T cells from spleens of naive mice. RNA-extraction, mRNA library preparation (non-stranded) and sequencing (75 bp single read using an Illumina NextSeq 500 V2 machine) as well as data analysis was performed by LGC Genomics GmbH. Illumina bcl2fastq 2.17.1.14 software was used for demultiplexing, adapter trimming and filtering of reads below 20 bases. Additionally, rRNA sequences were filtered using RiboPicker 0.4.3 and fastq files quality checked using fastQC. To determine differential expression, reads were aligned against the Mus musculus reference (GRCm38 release 91) using STAR 2.4., followed by analysis with edgeR 3.2.3., DESeq 1.12.0 and cuffdiff 2.1.1. The resulting raw p-values from these statistical tests were adjusted for multiple testing using the Benjamini-Hochberg false discovery rate (FDR) method. Then differentially expressed genes that were identified by more than one software were listed. Genes with an FDR of less than five percent were considered to be differentially expressed. Based on the resulting list of differentially expressed genes (upregulated on Treg compared to non- target cells), antibodies (mIgG2a) were analyzed for binding to transfected HEK cells overexpressing individual cell surface proteins and / or tested for protein binding by ELISA. 49 targets were tested in total, of which 5 were identified as Treg-specific in addition to several other T cell binders.

[0333] The specificity for some other clones was determined by mass spectrometry with cell lysate from cells that bound the antibody of interest. These experiments were performed using the LRC-TriCEPS methodology at Dualsystems Biotech according to their standard procedures. In vivo studies

[0334] C57BL / 6 and BALB / C mice were obtained from Charles River Laboratories or Envigo. FcRy- KO BALB / c mice were bred and maintained in local animal facilities. Young adult mice were sex- and age-matched and randomly assigned to experimental groups. Experiments were not blinded.

[0335] CT26 survival model: 8-12 week old WT BALB / c mice (female only) or FcRy-KO BALB / c mice (female and male) were inoculated with 5x105 cells in 100 pL PBS subcutaneously (s.c.) in the rear flank. A group size of 5 to 6 was used based on similar work showing n>5 was sufficient to see a statistical significance of p <0.05(Griffiths et al., 2020). Once tumors were palpable (5-6 days after injection) mice were distributed evenly across treatment groups, ensuring that the average tumor area was similar (30-40 mm2) across the groups. This was designated day 0. Mice received 200 pg mAb in 200 pL PBS intraperitoneally (i.p.) on day 0, 4 and 7. Tumor growth was monitored three times a week using digital callipers to record the length and width of the tumor. Tumor area was calculated by the equation Length * Width. When tumors reached 300-400 mm2in area mice were sacrificed, following approved Schedule 1 methods.

[0336] CT26 tumor phenotyping experiments: mice were treated in the same way as the CT26 survival model, except on Day 10 following the first dose of mAb, mice were sacrificed and the blood, spleen, td-LN and tumors were collected for processing.

[0337] MC38 survival model: 10-12 week old WT female C57BL / 6 mice were inoculated with 5x105 cells in 100 pL PBS subcutaneously in the rear flank (s.c.). A group size of n=6 was used based on similar work showing n>5 was sufficient to see a statistical significance of p <0.05(Griffiths et al., 2020). Once tumors were palpable (5-6 days after injection), mice were distributed evenly across treatment groups, ensuring that the average tumor area was similar (30-40 mm2) across the groups. Mice received 200 pg of 12-D10-mIgG2a, anti-CD25 mIgG2a (PC-61.5.3; Absolute Antibody) or anti-CTLA4 mIgG2a (9H10; BioInvent), all i.p., on days 0 and 7; 100 pg of anti-PD-1 (clone 29.F.A12, BioXcell / AssayGenie) i.p. on day 2 and day 7. Tumor growth was monitored three times a week using digital callipers to record the length and width of the tumor. Tumor area was calculated by the equation Length * Width. When tumors reached 300 mm2in area, had a tumor ulceration score >7 (Wolfensohn et al., Blackwell Publishing Ltd; 2003) or exhibited deteriorating signs of ill-health, mice were sacrificed, following approved Schedule 1 methods.

[0338] Tolerability assessment:

[0339] 4 week-old BALB / c mice were treated i.p. with isotype control IgG, anti-CTLA-4 mAb clone 9H10 (m!gG2a; BioInvent), or anti-ICAM-1 clone 12-D10 in the presence or absence of anti-PD-1 (RMP1-14; BioXcell) at a dose of 10 mg / kg per antibody and injection on days 0, 3, 7, 10, 14 and 18. Serum samples for cardiac troponin I analysis were collected on day 21 after treatment start. The cardiac troponin I ELISA kit was purchased from Nordic Biosite, and the assay performed according to manufacturer's instructions. The MOV analysis was performed on day 39 using a Scil Vet abc Plus analyzer, the spleen weight was measured when the mice were sacrificed on day 40.

[0340] TIL phenotypinq of mouse tissue:

[0341] Tumor tissue was processed into a single cell suspension by dicing and treating with 1 mg / mL collagenase V (Sigma Aldrich) or 0.5 Wunsch units of liberase TL (Roche) for 20- 30 mins at 37°C in a shaking incubator, before passing through a 100 pm cell strainer. Spleen and LN tissue was mechanically disaggregated into a single cell suspension without collagenase treatment. All cell suspensions were pelleted at 400g, supernatant removed and resuspended in a volume suitable for flow cytometry staining. Samples that were assessed for intracellular cytokine expression were treated with golgi-plug (BD Biosciences) for 4 hours at 37°C prior to staining.

[0342] Primary surface staining for flow cytometry was performed for 30 min at 4°C. Remaining red blood cells were lysed with 1 mL of BioRad PharmLyse (reconstituted in dH2O from a lOx stock solution) for 2 mins, before washing twice in 2mL wash buffer (PBS containing 1% BSA and 10 mM sodium azide). Supplier and clone information of all commercial mAbs used are in Table 4.

[0343] Intracellular staining was performed following an adapted protocol from eBioScience transcription factor staining kit (CAT 00-5523-00). Cells were fixed in 300 pL fixation buffer overnight at 4°C, washed twice with 1 mL permeabilsation buffer, then FoxP3 antibody added for 1 hour at 4°C. Cells were then washed in 2mL wash buffer, and kept in the dark at 4°C until acquisition. All samples were acquired on FACS Canto II or Fortessa x20 using FACSDIVA and data analysed using FlowJo (version 8.8). Analysed flow data was processed in GraphPad Prism version 9, and heatmap analysis performed in R (version 4.2.1) using the "ComplexHeatmap" package (Version 2.14.0) using the default euclidean clustering (Team RC. R: A language and environment for statistical computing. Vienna, Austria : R Foundation for Statistical Computing; 2022; Gu Z. iMeta. 2022; l(3):e43).

[0344] Table 4. Commercial antibodies used

[0345] Processing human tissue for mAb binding:

[0346] Cryo preserved, dissociated tumor cells (DTCs) were purchased from Discovery Life Sciences. Material from 4 different indications (liver carcinoma, melanoma, breast cancer and ovarian cancer) were included in the analyses and stained with the antibodies described in Table 5. Anti-ICAM-1 antibodies were produced in-house and labelled with Alexa Fluor 647.

[0347] Table 5. Commercial antibodies used for human flow cytometry experiments

[0348] Cloning of fusion constructs

[0349] Mouse and human ICAM-1 truncation mutants were generated by overlap PCR (Horton et al., Gene. 1989;77(l):61-8). An Rp3 peptide (recognised by rituximab (Perosa et al., Blood. 2006;107(3): 1070-7) was incorporated into the N' terminus and the sites of truncation chosen based on recorded annotations within the UniProt reference sequences (P05362 and P13597) and published structures within the Protein Data Bank (2OZ4 and HAM). All constructs were cloned into the pcDNA3.1 / - (neo) expression vector.

[0350] Site-directed mutagenesis Bespoke primers were designed (SeqBuilder vl4, Lasergene DNAStar) to induce point mutations and plasmid DNA was mutated using the Agilent SDM kit. Unmutated DNA was digested with Dpnl before transforming into XL-gold E.coli. Presence of mutation was confirmed by sanger sequencing at Source BioScience (Cambridge, UK).

[0351] Isolation of mouse ICAM-1 transcripts from spleen and tumor:

[0352] Spleen and tumor from CT26-bearing mice were processed into single cell suspension as described above. CD4+ cells were enriched using an EasySep mouse CD4 positive selection kit. The cell suspension was stained with anti-CD4 and anti-CD25 fluorescently labelled mAb before sorting the CD4+CD25- and CD4+CD25+ populations on a BD FACSMelody (BD Biosciences). RIMA was extracted using a RNAeasy kit (Qiagen) and RT-PCR performed using the SuperScriptHI first-strand synthesis system (Invitrogen). PCR was performed on the resulting cDNA using Pfu polymerase (Promega) and primers designed to amplify the full extracellular domain of mouse Icaml (mIcaml_E2F GTGATGCTCAGGTATCCATCCATC, mIcaml_E6R CAGGTACACATTCCTGGTGACATTC). The resulting PCR product was run on a 1% agarose gel. The resulting DNA band was extracted with a QIAquick gel extraction kit (Qiagen) and Sanger sequenced (Source Biosciences) before analysis using SeqMan Pro 18 (DNASTAR).

[0353] HEK293F and CHO-S transfections

[0354] 10x106 HEK293F or CHO-S cells (Invitrogen) were transfected with 10 pg of plasmid DNA using Freestyle Max (Invitrogen) lipofection reagent. 24 hours following transfection, cell surface expression was assessed using flow cytometry, with binding of rituximab-FITC to the Rp3 peptide used as a positive control for surface expression.

[0355] Cross blocking assays

[0356] HEK293F cells were transiently transfected with WT mouse ICAM-1 (cDNA purchased from R8iD) for 24 hours prior to assay. Cells were treated with 10 pg / mL of unlabelled mAb or ligand for 30 mins at 4°C before the secondary fluorescently labelled detection agent was added for 30 min at 4°C. Samples were then washed twice in wash buffer and stored in the dark at 4°C before acquisition on a flow cytometer.

[0357] ELISA for binding to deglvcosylated ICAM-1 :

[0358] Recombinant mouse ICAM-1 was obtained from R&D Systems. To deglycosylate, 100 pg was added to an equilibrated OmniGLYZOR column (Genovis) and incubated at 37°C for 6 hours. The deglycosylated protein was collected by centrifugation. For antibody binding ELISA, recombinant mouse ICAM-1 protein and the in-house deglycosylated form of the protein, were coated to assay plates at 2 pmol / well. Antibodies were titrated from 100 pg / ml to 0.0056 pg / ml and left to bind for 1 hour at room temperature. Bound antibodies were detected using HRP-labelled goat anti-mouse IgG, F(ab')2 fragment specific antibody (Jackson Immunoresearch, RRID:AB_2338507). A chemiluminescence substrate (Pierce 37070) was added and plate reading was performed with a Tecan Spark.

[0359] Statistical Analyses:

[0360] Statistical analyses were calculated in GraphPad Prism (vlO.1.0).

[0361] Discussion

[0362] Developing therapeutics that target tumor Treg cells is an attractive approach for cancer immunotherapy. Approaches to date have included the use of low dose metronomic chemotherapy with cyclophosphamide (Scurr et al., Clin Cancer Res. 2017;23(22):6771- 80), small molecule inhibitors, such as those targeting PI3K isoforms (Lauder et al., Br J Cancer. 2022; 127(9): 1595-602), and antibodies directed towards members of the TNFRSF, CD25, CCR4 (Zhang et al., BMC Cancer. 2021;21(l):618) or CCR8 (Gampa et al., Br J Pharmacol. 2024) which rely on the differential expression of targets to distinguish between Tregs and non-regulatory CD4+ T cells. Each of these approaches have their limitations, and only marginal gains in the clinic have been made. These approaches can also lead to the development of autoimmune conditions and toxicities in patients due to the systemic targeting (The L. End of the road for daclizumab in multiple sclerosis. Lancet. 2018;391(10125): 1000).

[0363] Here, we used a target-cell first approach to identify antibodies with selectivity towards tumor associated Tregs. These antibodies depleted Tregs efficiently in the tumor compartment and resulted in anti-tumor responses as a monotherapy in the CT26 model. Target identification revealed that 12-D10 recognised an epitope on ICAM-1, providing a promising new target. ICAM-1 is known to be upregulated in inflammatory conditions and has been linked to the migration and interaction of Treg cells with DCs (Bui TM et al., J Leukoc Biol. 2020;108(3):787-99; 46; Kohm et al., J Autoimmun. 2003;21(3):261-71), with potential impacts additional to target cell deletion. ICAM-1 has been previously investigated as a target for antibody immunotherapy for multiple myeloma (Dahlhoff et al., Leukemia. 2022;36(3):790-800). Those antibodies were well tolerated in phase 1 clinical trials but displayed little therapeutic benefit, leading to a pause in their clinical development (Wichert et al., PLoS One. 2017;12(2):e0171205). These agents were shown to bind the top two domains of ICAM-1 and were selected primarily for their ability to induce apoptosis of myeloma cells rather than for their impact on TILs. 12-D10 binds a different domain of ICAM-1 than those previous reagents, potentially explaining its differential selectivity towards Tregs, differentiating it from what has gone before. Moreover, we know that where an antibody binds in relation to the target cell membrane can have a profound impact on the effector mechanisms engaged by an antibody (Griffiths et al., Journal for Immunotherapy of Cancer. 2020;8(2), Cleary et al., The Journal of Immunology. 2017;198(10):3999-4011), and with 12-D10 binding far closer to the cell membrane compared to previous clinical candidates, it would be expected to have better depleting potential.

[0364] 12-D10 binding was specific and highest on Treg cells from the TME, in comparison to other T cells, although there were low levels of binding detected on NK cells in the tumor. Further phenotyping of the 12-D10+ tumor Tregs found co-expression of CCR.8, identified as a marker of functionally suppressive Treg subpopulations (Van Damme et al. J Immunother Cancer. 2021;9(2)) alongside other "Treg" targets including 4-1BB, CTLA-4 and GITR.( Buchan et al. Immunity. 2018;49(5):958-70 e7; Wegrzyn et al. Front Immunol. 2022;13: 1055805; Sobhani et al. Cancers (Basel). 2021;13(6)) Across a range of different human tumor samples (including liver carcinoma, breast cancer and ovarian cancer) a similar preferential binding for tumor Treg was observed above other T cells and Tregs from normal healthy PBMCs with two antibodies identified as binding to domain 4 of human ICAM-1 compared to the broader cellular binding pattern seen with the domain 1 ICAM-1 binder enlimomab. Further work is required to understand the basis behind the fine specificity of 12-D10 for ICAM-1 on tumor Tregs.

[0365] ICAM-1 is a large protein consisting of 5 globular Ig-like domains and is highly glycosylated. There are many isoforms of ICAM-1 (Hu et al., Mol Immunol. 2010;47(9): 1692-700; Ramos et al., J Immunol. 2014;192(10):4469-74), but what is unclear is whether there are cell-specific isoforms present, and how the tumor microenvironment (TME) can influence this. Our analysis indicated that the differential targeting of tumor Treg by 12- D10 is likely not due to differences in ICAM-1 isoform expression. However, it is known that the TME itself can influence the glycosylation patterns of proteins due to changes in metabolism and expression of glycan-processing enzymes (Peixoto et al., Front Oncol. 2019;9:380). It is also known that surface glycosylation differences are observed in both mouse and human Tregs (Cabral et al. Front Immunol. 2017;8:987) and that different glycoforms of ICAM-1 are evident in disease (Regal-McDonald et al. PLoS One. 2020; 15(3) :e0230358). These changes could therefore alter the accessibility of specific epitopes on ICAM-1 on tumor associated Tregs compared to ICAM-1 expressed on other cells in the same niche (e.g. the TME) and the same cells in other environments. In support of this suggestion, the Inventors showed that the binding of 12-D10 was sensitive to deglycosylation of ICAM-1 whereas a domain 1 binding anti-ICAM-1 antibody was entirely unaffected.

[0366] Other tumor Treg-specific molecules identified in our screen included Folate Receptor 4 (FolR4) and GITR. FolR4 was proposed as a Treg marker by Yamagutchi in 2007 and targeting with antibodies also reported similar levels of anti-tumor efficacy as seen with our panel (Yamaguchi et al., Immunity. 2007;27(l): 145-59). However, a direct human homologue of FolR4 is not apparent. Humans express a family of Folate Receptors (a, P, y and 6), however there is limited evidence of this being a Treg specific receptor and the family are expressed on different cell populations (Zhang et al., Dis Markers. 2022;2022:4351949; Young et al., Curr Prob Cancer. 2023;47(l)). FoIRo is upregulated on tumors and recently an antibody drug conjugate, mirvetuximab soravtansine, was approved for the treatment of ovarian cancers (Matulonis et al., J Clin Oncol. 2023;41(13):2436-45). Regardless of the disparity between mouse and human FoIR, our antibodies against FolR4 (such as 16-A03) should provide useful tools for pre-clinical studies, allowing the targeting of Tregs but not CD4 helper or CD8 cytotoxic cells.

[0367] Employing a target agnostic differential cell panning "cell-first" approach to identify tumor Treg specific mAb, a number of established and novel targets were identified. By subsequently taking a "function-first" approach testing for desired activity (tumor Treg depletion), before target identification, time and resources were focussed on mAb that demonstrated therapeutic potential. One drawback with this approach, is that the subsequent identification of the target can then be time-consuming. For human target identification, many techniques exist that allow for rapid protein screening to identify the target , but these platforms are currently more limited in the mouse. A lack of Treg cell lines also limits the resources available for protein heavy techniques, such as immunoprecipitations and mass spectrometry, such that primary cells and tissue are currently required for screening. Nevertheless, approaches to refine the pool of antibodies of interest are of benefit, meaning functional assays evaluating antibody activity are essential.

[0368] A key point here is that functional screening will only reveal mAb with the desired activity - for example in this case, depletion of tumor Tregs. Indeed, our choice of the mIgG2a isotype was predicated on this activity. It may well be that some of the clones identified in our initial Treg scFv pool would work as immunomodulatory antibodies; either in immune stimulation or checkpoint inhibition. In this case, expressing the clones in a mlgGl or mIgGl-N297A format, to engage the inhibitory FcyRII for greater receptor cross-linking or to obviate FcyR interaction, respectively, as we recently did with anti-PD-1 (Moreno-Vicente et al. J Immunother Cancer. 2022; 10(1)) may identify alternative activities.

[0369] Returning to 12-D10, and its potential human surrogate 5-A11, it is apparent that ICAM-1 is expressed on tumor associated Tregs in various sites and upregulated is compared to Tregs (and other T cell populations) in healthy PBMCs. The molecular basis behind this specificity requires further interrogation, however, it does highlight the benefit of undertaking a phenotypic approach to identifying novel immunotherapeutic targets. By following a function-first screening approach, many unexpected avenues for investigation are revealed, and even targets initially overlooked for being too widely expressed, such as ICAM-1, can be pursued in a new framework beyond expression levels alone.

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Claims

CLAIMS1. An antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1 for use in the treatment of cancer in a patient.

2. Use of an antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1, in the manufacture of a medicament for the treatment of cancer in a patient.

3. A method for treating cancer in a patient, the method comprising the step of administering an antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1.

4. An antibody molecule, or fragment thereof, for use according to Claim 1, or a use according to Claim 2, or a method according to Claim 3, wherein the ICAM-1 is present on the surface of Tregs.

5. An antibody molecule, or fragment thereof, for use, or a use, or a method according to Claim 4, wherein the Tregs are tumor-associated Tregs.

6. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the antibody molecule, or fragment thereof, is a Treg-depleting antibody molecule.

7. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein binding of the antibody, or fragment thereof, to ICAM-1 induces the depletion of Tregs in the patient.

8. An antibody molecule, or fragment thereof, for use, or a use, or a method according to Claim 7, wherein binding of the antibody, or fragment thereof, to ICAM-1 induces the specific depletion of tumor-associated Tregs in the patient.

9. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the antibody molecule, or fragment thereof, binds to a glycosylation dependent epitope in Domain 4 of ICAM-1.9310. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the antibody molecule, or fragment thereof, comprises an Fc region that binds to at least one activating Fey receptor.

11. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the antibody molecule, or fragment thereof, is a full-length antibody or a chimeric antibody.

12. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the antibody molecule or fragment thereof is IgGl or an Fc-engineered variant thereof.

13. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the antibody molecule, or fragment thereof, is a human IgGl antibody.

14. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the antibody molecule, or fragment thereof, is a monoclonal antibody.

15. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the antibody molecule, or fragment thereof, comprises 1-6 of the CDRs VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, wherein VH-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs:1, 9 and 17; wherein VH-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs:2, 10 and 18; wherein VH-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs:3, 11 and 19; wherein VL-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs:4, 12 and 20; wherein VL-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs:5, 13 and 21; andwherein VL-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs: 6, 14 and 22.

16. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the antibody molecule, or fragment thereof, comprises a variable heavy chain (VH) comprising the following CDRs:(i) SEQ. ID. NO: 1, SEQ. ID. NO: 2 and SEQ. ID. NO. : 3; or(ii) SEQ. ID. NO: 9, SEQ. ID. NO: 10 and SEQ. ID. NO: 11; or(iii) SEQ. ID. NO: 17, SEQ. ID. NO: 18 and SEQ. ID. NO: 19; and / or wherein the antibody molecule comprises a variable light chain (VL) comprising the following CDRs:(i) SEQ. ID. NO: 4, SEQ. ID. NO: 5 and SEQ. ID. NO: 6; or(ii) SEQ. ID. NO: 12, SEQ. ID. NO: 13 and SEQ. ID. NO: 14; or(iii) SEQ. ID. NO: 20, SEQ. ID. NO: 21 and SEQ. ID. NO: 22.

17. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the antibody molecule, or fragment thereof, comprises a variable heavy chain (VH) amino acid sequence selected from the group consisting of SEQ. ID. NOs 7, 15 and 23; and / or wherein the antibody molecule comprises a variable light chain (VL) amino acid sequence selected from the group consisting of SEQ. ID. NOs: 8, 16 and 24.

18. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the antibody molecule, or fragment thereof, is capable of competing for binding to Domain 4 of ICAM-1 with an antibody molecule as defined in any of the Claims 15 to 17.

19. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the ICAM-1 comprises or consists of a polypeptide sequence with at least 90% homology to SEQ. ID. NO: 27 or 28.9520. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein Domain 4 of ICAM-1 comprises or consists of a polypeptide sequence with at least 90% homology to SEQ. ID. NO: 29 or 30.

21. An antibody molecule for use, or a use, or a method according to any preceding claim, wherein the antibody molecule is administered with an anti-cancer agent, such as a therapeutic anti-cancer antibody molecule.

22. An antibody molecule, or fragment thereof, for use, or a use, or a method according to Claim 21, wherein the therapeutic anti-cancer antibody molecule is an immunostimulatory antibody molecule.

23. An antibody molecule, or fragment thereof, for use, or a use, or a method according to Claim 22, wherein the immunostimulatory antibody molecule is an anti-PD-1 antibody molecule; an anti-PD-Ll antibody molecule; or an anti-CTLA-4 antibody molecule.

24. An antibody molecule, or fragment thereof, for use, or a use, or a method according to Claim 20, wherein the anti-cancer agent is a chemotherapeutic agent.

25. An antibody molecule, or fragment thereof, for use, or a use, or a method according to Claim 23, wherein the chemotherapeutic agent is selected from the group consisting of: carboplatin and / or paclitaxel.

26. An antibody molecule, or fragment thereof, for use, or a use, or a method according to any preceding claim, wherein the cancer is a solid cancer, such as a sarcoma, and / or carcinoma.

27. An antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1, wherein the antibody molecule or fragment thereof comprises 1-6 of the CDRs VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, wherein VH-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs:1, 9 and 17; wherein VH-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs:2, 10 and 18;wherein VH-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs:3, 11 and 19; wherein VL-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs:4, 12 and 20; wherein VL-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs:5, 13 and 21; and wherein VL-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs:6, 14 and 22.

28. An antibody molecule, or fragment thereof, according to Claim 27, wherein the antibody molecule, or fragment thereof, comprises a variable heavy chain (VH) comprising the following CDRs:(i) SEQ. ID. NO: 1, SEQ. ID. NO: 2 and SEQ. ID. NO. : 3; or(ii) SEQ. ID. NO: 9, SEQ. ID. NO: 10 and SEQ. ID. NO: 11; or(iii) SEQ. ID. NO: 17, SEQ. ID. NO: 18 and SEQ. ID. NO: 19; and / or wherein the antibody molecule comprises a variable light chain (VL) comprising the following CDRs:(i) SEQ. ID. NO: 4, SEQ. ID. NO: 5 and SEQ. ID. NO: 6; or(ii) SEQ. ID. NO: 12, SEQ. ID. NO: 13 and SEQ. ID. NO: 14; or(iii) SEQ. ID. NO: 20, SEQ. ID. NO: 21 and SEQ. ID. NO: 22.

29. An antibody molecule, or fragment thereof, according to Claim 27 or 28, wherein the antibody molecule, or fragment thereof, comprises a variable heavy chain (VH) amino acid sequence selected from the group consisting of SEQ. ID. NOs 7, 15 and 23; and / or wherein the antibody molecule comprises a variable light chain (VL) amino acid sequence selected from the group consisting of SEQ. ID. NOs: 8, 16 and 24.

30. A polynucleotide molecule encoding an antibody, or fragment thereof, as defined in any preceding claim.

31. A vector comprising a polynucleotide molecule as defined in Claim 30.

32. A virus comprising a polynucleotide molecule as defined in Claim 30 and / or a vector as defined in Claim 31.

33. A cell comprising a polynucleotide molecule as defined in Claim 29, and / or a vector as defined in Claim 30, and / or a virus as defined in Claim 31.

34. An antibody molecule, or fragment thereof, that binds specifically to Domain 4 of ICAM-1 for use in medicine.

35. An antibody molecule, or fragment thereof, for use according to Claim 34, wherein the antibody molecule, or fragment thereof, binds to a glycosylation dependent epitope in Domain 4 of ICAM-1.

36. A pharmaceutical composition comprising or consisting of an antibody molecule, or fragment thereof, as defined in any of Claims 1-29, and optionally a pharmaceutically acceptable diluent, carrier, vehicle and / or excipient.

37. A pharmaceutical composition comprising or consisting of a polynucleotide molecule as defined in Claim 30, a vector as defined in Claim 31, a virus as defined in Claim 32 and / or a cell as defined in Claim 33, and optionally a pharmaceutically acceptable diluent, carrier, vehicle and / or excipient.

38. A pharmaceutical composition as defined in Claim 36 or 37, for use in the treatment of cancer.

39. A method for treating cancer in a patient, the method comprising the step of administering the pharmaceutical composition of Claim 36 or 37 to the patient.

40. A kit for the treatment of cancer, comprising an antibody molecule, or fragment thereof, as defined in any of Claims 1-29, and / or a polynucleotide molecule as defined in Claim 30, and / or a vector as defined in Claim 31, and / or virus as defined in Claim 32, and / or a cell as defined in Claim 33, and / or the pharmaceutical composition as defined in Claim 35 or 36.

41. A kit according to Claim 40, wherein the kit further comprises an anti-cancer agent, such as a therapeutic anti-cancer antibody molecule or a chemotherapeutic agent.

42. An antibody molecule, or fragment thereof, for use; or a use; or a method; or an antibody; or a polynucleotide molecule; or a vector; or a cell; or a pharmaceutical composition; or a kit; as described herein with reference to the accompanying description, examples and / or figures.99

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