F-actin binding agents

Abstract

Radiolabelled binding agents that specifically bind to F-actin are provided. In particular, the radiolabelled binding agents of the invention exhibit preferential binding for F-actin over G-actin, which enables them to be used in medical and research settings. The invention provides radiolabelled binding agents, medical uses thereof, methods of generating radiolabelled binding agents and kits.

Description

[0001] F-actin binding agents

[0002] This application claims priority from GB 2407700.0 filed 30 May 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Field of the Invention

[0004] The present invention relates to binding agents that comprise a molecule that binds specifically to F-actin and a cargo moiety. Specifically, it provides binding agents that comprise a molecule that binds specifically to F-actin and a radioactive cargo moiety, which find use in radio-immuno therapy (RIT) and radio-immuno imaging (RII) of solid tumours. Also provided are binding agents that comprise a molecule that binds specifically to F-actin and a cargo moiety which is a cytotoxic agent. The invention provides binding agents, medical uses thereof, particularly in the treatment of cancer, and use of the binding agents as an imaging agent for cancer.

[0005] The application contains a sequence listing which has been submitted herewith and is hereby incorporated by reference in its entirety.

[0006] Background

[0007] F-actin is a polymer of the monomeric globular-actin (G-actin). Each monomer of actin is a 375-amino acid polypeptide which folds into two major a / p domains separated by a deep cleft and consists of subdomains 1-4. F-actin assembles into two protofilaments that turn around each other to form a right- handed, two-chained long helix with approximately 13 molecules repeating every 6 turns in an axial distance of 35.9 nm (Dominguez & Holmes, 2011 , which is hereby incorporated by reference in its entirety). Actin is an ATPase and it is this nucleotide binding and hydrolysis that regulates the transition between F-actin and G-actin, resulting in highly dynamic actin filaments which enable cells to change shape.

[0008] Gelsolin (GSN) is a multifunctional protein which can act to sever, cap and nucleate actin filaments. It is expressed both in the extracellular fluids and in the cytoplasm of the majority of human cells, and it is implicated in a variety of both physiological and pathological processes. Both cytoplasmic and secreted forms are coded by the same gene, with the secreted form (sGSN) being made by use of an alternatively spliced exon in the GSN gene that encodes a signal peptide.. sGSN can also be found in other fluids such as the lymphatic and cerebrospinal fluid (CSF). sGSN is one of two abundant actin-binding proteins (ABPs) that are present in the serum and plasma of all mammals, the other being Gc globulin. These ABPs are thought to form part of an actin-scavenging system, which contributes to the removal of potentially pathological actin filaments released by dying cells following tissue damage. In this system, sGSN binds to F-actin in a Ca2+-dependent manner and severs the filaments for subsequent depolymerisation, which is facilitated by Ca2+-independent sequestering of monomeric G-actin by Gc globulin. Radio-immuno therapy (RIT) and radio-immuno imaging (RII) of solid tumours has shown great promise in recent years. However, treatment of solid tumours remains a great challenge due to a number of issues, such as tumour penetration by the agents, acquired resistance, and the tumour microenvironment. Treatment with RIT requires a delicate balance between targeted killing of cancer cells and on-target / off tissue or off-target killing of healthy cells. As tumours grow, the central areas can become hypoxic and undernourished - leading to necrosis. Hence, most tumours tend to have necrotic areas, with the core of the tumour often being necrotic.

[0009] Hitherto, most radio-immuno conjugates (RICs) have been directed at targets expressed on live cancer cells which typically reside on the outer margins and / or near blood vessels. Tumour penetration of agents against such targets is usually compromised by the “binding site barrier” (also referred to as “affinity barrier”): high affinity binders are retained perivascularly because the interaction with the target and internalisation by perivascular tumour cells prevents the agent from diffusing further into the tumour core (see Figure 1).

[0010] Such “outside-in” killing mechanism results in efficient killing of only the outer perivascular tumour cells and spares cells towards the centre of the tumour.

[0011] In addition, there is a diminishing curative potential as the target cell number is reduced, i.e. cell killing-led loss of target (Sgouros, 2020, which is hereby incorporated by reference in its entirety). Killing of target cells by traditional RICs reduces the available target antigen for the RICs, thus diminishing efficacy over time.

[0012] Therefore, there remains a need for how to achieve the benefits of RIC and RII whilst avoiding the drawbacks listed above.

[0013] In contrast to most RIC targets, filamentous-actin (F-actin) is normally intracellular, and is only exposed extracellularly upon atypical cell death (e.g. necrosis) whereby there is a rupture of plasma membrane.

[0014] Other cargo molecules besides radionuclides may also be targeted to specific areas, such as tumours, by conjugation to specific binding moieties. Examples of these include antibody-drug conjugates (ADCs) which have shown promise in cancer treatment. Nevertheless, the success of ADCs has been hampered by acquired resistance and treatment-related adverse effects.

[0015] Targeting necrotic cell antigens is known. For example, melanin is an intracellular pigment that is exposed following necrosis, and melanin has been targeted with radiolabelled antibodies (Staudacher, 2018, which is hereby incorporated by reference in its entirety). Other examples of necrotic cell targeting agents include the La / SSB protein that is also exposed following loss of membrane integrity during necrosis, and monoclonal antibodies against nuclear antigen (chTNT-1 / B that targets Histone / DNA complex) (Staudacher, 2018).

[0016] However, F-actin has unique advantages over other necrotic antigens, as will be described below.

[0017] The present invention has been devised in light of the above considerations. Summary of the Invention

[0018] The inventors have identified F-actin as a highly relevant target for therapy. Importantly, F-actin is only exposed extracellularly upon atypical cell death (e.g. necrosis) whereby there is a rupture of plasma membrane. This is a common feature of tumours, in which cancer cells often undergo primary or secondary necrosis as a result of restrictions in nutrient or oxygen supply, immune responses and / or cytotoxic therapies. Therefore, F-actin is a specific marker of dead cells.

[0019] F-actin is a polymeric molecule, thus affording increased target epitopes relative to other antigens. This can lead to a greatly improved avidity of the interaction between F-actin and F-actin binding agents. In the present context of F-actin binding agents, this means that high levels of drug (where the cargo moiety is a cytotoxic agent) or radioactivity (where the cargo moiety is a radionuclide) can be specifically targeted to a tumour. Unlike cytoplasmic proteins, F-actin is also retained for prolonged periods on dead and dying cells after loss of membrane integrity, providing for a more stable therapeutic target within tumours that is not rapidly lost upon cell death (WO 2024 / 018062, which is hereby incorporated by reference in its entirety).

[0020] Patients treated with targeted therapies in general, and antigen-targeted ADCs and RICs in particular, acquire resistance via loss of target antigen. Targeting F-actin exposed upon cell death will prevent such acquired resistance, firstly because F-actin is a critical protein for cell survival, and secondly because targeting dead or dying cells prevents evolutionary acquisition of resistance. A further cause of resistance to targeted therapies is tumour heterogeneity. As F-actin is ubiquitously expressed, anti-F actin agents will not be subject to these limitations.

[0021] Finally, due to the activity of secreted gelsolin (sGSN) there are very low levels of target epitopes (F- actin) in non-tumour tissues.

[0022] The inventors have surprisingly shown that despite the amount and F-actin-depolymerising activity of sGSN in serum and tissues (see Piktel, 2018, which is hereby incorporated by reference in its entirety), there is sufficient F-actin exposed within tumours to be a viable target. In addition, the depolymerising action of sGSN means that even small amounts of F-actin that is exposed in healthy tissues is neutralised. Based on this, it is hypothesised that on-target, off-tumour effects are reduced compared to other cytotoxic drug delivery or radioimmunotherapy (RIT) approaches.

[0023] Attaching a radioactive cargo moiety, such as a radionuclide, to an F-actin binding moiety has specific advantages. As F-actin is not exposed at peripheral oxygenated areas, high affinity anti-F-actin RIC molecules will not be sequestered at the periphery, and hence will have better tumour penetration towards the central necrotic core (Figure 2A). In other words, anti-F-actin RIC molecules do not have the issue of an “affinity barrier”, unlike RIC molecules directed towards other targets.

[0024] Once at the necrotic tumour core, the anti-F actin RICs bind the outer rim of the core with high affinity, which prevents further penetration. The anti-F actin RICs bound to the outer rim of the necrotic core elicit killing of neighbouring cells via bystander radioactive killing (Figure 2B). This initial round of killing of the peri-necrotic core cells exposes additional F-actin and enlarges the necrotic core: affording greater target substrate for binding of additional anti-F actin RICs, thus amplifying the initial killing efficacy and killing of tumours from inside out (Figure 2C). It is noted that although in Figure 2 the necrotic area is depicted at the central core of the tumour, tumours may have necrotic areas in other regions, such as at the periphery, and the same principles described herein apply regardless of the location of the necrotic area.

[0025] The present invention therefore provides an alternative mechanism of RIT: not only is the affinity barrier problem associated with other RIT overcome, the killing is amplified by a positive feedback mechanism. It is hypothesised that this means that a much smaller dose of RIC will be required compared to unlabelled F-actin agents or other RICs.

[0026] The present invention also provides agents which deliver a cargo of a cytotoxic agent to tumours. Such agents may be referred to herein as “non-radiolabelled binding agents” or “payload delivery agents” or “conjugates”. The advantages described above for radioactive cargo moieties also apply to cytotoxic agent cargo moieties used in these payload delivery agents.

[0027] In a first aspect, the invention provides a radiolabelled binding agent comprising: i) an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and ii) a radionuclide.

[0028] Preferably, the radiolabelled binding agent has a higher affinity for F-actin than for globular actin (G- actin). Preferably, the radiolabelled binding agent has an EC50 value for F-actin of about 5 nM or less.

[0029] In some embodiments, the radiolabelled binding agent has an EC50 value for F-actin of about 0.01 nM to about 5 nM, about 0.1 to about 4 nM, about 0.2 to about 3 nM, about 0.3 nM to about 2 nM, 0.4 nM to about 1 nM, about 0.5 nM to about 1 nM. In some embodiments, the radiolabelled binding agent has an EC50 value for F-actin of about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, or about 0.5 nM.

[0030] In some embodiments, the radiolabelled binding agent has at least 5-fold higher affinity for F-actin than for globular actin (G-actin). In some embodiments, the radiolabelled binding agent has at least 10-fold higher affinity for F-actin than for G-actin. In some embodiments, the specificity for F-actin is between 2- and 100-fold greater than for G-actin. In some embodiments, the specificity for F-actin is between 2- and 10-fold greater than for G-actin. In some embodiments, the specificity for F-actin is at least 2-fold greater than for G-actin. In some embodiments, the specificity for F-actin is at least 5-fold greater than for G- actin. In some embodiments, the specificity for F-actin is at least 10-fold greater than for G-actin. In some embodiments, the specificity for F-actin is at least 100-fold greater than for G-actin. Specificity is explicitly intended to encompass affinity and / or avidity.

[0031] In some embodiments, affinity is determined by bio-layer interferometry (BLI). In some embodiments, affinity is determined by ELISA.

[0032] Affinity of the radiolabelled binding agents (or of the non-radiolabelled binding agents) for F-actin and G- actin can be assessed by the following method. Immunoplates (MaxiSorp 96-well, Thermo Fisher Scientific) are coated with 5 pg / ml NeutrAvidinTM (Thermo Fisher Scientific) in PBS overnight at 4°C, washed, blocked with 2.5% BSA in PBS for 2h at room temperature and stored overnight at 4°C after addition of biotinylated F- or G-actin (2 nM). Capture of biotinylated F- and G-actin is verified using a commercial actin-specific mAb (AC-40, Sigma) that recognises both, F- and G-actin. To prevent the depolymerisation of F-actin or the polymerisation of G-actin during the assay, F- and G-actin is stabilised by adding phalloidin or cytochalasin D, respectively, during the preparation of the actin stocks. After washing to remove unbound actin, three-fold serial dilutions of radiolabelled binding agent or control reagent (Fc-DNGR-1 fusion, see Experimental section below) starting at 300 nM are added to the ELISA plate for 1 hr. Actin-bound radiolabelled binding agent or control reagent are detected using a horseradish peroxidase (HRP)-conjugated mouse-specific anti-IgG (Jackson ImmunoResearch) antibody (0.16 pg / ml) and developed with an ELISA substrate for HRP based detection (TMB, eBioscience). Substrate development is stopped by adding sulphuric acid (H2SO4, Fisher). Titration curves are plotted using GraphPad Prism7 and EC50 values for each radiolabelled binding agent are calculated by applying a nonlinear regression (curve fit) of a log (agonist) vs response - variable slope (four parameters).

[0033] Radionuclide

[0034] In embodiments where the binding agent comprises a radionuclide, the radionuclide may be selected from the group of radionuclides listed in Eckerman, K. F. & Enzo, A. MIRD: Radionuclide Data and Decay Schemes, 2ndedn. (Society of Nuclear Medicine, 2008); Chomet, M. et al., State of the Art in Radiolabeling of Antibodies with Common and Uncommon Radiometals for Preclinical and Clinical Immuno-PET. Bioconjugate Chemistry, 2021. 32:1315-1330; and Holik, H. A. et al., The Chemical Scaffold of Theranostic Radiopharmaceuticals: Radionuclide, Bifunctional Chelator, and Pharmacokinetics Modifying Linker. Molecules, 2022, 27, 3062, which are hereby incorporated by reference in their entirety.

[0035] In some embodiments, the radionuclide has a half-life of about 30 minutes, about 45 minutes, or about 60 minutes.

[0036] In some embodiments, the radionuclide has a half-life of about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.

[0037] In some embodiments, the radionuclide has a half-life of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days or about 20 days.

[0038] In some embodiments, the radionuclide is a radioisotope of a metal.

[0039] In some embodiments, the radionuclide is selected from the group consisting of:227Th,223Ra,212Pb,212Bi,

[0040] 244At,225Ac,213Bi ,230U,226Th,224Ra,213Po,149Tb,32P,47Sc,64Cu,67Cu,153Sm,177Lu,90Y,131l,199Au,166Ho,186Re,188Re,194lr,159Gd,149Pm,142Pr,109Pd,225Ra,213Bi,212Bi,161Tb,89Sr,77As,149Tb,123l,1311 ,203Pb,205Bi,99mTc, i33Xe,201Tl,51Cr,67Ga,166Ho,61Cu,64Cu,110mAg,90Y,188Re,166Ho,159Gd,137Cs,18F-AI,44Sc,52Mn,76Br,77Br,86Y, "Y,89Zr,124l,66Ga,68Ga,18F,11C,82Rb,13N,61Cu,64Cu,1111 n,18F,67Ga,123l,125l,61Cu,186Re,77As,211At,117mSn,67Ga,77Br and103Pd.

[0041] In some embodiments, the radionuclide is an alpha(a)-emitting radionuclide. In these embodiments, the radiolabelled binding agent may find use in targeted-alpha therapy (TAT). In some embodiments, the alpha(a)-emitting radionuclide is selected from the group consisting of:227Th,223Ra,212Pb,212Bi,211At,225Ac,213Bi,23°U,226Th,224Ra,213Po, and149Tb.

[0042] In some embodiments, the radionuclide is a beta (p-)-emitting radionuclide. In some embodiments, the beta (p-)-emitting radionuclide is selected from the group consisting of:32P,47Sc,64Cu,67Cu,153Sm,177Lu, "Y, 1311;199AU,166Ho,186Re,188Re,194lr,159Gd,149Pm,142Pr,109Pd,225Ra,213Bi,212Bi,161Tb,89Sr,77As and149Tb.

[0043] In some embodiments, the radionuclide is a gamma (y)-emitting radionuclide. In some embodiments, the gamma (y)-emitting radionuclide is selected from the group consisting of:123l,1311 ,203Pb,205Bi ,99mTc,133Xe,201Tl,51Cr,67Ga,166Ho,61Cu,64Cu,110mAg, "Y,188Re,166Ho,159Gd, and137Cs.

[0044] In some embodiments, the radionuclide is a high energy beta (p+)-emitting radionuclide. In some embodiments, the high energy beta (p+)-emitting radionuclide is selected from the group consisting of:18F-AI,44Sc,52Mn,76Br,77Br,86Y, "Y,89Zr,124l,66Ga,68Ga,18F,11C,82Rb,13N,61Cu, and64Cu.

[0045] In some embodiments, the radionuclide decays by electron capture. In some embodiments, the radionuclide is selected from the group consisting of:1111n ,18F,67Ga,123l,125l,61Cu,186Re,77As and211At.

[0046] In some embodiments, the radionuclide decays by isomeric transition. In some embodiments, the radionuclide is117mSn.

[0047] In some embodiments, the radionuclide is an Auger electron-emitting radionuclide. In some embodiments, the Auger electron-emitting radionuclide is selected from the group consisting of:67Ga,77Br and103Pd.

[0048] Radionuclides particularly useful in imaging applications may be radionuclides that are gamma emitters. Therefore, for imaging applications the radionuclide may be selected from the gamma-emitting radionuclides disclosed herein.

[0049] Radionuclides particularly useful in therapeutic applications may be radionuclides that are alpha or beta emitters. Therefore, for therapeutic applications the radionuclide may be selected from the alpha- and / or beta-emitting radionuclides disclosed herein.

[0050] In some embodiments, the radiolabelled binding agent may comprise multiple radionuclides. In some embodiments, the radiolabelled binding agent comprises two, three or four radionuclides.

[0051] For the purposes of the present invention, the term “radionuclide” encompasses and is interchangeable with the terms “radioactive nuclide”, “radioisotope” and “radioactive isotope”. Labelling

[0052] The radionuclide may be attached to the F-actin binding moiety at a tyrosine, lysine or cysteine residue.

[0053] The radionuclide may be attached to the F-actin binding moiety via direct or indirect labelling. Direct labelling may target a tyrosine residue in the F-actin binding moiety. For example, a halogenide e.g. Br, |- , At is oxidised by an appropriate oxidant, e.g. iodogen® (coated tubes), iodo-Beads, chloramine-T (sodium salt of N-chloro-p-toluenesulfonamide) etc. in the presence of the (unlabelled) F-actin binding moiety.

[0054] Indirect labelling with e.g. bromine, iodine or astatine may be performed by pre-labelling a bi-functional halogen carrier, preferably derived from e.g. benzoic acid derivatives, Bolton-Hunter derivatives, benzene derivatives etc. The carrier may be transformed into an activated species to be conjugated to the e-amino group of Lysine residues or the N-terminus of the (unlabelled) binding agent. This indirect method also provides a synthetic route to radiolabel the peptide compounds chemo-selectively at the sulfhydryl group of a cysteine moiety. The cysteine bridged molecules may first be reduced by an appropriate reducing agent e.g. stannous(ll)chloride, Tris(2-carboxyethyl)phosphine (TCEP) generating free cysteine SH- groups that can react with the halogen carrier. As functional groups for the binding maleimide and a- brom acetamide derivatives may be employed.

[0055] Chelator

[0056] In some embodiments, the radiolabelled binding agent further comprises a chelator that is co-ordinated to the radionuclide. In some embodiments, the chelator is a bidentate, tridentate, or tetradentate molecule.

[0057] In some embodiments, the chelator is selected from the group of chelators disclosed in Table 1 below, and salts or functional variants or derivatives thereof capable of chelating the metal.

[0058] Table 1 : Chelators that may be used in the present invention in the context of radiolabelled binding agents.

[0059] In some embodiments, the linker may be a bifunctional chelator.

[0060] In other embodiments, the radionuclide requires halogenation chemistry for conjugation to the F-actin binding agent. In these embodiments, a chelator is not required for conjugation to the F-actin binding agent. For example, Astatine-211 (211At) requires halogenation chemistry.

[0061] Linkers for radiolabelled binding agents of the invention

[0062] Any of the chelators listed above may be combined with a linker as described below. This combination may be referred to as a bifunctional chelator. Thus, in some embodiments, the radiolabelled binding agent further comprises a linker to couple the radionuclide or chelator to the F-actin binding moiety. In this way, the linker may act as a spacer between the F-actin binding moiety and the radionuclide or radionuclide-chelator complex.

[0063] The linker may be attached to a region of the binding agent that does not interact with F-actin or affect the binding interaction with F-actin. In embodiments where the F-actin binding moiety is an antibody, the linker may be attached to the Fc domain.

[0064] In some embodiments, the linker is a covalent linker.

[0065] In some embodiments, the linker is a pharmacokinetic-modifying (PKM) linker. PKM linkers can modify the distribution or excretion of radiolabelled molecules. For example, PKM linkers can reduce uptake by non-target organs and increase accumulation in tumours.

[0066] In some embodiments, the linker is selected from the group comprising: aminohexanoic acid, ethylene glycol bis(succinimidyl succinate) (EGS), disuccinimidyl subera (DSS), EMCS-Bz, MESS-Bz, MIH, 6- carboxy-1 ,4,8,11 -tetraazaundecane (N4), p-aminomethylaniline-diglycolic acid, polyethylene glycol (PEG), NCS, maleimide, N-suc-TFP-ester, maleimide-monoamide, NHS, and MMA. As the radiolabelled binding agents of the invention will operate by killing neighbouring cells via radiation, it is envisaged that in these embodiments the linker is a non-cleavable linker.

[0067] Also provided are non-radiolabelled binding agents in which an F-actin binding moiety is conjugated to a drug or cytotoxic agent. As used herein, the non-radiolabelled binding agent may be referred to as a payload delivery agent or a “conjugate”. In embodiments where the F-actin binding moiety is an antibody, the conjugate may be referred to as an “antibody drug conjugate” or ADC.

[0068] These conjugates find application in the treatment and / or diagnosis of diseases as described herein. As used herein, the drug or cytotoxic agent may be referred to as a “payload” or “warhead”.

[0069] A conjugate of the present invention may have the formula I:

[0070] A-(L-D)P(I) or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0071] A is an F-actin binding moiety described herein;

[0072] L is a linker;

[0073] D is a drug or cytotoxic agent; and p is 1 to n.

[0074] In some embodiments, the drug comprises a cytotoxin, an immunomodulator, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, a DNA, an RNA, an siRNA, an RNAi, a microRNA, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelating agent, or combinations thereof. For the avoidance of doubt, the term “immunomodulator” specifically includes immunostimulatory agents.

[0075] A cytotoxin is a compound that is able to induce death of the cell that is being targeted. Typically, in the context of antibody drug conjugates, a cytotoxin is delivered to a cell targeted by the antibody molecule, where it is released into the cell and induces cell death. The use of cytotoxins in antibody drug conjugates is described, for example, in Chalouni and Doll 2018 J Exp Clin Cancer Res. 37(1):20 (which is hereby incorporated by reference in its entirety). In some embodiments, the cytotoxin is a tubulysin, an auristatin, a maytansinoid, a topoisomerase inhibitor or a pyrrolobenzodiazepine (PBD).

[0076] The design and selection of linkers to be used in conjugates is known in the art and is described for example in Beck, 2017 Nat Rev Drug Discov 16(5):315-337 (which is hereby incorporated by reference in its entirety). The linker used herein may be any of the linkers described in Beck, 2017. In some embodiments, the linker is a cleavable linker. In these embodiments, the cleavable linker is designed to be cleaved exclusively in the extracellular tumour microenvironment. In some embodiments, the linker is an acid-cleavable linker, an enzyme-cleavable linker or a reducible disulfide. In some embodiments, the acid-cleavable linker is a carbonate, a hydrazone, or an ester. In some embodiments, the enzyme-cleavable linker is a dipeptide. In some embodiments, the dipeptide is Val-Cit. The linker may also be any of the cleavable linkers described in Bargh 2019 (which is hereby incorporated by reference in its entirety).

[0077] In some embodiments, the payload delivery agent further comprises a self-immolative spacer.

[0078] The drug loading (p) is the average number of drugs per F-actin binding moiety. Drug loading may range from 1 to 80 drugs (D) per F-actin binding moiety, although an upper limit of 40, 20, 10 or 8 may be preferred. Compositions of conjugates include collections of F-actin binding moieties, conjugated with a range of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.

[0079] The average number of drugs per F-actin binding moiety in preparations of conjugates from conjugation reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis. The quantitative distribution of conjugates in terms of p may also be determined. By ELISA, the averaged value of p in a particular preparation of conjugates may be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11 :843-852, which are both hereby incorporated by reference in their entirety). However, the distribution of p (drug) values is not discernible by the antibody-antigen binding and detection limitation of ELISA. Also, ELISA assay for detection of conjugates does not determine where the drug moieties are attached to the F-actin binding moiety, such as the heavy chain or light chain fragments of an antibody, or the particular amino acid residues. In some instances, separation, purification, and characterization of homogeneous conjugates where p is a certain value from conjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.

[0080] For some conjugates, p may be limited by the number of attachment sites on the F-actin binding moiety. For example, the F-actin binding moiety may be an antibody, and an antibody molecule may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached. Higher drug loading, e.g. p >5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain conjugates.

[0081] Typically, fewer than the theoretical maximum of drug are conjugated to an F-actin binding moiety during a conjugation reaction. An F-actin binding moiety may contain, for example, many lysine residues that do not react with the linker (L). Only the most reactive lysine groups may react with an amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibody molecules do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety. Most cysteine thiol residues in the antibody molecules of the conjugates exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP, under partial or total reducing conditions. The loading (drug / F-actin binding moiety ratio) of a conjugate may be controlled in several different manners, including: (i) limiting the molar excess of Drug Linker relative to F-actin binding moiety, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.

[0082] Certain antibody molecules have reducible interchain disulfides, i.e. cysteine bridges. Antibody molecules may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut’s reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues). US 7521541 teaches engineering antibodies by introduction of reactive cysteine amino acids.

[0083] Methods to prepare conjugates using direct conjugation at solvent-accessible thiols generated by reduction of the antibody molecule interchain disulphide bridges involving N-alkyl maleimide are known. Other methods conjugate the drug at primary amines of lysines using N-hydroxysuccinimide ester. Such methods are reviewed in, for example, Gebleux and Casi, Pharmacol Ther (2016) 167: 48-59, which is herein incorporated by reference in its entirety. These methods result in the preparation of heterogeneous conjugates with various drug load, efficacy, pharmacokinetics, and therapeutic properties.

[0084] Separately or in addition to the direct conjugation methods described above, it is also possible to use sitespecific conjugation, a method in which drug load and site of conjugation is controlled. This can be achieved by, for example, engineering cysteines at specific residues, replacement of residues with unnatural amino acids with bio-orthogonal reactivity or enzyme ligation approaches. One method of sitespecific conjugation is described in Dimasi et al. (2017) Mol Pharmaceuticals 14(5) 1501-1516, which is hereby incorporated by reference in its entirety, and involves inserting cysteines into, for example, antibody molecules at particular positions.

[0085] Cysteine amino acids may be engineered at reactive sites in an antibody molecule and which do not form intrachain or intermolecular disulfide linkages (Junutula, et al., (2008) Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721 -2729; US 7521541 ; US 7723485; W02009 / 052249, which are hereby incorporated by reference in their entirety). The engineered cysteine thiols may react with linkers or the drug-linker described herein which have thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form conjugates with cysteine engineered antibody molecules and the drug. The location of the drug can thus be designed, controlled, and known. The drug loading can be controlled since the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or drug-linker reagents in high yield. Engineering an IgG antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain gives two new cysteines on the symmetrical antibody. If required, a drug loading near 2 can be achieved with near homogeneity of the conjugation product.

[0086] Where more than one nucleophilic or electrophilic group of the antibody molecule reacts with a drug-linker intermediate, or linker reagent followed by drug reagent, then the resulting product is a mixture of conjugate compounds with a distribution of drug attached to an antibody, e.g. 1 , 2, 3, etc. Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate compounds in the mixture by drug loading value. Preparations of conjugate with a single drug loading value (p) may be isolated, however, these single loading value conjugates may still be heterogeneous mixtures because the drug may be attached, via the linker, at different sites on the antibody molecule.

[0087] Thus, the conjugate compositions of the invention include mixtures of F-actin binding moiety -drug conjugate compounds where the F-actin binding moiety has one or more drug moieties and where the drug moieties may be attached to the F-actin binding moiety at various amino acid residues.

[0088] F-actin bindinci moiety

[0089] In preferred embodiments, binding of the F-actin binding moiety of the radiolabelled binding agent to F- actin is inhibited by sGSN.

[0090] In some embodiments, the F-actin binding moiety is selected from the group consisting of: an antibody or antigen-binding fragment thereof, an affimer, an aptamer, a DarPin, the C-type lectin like domain (CTLD) of DNGR-1 , the extracellular domain of DNGR-1 , LifeAct peptide and phalloidin.

[0091] The anti-F-actin DarPin may have the sequence of a DarPin disclosed in Table S1 of Ivanova, 2024, which is hereby incorporated by reference in its entirety.

[0092] In preferred embodiments, the F-actin binding moiety is an antigen-binding fragment of an antibody.

[0093] In some embodiments, the antigen-binding fragment of an antibody is a Fab fragment, a single chain Fc fragment (ScFv), a single domain antibody (sdAb), a diabody or a variable domain (Fv).

[0094] In some embodiments, the radiolabelled binding agent binds to both F-actin and G-actin.

[0095] In preferred embodiments, binding of the F-actin binding moiety of the non-radiolabelled binding agent to F-actin is inhibited by sGSN.

[0096] In some embodiments, the non-radiolabelled binding agent binds to both F-actin and G-actin.

[0097] Antibody

[0098] In some embodiments, the F-actin binding moiety is an antibody. Preferably, the antibody is a human or humanised antibody. In some embodiments, the antibody is of the human lgG1 isotype.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof does not comprise an Fc domain.

[0100] In other embodiments, the antibody or antigen-binding fragment thereof comprises an Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence that has been mutated from that of a wild type Fc domain such that the mutated Fc domain binds to an Fc receptor with lower affinity and / or avidity than the affinity and / or avidity of the wild type Fc domain for the Fc receptor. In some embodiments, the affinity and / or avidity of the mutated Fc domain for an Fc receptor is between 2- and 100-fold lower than the affinity and / or avidity of the wild type Fc domain. In some embodiments, the affinity and / or avidity of the mutated Fc domain for an Fc receptor is between 2- and 10-fold lower than the affinity and / or avidity of the wild type Fc domain. In some embodiments, the affinity and / or avidity of the mutated Fc domain for an Fc receptor is at least 2-fold lower than the affinity and / or avidity of the wild type Fc domain. In some embodiments, the affinity and / or avidity of the mutated Fc domain for an Fc receptor is at least 5-fold lower than the affinity and / or avidity of the wild type Fc domain. In some embodiments, the affinity and / or avidity of the mutated Fc domain for an Fc receptor is at least 10-fold lower than the affinity and / or avidity of the wild type Fc domain. In some embodiments, the affinity and / or avidity of the mutated Fc domain for an Fc receptor is at least 100-fold lower than the affinity and / or avidity of the wild type Fc domain.

[0101] In some embodiments, the mutated Fc domain does not bind to an Fc receptor.

[0102] In some embodiments, the Fc domain comprises an amino acid sequence that has been mutated from that of a wild type Fc domain such that the mutated Fc domain binds to a neonatal Fc receptor (FcRn) with higher affinity and / or avidity than the affinity and / or avidity of the wild type Fc domain for the Fc receptor. This can extend the half-life of the binding agent. In some embodiments, the affinity and / or avidity of the mutated Fc domain for the neonatal Fc receptor is between 2- and 100-fold greater than the affinity and / or avidity of the wild type Fc domain. In some embodiments, the affinity and / or avidity of the mutated Fc domain for the neonatal Fc receptor is between 2- and 10-fold greater than the affinity and / or avidity of the wild type Fc domain. In some embodiments, the affinity and / or avidity of the mutated Fc domain for the neonatal Fc receptor is at least 2-fold greater than the affinity and / or avidity of the wild type Fc domain. In some embodiments, the affinity and / or avidity of the mutated Fc domain for the neonatal Fc receptor is at least 5-fold greater than the affinity and / or avidity of the wild type Fc domain. In some embodiments, the affinity and / or avidity of the mutated Fc domain for the neonatal Fc receptor is at least 10-fold greater than the affinity and / or avidity of the wild type Fc domain. In some embodiments, the affinity and / or avidity of the mutated Fc domain for the neonatal Fc receptor is at least 100-fold greater than the affinity and / or avidity of the wild type Fc domain.

[0103] In some embodiments, the radiolabelled (or non-radiolabelled) binding agent does not compete for binding to F-actin with any of the antibodies selected from the group consisting of: ab1 1003 (Abeam), ab205 (Abeam), ab130935 (Abeam), A2228 or A5316 (Sigma-Aldrich), NBP2-61610 (Novus Biologicals), MA1-80729 (ThermoFisher) and BS-1571 R (ThermoFisher).

[0104] In some embodiments, the radiolabelled (or non-radiolabelled) binding agent competes with human or mouse DNGR-1 for binding to F-actin. In other embodiments, the radiolabelled (or non-radiolabelled) binding agent does not compete with human or mouse DNGR-1 for binding to F-actin. Preferably, the radiolabelled (or non-radiolabelled) binding agent binds to the same epitope or overlaps with the same epitope as human or mouse DNGR-1. In other embodiments, the radiolabelled (or non-radiolabelled) binding agent does not bind to the same epitope as human or mouse DNGR-1 .

[0105] In some embodiments, binding of the radiolabelled (or non-radiolabelled) binding agent to F-actin is not inhibited by the presence of G-actin.

[0106] In some embodiments, the radiolabelled (or non-radiolabelled) binding agent specifically binds to a discontinuous, conformational epitope that includes amino acid residues present on at least 2 subunits of F-actin, wherein the discontinuous, conformational epitope comprises three or more residues selected from: R196 and / or E237 of a first subunit of F-actin on a first filament and A114, E117, Q121 , A365, G366 and P367 of a second subunit of F-actin on a separate filament of F-actin to the first subunit. In some embodiments, the F-actin binding moiety of the radiolabelled (or non-radiolabelled) binding agent binds to a conformational epitope that further comprises residue D80 of a third subunit of F-actin which is part of the same filament as the first subunit and adjacent to the first subunit.

[0107] CDRs

[0108] For embodiments where the F-actin binding moiety is an antibody or antigen-binding fragment thereof, the antibodies, or antigen-binding fragments thereof, can be defined in whole or in part by their amino acid sequences. For instance, the antibody or antigen-binding fragment may comprise the following heavy and light chain CDRs:

[0109] Heavy chain:

[0110] CDR1 comprises GGTFRSY (SEQ ID NO:1) or a variant thereof comprising one or two amino acid substitutions with respect to GGTFRSY (SEQ ID NO:1);

[0111] CDR2 comprises NPIFDT (SEQ ID NO:2) or a variant thereof comprising one or two amino acid substitutions with respect to NPIFDT (SEQ ID NO:2); and

[0112] CDR3 comprises TVIGAFDS (SEQ ID NO:3) or a variant thereof comprising one or two amino acid substitutions with respect to TVIGAFDS (SEQ ID NO:3);

[0113] Light chain:

[0114] CDR1 comprises TRTSGDIGGYNFVS (SEQ ID NO:4) or a variant thereof comprising one, two or three amino acid substitutions with respect to TRTSGDIGGYNFVS (SEQ ID NO:4)

[0115] CDR2 comprises DVNSRPS (SEQ ID NO:5) or a variant thereof comprising one or two amino acid substitutions with respect to DVNSRPS (SEQ ID NO:5); and

[0116] CDR3 comprises SSYTSRNTV (SEQ ID NO:6) or a variant thereof comprising one or two amino acid substitutions with respect to SSYTSRNTV (SEQ ID NO:6), wherein the CDRs are identified using Chothia numbering. Preferably, the one or more substitutions are conservative substitutions, i.e. a substitution within an amino acid class and / or a substitution that achieves a positive score in the BLOSUM62 matrix.

[0117] In some embodiments, the G at position 2 of CDRH1 is substituted with F or Y; the T at position 3 of CDRH1 is substituted with I or A, the R at position 5 of CDRH1 is substituted with S or T; the S at position 6 of CDRH1 is substituted with A or N; and / or the Y at position 7 of CDRH1 is substituted with S.

[0118] In some embodiments, the N at position 1 of CDRH2 is substituted with I; the P at position 2 of CDRH2 is substituted with S; the I at position 3 of CDRH2 is substituted with A, S or M; the F at position 4 of CDRH2 is substituted with Y or S; the D at position 5 of CDRH2 is substituted with N or G; and / or the T at position 6 of CDRH2 is substituted with G.

[0119] In some embodiments, the T at position 1 of CDRH3 is substituted with G or Y; the I at position 3 of CDRH3 is substituted with W or V; the G at position 4 of CDRH3 is substituted with M; the A at position 5 of CDRH3 is substituted with R, T or K; the F at position 6 of CDRH3 is substituted with T, Y or I; the D at position 7 of CDRH3 is substituted with P or S; and / or the S at position 8 of CDRH3 is substituted with Y.

[0120] In some embodiments, T at position 1 of CDRL1 is substituted with S; the R at position 2 of CDRL1 is substituted with G; the T at position 3 of CDRL1 is substituted with A, S or G; the S at position 4 of CDRL1 is substituted with T; the G at position 5 of CDRL1 is substituted with Q or S; wherein the D at position 6 of CDRL1 is substituted with S or N; the G at position 8 of CDRL1 is substituted with S; the G at position 9 of CDRL1 is substituted with S; the Y at position 10 of CDRL1 is substituted with R or K; the N at position 11 of CDRL1 is substituted with R or L; the F at position 12 of CDRL1 is substituted with Y, H or N; and / or the S at position 14 of CDRL1 is substituted with F.

[0121] In some embodiments, the D at position 1 of CDRL2 is substituted with A or R; the V at position 2 of CDRL2 is substituted with A, N or G; the N at position 3 of CDRL2 is substituted with S or D; the S at position 4 of CDRL2 is substituted with M or Q; the R at position 5 of CDRL2 is substituted with L; and / or the P at position 6 of CDRL2 is substituted with Q.

[0122] In some embodiments, the S at position 1 of CDRL3 is substituted with Q; the S at position 2 of CDRL3 is substituted with R or G; the Y at position 3 of CDRL3 is substituted with W; the T at position 4 of CDRL3 is substituted with S or V, the S at position 5 of CDRL3 is substituted with T ; the R at position 6 of CDRL3 is substituted with P; and / or the N at position 7 of CDRL3 is substituted with Y.

[0123] In another instance, the antibody or antigen-binding fragment comprises the following heavy and light chain CDRs:

[0124] Heavy chain:

[0125] CDR1 comprises GYIFTSY (SEQ ID NO:7) or a variant thereof comprising one or two amino acid substitutions with respect to GYIFTSY (SEQ ID NO:7);

[0126] CDR2 comprises SAYNGH (SEQ ID NO:8) or a variant thereof comprising one or two amino acid substitutions with respect to SAYNGH (SEQ ID NO:8); and CDR3 comprises GKISSWFVLED (SEQ ID NO:9) or a variant thereof comprising one, two or three amino acid substitutions with respect to GKISSWFVLED (SEQ ID NO:9);

[0127] Light chain:

[0128] CDR1 comprises SGGTSNIGKNYVS (SEQ ID NQ:10) or a variant thereof comprising one, two or three amino acid substitutions with respect to SGGTSNIGKNYVS (SEQ ID NQ:10)

[0129] CDR2 comprises DNNMRPS (SEQ ID NO:11) or a variant thereof comprising one or two amino acid substitutions with respect to DNNMRPS (SEQ ID NO:11); and

[0130] CDR3 comprises GMWIRSLSRWV (SEQ ID NO:12) or a variant thereof comprising one, two or three amino acid substitutions with respect to GMWIRSLSRWV (SEQ ID NO:12), wherein the CDRs are identified using Chothia numbering.

[0131] Preferably, the one or more substitutions are conservative substitutions, i.e. a substitution within an amino acid class and / or a substitution that achieves a positive score in the BLOSUM62 matrix.

[0132] In some embodiments, the Y at position 2 of CDRH1 is substituted with F or G; the I at position 3 of CDRH1 is substituted with T or A, the T at position 5 of CDRH1 is substituted with S or R; the S at position 6 of CDRH1 is substituted with A or N; and / or the Y at position 7 of CDRH1 is substituted with S.

[0133] In some embodiments, the S at position 1 of CDRH2 is substituted with P; the A at position 2 of CDRH2 is substituted with S, I or M; the Y at position 3 of CDRH2 is substituted with F or S; the N at position 4 of CDRH2 is substituted with D or G; the G at position 5 of CDRH2 is substituted with T; and / or the H at position 6 of CDRH2 is substituted with N.

[0134] In some embodiments, the G at position 1 of CDRH3 is substituted with M; the K at position 2 of CDRH3 is substituted with R, T or A; the I at position 3 of CDRH3 is substituted with T, Y or F; the S at position 4 of CDRH3 is substituted with P or D; the S at position 5 of CDRH3 is substituted with Y; the W at position 6 of CDRH3 is substituted with Y or A; the F at position 7 of CDRH3 is substituted with Y; the L at position 9 of CDRH3 is substituted with M; and / or the D at position 11 of CDRH3 is substituted with V.

[0135] In some embodiments, the S at position 1 of CDRL1 is substituted with T; the G at position 2 of CDRL1 is substituted with R; the G at position 3 of CDRL1 is substituted with A, S or T; the T at position 4 of CDRL1 is substituted with S; the S at position 5 of CDRL1 is substituted with Q or G; the N at position 6 of CDRL1 is substituted with S or D; the G at position 8 of CDRL1 is substituted with S; the K at position 9 of CDRL1 is substituted with R or Y; the N at position 10 of CDRL1 is substituted with R or L; the Y at position 11 of CDRL1 is substituted with H, F or N; and / or the S at position 13 of CDRL1 is substituted with F.

[0136] In some embodiments, the D at position 1 of CDRL2 is substituted with A or R; the N at position 2 of CDRL2 is substituted with A, V or G; the N at position 3 of CDRL2 is substituted with S, N or D; the M at position 4 of CDRL2 is substituted with S or Q; the R at position 5 of CDRL2 is substituted with L; and / or the P at position 6 of CDRL2 is substituted with Q. In some embodiments, the G at position 1 of CDRL3 is substituted with A; the M at position 2 of CDRL3 is substituted with T; the I at position 4 of CDRL3 is substituted with D; the R at position 5 of CDRL3 is substituted with D; the S at position 6 of CDRL3 is substituted with G; the L at position 7 of CDRL3 is substituted with Q; the S at position 8 of CDRL3 is substituted with Q; the R at position 9 of CDRL3 is substituted with S or G; the W at position 10 of CDRL3 is substituted with Y; and / or the V at position 11 of CDRL3 is substituted with S or T.

[0137] In another instance, the antibody or antigen-binding fragment comprises the following heavy and light chain CDRs:

[0138] Heavy chain:

[0139] CDR1 comprises GFTFSAY (SEQ ID NO:13) or a variant thereof comprising one or two amino acid substitutions with respect to GFTFSAY (SEQ ID NO:13);

[0140] CDR2 comprises SYDGNN (SEQ ID NO:14) or a variant thereof comprising one or two amino acid substitutions with respect to SYDGNN (SEQ ID NO:14); and

[0141] CDR3 comprises DFRDYVWGTYPSAY (SEQ ID NO:15) or a variant thereof comprising one, two or three amino acid substitutions with respect to DFRDYVWGTYPSAY (SEQ ID NO:15);

[0142] Light chain:

[0143] CDR1 comprises SGSSSNIGRRHVF (SEQ ID NO:16) or a variant thereof comprising one, two or three amino acid substitutions with respect to SGSSSNIGRRHVF (SEQ ID NO:16)

[0144] CDR2 comprises RGDQRPS (SEQ ID NO:17) or a variant thereof comprising one or two amino acid substitutions with respect to RGDQRPS (SEQ ID NO:17); and

[0145] CDR3 comprises ATWDDGLSGYV (SEQ ID NO:18) or a variant thereof comprising one, two or three amino acid substitutions with respect to ATWDDGLSGYV (SEQ ID NO:18), wherein the CDRs are identified using Chothia numbering.

[0146] Preferably, the one or more substitutions are conservative substitutions, i.e. a substitution within an amino acid class and / or a substitution that achieves a positive score in the BLOSUM62 matrix.

[0147] In some embodiments, the F at position 2 of CDRH1 is substituted with G or Y; the T at position 3 of CDRH1 is substituted with I or A; the S at position 5 of CDRH1 is substituted with R or T; the A at position 6 of CDRH1 is substituted with S or N; and / or the Y at position 7 of CDRH1 is substituted with S.

[0148] In some embodiments, the S at position 1 of CDRH2 is substituted with A, I or M; the Y at position 2 of CDRH2 is substituted with F or S; the D at position 3 of CDRH2 is substituted with N or G; the G at position 4 of CDRH2 is substituted with T; and / or the N at position 5 of CDRH2 is substituted with H.

[0149] In some embodiments, the D at position 1 of CDRH3 is substituted with K; the F position 2 of CDRH3 is substituted with E; the D at position 4 of CDRH3 is substituted with T; the Y at position 5 of CDRH3 is substituted with G or T; the W at position 7 of CDRH3 is substituted with I or V; the G at position 8 of CDRH3 is substituted with M; the T at position 9 of CDRH3 is substituted with R, A or K; the Y at position 10 of CDRH3 is substituted with T, F or I; the P at position 11 of CDRH3 is substituted with D or S; the S at position 12 of CDRH3 is substituted with Y ; the A at position 13 of CDRH3 is substituted with Y or W; and / or the Y at position 14 of CDRH3 is substituted with F.

[0150] In some embodiments, the S at position 1 of CDRL1 is substituted with T; the G at position 2 of CDRL1 is substituted with R; the S at position 3 of CDRL1 is substituted with A, T or G; the S at position 4 of CDRL1 is substituted with T; the S at position 5 of CDRL1 is substituted with Q or G; the N at position 6 of CDRL1 is substituted with S or D; the G at position 8 of CDRL1 is substituted with S; the R at position 9 of CDRL1 is substituted with Y or K; the R at position 10 of CDRL1 is substituted with L or N; the H at position 11 of CDRL1 is substituted with F, N or Y; and / or the F at position 13 of CDRL1 is substituted with S.

[0151] In some embodiments, the R at position 1 of CDRL2 is substituted with A or D; the G at position 2 of CDRL2 is substituted with A, N or V; the D at position 3 of CDRL2 is substituted with S or N; the Q at position 4 of CDRL2 is substituted with S or M; the R at position 5 of CDRL2 is substituted with L; and / or the P at position 6 of CDRL2 is substituted with Q.

[0152] In some embodiments, A at position 1 of CDRL3 is substituted with G; the T at position 2 of CDRL3 is substituted with M; the D at position 4 of CDRL3 is substituted with I; the D at position 5 of CDRL3 is substituted with R; the G at position 6 of CDRL3 is substituted with S; the L at position 7 of CDRL3 is substituted with Q; the S at position 8 of CDRL3 is substituted with Q; the G at position 9 of CDRL3 is substituted with S or R; the Y at position 10 of CDRL3 is substituted with W; and / or the V at position 11 of CDRL3 is substituted with S or T.

[0153] In another instance, the antibody or antigen-binding fragment thereof comprises the following heavy and light chain CDRs:

[0154] Heavy chain:

[0155] CDR1 comprises GGAFRNS (SEQ ID NO:19) or a variant thereof comprising one or two amino acid substitutions with respect to GGAFRNS (SEQ ID NO:19);

[0156] CDR2 comprises IPMSGT (SEQ ID NQ:20) or a variant thereof comprising one or two amino acid substitutions with respect to IPMSGT (SEQ ID NQ:20); and

[0157] CDR3 comprises EKERTFGVVMRTSYYYVMEV (SEQ ID NO:21) or a variant thereof comprising one, two, three or four amino acid substitutions with respect to EKERTFGWMRTSYYYVMEV (SEQ ID NO:21);

[0158] Light chain:

[0159] CDR1 comprises RASQSISSYLN (SEQ ID NO:22) or a variant thereof comprising one or two amino acid substitutions with respect to RASQSISSYLN (SEQ ID NO:22)

[0160] CDR2 comprises AASSLQS (SEQ ID NO:23) or a variant thereof comprising one or two amino acid substitutions with respect to AASSLQS (SEQ ID NO:23); and CDR3 comprises QQSYSTPYT (SEQ ID NO:24) or a variant thereof comprising one or two amino acid substitutions with respect to QQSYSTPYT (SEQ ID NO:24), wherein complementarity determining regions (CDRs) are identified using Chothia numbering.

[0161] Preferably, the one or more substitutions are conservative substitutions, i.e. a substitution within an amino acid class and / or a substitution that achieves a positive score in the BLOSUM62 matrix.

[0162] In some embodiments, the G at position 2 of CDRH1 is substituted with F or Y; the A at position 3 of CDRH1 is substituted with T or I; the R at position 5 of CDRH1 is substituted with S or T; the N at position 6 of CDRH1 is substituted with A or S; and / or the S at position 7 of CDRH1 is substituted with Y.

[0163] In some embodiments, the I at position 1 of CDRH2 is substituted with N; the P at position 2 of CDRH2 is substituted with S; the M at position 3 of CDRH2 is substituted with I, A or S; the S at position 4 of CDRH2 is substituted with F or Y; the G at position 5 of CDRH2 is substituted with D or N; and / or the T at position 6 of CDRH2 is substituted with G.

[0164] In some embodiments, the K at position 2 of CDRH3 is substituted with D; the E at position 3 of CDRH3 is substituted with F; the T at position 5 of CDRH3 is substituted with D; the G at position 7 of CDRH3 is substituted with Y or T; the V at position 9 of CDRH3 is substituted with W or I; the M at position 10 of CDRH3 is substituted with G; the R at position 11 of CDRH3 is substituted with A, T or K; the T at position 12 of CDRH3 is substituted with F, Y or I; the S at position 13 of CDRH3 is substituted with P or D; the Y at position 14 of CDRH3 is substituted with S; the Y at position 15 of CDRH3 is substituted with W or A; the Y at position 16 of CDRH3 is substituted with F; the M at position 18 of CDRH3 is substituted with L; and / or the V at position 20 of CDRH3 is substituted with D.

[0165] In some embodiments, the R at position 1 of CDRL1 is substituted with G; the A at position 2 of CDRL1 is substituted with S, T or G; the S at position 3 of CDRL1 is substituted with T; the Q at position 4 of CDRL1 is substituted with S or G; the S at position 5 of CDRL1 is substituted with D or N; the S at position 7 of CDRL1 is substituted with G; the S at position 8 of CDRL1 is substituted with G; the Y at position 9 of CDRL1 is substituted with R or K; the L at position 10 of CDRL1 is substituted with N or R; and / or the N at position 11 of CDRL1 is substituted with F, H or Y.

[0166] In some embodiments, the A at position 1 of CDRL2 is substituted with D or R; the A at position 2 of CDRL2 is substituted with V, N or G; the S at position 3 of CDRL2 is substituted with N or D; the S at position 4 of CDRL2 is substituted with M or Q; the L at position 5 of CDRL2 is substituted with R; and / or the Q at position 6 of CDRL2 is substituted with P.

[0167] In some embodiments, the Q at position 1 of CDRL3 is substituted with L; the Q at position 2 of CDRL3 is substituted with S; the S at position 3 of CDRL3 is substituted with R or G; the Y at position 4 of CDRL3 is substituted with W; the S at position 5 of CDRL3 is substituted with T or V; the T at position 6 of CDRL3 is substituted with S; the P at position 7 of CDRL3 is substituted with R; and / or the Y at position 8 of CDRL3 is substituted with N. In some embodiments, the six CDR sequences of the antibody correspond with the CDR-H1 , CDR-H2, CDR-H3, CDR-L1 , CDR-L2 and CDR-L3 sequences annotated herein.

[0168] Preferably, the one or more substitutions are conservative substitutions, i.e. a substitution within an amino acid class and / or a substitution that achieves a positive score in the BLOSUM62 matrix.

[0169] In another aspect, the invention provides radiolabelled binding agents as defined herein, for use in medicine. Relatedly, the radiolabelled binding agents of the invention may be used in methods of treating a subject, e.g. a cancer patient. In these embodiments, the radiolabelled binding agent is for use in a method of treating cancer, the method comprising administering the radiolabelled binding agent to a subject that has cancer. In some embodiments, the radionuclide comprises an ionising isotope.

[0170] In some embodiments, the cancer comprises a solid tumour. In some embodiments, the cancer is characterised by F-actin presentation on necrotic cancer cells. In some embodiments, the method of treating cancer comprises killing cancer cells via necrosis. The necrosis may be caused by the radiolabelled binding agent itself, for example via radiation from the radionuclide. The necrosis may be caused by another agent.

[0171] In some embodiments, the method comprises contacting the necrotic cancer cells with the radiolabelled binding agent. As described herein, the radiolabelled binding agent can lead to necrosis of cancer cells. As necrosis disrupts membrane integrity, more F-actin is exposed to the extracellular environment, allowing more radiolabelled binding agent to bind. Thus, a positive feedback loop is seen which leads to an amplified effect.

[0172] In some embodiments, the method of treating cancer comprises (i) administering a therapy to the subject to cause necrosis of the cancer cells, and then (ii) administering the radiolabelled binding agent to the subject and thereby contacting the necrotic cancer cells with the radiolabelled binding agent. This method has the advantage of increasing the amount of necrosis and therefore the amount of target antigen (F- actin) exposed before the radiolabelled binding agent is administered.

[0173] In some embodiments, the therapy administered to the subject in step (i) is selected from the group comprising: an F-actin binding agent, a chemotherapeutic agent or a radiotherapy. Therefore, in some embodiments the method of treatment comprises administering a “cold” anti-F-actin antibody (i.e. unlabelled antibody) and then administering a “hot” anti-F-actin antibody (i.e. a radiolabelled antibody).

[0174] In some embodiments, the radionuclide is selected from the group consisting of:227Th,223Ra,212Pb,212Bi,211At,225Ac,213Bi,23°U,226Th,224Ra,213Po,149Tb,32P,47Sc,64Cu,67Cu,153Sm,177Lu, "Y,131l,199Au,166Ho,186Re,188Re,194lr,159Gd,149Pm,142Pr,109Pd,225Ra,213Bi,212Bi,161Tb,89Sr,77As,149Tb,123l,1311 ,203Pb,205Bi, "mTc, i33Xe, 2oi Tl, 5icr,67Ga,166Ho,61Cu,64Cu,110mAg, "Y,188Re,166Ho,159Gd,137Cs,18F-AI,44Sc,52Mn,76Br,77Br,86Y, "Y,89Zr,124l,66Ga,68Ga,18F,11C,82Rb,13N,61Cu,64Cu,1111 n,18F,67Ga,123l,125l,61Cu,186Re,77As,211At,117mSn,67Ga,77Br and103Pd. In some embodiments of the radiolabelled binding agent for use in medicine, the radionuclide is a “therapeutic” radionuclide. As used herein, a therapeutic radionuclide is one which will be absorbed in sufficient quantities and retained for sufficiently long periods to deliver a sufficient dose of radiation to the target area (such as a tumour). In some embodiments where the radionuclide is an alpha (a)-emitting radionuclide, the radiolabelled binding agent is for use in targeted-alpha therapy (TAT).

[0175] In some embodiments, the use comprises administration of the specific binding agent in combination with an additional anticancer therapy. As used herein, the term “in combination with” is explicitly intended to encompass uses where the additional anticancer therapy and the radiolabelled binding agent are administered simultaneously, sequentially, or separately to the subject. In some embodiments, the additional anticancer therapy is an immune checkpoint inhibitor (ICI). In specific embodiments, the immune checkpoint inhibitor is an anti-PD1 monoclonal antibody.

[0176] In some embodiments, the additional anticancer therapy is a radiotherapy.

[0177] In some embodiments, the use comprises administration of the radiolabelled binding agent in combination with a chemotherapeutic agent. This approach has an additive effect, as the chemotherapeutic agent initiates necrosis in the tumour, which exposes further F-actin for the radiolabelled binding agent to bind to.

[0178] In some embodiments, the use comprises systemic administration of the antibody or antigen-binding fragment.

[0179] In another aspect, the invention provides non-radiolabelled binding agents as defined herein, for use in medicine. Relatedly, the non-radiolabelled binding agents of the invention may be used in methods of treating a subject, e.g. a cancer patient. In these embodiments, the non-radiolabelled binding agent is for use in a method of treating cancer, the method comprising administering the non-radiolabelled binding agent to a subject that has cancer.

[0180] In some embodiments, the cancer comprises a solid tumour. In some embodiments, the cancer is characterised by F-actin presentation on necrotic cancer cells. In some embodiments, the method of treating cancer comprises killing cancer cells via necrosis. The necrosis may be caused by the non- radiolabelled binding agent itself, for example via the cytotoxic agent. The necrosis may be caused by another agent.

[0181] In some embodiments, the method comprises contacting the necrotic cancer cells with the non- radiolabelled binding agent. As described herein, the non-radiolabelled binding agent can lead to necrosis of cancer cells. As necrosis disrupts membrane integrity, more F-actin is exposed to the extracellular environment, allowing more non-radiolabelled binding agent to bind. Thus, a positive feedback loop is seen which leads to an amplified effect. In some embodiments, the method of treating cancer comprises (i) administering a therapy to the subject to cause necrosis of the cancer cells, and then (ii) administering the non-radiolabelled binding agent to the subject and thereby contacting the necrotic cancer cells with the non-radiolabelled binding agent. This method has the advantage of increasing the amount of necrosis and therefore the amount of target antigen (F-actin) exposed before the non-radiolabelled binding agent is administered.

[0182] In some embodiments, the therapy administered to the subject in step (i) is selected from the group comprising: an F-actin binding agent, a chemotherapeutic agent or a radiotherapy.

[0183] In some embodiments, the use comprises administration of the non-radiolabelled binding agent in combination with an additional anticancer therapy. As used herein, the term “in combination with” is explicitly intended to encompass uses where the additional anticancer therapy and the non-radiolabelled binding agent are administered simultaneously, sequentially, or separately to the subject. In some embodiments, the additional anticancer therapy is an immune checkpoint inhibitor (ICI). In specific embodiments, the immune checkpoint inhibitor is an anti-PD1 monoclonal antibody.

[0184] In some embodiments, the additional anticancer therapy is a radiotherapy.

[0185] In some embodiments, the use comprises administration of the non-radiolabelled binding agent in combination with a chemotherapeutic agent. This approach has an additive effect, as the chemotherapeutic agent initiates necrosis in the tumour, which exposes further F-actin for the non- radiolabelled binding agent to bind to.

[0186] In some embodiments, the use comprises systemic administration of the antibody or antigen-binding fragment.

[0187] In another aspect, the invention provides radiolabelled binding agents as defined herein, for use in a method of radioimaging. As described herein, the radiolabelled binding agent may include a theranostic radionuclide, i.e. a radionuclide which can be used for both therapeutic and imaging applications. This has the advantage that the biodistribution of the therapeutic agent can be imaged in a precise and non- invasive manner (Sgouros, 2020 & Holik, 2022). For example, the radionuclide may be47Sc,90Y,131l,

[0188] 166Ho,177Lu,188Re, or213Bi. In some embodiments, the radionuclide comprises a non-ionising isotope.

[0189] In some embodiments, the radionuclide is selected from the group consisting of:227Th,223Ra,212Pb,212Bi,

[0190] 211At,225Ac,213Bi,23°U,226Th,224Ra,213Po,149Tb,32P,47Sc,64Cu,67Cu,153Sm,177Lu, "Y,131l,199Au,166Ho,

[0191] 186Re,188Re,194lr,159Gd,149Pm,142Pr,109Pd,225Ra,213Bi,212Bi,161Tb,89Sr,77As,149Tb,123l,1311 ,203Pb,205Bi,

[0192] 77As,211At,117mSn,67Ga,77Br and103Pd.

[0193] In some embodiments, the radioimaging comprises imaging a tumour or a cancer. In some embodiments, the cancer is characterised by F-actin presentation on necrotic cancer cells. In another aspect, the invention provides a method of radio-immuno imaging, comprising administering the radiolabelled binding agent as defined herein to a subject and detecting the radionuclide. In some embodiments, the method of radio-immuno imaging is a positron-emission tomography (PET) method and the radionuclide is a high energy beta (p+)-emitting radionuclide. In some embodiments, the method of radio-immuno imaging is a single-photon emission computed tomography (SPEC!) method.

[0194] In another aspect, the invention provides a radiolabelled binding agent as defined herein, for use in an in vivo method of diagnosis of cancer.

[0195] Methods of obtaining

[0196] In another aspect, the invention provides a method of selecting an antibody or antigen-binding fragment thereof that specifically binds to F-actin. The method comprises providing a population of candidate antibodies, contacting a binding substrate comprising F-actin with the population of candidate antibodies, contacting the binding substrate with trypsin or a competitor agent comprising the CTLD of DNGR-1 to displace one or more lead antibodies from the binding substrate. The antibody is then selected from said one or more lead antibodies; and then labelled with a cargo. The cargo may be a radionuclide (radiolabelled binding agent) or a cytotoxic agent (non-radiolabelled binding agent).

[0197] In some embodiments, the method comprises screening said one or more lead antibodies for 10-fold specificity for F-actin over G-actin, wherein the selected antibody has said 10-fold specificity.

[0198] In some embodiments, the method comprises screening said one or more lead antibodies for at least 3- fold specificity for binding necrotic cells over live cells, as measured by FACS or flow cytometry, wherein the selected antibody has said 3-fold specificity.

[0199] In another aspect, the invention provides a method of producing a radiolabelled binding agent as described herein. The method comprises obtaining an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and labelling the F-actin binding moiety with a radionuclide. In some embodiments, the radionuclide is selected from the group consisting of:227Th,223Ra,212Pb,212Bi,211At,225Ac,213Bi,23°U,226Th,224Ra,213Po,149Tb,32P,47Sc,64Cu,67Cu,153Sm,177Lu,"Y,131l,199Au,166Ho,186Re,188Re,194lr,159Gd,149Pm,142Pr,109Pd,225Ra,213Bi,212Bi,161Tb,89Sr,77As,149Tb,123l,1311 ,203Pb,205Bi , "mTc, i33Xe, 2oi Tl, 5icr,67Ga,166Ho,61Cu,64Cu,110mAg, "Y,188Re,166Ho,159Gd,137Cs,18F-AI,44Sc,52Mn,76Br,77Br,86Y, "Y,89Zr,124l,66Ga,68Ga,18F,11C,82Rb,13N,61Cu,64Cu,1111 n,18F,67Ga,123l,125l,61Cu,186Re,77As,211At,117mSn,67Ga,77Br and103Pd.

[0200] In another aspect, the invention provides a method of producing a non-radiolabelled binding agent as described herein. The method comprises obtaining an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and conjugating the F-actin binding moiety to a cytotoxic agent. In some embodiments, the cytotoxic agent is selected from the group consisting of: a cytotoxin, an immunomodulator, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, a DNA, an RNA, an siRNA, an RNAi, a microRNA, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelating agent, or combinations thereof.

[0201] Kits

[0202] In another aspect, the invention provides a kit comprising: an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and a radionuclide. In some embodiments, the kit further comprises instructions for radiolabelling the F-actin binding moiety that specifically binds to filamentous actin (F- actin) with the radionuclide. In some embodiments, the radionuclide is selected from the group consisting of:227Th,223Ra,212Pb,212Bi,211At,225Ac,213Bi,23°U,226Th,224Ra,213Po,149Tb,32P,47Sc,64Cu,67Cu,153Sm,177Lu,90Y,131l,199Au,166Ho,186Re,188Re,194lr,159Gd,149Pm,142Pr,109Pd,225Ra,213Bi,212Bi,161Tb,89Sr,77As,149Tb,123l,1311 ,203Pb,205Bi ,99mTc,133Xe,201TI,51Cr,67Ga,166Ho,61Cu,64Cu,110mAg, "Y,188Re,166Ho,159Gd,137Cs,18F-AI,44Sc,52Mn,76Br,77Br,86Y, "Y,89Zr,124l,66Ga,68Ga,18F,11C,82Rb,13N,61Cu,64Cu,1111 n,18F,67Ga,123l,125l,61Cu,186Re,77As,211At,117mSn,67Ga,77Br and103Pd.

[0203] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0204] Sequences

[0205] In some embodiments of the present invention, the F-actin binding moiety is an antibody. The following amino acid sequences relate to some antibodies that may be useful in the present invention.

[0206] Antibody A, VH

[0207] 1 QVQLVQSGAE VKKPGSSVKV SCKSSGGTFR SYI I SWVRQA PGQGLEWMGG FNPI FDTTIY AQKFQGRVTF

[0208] 71 TADESTSTAY MDLSSLRSED TAVYYCARTV IGAFDSWGQG TPVTVSS ( SEQ ID NO : 181 )

[0209] Antibody A, VL

[0210] 1 QSALTQPASV SGSPGQSITI SCTRTSGDIG GYNFVSWYQQ HPGKAPKLMI YDVNSRPSGV SDRFSGSKSG

[0211] 71 DTASLTISGL QAEDEADYYC SSYTSRNTVF GGGTKLTVL ( SEQ ID NO : 182 )

[0212] The FRs and CDRs of Antibody A can be defined as follows, using the Chothia system:

[0213] FR-Hl QVQLVQSGAEVKKPGSSVKVSCKSS ( SEQ ID NO : 25 )

[0214] CDR-H1 GGTFRSY ( SEQ ID NO : 1 )

[0215] FR-H2 I ISWVRQAPGQGLEWMGGF ( SEQ ID NO : 26 )

[0216] CDR-H2 NPI FDT ( SEQ ID NO : 2 )

[0217] FR-H3 TIYAQKFQGRVTFTADESTSTAYMDLSSLRSEDTAVYYCAR ( SEQ ID NO : 27 )

[0218] CDR-H3 TVIGAFDS ( SEQ ID NO : 3 )

[0219] FR-H4 WGQGTPVTVSS ( SEQ ID NO : 28 )

[0220] FR-L1 QSALTQPASVSGSPGQSITI SC ( SEQ ID NO : 29 ) CDR-Ll TRTSGDIGGYNFVS (SEQ ID NO: 4)

[0221] FR-L2 WYQQHPGKAPKLMIY (SEQ ID NO:30)

[0222] CDR-L2 DVNSRPS (SEQ ID NO : 5 )

[0223] FR-L3 GVSDRFSGSKSGDTASLTISGLQAEDEADYYC (SEQ ID NO: 31)

[0224] CDR-L3 SSYTSRNTV (SEQ ID NO: 6)

[0225] FR-L4 FGGGTKLTVL (SEQ ID NO: 32)

[0226] The FRs and CDRs of Antibody A can be defined as follows, using the Kabat system:

[0227] FR-Hl QVQLVQSGAEVKKPGSSVKVSCKSSGGTFR (SEQ ID NO: 33)

[0228] CDR-H1 SYIIS (SEQ ID NO:34)

[0229] FR-H2 WVRQAPGQGLEWMG (SEQ ID NO: 35)

[0230] CDR-H2 GFNPIFDTTIYAQKFQG (SEQ ID NO: 36)

[0231] FR-H3 RVTFTADESTSTAYMDLSSLRSEDTAVYYCAR (SEQ ID NO: 37)

[0232] CDR-H3 TVIGAFDS (SEQ ID NO: 3)

[0233] FR-H4 WGQGTPVTVSS (SEQ ID NO:28)

[0234] FR-L1 QSALTQPASVSGSPGQSITISC (SEQ ID NO: 29)

[0235] CDR-Ll TRTSGDIGGYNFVS (SEQ ID NO: 4)

[0236] FR-L2 WYQQHPGKAPKLMIY (SEQ ID NO:30)

[0237] CDR-L2 DVNSRPS (SEQ ID NO : 5 )

[0238] FR-L3 GVSDRFSGSKSGDTASLTISGLQAEDEADYYC (SEQ ID NO: 31)

[0239] CDR-L3 SSYTSRNTV (SEQ ID NO: 6)

[0240] FR-L4 FGGGTKLTVL (SEQ ID NO: 32)

[0241] The FRs and CDRs of Antibody A can be defined as follows, using the IMGT system:

[0242] FR-Hl QVQLVQSGAEVKKPGSSVKVSCKSS (SEQ ID NO: 25)

[0243] CDR-H1 GGTFRSYI (SEQ ID NO: 38)

[0244] FR-H2 ISWVRQAPGQGLEWMGG (SEQ ID NO: 39)

[0245] CDR-H2 FNPIFDTT (SEQ ID NO: 40)

[0246] FR-H3 IYAQKFQGRVTFTADESTSTAYMDLSSLRSEDTAVYYC (SEQ ID NO: 41)

[0247] CDR-H3 ARTVIGAFDS (SEQ ID NO:42)

[0248] FR-H4 WGQGTPVTVSS (SEQ ID NO:28)

[0249] FR-L1 QSALTQPASVSGSPGQSITISCTRT (SEQ ID NO: 43)

[0250] CDR-Ll SGDIGGYNF (SEQ ID NO: 44)

[0251] FR-L2 VSWYQQHPGKAPKLMIY (SEQ ID NO: 45)

[0252] CDR-L2 DVN FR-L3 SRPSGVSDRFSGSKSGDTASLTISGLQAEDEADYYC (SEQ ID NO: 46)

[0253] CDR-L3 SSYTSRNTV (SEQ ID NO: 6)

[0254] FR-L4 FGGGTKLTVL (SEQ ID NO: 32)

[0255] The FRs and CDRs of Antibody A can be defined as follows, using the Martin / Contact system:

[0256] FR-Hl QVQLVQSGAEVKKPGSSVKVSCKSSGGTF (SEQ ID NO: 47)

[0257] CDR-H1 RSYIIS (SEQ ID NO: 48)

[0258] FR-H2 WVRQAPGQGLE (SEQ ID NO: 49)

[0259] CDR-H2 WMGGFNPIFDTTI (SEQ ID NO:50)

[0260] FR-H3 YAQKFQGRVTFTADESTSTAYMDLSSLRSEDTAVYYC (SEQ ID NO:51)

[0261] CDR-H3 ARTVIGAFD (SEQ ID NO:52)

[0262] FR-H4 SWGQGTPVTVSS (SEQ ID NO: 53)

[0263] FR-L1 QSALTQPASVSGSPGQSITISCTRTSGD (SEQ ID NO: 54)

[0264] CDR-L1 IGGYNFVSWY (SEQ ID NO: 55)

[0265] FR-L2 QQHPGKAPK (SEQ ID NO: 56)

[0266] CDR-L2 LMIYDVNSRP (SEQ ID NO: 57)

[0267] FR-L3 SGVSDRFSGSKSGDTASLTISGLQAEDEADYYC (SEQ ID NO:58)

[0268] CDR-L3 SSYTSRNT (SEQ ID NO: 59)

[0269] FR-L4 VFGGGTKLTVL (SEQ ID NO: 60)

[0270] Antibody B, VH

[0271] 1 QMQLVQSGAE VKKPGASVKV SCKAPGYIFT SYGISWVRQA PGQGLEWMGR ISAYNGHTNY AQNLQDRVTL

[0272] 71 TTDTSTSTAY MELRSLRYDD TAIYYCATGK ISSWFVLEDW GQGTQVTVSS (SEQ ID NO: 183)

[0273] Antibody B, VL

[0274] 1 QSWTQPPSV SAAPGQKVTI SCSGGTSNIG KNYVSWYQQL PGTAPRLLIY DNNMRPSGIP DRFSGSKSGT

[0275] 71 SATLAITGLQ TGDEADYYCG MWIRSLSRWV FGGGTKLAVL (SEQ ID NO: 184)

[0276] The framework regions (FRs) and CDRs of Antibody B can be defined as follows, using the Chothia system:

[0277] FR-Hl QMQLVQSGAEVKKPGASVKVSCKAP (SEQ ID NO: 61)

[0278] CDR-H1 GYIFTSY (SEQ ID NO : 7 )

[0279] FR-H2 GISWVRQAPGQGLEWMGRI (SEQ ID NO: 62)

[0280] CDR-H2 SAYNGH (SEQ ID NO: 8)

[0281] FR-H3 TNYAQNLQDRVTLTTDTSTSTAYMELRSLRYDDTAI YYCAT (SEQ ID NO: 63)

[0282] CDR-H3 GKISSWFVLED (SEQ ID NO: 9)

[0283] FR-H4 WGQGTQVTVSS (SEQ ID NO: 64) FR-L1 QSWTQPPSVSAAPGQKVTI SC (SEQ ID NO: 65)

[0284] CDR-L1 SGGTSNIGKNYVS (SEQ ID NO: 10)

[0285] FR-L2 WYQQLPGTAPRLLIY (SEQ ID NO: 66)

[0286] CDR-L2 DNNMRPS (SEQ ID NO: 11)

[0287] FR-L3 GIPDRFSGSKSGTSATLAITGLQTGDEADYYC (SEQ ID NO: 67)

[0288] CDR-L3 GMWIRSLSRWV (SEQ ID NO: 12)

[0289] FR-L4 FGGGTKLAVL (SEQ ID NO: 68)

[0290] The FRs and CDRs of Antibody B can be defined as follows, using the Kabat system:

[0291] FR-Hl QMQLVQSGAEVKKPGASVKVSCKAPGYIFT (SEQ ID NO: 69)

[0292] CDR-H1 SYGIS (SEQ ID NO:70)

[0293] FR-H2 WVRQAPGQGLEWMG (SEQ ID NO: 35)

[0294] CDR-H2 RISAYNGHTNYAQNLQD (SEQ ID NO:71)

[0295] FR-H3 RVTLTTDTSTSTAYMELRSLRYDDTAIYYCAT (SEQ ID NO: 72)

[0296] CDR-H3 GKISSWFVLED (SEQ ID NO: 9)

[0297] FR-H4 WGQGTQVTVSS (SEQ ID NO: 64)

[0298] FR-L1 QSWTQPPSVSAAPGQKVTI SC (SEQ ID NO: 65)

[0299] CDR-L1 SGGTSNIGKNYVS (SEQ ID NO: 10)

[0300] FR-L2 WYQQLPGTAPRLLIY (SEQ ID NO: 66)

[0301] CDR-L2 DNNMRPS (SEQ ID NO: 11)

[0302] FR-L3 GIPDRFSGSKSGTSATLAITGLQTGDEADYYC (SEQ ID NO: 67)

[0303] CDR-L3 GMWIRSLSRWV (SEQ ID NO: 12)

[0304] FR-L4 FGGGTKLAVL (SEQ ID NO: 68)

[0305] The FRs and CDRs of Antibody B can be defined as follows, using the IMGT system:

[0306] FR-Hl QMQLVQSGAEVKKPGASVKVSCKAP (SEQ ID NO: 61)

[0307] CDR-H1 GYIFTSYG (SEQ ID NO: 73)

[0308] FR-H2 ISWVRQAPGQGLEWMGR (SEQ ID NO: 74)

[0309] CDR-H2 ISAYNGHT (SEQ ID NO: 75)

[0310] FR-H3 NYAQNLQDRVTLTTDTSTSTAYMELRSLRYDDTAIYYC (SEQ ID NO: 76)

[0311] CDR-H3 ATGKISSWFVLED (SEQ ID NO:77)

[0312] FR-H4 WGQGTQVTVSS (SEQ ID NO: 64)

[0313] FR-L1 QSWTQPPSVSAAPGQKVTI SCSGG (SEQ ID NO: 78)

[0314] CDR-L1 TSNIGKNY (SEQ ID NO: 79)

[0315] FR-L2 VSWYQQLPGTAPRLLIY (SEQ ID NO: 80) CDR-L2 DNN

[0316] FR-L3 MRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYC (SEQ ID NO: 81)

[0317] CDR-L3 GMWIRSLSRWV (SEQ ID NO: 12)

[0318] FR-L4 FGGGTKLAVL (SEQ ID NO: 68)

[0319] The FRs and CDRs of Antibody B can be defined as follows, using the Martin / Contact system:

[0320] FR-Hl QMQLVQSGAEVKKPGASVKVSCKAPGYIF (SEQ ID NO: 82)

[0321] CDR-H1 TSYGIS (SEQ ID NO: 83)

[0322] FR-H2 WVRQAPGQGLE (SEQ ID NO: 49)

[0323] CDR-H2 WMGRI SAYNGHTN (SEQ ID NO:84)

[0324] FR-H3 YAQNLQDRVTLTTDTSTSTAYMELRSLRYDDTAI YYC (SEQ ID NO: 85)

[0325] CDR-H3 ATGKISSWFVLE (SEQ ID NO: 86)

[0326] FR-H4 DWGQGTQVTVSS (SEQ ID NO: 87)

[0327] FR-L1 QSWTQPPSVSAAPGQKVTI SCSGGTSN (SEQ ID NO: 88)

[0328] CDR-L1 IGKNYVSWY (SEQ ID NO: 89)

[0329] FR-L2 QQLPGTAPR (SEQ ID NO: 90)

[0330] CDR-L2 LLIYDNNMRP (SEQ ID NO: 91)

[0331] FR-L3 SGIPDRFSGSKSGTSATLAITGLQTGDEADYYC (SEQ ID NO: 92)

[0332] CDR-L3 GMWIRSLSRW (SEQ ID NO: 93)

[0333] FR-L4 VFGGGTKLAVL (SEQ ID NO: 94)

[0334] Antibody C, VH

[0335] 1 QVQLVESGGG WQPGRSLRL SCAASGFTFS AYAMHWVRQA PGKGLEWMAV ISYDGNNIHY ADSVKGRFTV

[0336] 71 SRDNSKNTLF LQMDGLRTED TAVYYCARDF RDYVWGTYPS AYWGQGTLVT VSS (SEQ ID NO: 185)

[0337] Antibody C, VL

[0338] 1 QSVLTQPPSV SGTPGQRVII SCSGSSSNIG RRHVFWYQQF PESAPKLLIY RGDQRPSGVP ERYSGSKSGT

[0339] 71 SASLAISGLR SEDEADYYCA TWDDGLSGYV FGTGTRVTVL (SEQ ID NO: 186)

[0340] The FRs and CDRs of Antibody C can be defined as follows, using the Chothia system:

[0341] FR-Hl QVQLVESGGGWQPGRSLRLSCAAS (SEQ ID NO: 95)

[0342] CDR-H1 GFTFSAY (SEQ ID NO: 13)

[0343] FR-H2 AMHWVRQAPGKGLEWMAVI (SEQ ID NO: 96)

[0344] CDR-H2 SYDGNN (SEQ ID NO: 14)

[0345] FR-H3 IHYADSVKGRFTVSRDNSKNTLFLQMDGLRTEDTAVYYCAR (SEQ ID NO: 97)

[0346] CDR-H3 DFRDYVWGTYPSAY (SEQ ID NO: 15)

[0347] FR-H4 WGQGTLVTVSS (SEQ ID NO: 98) FR-L1 QSVLTQPPSVSGTPGQRVI I SC (SEQ ID NO: 99)

[0348] CDR-L1 SGSSSNIGRRHVF (SEQ ID NO: 16)

[0349] FR-L2 WYQQFPESAPKLLIY (SEQ ID NO: 100)

[0350] CDR-L2 RGDQRPS (SEQ ID NO: 17)

[0351] FR-L3 GVPERYSGSKSGTSASLAISGLRSEDEADYYC (SEQ ID NO: 101)

[0352] CDR-L3 ATWDDGLSGYV (SEQ ID NO:18)

[0353] FR-L4 FGTGTRVTVL (SEQ ID NO: 102)

[0354] The FRs and CDRs of Antibody C can be defined as follows, using the Kabat system:

[0355] FR-Hl QVQLVESGGGWQPGRSLRLSCAASGFTFS (SEQ ID NO: 103)

[0356] CDR-H1 AYAMH (SEQ ID NO:104)

[0357] FR-H2 WVRQAPGKGLEWMA (SEQ ID NO: 105)

[0358] CDR-H2 VISYDGNNIHYADSVKG (SEQ ID NO:106)

[0359] FR-H3 RFTVSRDNSKNTLFLQMDGLRTEDTAVYYCAR (SEQ ID NO: 107)

[0360] CDR-H3 DFRDYVWGTYPSAY (SEQ ID NO: 15)

[0361] FR-H4 WGQGTLVTVSS (SEQ ID NO: 98)

[0362] FR-L1 QSVLTQPPSVSGTPGQRVI I SC (SEQ ID NO: 99)

[0363] CDR-L1 SGSSSNIGRRHVF (SEQ ID NO: 16)

[0364] FR-L2 WYQQFPESAPKLLIY (SEQ ID NO: 100)

[0365] CDR-L2 RGDQRPS (SEQ ID NO: 17)

[0366] FR-L3 GVPERYSGSKSGTSASLAISGLRSEDEADYYC (SEQ ID NO: 101)

[0367] CDR-L3 ATWDDGLSGYV (SEQ ID NO:18)

[0368] FR-L4 FGTGTRVTVL (SEQ ID NO: 102)

[0369] The FRs and CDRs of Antibody C can be defined as follows, using the IMGT system:

[0370] FR-Hl QVQLVESGGGWQPGRSLRLSCAAS (SEQ ID NO: 95)

[0371] CDR-H1 GFTFSAYA (SEQ ID NO: 108)

[0372] FR-H2 MHWVRQAPGKGLEWMAV (SEQ ID NO: 109)

[0373] CDR-H2 ISYDGNNI (SEQ ID NO: 110)

[0374] FR-H3 HYADSVKGRFTVSRDNSKNTLFLQMDGLRTEDTAVYYC (SEQ ID NO: 111)

[0375] CDR-H3 ARDFRDYVWGTYPSAY (SEQ ID NO:112)

[0376] FR-H4 WGQGTLVTVSS (SEQ ID NO: 98)

[0377] FR-L1 QSVLTQPPSVSGTPGQRVI I SCSGS (SEQ ID NO: 113)

[0378] CDR-L1 SSNIGRRH (SEQ ID NO: 114)

[0379] FR-L2 VFWYQQFPESAPKLLIY (SEQ ID NO: 115) CDR-L2 RGD

[0380] FR-L3 QRPSGVPERYSGSKSGTSASLAISGLRSEDEADYYC (SEQ ID NO: 116)

[0381] CDR-L3 ATWDDGLSGYV (SEQ ID NO:18)

[0382] FR-L4 FGTGTRVTVL (SEQ ID NO: 102)

[0383] FRs and CDRs of Antibody C can be defined as follows, using the Contact system:

[0384] FR-Hl QVQLVESGGGWQPGRSLRLSCAASGFTF (SEQ ID NO: 117)

[0385] CDR-H1 SAYAMH (SEQ ID NO: 118)

[0386] FR-H2 WVRQAPGKGLE (SEQ ID NO: 119)

[0387] CDR-H2 WMAVISYDGNNIH (SEQ ID NO:120)

[0388] FR-H3 YADSVKGRFTVSRDNSKNTLFLQMDGLRTEDTAVYYC (SEQ ID NO: 121)

[0389] CDR-H3 ARDFRDYVWGTYPSA (SEQ ID NO:122)

[0390] FR-H4 YWGQGTLVTVSS (SEQ ID NO: 123)

[0391] FR-L1 QSVLTQPPSVSGTPGQRVIISCSGSSSN (SEQ ID NO: 124)

[0392] CDR-L1 IGRRHVFWY (SEQ ID NO: 125)

[0393] FR-L2 QQFPESAPK (SEQ ID NO: 126)

[0394] CDR-L2 LLIYRGDQRP (SEQ ID NO: 127)

[0395] FR-L3 SGVPERYSGSKSGTSASLAISGLRSEDEADYYC (SEQ ID NO: 128)

[0396] CDR-L3 ATWDDGLSGY (SEQ ID NO: 129)

[0397] FR-L4 VFGTGTRVTVL (SEQ ID NO: 130)

[0398] Antibody D, VH

[0399] 1 QVQLVQSGAE VKKTGSSVKV SCKVSGGAFR NSAINWVRQA PGRGLEWMGV IIPMSGTTNY ARNFQGRVTI

[0400] 71 SADESTSTAY MELSTLTSGD TADYYCAREK ERTFGWMRT SYYYVMEVWG QGTTVTVSS (SEQ ID

[0401] NO: 187)

[0402] Antibody D, VL

[0403] 1 AIRMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD

[0404] 71 FTLTISSLQP EDFATYYCQQ SYSTPYTFGQ GTKLEIK (SEQ ID NO: 188)

[0405] The FRs and CDRs of Antibody D can be defined as follows, using the Chothia system:

[0406] FR-Hl QVQLVQSGAEVKKTGSSVKVSCKVS (SEQ ID NO: 131)

[0407] CDR-H1 GGAFRNS (SEQ ID NO: 19)

[0408] FR-H2 AINWVRQAPGRGLEWMGVI (SEQ ID NO: 132)

[0409] CDR-H2 IPMSGT (SEQ ID NO: 20)

[0410] FR-H3 TNYARNFQGRVTISADESTSTAYMELSTLTSGDTADYYCAR (SEQ ID NO: 133)

[0411] CDR-H3 EKERTFGWMRTSYYYVMEV (SEQ ID NO: 21)

[0412] FR-H4 WGQGTTVTVSS (SEQ ID NO: 134) FR-L1 IRMTQSPSSLSASVGDRVTITC (SEQ ID NO: 135)

[0413] CDR-L1 RASQSISSYLN (SEQ ID NO:22)

[0414] FR-L2 WYQQKPGKAPKLLIY (SEQ ID NO: 136)

[0415] CDR-L2 AASSLQS (SEQ ID NO:23)

[0416] FR-L3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 137)

[0417] CDR-L3 QQSYSTPYT (SEQ ID NO:24)

[0418] FR-L4 FGQGTKLEIK (SEQ ID NO: 138)

[0419] The FRs and CDRs of Antibody D can be defined as follows, using the Kabat system:

[0420] FR-Hl QVQLVQSGAEVKKTGSSVKVSCKVSGGAFR (SEQ ID NO: 139)

[0421] CDR-H1 NSAIN (SEQ ID NO: 140)

[0422] FR-H2 WVRQAPGRGLEWMG (SEQ ID NO: 141)

[0423] CDR-H2 VIIPMSGTTNYARNFQG (SEQ ID NO:142)

[0424] FR-H3 RVTISADESTSTAYMELSTLTSGDTADYYCAR (SEQ ID NO: 143)

[0425] CDR-H3 EKERTFGWMRTSYYYVMEV (SEQ ID NO: 21)

[0426] FR-H4 WGQGTTVTVSS (SEQ ID NO: 134)

[0427] FR-L1 IRMTQSPSSLSASVGDRVTITC (SEQ ID NO: 135)

[0428] CDR-L1 RASQSISSYLN (SEQ ID NO:22)

[0429] FR-L2 WYQQKPGKAPKLLIY (SEQ ID NO: 136)

[0430] CDR-L2 AASSLQS (SEQ ID NO:23)

[0431] FR-L3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 137)

[0432] CDR-L3 QQSYSTPYT (SEQ ID NO:24)

[0433] FR-L4 FGQGTKLEIK (SEQ ID NO: 138)

[0434] The FRs and CDRs of Antibody D can be defined as follows, using the IMGT system:

[0435] FR-Hl QVQLVQSGAEVKKTGSSVKVSCKVS (SEQ ID NO: 131)

[0436] CDR-H1 GGAFRNSA (SEQ ID NO: 144)

[0437] FR-H2 INWVRQAPGRGLEWMGV (SEQ ID NO: 145)

[0438] CDR-H2 IIPMSGTT (SEQ ID NO: 146)

[0439] FR-H3 NYARNFQGRVTISADESTSTAYMELSTLTSGDTADYYC (SEQ ID NO: 147)

[0440] CDR-H3 AREKERTFGWMRTSYYYVMEV (SEQ ID NO:148)

[0441] FR-H4 WGQGTTVTVSS (SEQ ID NO: 134)

[0442] FR-L1 IRMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 149)

[0443] CDR-L1 QSISSY (SEQ ID NO: 150)

[0444] FR-L2 LNWYQQKPGKAPKLLIY (SEQ ID NO: 151) CDR-L2 AAS

[0445] FR-L3 SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 152)

[0446] CDR-L3 QQSYSTPYT (SEQ ID NO:24)

[0447] FR-L4 FGQGTKLEIK (SEQ ID NO: 138)

[0448] The FRs and CDRs of Antibody D can be defined as follows, using the Martin / Contact system:

[0449] FR-Hl QVQLVQSGAEVKKTGSSVKVSCKVSGGAF (SEQ ID NO: 153)

[0450] CDR-H1 RNSAIN (SEQ ID NO: 154)

[0451] FR-H2 WVRQAPGRGLE (SEQ ID NO: 155)

[0452] CDR-H2 WMGVIIPMSGTTN (SEQ ID NO:156)

[0453] FR-H3 YARNFQGRVTISADESTSTAYMELSTLTSGDTADYYC (SEQ ID NO: 157)

[0454] CDR-H3 AREKERTFGWMRTSYYYVME (SEQ ID NO:158)

[0455] FR-H4 WGQGTTVTVSS (SEQ ID NO: 159)

[0456] FR-L1 IRMTQSPSSLSASVGDRVTITCRASQSI (SEQ ID NO: 160)

[0457] CDR-L1 SSYLNWY (SEQ ID NO: 161)

[0458] FR-L2 QQKPGKAPK (SEQ ID NO:162)

[0459] CDR-L2 LLIYAASSLQ (SEQ ID NO: 163)

[0460] FR-L3 SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:164)

[0461] CDR-L3 QQSYSTPY (SEQ ID NO: 165)

[0462] FR-L4 TFGQGTKLEIK (SEQ ID NO: 166)

[0463] Antibody E, VH

[0464] 1 QVQLVQSGAE VKKTGSSVKV SCKVFGGAFR NSAINWVRQA PGRGLEWMGV LIPMSGTTNY ARNFQGRVTI

[0465] 71 SADESTSTAY MELSTLTSGD TAVYYCAREK ERTFGWMRT SYYYVMEWG QGTTVTVSS (SEQ ID

[0466] NO: 189)

[0467] Antibody E, VL

[0468] 1 AIRMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD

[0469] 71 FTLTISSLQP EDFATYYCQQ SYSTPYTFGQ GTKLEIK (SEQ ID NO: 188)

[0470] The FRs and CDRs of Antibody E can be defined as follows, using the Chothia system:

[0471] FR-Hl QVQLVQSGAEVKKTGSSVKVSCKVF (SEQ ID NO: 167)

[0472] CDR-H1 GGAFRNS (SEQ ID NO: 19)

[0473] FR-H2 AINWVRQAPGRGLEWMGVL (SEQ ID NO:168)

[0474] CDR-H2 IPMSGT (SEQ ID NO: 20)

[0475] FR-H3 TNYARNFQGRVTISADESTSTAYMELSTLTSGDTAVYYCAR (SEQ ID NO: 169)

[0476] CDR-H3 EKERTFGWMRTSYYYVMEV (SEQ ID NO: 21)

[0477] FR-H4 WGQGTTVTVSS (SEQ ID NO: 134) FR-L1 IRMTQSPSSLSASVGDRVTITC (SEQ ID NO: 135)

[0478] CDR-L1 RASQSISSYLN (SEQ ID NO:22)

[0479] FR-L2 WYQQKPGKAPKLLIY (SEQ ID NO: 136)

[0480] CDR-L2 AASSLQS (SEQ ID NO:23)

[0481] FR-L3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 137)

[0482] CDR-L3 QQSYSTPYT (SEQ ID NO:24)

[0483] FR-L4 FGQGTKLEIK (SEQ ID NO: 138)

[0484] The FRs and CDRs of Antibody E can be defined as follows, using the Kabat system:

[0485] FR-Hl QVQLVQSGAEVKKTGSSVKVSCKVFGGAFR (SEQ ID NO: 170)

[0486] CDR-H1 NSAIN (SEQ ID NO: 140)

[0487] FR-H2 WVRQAPGRGLEWMG (SEQ ID NO: 141)

[0488] CDR-H2 VLIPMSGTTNYARNFQG (SEQ ID NO:171)

[0489] FR-H3 RVTISADESTSTAYMELSTLTSGDTAVYYCAR (SEQ ID NO: 172)

[0490] CDR-H3 EKERTFGWMRTSYYYVMEV (SEQ ID NO: 21)

[0491] FR-H4 WGQGTTVTVSS (SEQ ID NO: 134)

[0492] FR-L1 IRMTQSPSSLSASVGDRVTITC (SEQ ID NO: 135)

[0493] CDR-L1 RASQSISSYLN (SEQ ID NO:22)

[0494] FR-L2 WYQQKPGKAPKLLIY (SEQ ID NO: 136)

[0495] CDR-L2 AASSLQS (SEQ ID NO:23)

[0496] FR-L3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 137)

[0497] CDR-L3 QQSYSTPYT (SEQ ID NO:24)

[0498] FR-L4 FGQGTKLEIK (SEQ ID NO: 138)

[0499] The FRs and CDRs of Antibody E can be defined as follows, using the IMGT system:

[0500] FR-Hl QVQLVQSGAEVKKTGSSVKVSCKVF (SEQ ID NO: 167)

[0501] CDR-H1 GGAFRNSA (SEQ ID NO: 144)

[0502] FR-H2 INWVRQAPGRGLEWMGV (SEQ ID NO: 145)

[0503] CDR-H2 LIPMSGTT (SEQ ID NO: 173)

[0504] FR-H3 NYARNFQGRVTISADESTSTAYMELSTLTSGDTAVYYC (SEQ ID NO: 174)

[0505] CDR-H3 AREKERTFGWMRTSYYYVMEV (SEQ ID NO:148)

[0506] FR-H4 WGQGTTVTVSS (SEQ ID NO: 134)

[0507] FR-L1 IRMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 149)

[0508] CDR-L1 QSISSY (SEQ ID NO: 150)

[0509] FR-L2 LNWYQQKPGKAPKLLIY (SEQ ID NO: 151) CDR-L2 AAS

[0510] FR-L3 SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 152)

[0511] CDR-L3 QQSYSTPYT (SEQ ID NO:24)

[0512] FR-L4 FGQGTKLEIK (SEQ ID NO: 138)

[0513] The FRs and CDRs of Antibody E can be defined as follows, using the Contact system:

[0514] FR-Hl QVQLVQSGAEVKKTGSSVKVSCKVFGGAF (SEQ ID NO: 175)

[0515] CDR-H1 RNSAIN (SEQ ID NO: 154)

[0516] FR-H2 WVRQAPGRGLE (SEQ ID NO: 155)

[0517] CDR-H2 WMGVLIPMSGTTN (SEQ ID NO:176)

[0518] FR-H3 YARNFQGRVTISADESTSTAYMELSTLTSGDTAVYYC (SEQ ID NO: 177)

[0519] CDR-H3 AREKERTFGWMRTSYYYVME (SEQ ID NO:158)

[0520] FR-H4 VWGQGTTVTVSS (SEQ ID NO: 159)

[0521] FR-L1 IRMTQSPSSLSASVGDRVTITCRASQSI (SEQ ID NO: 160)

[0522] CDR-L1 SSYLNWY (SEQ ID NO: 161)

[0523] FR-L2 QQKPGKAPK (SEQ ID NO:162)

[0524] CDR-L2 LLIYAASSLQ (SEQ ID NO: 163)

[0525] FR-L3 SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:164)

[0526] CDR-L3 QQSYSTPY (SEQ ID NO: 165)

[0527] FR-L4 TFGQGTKLEIK (SEQ ID NO: 166)

[0528] Some antibodies of the invention can be defined as having six CDR sequences that correspond to the CDR sequences of one of the clones defined above.

[0529] Summary of the Figures

[0530] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0531] Figure 1. Standard RIC tumour penetration is hampered by affinity barrier. Schematic depicting the affinity barrier which prevents tumour penetration of standard RICs to the necrotic core.

[0532] Figure 2. Anti-F-actin mAbs are able to penetrate to the necrotic core of tumours. Schematic depicting the ability of anti-F-actin mAbs to penetrate to the necrotic core of tumours and kill tumours from “the inside-out”. A) Anti-F-actin mAbs escape the affinity barrier and penetrate to tumour necrotic core. B) Anti-F-actin RIC-led killing of peri-necrotic cells increases F-actin exposure, allowing greater anti-F-actin RIC binding. C) Sequential rounds of steps A and B kills tumours from the inside-out.

[0533] Figure 3. Revealing necrotic cells in tumour spheroids with Fc-DNGR-1. (A) Confocal microscopy of 20 pm sections from Braf'7600Emelanoma tumour spheroids cultured with 2WA or WT Fc-DNGR-1 overnight. Fc-DNGR-1 localisation (depicted by arrows) was revealed using anti-mouse lgG2a secondary antibody. (B) Confocal microscopy of tumour spheroids as in A cultured in vitro with intracellular binding reagents. (C) Representative confocal images of co-localisation of actin cytoskeleton- and DNA-binding reagents from B. (D) Example filamentous staining pattern of Fc-DNGR-1 on necrotic cells within tumour spheroids, prepared as in A. Data are representative of three independent experiments.

[0534] Figure 4. Revealing necrotic cells in tumours in vivo with local Fc-DNGR-1. (A) Schematic of MCA205-LifeAct (LA)-OVA-mCherry tumour experiments in C57BL / 6 mice showing route, dose, and frequency of treatment with 2WA / WT Fc-DNGR-1. (B) Confocal microscopy comparing MCA205 LA- OVA-mCherry tumours 24 h after injection with either 2WA or WT Fc-DNGR-1 (200 pg peri-tumoural). Necrotic area stained by WT Fc-DNGR-1 is denoted by “X”. (C) Quantification of Fc-DNGR-1 staining shown in B. N = 9 independent tumours. Data pooled from multiple experiments and mean ± SEM is plotted. * P < 0.05; ** P < 0.01 ; *** P < 0.001 ; **** P < 0.0001 .

[0535] Figure 5. Revealing necrotic cells in tumours in vivo with systemic anti-F-actin lgG2a. (A) Schematic of MCA205 tumour experiments in C57BL / 6 mice showing route, dose, and frequency of treatment with anti-F-actin / irrelevant lgG2a and doxorubicin chemotherapy. (B) Confocal microscopy comparing MCA205 tumours on day 21 post-cell inoculation and 72 h after last lgG2a injection. Areas stained by anti-F-actin lgG2a are highlighted. (C) Quantification of anti-F-actin lgG2a staining shown in B. N = 3 tumours from individual mice. Mean ± SEM is plotted. * P < 0.05; ** P < 0.01 ; *** P < 0.001 ; *“* P < 0.0001.

[0536] Figure 6. Phage selection funnel for F-actin monoclonal antibodies.

[0537] Figure 7. F- vs G-actin ELISA for candidate anti-F-actin clones. Binding and F-actin specificity of selected mAbs was measured by ELISA using immobilised F- and G-actin.

[0538] Figure 8. Necrotic cell binding of candidate anti-F-actin clones. Flow cytometric analysis of UV- irradiated necrotic mouse 5555 Braf'7600Emelanoma cells (A) or necrotic human HEK293T cells (B) incubated with serial dilutions of anti-F-actin lgG2a clones or Fc-DNGR-1 fusion proteins (2WA [negative control] or WT [positive control]) and stained with secondary AF488-conjugated anti-mouse IgG. Mean + / - SEM is shown from duplicate measurements.

[0539] Figure 9. Necrotic cell binding of candidate commercial anti-actin clones. Flow cytometric analysis of UV-irradiated necrotic mouse 5555 Braf760^ melanoma cells incubated with serial dilutions of anti-actin lgG2a clones (AC-40 or AC-74) or Fc-DNGR-1 fusion proteins (2WA or WT) and stained with secondary AF488-conjugated anti-mouse IgG.

[0540] Figure 10. G-actin competition for necrotic cell binding by anti-actin antibodies. Flow cytometric analysis of UV-irradiated necrotic mouse 5555 Braf'7600Emelanoma cell binding by anti-actin antibodies (10 nM) in the presence of increasing doses of soluble cytochalasin D-stabilised human platelet G-actin. (A) Solid black line = no G-actin; dashed black line = 256 nM G-actin. (B) Single points per condition are plotted.

[0541] Figure 11 . Anti-F-actin mAbs vary in their ability to inhibit DNGR-1 binding to F-actin. Comparison of three anti-F-actin mAbs for their ability to compete with DNGR-1 extracellular domain (ECD) binding to F-actin. Anti-F-actin mAbs were mixed at various doses as indicated with 10 nM FLAG-tagged DNGR-1 ECD and added to immobilised F-actin. Graph shows inhibition curves for each competitor. Percent inhibition was calculated from the measured OD405 values as described in the Material & Methods section.

[0542] Figure 12. Mouse serum and hGSN block binding of anti-F-actin mAbs to immobilised F-actin. Comparison of binding of mFc-mDNGR-1 with three anti-F-actin mAbs to immobilised F-actin in the presence of (A) 1% BSA, (B) 100% mouse serum and (C) hGSN (100 pg / ml). 10-fold dilutions of anti-F- actin mAbs or mFc-mDNGR-1 in either mouse serum (B), PBS containing 100pg / ml hGSN (C) or 1% BSA (A), which was used as negative control, were added to wells pre-coated with F-actin. Binding was measured by ELISA and graphs show titration curves for each ligand.

[0543] Figure 13. Mouse serum blocks binding of anti-F-actin mAbs to necrotic cells. Comparison of binding of mFc-mDNGR-1 with three anti-F-actin lgG2a mAbs to necrotic cells in the presence of a 5-fold dilution series of mouse serum. Binding was detected with a secondary goat anti-mouse lgG-AF488 and measured by flow cytometry.

[0544] Figure 14. Affimer-Fc binding properties. (A, B) Binding of mC9-Fc (in-house) and Affimer-Fc to F- actin was investigated by incubating the Fc-fusion proteins with UV-treated 5555 dead tumour cells, for 1 hour at equimolar concentrations. Binding was detected using anti-mouse lgG-A647 (GMFI). (C) Inhibition of Affimer-Fc binding to dead cells by sGSN was investigated by incubating UV-5555 dead cells with 10 pg / ml sGSN for 1 h at 4°C, followed by incubation with 10 nM Affimer-Fc. Binding of all Affimer-Fc to dead cells was inhibited by sGSN. (D) Inhibition of Affimer-Fc binding to dead cells by sGSN was quantified by incubating UV-5555 dead cells with various concentrations of sGSN for 1 h at 4°C, followed by incubation with 10 nM Affimer-Fc.

[0545] Figure 15. Systemic anti-F-actin antibody promotes therapeutic tumour control. (A) Experimental schematic and tumour growth profiles of C57BL / 6 mice implanted with MCA205 cells (0.5 x 106cells) and treated with WT or 2WA Fc-DNGR-1 (p.t. 100 pg) or antibody A (p.t. 150 pg) in combination doxorubicin (i.t. 2.5 mg / kg, day 7) (n = 10 per group). Mean ± SEM is plotted. (B) ELISA of serum antibody A or Fc- DNGR-1 levels following systemic administration (n = 2 per timepoint). Mean ± SEM is plotted. (C) Confocal microscopy of doxorubicin-treated MCA205 tumours three days after systemic injection of antibody A or isotype lgG2a (400 pg i.p.). Antibody A was detected using either anti-mouse lgG2a (top row) or anti-CH1-lgG (bottom row). Mean ± SEM is plotted. (D) Tumour growth profiles from MC38- implanted C57BL / 6 mice treated systemically with anti-F-actin or isotype lgG2a (400 pg i.p., biweekly from day 4) (n = 10). Mean ± SEM is plotted. (E) Relative weight change for mice treated in D. Mean ± SEM is plotted. Data are representative of two independent experiments. Data were analysed using a Bonferroni-corrected two-way ANOVA (A, D). * P < 0.05; ** P < 0.01 ; *** P < 0.001 ; **** P < 0.0001 . (F) Schematics and standard curves of anti-DNGR-1 (left) and anti-human CH1 (right) ELISAs for PK analysis. Data are representative of two independent experiments.

[0546] Figure 16. Necrotic cells localise close to FcyR+APC in tumours. (A) Confocal microscopy to identify

[0547] Fc-DNGR-1+necrotic cells and MHC-II+APC populations (CD103+cDC1s vs CD103- non-cDC1s) in tumours from D. Each dot represents a single cell within one tumour and only APCs < 500 pm distance to necrotic core analysed. Mean is plotted. Data are representative of two independent experiments. Data were analysed using an unpaired Student’s t test (D-E). * P < 0.05; “ P < 0.01 ; *** P < 0.001 ; ““ P < 0.0001 . (B) Confocal microscopy showing staining of MCA205 LA-OVA-mCherry tumours with antimouse FcyRI. Arrows indicate cells co-staining for MHC-II and FcyRI. Data are representative of three or more independent experiments.

[0548] Figure 17. Orthogonal approach to cross-training using anti-actin affimer-Fc. (A) Schematic of antiactin and control affimer-Fc fusion proteins. (B-C) Flow cytometric analysis of Fc-DNGR-1 (red) and anti- actin affimer-Fc (blue) binding to G-actin (dashed) or F-actin (solid line) coated latex beads (B) and UV- irradiated necrotic tumour cells (C). (D) Flow cytometric pulse-chase experiment of necrotic tumour cells incubated with Fc- DNGR-1 or affimer-Fc for 1 h followed by DNGR-1-FLAG for 30 min. Binding of the primary fusion protein (bottom left) and the DNGR-1 -FLAG (bottom right) was assessed. Mean ± SEM from duplicate measurements is plotted. (E) ELISA for IFNy release from OT-I CD8+ T cells co-cultured with primary FLT3L-cDCs and OVA-soaked necrotic cells ± affimer-Fc. Mean ±SEM from duplicate measurements is plotted. Data are representative of two (B-E) independent experiments. Data were analysed using a Tukey-corrected two-way ANOVA. * P < 0.05; ** P < 0.01 ; *** P < 0.001 ; *“* P <0.0001 .

[0549] Detailed Description of the Invention

[0550] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0551] Radionuclides

[0552] For therapeutic applications, it is desired that the radiolabelled binding agent will be absorbed in large quantities and retained for sufficiently long periods to deliver the highest possible dose of radiation to the target area (such as a tumour) (Holik, 2022, which is hereby incorporated by reference in its entirety). Therefore, radionuclides with longer half-lives are generally applicable for therapeutic applications (Table 2). Therefore, in general, for therapeutic applications the F-actin binding agent will be labelled with a radionuclide that is an alpha (a)-emitter or a high energy beta (p~)-emitter. Nevertheless, certain y- emitting radionuclides, positron p+ emitting radionuclides, radionuclides that decay by electron capture (EC), radionuclides that decay via isomeric transition (IT) and auger-electron-emitting radionuclides are also therapeutic radionuclides.

[0553] In contrast, for imaging applications it is generally desired that the radionuclide will be quickly absorbed and rapidly cleared from the body, to avoid non-specific binding in the patient. Therefore, in general, for imaging applications the F-actin binding agent will be labelled with a radionuclide that is a gamma (y)- emitter or beta (p+)-emitter. Nevertheless, certain high energy beta (p~)-emitting radionuclides, and radionuclides that decay by electron capture (EC) are also diagnostic radionuclides. The radionuclide may be a theranostic radionuclide. Theranostic radionuclides can be used for both therapeutic and imaging applications. This has the advantage that the biodistribution of the therapeutic agent can be imaged in a precise and non-invasive manner (Sgouros, 2020 & Holik, 2022). For example, the radionuclide may be47Sc,90Y,131l,166Ho,177Lu,188Re, or213Bi.

[0554] Table 2: Characteristics of alpha-, beta- and gamma- radiation.1LET: Linear Energy Transfer;2RBE: Relative Biological Effectiveness. Adapted from Holik, 2022.

[0555] Non-limiting Examples of Diaqnostic / lmaqinq Radionuclides

[0556] Y-emitting Radionuclides =99mTc,133Xe,201Tl,51Cr,67Ga,166Ho,61Cu,64Cu,110mAg

[0557] P+emitter Radionuclides =18F,11C,68Ga,82Rb,13N,89Zr,61Cu,64Cu

[0558] P" emitter Radionuclides =166Ho,64Cu

[0559] Electron capture (EC)-Radionuclides =1111n,18F,67Ga,123l,125l,61Cu

[0560] Non-limiting Examples of Therapeutic Radionuclides

[0561] Y-emitting Radionuclides = "Y,131l,188Re,166Ho,159Gd,110mAg,137Cs

[0562] Positron p+emitter Radionuclides = "Y,124l

[0563] P" emitter Radionuclides = "Y,131l,153Sm,177Lu,186Re,188Re,166Ho,199Au,194lr,159Gd,149Pm,142Pr,109Pd,225Ra,213Bi,212Bi,161Tb,89Sr,149Tb q emitter Radionuclides =225Ac,223Ra,230U,226Th,224Ra,213Po,149Tb

[0564] Electron capture (EC)-Radionuclides =186Re,77As,211At

[0565] Isomeric transition (IT)-Radionuclides =117mSn

[0566] Auger electrons =67Ga,77Br,103Pd

[0567] Chelating agent

[0568] As used herein, the term "chelating agent" refers to an organic moiety comprising functional groups that are able to form non-covalent bonds with the radionuclide and, thereby, form a stable radionuclide complex. Such chelating agents may be directly linked to the F-actin binding moiety or they may be connected via a linker molecule. The linking bond(s) is (are) either covalent or non-covalent bond(s) between the F-actin binding moiety (and the linker) and the chelating agent.

[0569] The chelating agent may be a bifunctional chelating agent (BFCA), which consists of a chelator to coordinate the radiometal and a linker to couple to the F-actin binding agent. As used herein, the terms bifunctional chelator and BFCA are interchangeable with the term bifunctional chelator (BFC).

[0570] Ideally, the BFCA is coupled to the F-actin binding moiety in an inert and stable manner, such that the protein integrity, immunoreactivity and in vivo biodistribution of the binding agent are not affected, and also to avoid release of the radionuclide (Chomet, 2021 , which is hereby incorporated by reference in its entirety).

[0571] Di agnostic imaging applications

[0572] Radiolabelled binding agents of the present invention find use in diagnostic imaging applications. For example, this may involve administering an effective dose of the radiolabelled binding agent; imaging the subject by positron emission tomography (PET) scan or single-photon emission computed tomography (SPECT); analysing the image obtained by PET or SPECT scan to determine the presence and / or localisation of a tumour with F-actin exposure in the subject.

[0573] The PET scan may be a PET / CT (PET / computed tomography) or PET / MRI scan. Methods for acquiring images by PET / MRI, PET / CT and SPECT are well known in the art.

[0574] Antibody-drug conjugates (ADCs)

[0575] In embodiments where the F-actin binding moiety is an antibody and the cargo is a radionuclide, the radiolabelled binding agent may be seen as a radiolabelled F-actin antibody-drug conjugate (ADC).

[0576] In embodiments where the F-actin binding moiety is an antibody and the cargo is a cytotoxic agent, the non-radiolabelled binding agent may be seen as an F-actin antibody-drug conjugate (ADC).

[0577] ADCs combine the specific targeting benefits of antibodies with the cytotoxicity of small-molecule drugs. This combination can reduce the severity of side effects as the payload drug is specifically targeted to the tissue or cell of interest. Thus, in the context of the radiolabelled (or non-radiolabelled) binding agents of the present invention, the radionuclide (or cytotoxic agent) is specifically targeted to areas with exposed F-actin, such as necrotic areas of tumours. For radiolabelled binding agents, this means the radiation emitted by the radionuclide is thus targeted and exposure to healthy cells is limited.

[0578] ADCs comprise an antibody conjugated to a cytotoxic payload (a drug), via a linker. The selection of antibody, linker and cytotoxic payload all have an impact on both safety and efficacy of the ADC in vivo. In the context of radiolabelled ADCs of the present invention, the radionuclide is the cytotoxic payload. Linkers

[0579] Depending on whether the binding agent is radiolabelled or non-radiolabelled, different linkers are envisaged.

[0580] Importantly, in the context of radiolabelled binding agents of the present invention, the linker must be stable such that it is not cleaved, to prevent release of the cytotoxic radionuclide. This allows the binding agent to exert its radiation-induced killing via bystander radioactive killing (Figure 2B). In contrast, in the context of non-radiolabelled binding agents of the present invention, the linker is a cleavable linker. The cleavable linker must nonetheless be stable under certain conditions, to ensure it is not cleaved until the binding agent reaches its target, to prevent off-target release of the cytotoxic payload. The linker may also play a role in maintaining the binding agent in an inactive, nontoxic state whilst bound to the F-actin binding moiety such as an antibody (Khongorzul, 2020, which is hereby incorporated by reference in its entirety).

[0581] The majority of ADCs are “internalising” ADCs, meaning that after binding to their target antigen (e.g. on a cancer cell) they are internalised by endocytosis and subsequently degraded in the lysosome, thus releasing the payload. The “free” payload may either exert its effect in that initial cell alone, or may diffuse out of this cell and into nearby “bystander” cancer cells, which is known as the bystander effect. Internalising ADCs generally require a cleavable linker, which is designed to remain stable outside the cell, but to be cleaved under specific conditions, such as the lysosome.

[0582] A more recent area of research is “non-internalising” ADCs, which do not require uptake into their target cell (Ashman, 2022, which is hereby incorporated by reference in its entirety). Instead, these ADCs generally make use of cleavable linkers to release their payload extracellularly. In contrast to the cleavable linkers designed for internalising ADCs, the cleavable linkers for non-internalising ADCs are intended to be cleaved extracellularly, albeit in specific environments to limit off-target effects.

[0583] The binding agents of the present invention are directed towards an extracellular target (F-actin exposed following necrosis). Thus, the non-radiolabelled binding agents may comprise a linker that is designed to be cleaved extracellularly such that the cytotoxic payload can be released to exert its cytotoxic effect. The linkers may be designed to be cleaved in the specific conditions of the tumour microenvironment (Ashman, 2022). In embodiments where the F-actin binding moiety is an antibody, the non-radiolabelled binding agent may be considered to be non-internalising ADCs.

[0584] Affimers®

[0585] The radiolabelled (or non-radiolabelled) binding agent may comprise an F-actin binding moiety that is an affimer®. Affimers® are small binding proteins which mimic antibodies in terms of molecular recognition characteristics. They exhibit higher stability in comparison to antibodies. They are recombinant proteins which can be engineered to bind a target of interest. All affimers® consist of an alpha-helix on top of an anti-parallel beta-sheet, as well as two peptide loops. These peptide loops can be randomised to bind to the desired target. The affimer may have affinity and / or avidity for both F-actin and G-actin. The affimer may be specific for F-actin.

[0586] The F-actin affimer may be an affimer disclosed in Lopata (2018) (which is hereby incorporated by reference in its entirety).

[0587] The F-actin affimer may be attached to an Fc domain. Exemplary F-actin affimer-Fc sequences include:

[0588] Affimer 6

[0589] The amino acid sequence of the in-house affimer 6-mFc construct is set forth below:

[0590] MYRMQLLSCIALSLALVTNSTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVWDVSEDDPD

[0591] VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGS

[0592] VRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLR

[0593] VEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGARTGGGGSGGGGSGGGGSDIASNSLEIEELARF

[0594] AVDEHNKKENALLEFVRVVKAKEQSSVPHWWWTTMYYLTLEAKDGGKKKLYEAKVWVKRDPNMIFKINF

[0595] KELQEFKPVGDAAGSTGSR (SEQ ID NO:191)

[0596] Control Affimer

[0597] The amino acid sequence of the control affimer-Fc construct is set forth below:

[0598] MYRMQLLSCIALSLALVTNSTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVWDVSEDDPD

[0599] VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGS

[0600] VRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLR

[0601] VEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGARTGGGGSGGGGSGGGGSDIASNSLEIEELARF

[0602] AVDEHNKKENALLEFVRVVKAKEQVVAGTMYYLTLEAKDGGKKKLYEAKVVWKPWENFKELQEFKPVG

[0603] DA (SEQ ID NO: 192)

[0604] Affimer 14

[0605] The amino acid sequence of the affimer 14-mFc construct is set forth below:

[0606] MYRMQLLSCIALSLALVTNSTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVWDVSEDDPD

[0607] VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGS

[0608] VRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLR

[0609] VEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGARTGGGGSGGGGSGGGGSDIASNSLEIEELARF

[0610] AVDEHNKKENALLEFVRVVKAKEQSDTPHWWWTTMYYLTLEAKDGGKKKLYEAKVWVKESPVHPKRLN

[0611] FKDLQEFKPVGDAAGSTGSR (SEQ ID NO:193)

[0612] Affimer 24

[0613] The amino acid sequence of the affimer 24-mFc construct is set forth below:

[0614] MYRMQLLSCIALSLALVTNSTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVWDVSEDDPD

[0615] VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGS VRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLR VEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGARTGGGGSGGGGSGGGGSDIASNSLEIEELARF AVDEHNKKENALLEFVRVVKAKEQMDMIGEYVSTMYYLTLEAKDGGKKKLYEAKVVWKGWMPLYSRQN FKELQEFKPVGDAAGSTGSR (SEQ ID NO:194)

[0616] F-actin

[0617] Actin is found in all eukaryotic cells and is one of the most highly conserved proteins known. It is a component of the cytoskeleton and also forms part of the contractile apparatus of muscle cells.

[0618] Higher eukaryotes have several different isoforms of actin, each classified as alpha, beta or gamma. Any given organism may have more than one isoform of any particular class. For example, mammals express at least 6 different actin isoforms, each encoded by separate genes, while lower eukaryotes may have fewer isoforms. Most yeasts, for example, have only one.

[0619] In general, alpha and gamma2 isotypes are found in muscle (alpha-skeletal, alpha-aortic smooth, alphacardiac and gamma2-enteric smooth) while beta and gammal isotypes are found in non-muscle cells (beta-cytoplasmic and gammal -cytoplasmic).

[0620] Actin molecules have ATPase activity. They possess a deep nucleotide-binding cleft capable of binding either ATP or ADP and of hydrolysing ATP to ADP.

[0621] The monomeric (globular) forms of actin molecules, of any isoform is generally designated “G-actin”. G- actin is capable of polymerising into strands, and the polymerised form is designated “F-actin”.

[0622] Filamentous actin (F-actin) consists of two parallel actin strands (filaments) in a helical configuration, approximately 7 nm in diameter and with a pitch (i.e. distance along the axis for one complete turn) of approximately 37 nm.

[0623] Polymerisation of G-actin into F-actin can be achieved by incubation of the monomeric form in suitable buffer containing physiological salt concentrations and ATP. In addition, F actin filaments of defined length (circa 100 subunits) can be made by subjecting F-actin to the action of actin-severing and capping proteins such as gelsolin.

[0624] F-actin can be stabilised by various molecules which bind to it and inhibit depolymerisation, such as phalloidin, jasplakinolide or tropomyosin.

[0625] For the purposes of the present specification, the term “F-actin” can be taken to refer to any substance containing two or more actin subunits associated covalently or non-covalently in a conformation which mimics that of the actin subunits in physiological F-actin strands or filaments. The substance may comprise any suitable number of associated monomers, e.g. 5 or more, 10 or more, 20 or more, 50 or more, or 100 or more monomer units. For example, it may comprise between 2 and 100 monomer units or more, e.g. 2-10 monomer units, 10-50 monomer units, 50-100 monomer units. F-actin may consist of one F-actin isoform, or may be composed of a mixture of different F-actin isoforms.

[0626] For example, it may be composed entirely of alpha, beta or gamma subunits, or it may be a mixture thereof. It may be composed entirely of non-muscle (NM) isoforms (NM F-actin) or may be entirely composed of muscle isoforms (muscle actin).

[0627] Exemplary human actin sequences include:

[0628] Alpha Actin (alpha skeletal muscle)

[0629] MCDEDETTALVCDNGSGLVKAGEFAGDDAPRAVEPSIVCGRPRHOGVMVGMGQKDSYVGDEAQSKRG ILTLKYPIEHGIITNWDDMEKIWHHTFYNELRVAPEEHPTLLTEAPLNPKANREKMIQIMEETENVPAMYVAI QAVLSLYASGRTTGIVLDSGDGVTHNVPIYEGYALPHAIMRLDLAGRDLTDYLMKILTERGYSEVITAEREI

[0630] VRDIKEKLCYVALDFENEMATAASSSSLEKSYELPDGQVITIGNERFRCPETLEFQPSEFIGMESAGIHETT YNSIMKCDIDIRKDLYANNVMSGGTTMYPGIADRMOKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLST EFQOMWITKOEYDEAGPSIVHRKCE (SEQ ID NO:178)

[0631] Beta Actin (cytoplasmic 1)

[0632] MDDDIAALVVDNGSGMCKAGEFAGDDAPRAVEPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTL KYPIEHGIVINWDDMEKIWHHTEYNELRVAPEEHPVLLTEAPLNPKANREKMTQIMFETENTPAMYVAIQA VLSLYASGRTTGIVMDSGDGVTHIVPIYEGYALPHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVRD IKEKLCYVALDFEQEMATAASSSSLEKSYBELPDGOVITIGNERFRCPEALFQPSEFLGMESCGIHETTEN

[0633] SIMKCDVDIRKDLYANTVLSGGTTMYPGIADRMOKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTEQ OMWISKQEYDESGPSIVHRKCE (SEQ ID NO:179)

[0634] Gamma Actin (cytoplasmic 2)

[0635] MEEEIAALVIDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHQOGVMVGMGQOKDSYVGDEAQSKRGILTI LKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANREKMTQIMFETFNTPAMYVAIQ AVLSLYASGRTTGIVMDSGDGVIHIVPIYEGYALPHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVR

[0636] DIKEKLCYVALDFEQEMATAASSSSLEKSYELPDGQVITIGNERFRCPEALFQPSFLGMESCGIHETTFNSI MKCDVDIRKDLYANTVLSGGITMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQO

[0637] MWISKQEYDESGPSIVHRKCE (SEQ ID NQ:180)

[0638] Actin is a highly conserved protein, with 90% identity between yeast and humans (Srinivasan, 2016).

[0639] Each monomer of actin is a 375-amino acid polypeptide which folds into two major a / p domains separated by a deep cleft and consists of subdomains 1-4. Subdomains 1 and 3 are structurally related, in contrast to subdomains 2 and 4 which are considered to be insertions into domains 1 and 3 respectively (Dominguez & Holmes, 2011 , which is hereby incorporated by reference in its entirety).

[0640] F-actin assembles into two protofilaments that turn around each other to form a right-handed, two- chained long helix with approximately 13 molecules repeating every 6 turns in an axial distance of 35.9 nm (Dominguez & Holmes, 2011). F-actin refers to the filamentous form of actin. As such, the term “F-actin” refers to a heterogeneous molecule of varying length.

[0641] Anti-F-actin antibodies and antigen binding fragments

[0642] Monoclonal antibodies (mAbs) are useful in the methods of the invention and are a homogenous population of antibodies specifically targeting a single epitope on an antigen. Suitable monoclonal antibodies can be prepared using methods well known in the art (e.g. see Kohler, G.; Milstein, C. (1975) "Continuous cultures of fused cells secreting antibody of predefined specificity”. Nature 256 (5517): 495; Siegel DL (2002). "Recombinant monoclonal antibody technology”. Schmitz U, Versmold A, Kaufmann P, Frank HG (2000); "Phage display: a molecular tool for the generation of antibodies-a review". Placenta. 21 Suppl A: S106-12. Helen E. Chadd and Steven M. Chamow; “Therapeutic antibody expression technology,” Current Opinion in Biotechnology 12, no. 2 (April 1 , 2001): 188-194; McCafferty, J.; Griffiths, A.; Winter, G.; Chiswell, D. (1990). "Phage antibodies: filamentous phage displaying antibody variable domains". Nature 348 (6301): 552-554; "Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982). Chimeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799), each of which are incorporated by reference in their entirety).

[0643] Polyclonal antibodies are useful in certain methods of the invention. However, monospecific polyclonal antibodies are preferred. Suitable polyclonal antibodies can be prepared using methods well known in the art.

[0644] Fragments of antibodies, such as Fab and Fab2 fragments may also be used, as can genetically engineered antibodies and antibody fragments.

[0645] As used herein, an “antigen binding fragment” of an antibody is a fragment that comprises the antigen binding portion of the VH / L region, such that it retains essentially the same specific binding activity of the full length antibody. (In the fragment, one or more amino acid residues that are present in the full length antibody have been removed, but this does not substantially affect the antigen binding activity of the fragment).

[0646] The variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81 , 6851-6855, which is incorporated by reference in its entirety).

[0647] That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sd. USA 85, 5879) and single domain antibodies (sdAbs) comprising isolated V domains (Ward et al (1989) Nature 341 , 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293- 299.

[0648] By "ScFv molecules" we mean molecules wherein the VH and VL partner domains are covalently linked, e.g. directly, by a peptide or by a flexible oligopeptide. Fab, Fv, ScFv and sdAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.

[0649] Whole antibodies, and F(ab')2 fragments are "bivalent". By "bivalent" we mean that the said antibodies and F(ab')2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and sdAb fragments are monovalent, having only one antigen combining site. Synthetic antibodies which bind to a target discussed herein may also be made using phage display technology as is well known in the art (e.g. see "Phage display: a molecular tool for the generation of antibodies-a review". Placenta. 21 Suppl A: 106- 12. Helen E. Chadd and Steven M. Chamow; "Phage antibodies: filamentous phage displaying antibody variable domains". Nature 348 (6301): 552-554).

[0650] Preparation of anti-F-actin antibodies

[0651] Antibodies of the present invention may be prepared using any suitable method, which are well known in the art. These include hybridoma, phage display and panning. These are described for example in Kohler, G.; Milstein, C. (1975) "Continuous cultures of fused cells secreting antibody of predefined specificity". Nature 256 (5517): 495; Siegel DL (2002) "Recombinant monoclonal antibody technology"; Schmitz U, Versmold A, Kaufmann P, Frank HG (2000); "Phage display: a molecular tool for the generation of antibodies-a review". Placenta. 21 Suppl A: S106-12; Helen E. Chadd and Steven M. Chamow;

[0652] “Therapeutic antibody expression technology,” Current Opinion in Biotechnology 12, no. 2 (April 1 , 2001): 188-194; McCafferty, J.; Griffiths, A.; Winter, G.; Chiswell, D. (1990). "Phage antibodies: filamentous phage displaying antibody variable domains". Nature 348 (6301): 552-554; "Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982), each of which are incorporated by reference in their entirety. Chimeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799).

[0653] For any of the above methods, elution may be performed using either standard elution buffer containing Trypsin (for example, at 1 mg / ml) or via competition with Fc-DNGR1 (for example, at 1000 nM). Fc domains

[0654] The Fc domain (fragment, crystallizable region) is the region of an antibody that allows it to interact with other proteins, including Fc receptors and some proteins of the complement system. Therefore, the Fc domain allows antibodies to activate the immune system. IgG Fc domains contain a highly conserved N- glycosylation site, which is essential for Fey receptor-mediated activity. Fc domains can be mutated to alter the binding characteristics with their Fc receptors. Antibodies of the invention may have an Fc domain.

[0655] Fey receptors

[0656] Preferably, the Fc domain of the antibody binds to an activating Fey receptor. Human activating Fey receptors include FcyRI, FcyRIIA, FcyRIIC and FcyRIIIB, whilst FcyRIIB is an inhibitory FcyR. Mouse activating Fey receptors include FcyRI, FcyRIII, and FcyRIV. Mouse FcyRIIB is an inhibitory FcyR. Both mice and human FcyRs display different affinities for different IgG Fc domains. Table 3 displays the binding affinities of various human FcyRs for different human IgG Fc domains and Table 4 displays the binding affinities of various mouse FcyRs for different mouse IgG Fc domains.

[0657] Table 3: Affinities of human FcyR for human IgG Fc domains. Adapted from Castro-Dopico, 2019 (which is hereby incorporated by reference in its entirety). Binding affinities are indicated as KA (M-1).

[0658] Table 4: Affinities of mouse FcyR for mouse IgG Fc domains. Adapted from Castro-Dopico, 2019.

[0659] Binding affinities are indicated as KA (M-1). Substitutions and sequence identity

[0660] A conservative substitution may be defined as a substitution within an amino acid class and / or a substitution that scores positive in the BLOSUM62 matrix.

[0661] According to one classification, the amino acid classes are acidic, basic, uncharged polar and nonpolar, wherein acidic amino acids are Asp and Glu; basic amino acids are Arg, Lys and His; uncharged polar amino acids are Asn, Gin, Ser, Thr and Tyr; and non-polar amino acids are Ala, Gly, Vai, Leu, He, Pro, Phe, Met, Trp and Cys.

[0662] According to another classification, the amino acid classes are small hydrophilic, acid / acid amide / hydrophilic, basic, small hydrophobic and aromatic, wherein small hydrophilic amino acids are Ser, Thr, Pro, Ala and Gly; acid / acidamide / hydrophilic amino acids are Asn, Asp, Glu and Gin; basic amino acids are His, Arg and Lys; small hydrophobic amino acids are Met, lie, Leu and Vai; and aromatic amino acids are Phe, Tyr and Trp.

[0663] Substitutions which score positive in the BLOSUM62 matrix are as follows:

[0664] Percent (%) amino acid sequence identity with respect to a reference sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. % identity values may be determined by WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span = 1 , overlap fraction = 0.125, word threshold (T) = 11 . A % amino acid sequence identity value is determined by the number of matching identical residues as determined by WU-BLAST-2, divided by the total number of residues of the reference sequence (gaps introduced by WU-BLAST-2 into the reference sequence to maximize the alignment score being ignored), multiplied by 100.

[0665] Alternatively, a specific pairwise alignment program may be used. A suitable example is ‘lalign ’ (implementing the algorithm of Huang and Miller; Adv. Appl. Math. (1991) 12:337-357) using default parameters. Professional / non-i cells

[0666] Certain immune cells, such as dendritic cells and particular macrophage populations, are considered "professional" antigen presenting cells (professional APCs). While most cell types can perform antigen presentation on MHC class I molecules (specifically when the antigen has been synthesised intracellularly (endogenous antigen)), professional APCs can additionally process and present exogenous antigens on MHC class II and / or cross-present exogenous antigens on MHC class I molecules. Whether a professional APC engages in antigen presentation on MHC-II or MHC-I is also affected by their cell type and the nature of the antigen. Importantly, cDC1 , a type of DC, are particularly adept at XP of cell- associated antigens, such as tumour antigens, due in part to their unique receptor expression pattern and have critical roles in anti-tumour immunity.

[0667] Pharmaceutical compositions

[0668] Pharmaceutical compositions may be prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and the like, when administered to a human. In some embodiments, this term refers to molecular entities and compositions approved by a regulatory agency of the US federal or a state government, as the GRAS list under section 204(s) and 409 of the Federal Food, Drug and Cosmetic Act, that is subject to premarket review and approval by the FDA or similar lists, the U.S. Pharmacopeia or another generally recognised pharmacopeia for use in animals, and more particularly in humans.

[0669] The term “carrier” refers to diluents, binders, lubricants and disintegrants. Those with skill in the art are familiar with such pharmaceutical carriers and methods of compounding pharmaceutical compositions using such carriers.

[0670] The pharmaceutical compositions provided herein may include one or more excipients, e.g., solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, antioxidants or antimicrobial preservatives. When used, the excipients of the compositions will not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredients, i.e. the vectors, cells and or chimeric receptors, used in the composition. Thus, the skilled person will appreciate that compositions are provided wherein there is no incompatibility between any of the components of the dosage form. Excipients may be selected from the group consisting of buffering agents, solubilizing agents, tonicity agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

[0671] Routes of Administration

[0672] Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by a number of routes, including but not limited to, systemic, parenteral, intravenous, intra-arterial, intramuscular, intratumoural, oral and nasal. The medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body.

[0673] Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0674] Combinations with other Anticancer treatments

[0675] As described herein, the medical methods, medical uses and pharmaceutical compositions of the invention may involve the radiolabelled (or non-radiolabelled) binding agent in combination with another anticancer treatment. In some embodiments, the anticancer treatment is an additional immunotherapy.

[0676] Currently, the most common cancer immunotherapies are checkpoint inhibitors. The radiolabelled (or non-radiolabelled) binding agent of the invention may be used in combination with a checkpoint inhibitor. Checkpoint inhibitors suitable for use in combination with the radiolabelled (or non-radiolabelled) binding agent of the invention include a checkpoint inhibitor that inhibits CTLA4, cytotoxic T-lymphocyte- associated antigen 4; e.g. anti-CTLA4; anti-LAG3, lymphocyte activation gene 3; anti-PD1 , programmed cell death protein 1 (e.g., KEYTRUDA); PDL, anti-PD1 ligand; anti-TIM3, T cell membrane protein 3, anti- CD40L, anti-A2aR, adenosine A2a receptor; anti-B7RP1 , B7-related protein 1 ; anti-BTLA, B and T lymphocyte attenuator; anti-GAL9, galectin 9; anti-HVEM, herpesvirus entry mediator; anti-ICOS, inducible T cell co-stimulator; anti-IL, interleukin; anti-KIR, killer cell immunoglobulin-like receptor; anti- LAG3, lymphocyte activation gene 3; anti-VISTA, V domain Ig Suppressor of T cell Activation; anti-B7-H3; anti-B7-H4; anti-TGFp, transforming growth factor-p; anti-TIM3, T cell membrane protein 3; or anti-CD27.

[0677] Other immunotherapies, such as T cell therapy, can be used in conjunction with the radiolabelled (or non- radiolabelled) binding agents disclosed herein. T cell therapies include administration of autologous or allogeneic T cells. In some embodiments, the radiolabelled (or non-radiolabelled) binding agent is administered in combination with a CAR-T cell (a T cell that expresses a chimeric antigen receptor).

[0678] In some embodiments, the anticancer treatment is a cytotoxic chemotherapeutic, meaning that the radiolabelled (or non-radiolabelled) binding agent of the invention may be used in combination with a cytotoxic chemotherapeutic. Combination with a cytotoxic chemotherapeutic has the advantage of leading to necrosis and exposure of F-actin on tumour cells, thus allowing the radiolabelled (or non- radiolabelled) binding agent to bind to the tumour cells. Cytotoxic chemotherapeutic agents non- exclusively relates to alkylating agents, anti-metabolites, plant alkaloids, topoisomerase inhibitors, antineoplastics and arsenic trioxide, carmustine, fludarabine, IDA ara-C, myalotang, GO, mustargen, cyclophosphamide, gemcitabine, bendamustine, total body irradiation, cytarabine, etoposide, melphalan, pentostatin and radiation.

[0679] In some embodiments, the anticancer treatment is radiotherapy. In some embodiments, the anticancer treatment is surgery.

[0680] The subject to be treated may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient. Therapeutic uses may be in human or animals (veterinary use).

[0681] Cancers

[0682] A "cancer" can comprise any one or more of the following: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical cancer, anal cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, cancer of the female genital system, cancer of the male genital system, central nervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colon and rectal cancer, colon cancer, endometrial cancer, endometrial sarcoma, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal tract cancer, hairy cell leukemia, head and neck cancer, hepatocellular cancer, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, leukemia, liver cancer, lung cancer, malignant fibrous histiocytoma, malignant thymoma, melanoma, mesothelioma, multiple myeloma, myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nervous system cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, plasma cell neoplasm, primary CNS lymphoma, prostate cancer, rectal cancer, respiratory system, retinoblastoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, stomach cancer, testicular cancer, thyroid cancer, urinary system cancer, uterine sarcoma, vaginal cancer, vascular system, Waldenstrom's macroglobulinemia and Wilms' tumor. In some embodiments, the cancer is not a prostate cancer.

[0683] Cancers may be of a particular type. Examples of types of cancer include astrocytoma, carcinoma (e.g. adenocarcinoma, hepatocellular carcinoma, medullary carcinoma, papillary carcinoma, squamous cell carcinoma), glioma, lymphoma, medulloblastoma, melanoma, myeloma, meningioma, neuroblastoma, sarcoma (e.g. angiosarcoma, chrondrosarcoma, osteosarcoma). Some cancers cause solid tumours. Such solid tumours may be located in any tissue, for example the pancreas, lung, breast, uterus, stomach, kidney or testis. In contrast, cancers of the blood, such as leukaemias, may not cause solid tumours - and may be referred to as liquid tumours.

[0684] The cancer that is the subject of the treatments and medical uses of the present invention may be selected from the lists provided above.

[0685] Epitope determination

[0686] The epitope of an antibody or binding agent may be determined using methods apparent to persons skilled in the art. For example, the epitope may be determined by X-ray crystallography, and / or by electron cryomicroscopy (Cryo-EM) (Hanc, et al, 2015).

[0687] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0688] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0689] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0690] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0691] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0692] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0693] Numbered paragraphs

[0694] 1 . A radiolabelled binding agent comprising: i. an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and ii. a radionuclide.

[0695] 2. The radiolabelled binding agent according to paragraph 1 , wherein the radiolabelled binding agent has at least 5-fold higher affinity for F-actin than for globular actin (G-actin).

[0696] 3. The radiolabelled binding agent according to any preceding paragraph, wherein binding of the radiolabelled binding agent to F-actin is inhibited by secreted gelsolin (sGSN).

[0697] 4. The radiolabelled binding agent according to any preceding paragraph, wherein the radionuclide is a radioisotope of a metal.

[0698] 5. The radiolabelled binding agent according to any preceding paragraph, wherein the radionuclide is an alpha-emitting radionuclide.

[0699] 6. The radiolabelled binding agent according to paragraph 5, wherein the alpha-emitting radionuclide is selected from the group consisting of:227Th,223Ra,212Pb,212Bi,211At,225Ac,213Bi,230U,226Th, 224Ra,213Po, and149Tb.

[0700] 7. The radiolabelled binding agent according to any preceding paragraph, wherein the radionuclide is a beta (p_)-emitting radionuclide.

[0701] 8. The radiolabelled binding agent according to paragraph 7, wherein the beta (p~)-emitting radionuclide is selected from the group consisting of:32P,47Sc,64Cu,67Cu,153Sm,177Lu,90Y,131l,199Au,166Ho, 186Re,188Re,194lr,159Gd,149Pm,142Pr,109Pd,225Ra,213Bi,212Bi,161Tb,89Sr, and149Tb.

[0702] 9. The radiolabelled binding agent according to any preceding paragraph, wherein the radionuclide is a gamma (y)-emitting radionuclide.

[0703] 10. The radiolabelled binding agent according to paragraph 9, wherein the gamma (y)-emitting radionuclide is selected from the group consisting of:123l,1311 ,203Pb,205Bi ,99mTc,133Xe,201Tl,51Cr, 67Ga,166Ho,61Cu,64Cu,110mAg, "Y,188Re,166Ho,159Gd, and137Cs.

[0704] 11. The radiolabelled binding agent according to any preceding paragraph, wherein the radionuclide is a high energy beta (p+)-emitting radionuclide. 12. The radiolabelled binding agent according paragraph 1 1 , wherein the high energy beta (p+)-emitting radionuclide is selected from the group consisting of:18F-AI,44Sc,52Mn,76Br,77Br,86Y,90Y,89Zr,124l, 66Ga,68Ga,18F,11C,82Rb,18N,61Cu, and64Cu.

[0705] 13. The radiolabelled binding agent according to any preceding paragraph, wherein the radionuclide decays by electron capture.

[0706] 14. The radiolabelled binding agent according to paragraph 13, wherein the radionuclide is selected from the group consisting of:111In,18F,67Ga,123l,125l,61Cu,186Re,77As, and211At.

[0707] 15. The radiolabelled binding agent according to any preceding paragraph, wherein the radionuclide decays by isomeric transition.

[0708] 16. The radiolabelled binding agent according to paragraph 15, wherein the radionuclide is117mSn.

[0709] 17. The radiolabelled binding agent according to any preceding paragraph, wherein the radionuclide is an Auger electron-emitting radionuclide.

[0710] 18. The radiolabelled binding agent according to paragraph 17, wherein the Auger electron-emitting radionuclide is selected from the group consisting of:67Ga,77Br and103Pd.

[0711] 19. The radiolabelled binding agent according to any preceding paragraph, wherein the radiolabelled binding agent comprises two, three or four radionuclides.

[0712] 20. The radiolabelled binding agent according to any preceding paragraph, wherein the radiolabelled binding agent further comprises a chelator that is co-ordinated to the radionuclide.

[0713] 21. The radiolabelled binding agent according to paragraph 20, wherein the chelator is a bidentate, tridentate, or tetradentate molecule.

[0714] 22. The radiolabelled binding agent according to any of paragraphs 20 or 21 , wherein the chelator is selected from the group of chelators disclosed in Table 1 , and salts or functional variants or derivatives thereof capable of chelating the metal.

[0715] 23. The radiolabelled binding agent according to paragraph 22, wherein the chelator is selected from the group comprising DFO, DOTA and NOTA.

[0716] 24. The radiolabelled binding agent according to any preceding paragraph, wherein the radiolabelled binding agent further comprises a linker to couple the radionuclide to the F-actin binding moiety. 25. The radiolabelled binding agent according to any preceding paragraph, wherein the linker is a covalent linker.

[0717] 26. The radiolabelled binding agent according to any of paragraphs 24 or 25, wherein the linker is selected from the group comprising: ethylene glycol bis(succinimidyl succinate) (EGS), disuccinimidyl subera (DSS), EMCS-Bz, MESS-Bz, MIH, 6-carboxy-1 ,4,8,11 -tetraazaundecane (N4), p- aminomethylaniline-diglycolic acid, PEG, NCS, maleimide, N-suc-TFP-ester, maleimide-monoamide, NHS, and MMA.

[0718] 27. The radiolabelled binding agent according to any preceding paragraph, wherein the radiolabelled binding agent comprises a bifunctional chelator.

[0719] 28. The radiolabelled binding agent according to paragraph 27, wherein the bifunctional chelator is selected from the group comprising: DFO(-NCS), DFO(-maleimide), DFO(-N-suc-TFP ester).

[0720] 29. The radiolabelled binding agent according to any preceding paragraph, wherein the F-actin binding moiety is selected from the group consisting of: an antibody or antigen-binding fragment thereof, an affimer, an aptamer, a DarPin, the CTLD of DNGR-1 , LifeAct peptide and phalloidin.

[0721] 30. The radiolabelled binding agent of paragraph 29, wherein the F-actin binding moiety is an antigenbinding fragment of an antibody.

[0722] 31. The radiolabelled binding agent of paragraph 30, wherein the antigen-binding fragment of an antibody is a Fab fragment, a single chain Fc fragment (ScFv), a single domain antibody (sdAb), a diabody or a variable domain (Fv).

[0723] 32. The radiolabelled binding agent of paragraph 29, wherein the F-actin binding moiety is an antibody.

[0724] 33. The radiolabelled binding agent according to paragraph 32, wherein the antibody is a human or humanised antibody.

[0725] 34. The radiolabelled binding agent according to paragraph 33, wherein the antibody is of the human lgG1 isotype.

[0726] 35. The radiolabelled binding agent according to any of paragraphs 29 to 34, wherein the antibody or antigen-binding fragment thereof does not comprise an Fc domain.

[0727] 36. The radiolabelled binding agent according to any of paragraphs 29 to 34, wherein the antibody or antigen-binding fragment thereof comprises an Fc domain. 37. The radiolabelled binding agent according to paragraph 36, wherein the Fc domain comprises an amino acid sequence that has been mutated from that of a wild type Fc domain such that the mutated Fc domain binds to an Fc receptor with lower affinity and / or avidity than the affinity and / or avidity of the wild type Fc domain for the Fc receptor.

[0728] 38. The radiolabelled binding agent according to paragraph 37, wherein the mutated Fc domain does not bind to an Fc receptor.

[0729] 39. The radiolabelled binding agent according to any of paragraphs 36 or 37, wherein the Fc domain comprises an amino acid sequence that has been mutated from that of a wild type Fc domain such that the mutated Fc domain binds to a neonatal Fc receptor (FcRn) with higher affinity and / or avidity than the affinity and / or avidity of the wild type Fc domain for the Fc receptor.

[0730] 40. The radiolabelled binding agent according to any preceding paragraph, wherein the F-actin binding moiety does not compete for binding to F-actin with any of the antibodies selected from the group consisting of: ab11003 (Abeam), ab205 (Abeam), ab130935 (Abeam), A2228 or A5316 (Sigma- Aldrich), NBP2-61610 (Novus Biologicals), MA1 -80729 (ThermoFisher) and BS-1571 R (ThermoFisher).

[0731] 41. The radiolabelled binding agent according to any one of the preceding paragraphs, wherein the F- actin binding moiety competes with human DNGR-1 for binding to F-actin.

[0732] 42. The radiolabelled binding agent according to any one of the preceding paragraphs, wherein the F- actin binding moiety binds to the same epitope or overlaps with the same epitope as human DNGR-1 .

[0733] 43. The radiolabelled binding agent according to any one of the preceding paragraphs, wherein binding of the F-actin binding moiety to F-actin is not inhibited by the presence of G-actin.

[0734] 44. The radiolabelled binding agent according to any one of the preceding paragraphs, wherein the F- actin binding moiety specifically binds to a discontinuous, conformational epitope that includes amino acid residues present on at least 2 subunits of F-actin, wherein the discontinuous, conformational epitope comprises three or more residues selected from: R196 and / or E237 of a first subunit of F- actin on a first filament and A114, E117, Q121 , A365, G366 and P367 of a second subunit of F-actin on a separate filament of F-actin to the first subunit.

[0735] 45. The radiolabelled binding agent according to paragraph 44, wherein the F-actin binding moiety binds to a conformational epitope that further comprises residue D80 of a third subunit of F-actin which is part of the same filament as the first subunit and adjacent to the first subunit. 46. The radiolabelled binding agent according to any of paragraphs 29 to 45, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: i. a CDR1 having a sequence GGTFRSY (SEQ ID NO:1) or a variant thereof comprising one or two amino acid substitutions with respect to GGTFRSY (SEQ ID NO:1); ii. a CDR2 having a sequence NPIFDT (SEQ ID NO:2) or a variant thereof comprising one or two amino acid substitutions with respect to NPIFDT (SEQ ID NO:2); and

[0736] Hi. a CDR3 having a sequence TVIGAFDS (SEQ ID NO:3) or a variant thereof comprising one or two amino acid substitutions with respect to TVIGAFDS (SEQ ID NO:3); and wherein the light chain comprises: iv. a CDR1 having a sequence TRTSGDIGGYNFVS (SEQ ID NO:4) or a variant thereof comprising one, two or three amino acid substitutions with respect to TRTSGDIGGYNFVS (SEQ ID NO:4) v. a CDR2 having a sequence DVNSRPS (SEQ ID NO:5) or a variant thereof comprising one or two amino acid substitutions with respect to DVNSRPS (SEQ ID NO:5); and vi. a CDR3 having a sequence SSYTSRNTV (SEQ ID NO:6) or a variant thereof comprising one or two amino acid substitutions with respect to SSYTSRNTV (SEQ ID NO:6), wherein complementarity determining regions (CDRs) are identified using Chothia numbering.

[0737] 47. The radiolabelled binding agent according to paragraph 46, wherein the G at position 2 of CDRH1 is substituted with F or Y; wherein the T at position 3 of CDRH1 is substituted with I or A, wherein the R at position 5 of CDRH1 is substituted with S or T; wherein the S at position 6 of CDRH1 is substituted with A or N; and / or wherein the Y at position 7 of CDRH1 is substituted with S; and / or wherein the N at position 1 of CDRH2 is substituted with I; wherein the P at position 2 of CDRH2 is substituted with S; wherein the I at position 3 of CDRH2 is substituted with A, S or M; wherein the F at position 4 of CDRH2 is substituted with Y or S; wherein the D at position 5 of CDRH2 is substituted with N or G; and / or wherein the T at position 6 of CDRH2 is substituted with G; and / or wherein the T at position 1 of CDRH3 is substituted with G or Y; wherein the I at position 3 of CDRH3 is substituted with W or V; wherein the G at position 4 of CDRH3 is substituted with M; wherein the A at position 5 of CDRH3 is substituted with R, T or K; wherein the F at position 6 of CDRH3 is substituted with T, Y or I; wherein the D at position 7 of CDRH3 is substituted with P or S; and / or wherein the S at position 8 of CDRH3 is substituted with Y; and / or wherein the T at position 1 of CDRL1 is substituted with S; wherein the R at position 2 of CDRL1 is substituted with G; wherein the T at position 3 of CDRL1 is substituted with A, S or G; wherein the S at position 4 of CDRL1 is substituted with T; wherein the G at position 5 of CDRL1 is substituted with Q or S; wherein the D at position 6 of CDRL1 is substituted with S or N; wherein the G at position 8 of CDRL1 is substituted with S; wherein the G at position 9 of CDRL1 is substituted with S; wherein the Y at position 10 of CDRL1 is substituted with R or K; wherein the N at position 11 of CDRL1 is substituted with R or L; wherein the F at position 12 of CDRL1 is substituted with Y, H or N; and / or wherein the S at position 14 of CDRL1 is substituted with F; and / or wherein the D at position 1 of CDRL2 is substituted with A or R; wherein the V at position 2 of CDRL2 is substituted with A, N or G; wherein the N at position 3 of CDRL2 is substituted with S or D; wherein the S at position 4 of CDRL2 is substituted with M or Q; wherein the R at position 5 of CDRL2 is substituted with L; and / or wherein the P at position 6 of CDRL2 is substituted with Q; and / or wherein the S at position 1 of CDRL3 is substituted with Q; wherein the S at position 2 of CDRL3 is substituted with R or G; wherein the Y at position 3 of CDRL3 is substituted with W; wherein the T at position 4 of CDRL3 is substituted with S or V, wherein the S at position 5 of CDRL3 is substituted with T; wherein the R at position 6 of CDRL3 is substituted with P; and / or wherein the N at position 7 of CDRL3 is substituted with Y. The radiolabelled binding agent according to any of paragraphs 29 to 47, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: i. a CDR1 having a sequence GYIFTSY (SEQ ID NO:7) or a variant thereof comprising one or two amino acid substitutions with respect to GYIFTSY (SEQ ID NO:7); ii. a CDR2 having a sequence SAYNGH (SEQ ID NO:8) or a variant thereof comprising one or two amino acid substitutions with respect to SAYNGH (SEQ ID NO:8); and

[0738] Hi. a CDR3 having a sequence GKISSWFVLED (SEQ ID NO:9) or a variant thereof comprising one, two or three amino acid substitutions with respect to GKISSWFVLED (SEQ ID NO:9); and wherein the light chain comprises: iv. a CDR1 having a sequence SGGTSNIGKNYVS (SEQ ID NQ:10) or a variant thereof comprising one or two or three amino acid substitutions with respect to SGGTSNIGKNYVS (SEQ ID NQ:10) v. a CDR2 having a sequence DNNMRPS (SEQ ID NO:11) or a variant thereof comprising one or two amino acid substitutions with respect to DNNMRPS (SEQ ID NO:11); and vi. a CDR3 having a sequence GMWIRSLSRWV (SEQ ID NO:12) or a variant thereof comprising one, two or three amino acid substitutions with respect to GMWIRSLSRWV (SEQ ID NO:12), wherein complementarity determining regions (CDRs) are identified using Chothia numbering. The radiolabelled binding agent according to paragraph 48, wherein the Y at position 2 of CDRH1 is substituted with F or G; wherein the I at position 3 of CDRH1 is substituted with T or A, wherein the T at position 5 of CDRH1 is substituted with S or R; wherein the S at position 6 of CDRH1 is substituted with A or N; and / or wherein the Y at position 7 of CDRH1 is substituted with S; wherein the S at position 1 of CDRH2 is substituted with P; wherein the A at position 2 of CDRH2 is substituted with S, I or M; wherein the Y at position 3 of CDRH2 is substituted with F or S; wherein the N at position 4 of CDRH2 is substituted with D or G; wherein the G at position 5 of CDRH2 is substituted with T; and / or wherein the H at position 6 of CDRH2 is substituted with N; wherein the G at position 1 of CDRH3 is substituted with M; wherein the K at position 2 of CDRH3 is substituted with R, T or A; wherein the I at position 3 of CDRH3 is substituted with T, Y or F; wherein the S at position 4 of CDRH3 is substituted with P or D; wherein the S at position 5 of CDRH3 is substituted with Y; wherein the W at position 6 of CDRH3 is substituted with Y or A; wherein the F at position 7 of CDRH3 is substituted with Y; wherein the L at position 9 of CDRH3 is substituted with M; and / or wherein the D at position 11 of CDRH3 is substituted with V; wherein the S at position 1 of CDRL1 is substituted with T; wherein the G at position 2 of CDRL1 is substituted with R; wherein the G at position 3 of CDRL1 is substituted with A, S or T; wherein the T at position 4 of CDRL1 is substituted with S; wherein the S at position 5 of CDRL1 is substituted with Q or G; wherein the N at position 6 of CDRL1 is substituted with S or D; wherein the G at position 8 of CDRL1 is substituted with S; wherein the K at position 9 of CDRL1 is substituted with R or Y; wherein the N at position 10 of CDRL1 is substituted with R or L; wherein the Y at position 11 of CDRL1 is substituted with H, F or N; and / or wherein the S at position 13 of CDRL1 is substituted with F; wherein the D at position 1 of CDRL2 is substituted with A or R; wherein the N at position 2 of CDRL2 is substituted with A, V or G; wherein the N at position 3 of CDRL2 is substituted with S or D; wherein the M at position 4 of CDRL2 is substituted with S or Q; wherein the R at position 5 of CDRL2 is substituted with L; and / or wherein the P at position 6 of CDRL2 is substituted with Q; and / or wherein the G at position 1 of CDRL3 is substituted with A; wherein the M at position 2 of CDRL3 is substituted with T; wherein the I at position 4 of CDRL3 is substituted with D; wherein the R at position 5 of CDRL3 is substituted with D; wherein the S at position 6 of CDRL3 is substituted with G; wherein the L at position 7 of CDRL3 is substituted with Q; wherein the S at position 8 of CDRL3 is substituted with Q; wherein the R at position 9 of CDRL3 is substituted with S or G; wherein the W at position 10 of CDRL3 is substituted with Y; and / or wherein the V at position 11 of CDRL3 is substituted with S or T. The radiolabelled binding agent according to any of paragraphs 29 to 49, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: i. a CDR1 having a sequence GFTFSAY (SEQ ID NO:13) or a variant thereof comprising one or two amino acid substitutions with respect to GFTFSAY (SEQ ID NO:13); ii. a CDR2 having a sequence SYDGNN (SEQ ID NO:14) or a variant thereof comprising one or two amino acid substitutions with respect to SYDGNN (SEQ ID NO:14); and

[0739] Hi. a CDR3 having a sequence DFRDYVWGTYPSAY (SEQ ID NO:15) or a variant thereof comprising one, two or three amino acid substitutions with respect to DFRDYVWGTYPSAY (SEQ ID NO:15); iv. and wherein the light chain comprises: v. a CDR1 having a sequence SGSSSNIGRRHVF (SEQ ID NO:16) or a variant thereof comprising one, two or three amino acid substitutions with respect to SGSSSNIGRRHVF (SEQ ID NO:16) vi. a CDR2 having a sequence RGDQRPS (SEQ ID NO:17) or a variant thereof comprising one or two amino acid substitutions with respect to RGDQRPS (SEQ ID NO:17); and vii. a CDR3 having a sequence ATWDDGLSGYV (SEQ ID NO:18) or a variant thereof comprising one, two or three amino acid substitutions with respect to ATWDDGLSGYV (SEQ ID NO:18), wherein complementarity determining regions (CDRs) are identified using Chothia numbering. The radiolabelled binding agent according to paragraph 50, wherein the F at position 2 of CDRH1 is substituted with G or Y; wherein the T at position 3 of CDRH1 is substituted with I or A, wherein the S at position 5 of CDRH1 is substituted with R or T; wherein the A at position 6 of CDRH1 is substituted with S or N; and / or wherein the Y at position 7 of CDRH1 is substituted with S; and / or wherein the S at position 1 of CDRH2 is substituted with A, I or M; wherein the Y at position 2 of CDRH2 is substituted with F or S; wherein the D at position 3 of CDRH2 is substituted with N or G; wherein the G at position 4 of CDRH2 is substituted with T; and / or wherein the N at position 5 of CDRH2 is substituted with H; wherein the D at position 1 of CDRH3 is substituted with K; wherein the F position 2 of CDRH3 is substituted with E; wherein the D at position 4 of CDRH3 is substituted with T; wherein the Y at position 5 of CDRH3 is substituted with G or T; wherein the W at position 7 of CDRH3 is substituted with I or V; wherein the G at position 8 of CDRH3 is substituted with M; wherein the T at position 9 of CDRH3 is substituted with R, A or K; wherein the Y at position 10 of CDRH3 is substituted with T, F or I; wherein the P at position 11 of CDRH3 is substituted with D or S; wherein the S at position 12 of CDRH3 is substituted with Y; wherein the A at position 13 of CDRH3 is substituted with Y or W; and / or wherein the Y at position 14 of CDRH3 is substituted with F; and / or wherein the S at position 1 of CDRL1 is substituted with T; wherein the G at position 2 of CDRL1 is substituted with R; wherein the S at position 3 of CDRL1 is substituted with A, T or G; wherein the S at position 4 of CDRL1 is substituted with T; wherein the S at position 5 of CDRL1 is substituted with Q or G; wherein the N at position 6 of CDRL1 is substituted with S or D; wherein the G at position 8 of CDRL1 is substituted with S; wherein the R at position 9 of CDRL1 is substituted with Y or K; wherein the R at position 10 of CDRL1 is substituted with L or N; wherein the H at position 11 of CDRL1 is substituted with F, N or Y; and / or wherein the F at position 13 of CDRL1 is substituted with S; and / or wherein the R at position 1 of CDRL2 is substituted with A or D; wherein the G at position 2 of CDRL2 is substituted with A, N or V; wherein the D at position 3 of CDRL2 is substituted with S or N; wherein the Q at position 4 of CDRL2 is substituted with S or M; wherein the R at position 5 of CDRL2 is substituted with L; and / or wherein the P at position 6 of CDRL2 is substituted with Q; and / or wherein the A at position 1 of CDRL3 is substituted with G; wherein the T at position 2 of CDRL3 is substituted with M; wherein the D at position 4 of CDRL3 is substituted with I; wherein the D at position 5 of CDRL3 is substituted with R; wherein the G at position 6 of CDRL3 is substituted with S; wherein the L at position 7 of CDRL3 is substituted with Q; wherein the S at position 8 of CDRL3 is substituted with Q; wherein the G at position 9 of CDRL3 is substituted with S or R; wherein the Y at position 10 of CDRL3 is substituted with W; and / or wherein the V at position 11 of CDRL3 is substituted with S or T. The radiolabelled binding agent according to any of paragraphs 29 to 51 , wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: i. a CDR1 having a sequence GGAFRNS (SEQ ID NO:19) or a variant thereof comprising one or two amino acid substitutions with respect to GGAFRNS (SEQ ID NO:19); ii. a CDR2 having a sequence IPMSGT (SEQ ID NQ:20) or a variant thereof comprising one or two amino acid substitutions with respect to IPMSGT (SEQ ID NQ:20); and

[0740] Hi. a CDR3 having a sequence EKERTFGVVMRTSYYYVMEV (SEQ ID NO:21) or a variant thereof comprising one, two, three or four amino acid substitutions with respect to EKERTFGVVMRTSYYYVMEV (SEQ ID NO:21); and wherein the light chain comprises: iv. a CDR1 having a sequence RASQSISSYLN (SEQ ID NO:22) or a variant thereof comprising one or two amino acid substitutions with respect to RASQSISSYLN (SEQ ID NO:22) v. a CDR2 having a sequence AASSLQS (SEQ ID NO:23) or a variant thereof comprising one or two amino acid substitutions with respect to AASSLQS (SEQ ID NO:23); and vi. a CDR3 having a sequence QQSYSTPYT (SEQ ID NO:24) or a variant thereof comprising one or two amino acid substitutions with respect to QQSYSTPYT (SEQ ID NO:24), wherein complementarity determining regions (CDRs) are identified using Chothia numbering. The radiolabelled binding agent according to paragraph 52, wherein the G at position 2 of CDRH1 is substituted with F or Y; wherein the A at position 3 of CDRH1 is substituted with T or I; wherein the R at position 5 of CDRH1 is substituted with S or T; wherein the N at position 6 of CDRH1 is substituted with A or S; and / or wherein the S at position 7 of CDRH1 is substituted with Y; and / or wherein the I at position 1 of CDRH2 is substituted with N; wherein the P at position 2 of CDRH2 is substituted with S; wherein the M at position 3 of CDRH2 is substituted with I, A or S; wherein the S at position 4 of CDRH2 is substituted with F or Y; wherein the G at position 5 of CDRH2 is substituted with D or N; and / or wherein the T at position 6 of CDRH2 is substituted with G; and / or wherein the K at position 2 of CDRH3 is substituted with D; wherein the E at position 3 of CDRH3 is substituted with F; wherein the T at position 5 of CDRH3 is substituted with D; wherein the G at position 7 of CDRH3 is substituted with Y or T; wherein the V at position 9 of CDRH3 is substituted with W or I; wherein the M at position 10 of CDRH3 is substituted with G; wherein the R at position 11 of CDRH3 is substituted with A, T or K; wherein the T at position 12 of CDRH3 is substituted with F, Y or I; wherein the S at position 13 of CDRH3 is substituted with P or D; wherein the Y at position 14 of CDRH3 is substituted with S; wherein the Y at position 15 of CDRH3 is substituted with W or A; wherein the Y at position 16 of CDRH3 is substituted with F; wherein the M at position 18 of CDRH3 is substituted with L; and / or wherein the V at position 20 of CDRH3 is substituted with D; and / or wherein the R at position 1 of CDRL1 is substituted with G; wherein the A at position 2 of CDRL1 is substituted with S, T or G; wherein the S at position 3 of CDRL1 is substituted with T; wherein the Q at position 4 of CDRL1 is substituted with S or G; wherein the S at position 5 of CDRL1 is substituted with D or N; wherein the S at position 7 of CDRL1 is substituted with G; wherein the S at position 8 of CDRL1 is substituted with G; wherein the Y at position 9 of CDRL1 is substituted with R or K; wherein the L at position 10 of CDRL1 is substituted with N or R; and / or wherein the N at position 11 of CDRL1 is substituted with F, H or Y; and / or wherein the A at position 1 of CDRL2 is substituted with D or R; wherein the A at position 2 of CDRL2 is substituted with V, N or G; wherein the S at position 3 of CDRL2 is substituted with N or D; wherein the S at position 4 of CDRL2 is substituted with M or Q; wherein the L at position 5 of CDRL2 is substituted with R; and / or wherein the Q at position 6 of CDRL2 is substituted with P; and / or wherein the Q at position 1 of CDRL3 is substituted with L; wherein the Q at position 2 of CDRL3 is substituted with S; wherein the S at position 3 of CDRL3 is substituted with R or G; wherein the Y at position 4 of CDRL3 is substituted with W; wherein the S at position 5 of CDRL3 is substituted with T or V; wherein the T at position 6 of CDRL3 is substituted with S; wherein the P at position 7 of CDRL3 is substituted with R; and / or wherein the Y at position 8 of CDRL3 is substituted with N.

[0741] 54. The radiolabelled binding agent according to any of paragraphs 46 to 53, wherein said one or more substitutions are conservative substitutions.

[0742] 55. The radiolabelled binding agent according to any one of the preceding paragraphs, having at least 3- fold specificity for binding necrotic cells over live cells, as measured by FACS or flow cytometry.

[0743] 56. The radiolabelled binding agent according to paragraph 55, wherein the necrotic cell binding is not inhibited by the presence of G-actin.

[0744] 57. The radiolabelled binding agent according to any one of the preceding paragraphs, for use in medicine.

[0745] 58. The radiolabelled binding agent according to any one of paragraphs 1 to 56, for use in a method of treating cancer, the method comprising administering the radiolabelled binding agent to a subject that has cancer.

[0746] 59. The radiolabelled binding agent according to paragraph 58, wherein the cancer comprises a solid tumour.

[0747] 60. The radiolabelled binding agent for the use according to any one of paragraphs 58 or 59, wherein the cancer is characterised by F-actin presentation on necrotic cancer cells.

[0748] 61. The radiolabelled binding agent for the use according to any one of paragraphs 58 to 60, wherein the method of treating cancer comprises killing cancer cells via necrosis.

[0749] 62. The radiolabelled binding agent for the use according to paragraph 61 , wherein the method comprises contacting the necrotic cancer cells with the radiolabelled binding agent. 63. The radiolabelled binding agent for the use according to any one of paragraphs 58 to 62, wherein the method of treating cancer comprises (i) administering a therapy to the subject to cause necrosis of the cancer cells, and then (ii) administering the radiolabelled binding agent to the subject and thereby contacting the necrotic cancer cells with the radiolabelled binding agent.

[0750] 64. The radiolabelled binding agent for the use according to paragraph 63, wherein the therapy administered to the subject is selected from the group comprising: an F-actin binding agent, a chemotherapeutic agent or a radiotherapy.

[0751] 65. The radiolabelled binding agent for the use according to any one of paragraphs 58 to 64, wherein the use comprises administration of the radiolabelled binding agent in combination with an additional anticancer therapy.

[0752] 66. The radiolabelled binding agent for the use according to paragraph 65, wherein the additional anticancer therapy is an immune checkpoint inhibitor.

[0753] 67. The radiolabelled binding agent for the use according to paragraph 66, wherein the immune checkpoint inhibitor is an anti-PD1 monoclonal antibody.

[0754] 68. The radiolabelled binding agent for the use according to any one of paragraphs 58 to 67, wherein the additional anticancer therapy is a radiotherapy.

[0755] 69. The radiolabelled binding agent for the use according to any one of paragraphs 58 to 68, wherein the use comprises administration of the radiolabelled binding agent in combination with a chemotherapeutic agent.

[0756] 70. The radiolabelled binding agent for the use according to any one of any one of paragraphs 58 to 69, wherein the use comprises co-administration of the radiolabelled binding agent with an anti-F-actin antibody that is not radiolabelled.

[0757] 71. The radiolabelled binding agent according to any one of paragraphs 1 to 56, for use in a method of radioimaging.

[0758] 72. The radiolabelled binding agent for the use according to paragraph 71 , wherein the radionuclide comprises a non-ionising isotope.

[0759] 73. The radiolabelled binding agent for the use according to any one of paragraphs 71 or 72, wherein the radioimaging comprises imaging a tumour or a cancer.

[0760] 74. The radiolabelled binding agent for the use according to paragraph 73, wherein the cancer is characterised by F-actin presentation on necrotic cancer cells. 75. A method of radio-immuno imaging, comprising administering the radiolabelled binding agent according to any one of paragraphs 1 to 56 to a subject and detecting the radionuclide.

[0761] 76. The method of paragraph 75, wherein the method of radio-immuno imaging is a positron-emission tomography (PET) method and the radionuclide is a high energy beta (p+)-emitting radionuclide.

[0762] 77. The method of paragraph 75, wherein the method of radio-immuno imaging is a single-photon emission computed tomography (SPECT) method.

[0763] 78. The radiolabelled binding agent according to any one of paragraphs 1 to 56, for use in an in vivo method of diagnosis of cancer.

[0764] 79. A method of selecting an antibody or antigen-binding fragment thereof that specifically binds to F- actin, the method comprising: i. providing a population of candidate antibodies, ii. contacting a binding substrate comprising F-actin with the population of candidate antibodies, Hi. contacting the binding substrate with trypsin or a competitor agent comprising the CTLD of

[0765] DNGR-1 to displace one or more lead antibodies from the binding substrate, then iv. selecting an antibody from said one or more lead antibodies; and then v. labelling the selected antibody with a radionuclide.

[0766] 80. The method according to paragraph 79, wherein the method comprises screening said one or more lead antibodies for 10-fold specificity for F-actin over G-actin, wherein the selected antibody has said 10-fold specificity.

[0767] 81. The method according to any one of paragraphs 79 or 80, wherein the method comprises screening said one or more lead antibodies for at least 3-fold specificity for binding necrotic cells over live cells, as measured by FACS or flow cytometry, wherein the selected antibody has said 3-fold specificity.

[0768] 82. A method of producing a radiolabelled binding agent according to any one of paragraphs 1 to 56, the method comprising: i. obtaining an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and ii. labelling the F-actin binding moiety with a radionuclide.

[0769] 83. The method according to paragraph 82, wherein the radionuclide is selected from the group consisting of:227Th,223Ra,212Pb,212Bi,211At,225Ac,213Bi,230U,226Th,224Ra,213Po,149Tb,32P,47Sc, 64Cu,67Cu,153Sm,177Lu, "Y,131l,199Au,166Ho,186Re,188Re,194lr,159Gd,149Pm,142Pr,109Pd,225Ra, 213Bi,212Bi,161Tb,89Sr,77As,149Tb,123l ,1311 ,203Pb,205Bi,99mTc,133Xe,201TI,51Cr,67Ga,166Ho,61Cu,64Cu, 110mAg, "Y,188Re,166Ho,159Gd,137Cs,18F-AI,44Sc,52Mn,76Br,77Br,86Y, "Y,89Zr,124l,66Ga,68Ga,18F,11C,82Rb,13N,61Cu,64Cu,1111n,18F,67Ga,123l,125l,61Cu,186Re,77As,211At,117mSn,67Ga,77Br and

[0770] 103Pd.

[0771] 84. A kit comprising: i. an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and ii. a radionuclide.

[0772] 85. A kit according to paragraph 84, further comprising instructions for radiolabelling the F-actin binding moiety that specifically binds to filamentous actin (F-actin) with the radionuclide.

[0773] 86. A payload delivery agent comprising: i. an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and ii. a cargo moiety.

[0774] 87. The payload delivery agent according to paragraph 86, wherein the cargo moiety is a radionuclide and the payload delivery agent is a radiolabelled binding agent according to any one of paragraphs 1 to 56.

[0775] 88. The payload delivery agent according to paragraph 86, wherein the cargo moiety is a cytotoxic agent.

[0776] 89. The payload delivery agent according to paragraph 88, wherein the payload delivery agent has at least 5-fold higher affinity for F-actin than for globular actin (G-actin).

[0777] 90. The pay load delivery agent according to paragraph 88 or 89, wherein binding of the payload delivery agent to F-actin is inhibited by secreted gelsolin (sGSN).

[0778] 91. The payload delivery agent according to any of paragraphs 88 to 90, wherein the cytotoxic agent is a cytotoxin, an immunomodulator, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, a DNA, an RNA, an siRNA, an RNAi, a microRNA, a photoactive therapeutic agent, an anti-angiogenic agent, a pro- apoptotic agent, a peptide, a lipid, a carbohydrate, a chelating agent, or combinations thereof.

[0779] 92. The payload delivery agent according to any of paragraphs 88 to 91 , wherein the linker is a cleavable linker.

[0780] 93. The payload delivery agent according to any of paragraphs 88 to 92, wherein the linker is an acid- cleavable linker, an enzyme-cleavable linker or a reducible disulfide.

[0781] 94. The payload delivery agent according to paragraph 93, wherein the acid-cleavable linker is a carbonate, a hydrazone, or an ester. 95. The payload delivery agent according to paragraph 93, wherein the enzyme-cleavable linker is a dipeptide, optionally wherein the dipeptide is Val-Cit.

[0782] 96. The payload delivery agent according to any of paragraphs 88 to 95, wherein the payload delivery agent further comprises a self-immolative spacer.

[0783] 97. The payload delivery agent according to any of paragraphs 88 to 96, wherein the F-actin binding moiety is selected from the group consisting of: an antibody or antigen-binding fragment thereof, an affimer, an aptamer, a DarPin, the CTLD of DNGR-1 , LifeAct peptide and phalloidin.

[0784] 98. The payload delivery agent of paragraph 97, wherein the F-actin binding moiety is an antigen-binding fragment of an antibody.

[0785] 99. The payload delivery agent of paragraph 98, wherein the antigen-binding fragment of an antibody is a Fab fragment, a single chain Fc fragment (ScFv), a single domain antibody (sdAb), a diabody or a variable domain (Fv).

[0786] 100. The payload delivery agent of paragraph 99, wherein the F-actin binding moiety is an antibody.

[0787] 101. The payload delivery agent according to paragraph 100, wherein the antibody is a human or humanised antibody.

[0788] 102. The pay load delivery agent according to paragraph 101 , wherein the antibody is of the human lgG1 isotype.

[0789] 103. The payload delivery agent according to any of paragraphs 97 to 102, wherein the antibody or antigen-binding fragment thereof does not comprise an Fc domain.

[0790] 104. The payload delivery agent according to any of paragraphs 97 to 103, wherein the antibody or antigen-binding fragment thereof comprises an Fc domain.

[0791] 105. The payload delivery agent according to paragraph 104, wherein the Fc domain comprises an amino acid sequence that has been mutated from that of a wild type Fc domain such that the mutated Fc domain binds to an Fc receptor with lower affinity and / or avidity than the affinity and / or avidity of the wild type Fc domain for the Fc receptor.

[0792] 106. The payload delivery agent according to paragraph 105, wherein the mutated Fc domain does not bind to an Fc receptor.

[0793] 107. The payload delivery agent according to any of paragraphs 104 to 106, wherein the Fc domain comprises an amino acid sequence that has been mutated from that of a wild type Fc domain such that the mutated Fc domain binds to a neonatal Fc receptor (FcRn) with higher affinity and / or avidity than the affinity and / or avidity of the wild type Fc domain for the Fc receptor. . The payload delivery agent according to any of paragraphs 88 to 107, wherein the F-actin binding moiety does not compete for binding to F-actin with any of the antibodies selected from the group consisting of: ab11003 (Abeam), ab205 (Abeam), ab130935 (Abeam), A2228 or A5316 (Sigma- Aldrich), NBP2-61610 (Novus Biologicals), MA1 -80729 (ThermoFisher) and BS-1571 R (ThermoFisher). . The payload delivery agent according to any of paragraphs 88 to 108, wherein the F-actin binding moiety competes with human DNGR-1 for binding to F-actin. . The payload delivery agent according to any one of paragraphs 88 to 108, wherein the F-actin binding moiety binds to the same epitope or overlaps with the same epitope as human DNGR-1 . . The pay load delivery agent according to any one of paragraphs 88 to 110, wherein binding of the F-actin binding moiety to F-actin is not inhibited by the presence of G-actin. . The payload delivery agent according to any one of paragraphs 88 to 111 , wherein the F-actin binding moiety specifically binds to a discontinuous, conformational epitope that includes amino acid residues present on at least 2 subunits of F-actin, wherein the discontinuous, conformational epitope comprises three or more residues selected from: R196 and / or E237 of a first subunit of F-actin on a first filament and A114, E117, Q121 , A365, G366 and P367 of a second subunit of F-actin on a separate filament of F-actin to the first subunit. . The payload delivery agent according to paragraph 112, wherein the F-actin binding moiety binds to a conformational epitope that further comprises residue D80 of a third subunit of F-actin which is part of the same filament as the first subunit and adjacent to the first subunit. . The payload delivery agent according to any of paragraphs 97 to 113, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: i. a CDR1 having a sequence GGTFRSY (SEQ ID NO:1) or a variant thereof comprising one or two amino acid substitutions with respect to GGTFRSY (SEQ ID NO:1); ii. a CDR2 having a sequence NPIFDT (SEQ ID NO:2) or a variant thereof comprising one or two amino acid substitutions with respect to NPIFDT (SEQ ID NO:2); and

[0794] Hi. a CDR3 having a sequence TVIGAFDS (SEQ ID NO:3) or a variant thereof comprising one or two amino acid substitutions with respect to TVIGAFDS (SEQ ID NO:3); and wherein the light chain comprises: iv. a CDR1 having a sequence TRTSGDIGGYNFVS (SEQ ID NO:4) or a variant thereof comprising one, two or three amino acid substitutions with respect to TRTSGDIGGYNFVS (SEQ ID NO:4) v. a CDR2 having a sequence DVNSRPS (SEQ ID NO:5) or a variant thereof comprising one or two amino acid substitutions with respect to DVNSRPS (SEQ ID NO:5); and vi. a CDR3 having a sequence SSYTSRNTV (SEQ ID NO:6) or a variant thereof comprising one or two amino acid substitutions with respect to SSYTSRNTV (SEQ ID NO:6), wherein complementarity determining regions (CDRs) are identified using Chothia numbering. . The payload delivery agent according to paragraph 114, wherein the G at position 2 of CDRH1 is substituted with F or Y; wherein the T at position 3 of CDRH1 is substituted with I or A, wherein the R at position 5 of CDRH1 is substituted with S or T; wherein the S at position 6 of CDRH1 is substituted with A or N; and / or wherein the Y at position 7 of CDRH1 is substituted with S; and / or wherein the N at position 1 of CDRH2 is substituted with I; wherein the P at position 2 of CDRH2 is substituted with S; wherein the I at position 3 of CDRH2 is substituted with A, S or M; wherein the F at position 4 of CDRH2 is substituted with Y or S; wherein the D at position 5 of CDRH2 is substituted with N or G; and / or wherein the T at position 6 of CDRH2 is substituted with G; and / or wherein the T at position 1 of CDRH3 is substituted with G or Y; wherein the I at position 3 of CDRH3 is substituted with W or V; wherein the G at position 4 of CDRH3 is substituted with M; wherein the A at position 5 of CDRH3 is substituted with R, T or K; wherein the F at position 6 of CDRH3 is substituted with T, Y or I; wherein the D at position 7 of CDRH3 is substituted with P or S; and / or wherein the S at position 8 of CDRH3 is substituted with Y; and / or wherein the T at position 1 of CDRL1 is substituted with S; wherein the R at position 2 of CDRL1 is substituted with G; wherein the T at position 3 of CDRL1 is substituted with A, S or G; wherein the S at position 4 of CDRL1 is substituted with T; wherein the G at position 5 of CDRL1 is substituted with Q or S; wherein the D at position 6 of CDRL1 is substituted with S or N; wherein the G at position 8 of CDRL1 is substituted with S; wherein the G at position 9 of CDRL1 is substituted with S; wherein the Y at position 10 of CDRL1 is substituted with R or K; wherein the N at position 11 of CDRL1 is substituted with R or L; wherein the F at position 12 of CDRL1 is substituted with Y, H or N; and / or wherein the S at position 14 of CDRL1 is substituted with F; and / or wherein the D at position 1 of CDRL2 is substituted with A or R; wherein the V at position 2 of CDRL2 is substituted with A, N or G; wherein the N at position 3 of CDRL2 is substituted with S or D; wherein the S at position 4 of CDRL2 is substituted with M or Q; wherein the R at position 5 of CDRL2 is substituted with L; and / or wherein the P at position 6 of CDRL2 is substituted with Q; and / or wherein the S at position 1 of CDRL3 is substituted with Q; wherein the S at position 2 of CDRL3 is substituted with R or G; wherein the Y at position 3 of CDRL3 is substituted with W; wherein the T at position 4 of CDRL3 is substituted with S or V, wherein the S at position 5 of CDRL3 is substituted with T; wherein the R at position 6 of CDRL3 is substituted with P; and / or wherein the N at position 7 of CDRL3 is substituted with Y. . The payload delivery agent according to any of paragraphs 97 to 115, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: i. a CDR1 having a sequence GYIFTSY (SEQ ID NO:7) or a variant thereof comprising one or two amino acid substitutions with respect to GYIFTSY (SEQ ID NO:7); ii. a CDR2 having a sequence SAYNGH (SEQ ID NO:8) or a variant thereof comprising one or two amino acid substitutions with respect to SAYNGH (SEQ ID NO:8); and

[0795] Hi. a CDR3 having a sequence GKISSWFVLED (SEQ ID NO:9) or a variant thereof comprising one, two or three amino acid substitutions with respect to GKISSWFVLED (SEQ ID NO:9); and wherein the light chain comprises: iv. a CDR1 having a sequence SGGTSNIGKNYVS (SEQ ID NQ:10) or a variant thereof comprising one or two or three amino acid substitutions with respect to SGGTSNIGKNYVS (SEQ ID NQ:10) v. a CDR2 having a sequence DNNMRPS (SEQ ID NO:11) or a variant thereof comprising one or two amino acid substitutions with respect to DNNMRPS (SEQ ID NO:11); and vi. a CDR3 having a sequence GMWIRSLSRWV (SEQ ID NO:12) or a variant thereof comprising one, two or three amino acid substitutions with respect to GMWIRSLSRWV (SEQ ID NO:12), wherein complementarity determining regions (CDRs) are identified using Chothia numbering. . The payload delivery agent according to paragraph 116, wherein the Y at position 2 of CDRH1 is substituted with F or G; wherein the I at position 3 of CDRH1 is substituted with T or A, wherein the T at position 5 of CDRH1 is substituted with S or R; wherein the S at position 6 of CDRH1 is substituted with A or N; and / or wherein the Y at position 7 of CDRH1 is substituted with S; wherein the S at position 1 of CDRH2 is substituted with P; wherein the A at position 2 of CDRH2 is substituted with S, I or M; wherein the Y at position 3 of CDRH2 is substituted with F or S; wherein the N at position 4 of CDRH2 is substituted with D or G; wherein the G at position 5 of CDRH2 is substituted with T; and / or wherein the H at position 6 of CDRH2 is substituted with N; wherein the G at position 1 of CDRH3 is substituted with M; wherein the K at position 2 of CDRH3 is substituted with R, T or A; wherein the I at position 3 of CDRH3 is substituted with T, Y or F; wherein the S at position 4 of CDRH3 is substituted with P or D; wherein the S at position 5 of CDRH3 is substituted with Y; wherein the W at position 6 of CDRH3 is substituted with Y or A; wherein the F at position 7 of CDRH3 is substituted with Y; wherein the L at position 9 of CDRH3 is substituted with M; and / or wherein the D at position 11 of CDRH3 is substituted with V; wherein the S at position 1 of CDRL1 is substituted with T; wherein the G at position 2 of CDRL1 is substituted with R; wherein the G at position 3 of CDRL1 is substituted with A, S or T; wherein the T at position 4 of CDRL1 is substituted with S; wherein the S at position 5 of CDRL1 is substituted with Q or G; wherein the N at position 6 of CDRL1 is substituted with S or D; wherein the G at position 8 of CDRL1 is substituted with S; wherein the K at position 9 of CDRL1 is substituted with R or Y; wherein the N at position 10 of CDRL1 is substituted with R or L; wherein the Y at position 11 of CDRL1 is substituted with H, F or N; and / or wherein the S at position 13 of CDRL1 is substituted with F; wherein the D at position 1 of CDRL2 is substituted with A or R; wherein the N at position 2 of CDRL2 is substituted with A, V or G; wherein the N at position 3 of CDRL2 is substituted with S or D; wherein the M at position 4 of CDRL2 is substituted with S or Q; wherein the R at position 5 of CDRL2 is substituted with L; and / or wherein the P at position 6 of CDRL2 is substituted with Q; and / or wherein the G at position 1 of CDRL3 is substituted with A; wherein the M at position 2 of CDRL3 is substituted with T; wherein the I at position 4 of CDRL3 is substituted with D; wherein the R at position 5 of CDRL3 is substituted with D; wherein the S at position 6 of CDRL3 is substituted with G; wherein the L at position 7 of CDRL3 is substituted with Q; wherein the S at position 8 of CDRL3 is substituted with Q; wherein the R at position 9 of CDRL3 is substituted with S or G; wherein the W at position 10 of CDRL3 is substituted with Y; and / or wherein the V at position 11 of CDRL3 is substituted with S or T. . The payload delivery agent according to any of paragraphs 97 to 117, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: i. a CDR1 having a sequence GFTFSAY (SEQ ID NO:13) or a variant thereof comprising one or two amino acid substitutions with respect to GFTFSAY (SEQ ID NO:13); ii. a CDR2 having a sequence SYDGNN (SEQ ID NO:14) or a variant thereof comprising one or two amino acid substitutions with respect to SYDGNN (SEQ ID NO:14); and

[0796] Hi. a CDR3 having a sequence DFRDYVWGTYPSAY (SEQ ID NO:15) or a variant thereof comprising one, two or three amino acid substitutions with respect to DFRDYVWGTYPSAY (SEQ ID NO:15); iv. and wherein the light chain comprises: v. a CDR1 having a sequence SGSSSNIGRRHVF (SEQ ID NO:16) or a variant thereof comprising one, two or three amino acid substitutions with respect to SGSSSNIGRRHVF (SEQ ID NO:16) vi. a CDR2 having a sequence RGDQRPS (SEQ ID NO:17) or a variant thereof comprising one or two amino acid substitutions with respect to RGDQRPS (SEQ ID NO:17); and vii. a CDR3 having a sequence ATWDDGLSGYV (SEQ ID NO:18) or a variant thereof comprising one, two or three amino acid substitutions with respect to ATWDDGLSGYV (SEQ ID NO:18), wherein complementarity determining regions (CDRs) are identified using Chothia numbering. . The payload delivery agent according to paragraph 118, wherein the F at position 2 of CDRH1 is substituted with G or Y; wherein the T at position 3 of CDRH1 is substituted with I or A, wherein the S at position 5 of CDRH1 is substituted with R or T; wherein the A at position 6 of CDRH1 is substituted with S or N; and / or wherein the Y at position 7 of CDRH1 is substituted with S; and / or wherein the S at position 1 of CDRH2 is substituted with A, I or M; wherein the Y at position 2 of CDRH2 is substituted with F or S; wherein the D at position 3 of CDRH2 is substituted with N or G; wherein the G at position 4 of CDRH2 is substituted with T; and / or wherein the N at position 5 of CDRH2 is substituted with H; wherein the D at position 1 of CDRH3 is substituted with K; wherein the F position 2 of CDRH3 is substituted with E; wherein the D at position 4 of CDRH3 is substituted with T; wherein the Y at position 5 of CDRH3 is substituted with G or T; wherein the W at position 7 of CDRH3 is substituted with I or V; wherein the G at position 8 of CDRH3 is substituted with M; wherein the T at position 9 of CDRH3 is substituted with R, A or K; wherein the Y at position 10 of CDRH3 is substituted with T, F or I; wherein the P at position 11 of CDRH3 is substituted with D or S; wherein the S at position 12 of CDRH3 is substituted with Y; wherein the A at position 13 of CDRH3 is substituted with Y or W; and / or wherein the Y at position 14 of CDRH3 is substituted with F; and / or wherein the S at position 1 of CDRL1 is substituted with T; wherein the G at position 2 of CDRL1 is substituted with R; wherein the S at position 3 of CDRL1 is substituted with A, T or G; wherein the S at position 4 of CDRL1 is substituted with T; wherein the S at position 5 of CDRL1 is substituted with Q or G; wherein the N at position 6 of CDRL1 is substituted with S or D; wherein the G at position 8 of CDRL1 is substituted with S; wherein the R at position 9 of CDRL1 is substituted with Y or K; wherein the R at position 10 of CDRL1 is substituted with L or N; wherein the H at position 11 of CDRL1 is substituted with F, N or Y; and / or wherein the F at position 13 of CDRL1 is substituted with S; and / or wherein the R at position 1 of CDRL2 is substituted with A or D; wherein the G at position 2 of CDRL2 is substituted with A, N or V; wherein the D at position 3 of CDRL2 is substituted with S or N; wherein the Q at position 4 of CDRL2 is substituted with S or M; wherein the R at position 5 of CDRL2 is substituted with L; and / or wherein the P at position 6 of CDRL2 is substituted with Q; and / or wherein the A at position 1 of CDRL3 is substituted with G; wherein the T at position 2 of CDRL3 is substituted with M; wherein the D at position 4 of CDRL3 is substituted with I; wherein the D at position 5 of CDRL3 is substituted with R; wherein the G at position 6 of CDRL3 is substituted with S; wherein the L at position 7 of CDRL3 is substituted with Q; wherein the S at position 8 of CDRL3 is substituted with Q; wherein the G at position 9 of CDRL3 is substituted with S or R; wherein the Y at position 10 of CDRL3 is substituted with W; and / or wherein the V at position 11 of CDRL3 is substituted with S or T. . The payload delivery agent according to any of paragraphs 97 to 119, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: i. a CDR1 having a sequence GGAFRNS (SEQ ID NO:19) or a variant thereof comprising one or two amino acid substitutions with respect to GGAFRNS (SEQ ID NO:19); ii. a CDR2 having a sequence IPMSGT (SEQ ID NQ:20) or a variant thereof comprising one or two amino acid substitutions with respect to IPMSGT (SEQ ID NQ:20); and iii. a CDR3 having a sequence EKERTFGVVMRTSYYYVMEV (SEQ ID NO:21) or a variant thereof comprising one, two, three or four amino acid substitutions with respect to EKERTFGVVMRTSYYYVMEV (SEQ ID NO:21); and wherein the light chain comprises: iv. a CDR1 having a sequence RASQSISSYLN (SEQ ID NO:22) or a variant thereof comprising one or two amino acid substitutions with respect to RASQSISSYLN (SEQ ID NO:22) v. a CDR2 having a sequence AASSLQS (SEQ ID NO:23) or a variant thereof comprising one or two amino acid substitutions with respect to AASSLQS (SEQ ID NO:23); and vi. a CDR3 having a sequence QQSYSTPYT (SEQ ID NO:24) or a variant thereof comprising one or two amino acid substitutions with respect to QQSYSTPYT (SEQ ID NO:24), wherein complementarity determining regions (CDRs) are identified using Chothia numbering. . The payload delivery agent according to paragraph 120, wherein the G at position 2 of CDRH1 is substituted with F or Y; wherein the A at position 3 of CDRH1 is substituted with T or I; wherein the R at position 5 of CDRH1 is substituted with S or T; wherein the N at position 6 of CDRH1 is substituted with A or S; and / or wherein the S at position 7 of CDRH1 is substituted with Y; and / or wherein the I at position 1 of CDRH2 is substituted with N; wherein the P at position 2 of CDRH2 is substituted with S; wherein the M at position 3 of CDRH2 is substituted with I, A or S; wherein the S at position 4 of CDRH2 is substituted with F or Y; wherein the G at position 5 of CDRH2 is substituted with D or N; and / or wherein the T at position 6 of CDRH2 is substituted with G; and / or wherein the K at position 2 of CDRH3 is substituted with D; wherein the E at position 3 of CDRH3 is substituted with F; wherein the T at position 5 of CDRH3 is substituted with D; wherein the G at position 7 of CDRH3 is substituted with Y or T; wherein the V at position 9 of CDRH3 is substituted with W or I; wherein the M at position 10 of CDRH3 is substituted with G; wherein the R at position 11 of CDRH3 is substituted with A, T or K; wherein the T at position 12 of CDRH3 is substituted with F, Y or I; wherein the S at position 13 of CDRH3 is substituted with P or D; wherein the Y at position 14 of CDRH3 is substituted with S; wherein the Y at position 15 of CDRH3 is substituted with W or A; wherein the Y at position 16 of CDRH3 is substituted with F; wherein the M at position 18 of CDRH3 is substituted with L; and / or wherein the V at position 20 of CDRH3 is substituted with D; and / or wherein the R at position 1 of CDRL1 is substituted with G; wherein the A at position 2 of CDRL1 is substituted with S, T or G; wherein the S at position 3 of CDRL1 is substituted with T; wherein the Q at position 4 of CDRL1 is substituted with S or G; wherein the S at position 5 of CDRL1 is substituted with D or N; wherein the S at position 7 of CDRL1 is substituted with G; wherein the S at position 8 of CDRL1 is substituted with G; wherein the Y at position 9 of CDRL1 is substituted with R or K; wherein the L at position 10 of CDRL1 is substituted with N or R; and / or wherein the N at position 11 of CDRL1 is substituted with F, H or Y; and / or wherein the A at position 1 of CDRL2 is substituted with D or R; wherein the A at position 2 of CDRL2 is substituted with V, N or G; wherein the S at position 3 of CDRL2 is substituted with N or D; wherein the S at position 4 of CDRL2 is substituted with M or Q; wherein the L at position 5 of CDRL2 is substituted with R; and / or wherein the Q at position 6 of CDRL2 is substituted with P; and / or wherein the Q at position 1 of CDRL3 is substituted with L; wherein the Q at position 2 of CDRL3 is substituted with S; wherein the S at position 3 of CDRL3 is substituted with R or G; wherein the Y at position 4 of CDRL3 is substituted with W; wherein the S at position 5 of CDRL3 is substituted with T or V; wherein the T at position 6 of CDRL3 is substituted with S; wherein the P at position 7 of CDRL3 is substituted with R; and / or wherein the Y at position 8 of CDRL3 is substituted with N.

[0797] 122. The payload delivery agent according to any of paragraphs 114 to 121 , wherein said one or more substitutions are conservative substitutions.

[0798] 123. The pay load delivery agent according to any one of paragraphs 88 to 122, having at least 3-fold specificity for binding necrotic cells over live cells, as measured by FACS or flow cytometry.

[0799] 124. The payload delivery agent according to paragraph 123, wherein the necrotic cell binding is not inhibited by the presence of G-actin.

[0800] 125. The pay load delivery agent according to any one of paragraphs 88 to 124, for use in medicine.

[0801] 126. The pay load delivery agent according to any one of paragraphs 88 to 124, for use in a method of treating cancer, the method comprising administering the radiolabelled binding agent to a subject that has cancer.

[0802] 127. The payload delivery agent according to paragraph 126, wherein the cancer comprises a solid tumour.

[0803] 128. The pay load delivery agent for the use according to any one of paragraphs 126 or 127, wherein the cancer is characterised by F-actin presentation on necrotic cancer cells.

[0804] 129. The pay load delivery agent for the use according to any one of paragraphs 126 to 128, wherein the method of treating cancer comprises killing cancer cells via necrosis.

[0805] 130. The pay load delivery agent for the use according to paragraph 129, wherein the method comprises contacting the necrotic cancer cells with the payload delivery agent.

[0806] 131 . The pay load delivery agent for the use according to any one of paragraphs 126 to 130, wherein the method of treating cancer comprises (i) administering a therapy to the subject to cause necrosis of the cancer cells, and then (ii) administering the pay load delivery agent to the subject and thereby contacting the necrotic cancer cells with the payload delivery agent.

[0807] 132. The payload delivery agent for the use according to paragraph 131 , wherein the therapy administered to the subject is selected from the group comprising: an F-actin binding agent, a chemotherapeutic agent or a radiotherapy. 133. The pay load delivery agent for the use according to any one of paragraphs 126 to 132, wherein the use comprises administration of the payload delivery agent in combination with an additional anticancer therapy.

[0808] 134. The payload delivery agent for the use according to paragraph 133, wherein the additional anticancer therapy is an immune checkpoint inhibitor.

[0809] 135. The payload delivery agent for the use according to paragraph 134, wherein the immune checkpoint inhibitor is an anti-PD1 monoclonal antibody.

[0810] 136. The pay load delivery agent for the use according to any one of paragraphs 126 to 135, wherein the additional anticancer therapy is a radiotherapy.

[0811] 137. The pay load delivery agent for the use according to any one of paragraphs 126 to 136, wherein the use comprises administration of the payload delivery agent in combination with a chemotherapeutic agent.

[0812] 138. The pay load delivery agent for the use according to any one of any one of paragraphs 126 to 137, wherein the use comprises co-administration of the payload delivery agent with an anti-F-actin antibody that is not radiolabelled.

[0813] 139. A method of selecting an antibody or antigen-binding fragment thereof that specifically binds to F- actin, the method comprising: i. providing a population of candidate antibodies, ii. contacting a binding substrate comprising F-actin with the population of candidate antibodies, Hi. contacting the binding substrate with trypsin or a competitor agent comprising the CTLD of

[0814] DNGR-1 to displace one or more lead antibodies from the binding substrate, then iv. selecting an antibody from said one or more lead antibodies; and then v. conjugating the selected antibody with a cytotoxic agent.

[0815] 140. The method according to paragraph 139, wherein the method comprises screening said one or more lead antibodies for 10-fold specificity for F-actin over G-actin, wherein the selected antibody has said 10-fold specificity.

[0816] 141. The method according to any one of paragraphs 139 or 140, wherein the method comprises screening said one or more lead antibodies for at least 3-fold specificity for binding necrotic cells over live cells, as measured by FACS or flow cytometry, wherein the selected antibody has said 3-fold specificity. . A method of producing a payload delivery agent according to any one of paragraphs 86 to 124, the method comprising: i. obtaining an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and ii. conjugating the F-actin binding moiety with a cytotoxic agent.

[0817] Examples

[0818] MATERIALS & METHODS

[0819] Mice

[0820] C57BL / 6Jax mice were bred at the Francis Crick Institute under specific-pathogen-free conditions.

[0821] Female mice were used at 6-8 weeks of age for in vivo tumour experiments. All animal experiments were performed in accordance with national and institutional guidelines for animal care and were approved by the Francis Crick Institute Biological Resources Facility Strategic Oversight Committee (incorporating the Animal Welfare and Ethical Review Body) and by the Home Office, United Kingdom.

[0822] Primary cells and cell lines

[0823] RPMI 1640 supplemented with 2 mM glutamine, 100 units / ml penicillin, 100 pg / ml streptomycin, non- essential amino acids, 10 mM HEPES, 50 pM 2-mercaptoethanol (all from Gibco) and 10% heat- inactivated fetal calf serum (FCS) (R10+medium) was used for all cell culture unless otherwise stated. Braf / sooE melanoma was a kind gift from George Kassiotis. MCA205 fibrosarcoma was obtained from the Francis Crick Institute Cell Services Science Technology Platform. MCA205 LA-OVA was generated previously (Giampazolias, 2021).

[0824] Fc-DNGR-1 fusion proteins

[0825] Fc-DNGR-1 variant fusion proteins were generated at the Francis Crick Institute (small scale) or contracted commercially (large scale at ImmunoPrecise, Netherlands). The 2WA Fc-DNGR-1 variant fusion protein contains 2 mutations in the CTLD DNGR-1 domain that prevent binding to F-actin (see WO 2023 / 203198). Briefly, WT and 2WA mouse DNGR-1 isoform 4 (long) ECD DNA sequences were amplified from existing pFB neo plasmids using Infusion-designed primers (Sigma Aldrich). Amplified pFUSEN-mG2aFc plasmid (InvivoGen) was linearised using Nhel and EcoRV restriction enzymes, gel purified, and subjected to Infusion reaction (Takara) with amplified 2WA or WT mouse DNGR-1 ECD DNA sequences according to manufacturer’s instructions. Plasmids were transformed into Stellar competent cells and subjected to overnight selection on zeocin agar plates. Single colonies were cloned, sequenced, and used for downstream expression. For mouse lgG2a N297A Fc mutagenesis, sequence- verified WT DNGR-1 -Fc was subjected to Quickchange Lightning Site-directed Mutagenesis Kit (Agilent Technologies) according to manufacturer’s instructions, using designed primers spanning mutation site (Sigma). Plasmids were transformed into XL10 Gold bacteria (Agilent Technologies) and grown overnight on LB agar plates, single colonies grown in zeocin-containing LB broth, plasmid DNA extracted using QIAprep Spin Miniprep Kit (QIAGEN), and sequenced.

[0826] Amplified sequence-verified plasmids were transiently transfected into Expi293F cells and supernatants harvested over several days. Fc-DNGR-1 fusion proteins were purified using protein A beads (Generon; M1300-5) following Pierce gentle Ag / Ab Binding and Elution Buffer Kit (Thermo Fisher Scientific, 21030) protocol. Proteins were then dialysed in endotoxin-free 25mM Tris pH7.2, 150mM NaCI (BupH Tris Buffered Saline Packs, Thermo Fisher Scientific). Samples were tested for endotoxin (Pierce Chromogenic Endotoxin Quant Kit-60 reactions, Thermo Fisher Scientific) and confirmed to be < 0.05 EU / ml. Non-reducing SDS-PAGE was performed on purified proteins by heating to 95°C for 5 min in Laemmli buffer and running on 7.5 % mini-PROTEAN TGX pre-cast gels (Bio-rad) with Precision Plus Dual Color Standards. Gels were developed with Coomassie Brilliant Blue R-250 Staining Solution (Biorad) and analysed on ImageQuant 800 (Amersham) imaging system. Large-scale production of Fc- DNGR-1 was contracted commercially (ImmunoPrecise (Netherlands) and FairJourney Biologies (Portugal)).

[0827] Anti-F-actin IciG

[0828] Candidate anti-F-actin lgG2a antibody was identified previously from a phage display library selected for preferential binding to F-actin over G-actin. The candidate clone was selected based on high relative affinity for F- over G-actin, low non-specific protein binding, and high necrotic cell binding (human and mouse) (pending patent application: GB2401381 .5). Large-scale production of this antibody was contracted commercially (ImmunoPrecise (Netherlands)).

[0829] Tumour spheroids and in vivo tumour experiments

[0830] For tumour spheroids (tumouroids), 5 x 103Braf'7600Emelanoma cells were seeded into Corning Matrigel Basement Membrane Matrix (VWR) in R10+medium and cultured for 5 days with constant agitation. Tumouroids were dissociated from Matrigel using Corning Cell Recovery Solution (Fisher Scientific) and incubated with 10 pg / ml Fc-DNGR-1 , Alexa Fluor 555 phalloidin (1 :400, Fisher Scientific), or 2 pg / ml DAPI for 24 h in R10+medium. Tumouroids were washed in DPBS for 1 h, fixed in 4% PFA for 1 h, then incubated in 30% sucrose overnight.

[0831] For MCA205-LifeAct (LA)-OVA-mCherry tumours and Fc-DNGR-1 injection, 2.5 x 106tumour cells were injected into the shaved flanks of C57BL / 6 mice. After 7 days, Fc-DNGR-1 was administered via peri- tumoural injection (50 pg) in DPBS and tumours harvested after 6-24 h.

[0832] For MCA205 tumours and anti-F-actin IgG injection, 0.5 x 106tumour cells were injected as above and anti-F-actin lgG2a or irrelevant lgG2a was injected intra-peritoneally (i.p.) (400 pg) every 3-5 days between days 0-18. Mice were additionally treated with 1.25 mg / kg doxorubicin (Merck Life Science UK) on day 6 via intra-tumoural (i.t.) injection. Tumours were harvested on day 21 (72 h post final injection). All tumours were fixed in 4% PFA overnight at 4°C, followed by incubation in 30% sucrose overnight at 4°C.

[0833] Microscopy

[0834] Tumouroids and tumours were embedded in O.C.T. (Tissue-Tek), sectioned (10-30 pm) using a cryostat (Leica), mounted onto SuperFrost Plus glass slides (Thermo Fisher Scientific), and stored at -80°C. Sections were thawed to RT, rehydrated in DPBS for 10 min, and blocked for 1 h in DPBS containing 0.3% Triton-X and 2-5% normal goat serum or 3% BSA (blocking buffer). Sections were stained (all at 1 :400 in blocking buffer, unless otherwise stated) with a combination of goat anti-mouse lgG2a-AF647 (Thermo Fisher Scientific), AF555-conjugated phalloidin (Fisher Scientific), DAPI and Hoechst (Thermo Fisher Scientific) for 1 h at RT. Slides were washed and mounted using Prolong Diamond Antifade Mountant (Fisher Scientific). Samples were imaged on a Zeiss LSM880 inverted confocal microscope.

[0835] Tile scans and Z-stacks of tumour sections were stitched using Fiji and z-projected using maximal projection. Based on tumour or Hoechst signal, non-tumour tissues were cropped out and a Gaussian blur filter (1 pm) applied. The channels were split and that corresponding to lgG2a was analysed. The image was binarized to keep the signal higher than a set threshold and particles were analysed to measure the area positive for the signal. The particles were summed up per section.

[0836] All statistical analyses were performed using GraphPad Prism software version 8. Statistical significance between two groups was determined using a paired or unpaired two-tailed Student’s t test. Data is plotted as mean ± SEM. The following scheme was used to represent statistical significance: *P < 0.05; **P < 0.01 ; ***P < 0.001 , ****P < 0.0001 .

[0837] As described above, the present invention is based on the hypothesis that F-actin is primarily exposed in vivo as a result of necrosis (i.e. pathological cell death). The F-actin may be exposed on necrotic cell debris following necrotic cell death, such as in the necrotic core of tumours.

[0838] The inventors have previously described the generation of an Fc-DNGR-1 fusion protein that specifically binds to F-actin (WO 2023 / 203198) as well as a mutant fusion protein (2WA Fc-DNGR-1) that does not bind to F-actin. This fusion protein was used as a representative of other actin-binding agents described herein, and the tumour penetration ability of this fusion protein was investigated.

[0839] To determine whether the Fc-DNGR-1 fusion protein is able to penetrate tumours and bind to necrotic cells within the core, tumour spheroids were cultured with either 2WA or WT Fc-DNGR-1 . As expected, no binding to F-actin was seen with the 2WA mutant (Figure 3A, left). In contrast, the WT Fc-DNGR-1 fusion protein localised to F-actin within the tumour (Figure 3A, right, depicted by arrows).

[0840] To further characterise this finding, confocal microscopy was performed on tumour spheroids cultured in vitro with intracellular binding reagents. DAPI was used for nuclear staining and phalloidin to stain the actin cytoskeleton. The WT Fc-DNGR-1 fusion protein is able to penetrate beyond the periphery of the tumour spheroid and localise to the necrotic core of the tumour (Figure 3B-D).

[0841] This Example provides proof-of-concept that an actin-binding agent can escape the affinity barrier that hinders standard radioimmunoconjugates and reach its target (F-actin) in the core of the tumour. In turn, this provides proof-of-concept that F-actin binding agents are able to identify necrotic cells. This indicates that the radiolabelled F-actin binding agents of the invention are suitable for diagnostic imaging purposes.

[0842] The data described above establish that the F-actin binding agent is able to overcome the affinity barrier. This indicates that the radiolabelled F-actin binding agents described herein will be useful therapeutically because they are able to deliver a radionuclide to sites of necrosis (e.g. a tumour).

[0843] EXAMPLE 2 - Fc-DNGR-1 penetrates tumours in vivo via peri-tumoural administration

[0844] After establishing that the F-actin binding agent Fc-DNGR-1 is able to penetrate to the necrotic core of tumours in vitro, the inventors sought to determine if this property was also possible in vivo.

[0845] To this end, C57BL / 6 mice were injected with MCA205 LA-OVA-mCherry tumour cells as described above. After 7 days, either 2WA or WT Fc-DNGR-1 fusion protein was administered via peri-tumoral injection. Tumours were harvested and Fc-DNGR-1 was detected by anti-lgG2a staining. A schematic of the experiment is shown in Figure 4A.

[0846] As expected, there was limited binding of the 2WA mutant Fc-DNGR-1 fusion protein to the tumour (Figure 4B, top and 4C). In contrast, the WT Fc-DNGR-1 fusion protein could penetrate the tumour periphery and bind to the F-actin exposed in the necrotic core (Figure 4B, bottom and 4C).

[0847] EXAMPLE 3 - Anti-F-actin antibody penetrates tumours in vivo via systemic administration

[0848] This experiment was to determine the level of tumour penetration of an anti-F-actin antibody following systemic administration.

[0849] As described above, mice were injected with MCA205 tumour cells. Every 3-5 days either 400 pg irrelevant lgG2a antibody (control) or 400 pg anti-F-actin lgG2a antibody were injected via intra-peritoneal injection. A schematic of the experiment is shown in Figure 5A.

[0850] Tumours were harvested on day 21 and F-actin was detected via anti-lgG2a staining. The irrelevant antibody demonstrated limited binding (Figure 5B, top and Figure 5C), whereas the anti-F-actin lgG2a antibody was able to bind F-actin within the tumour (Figure 5B, bottom, encircled areas and Figure 5C).

[0851] Anti-F-actin Antibodies Examples

[0852] The following examples demonstrate the generation of a class of antibodies that bind to F-actin with high affinity, avidity, and / or specificity. It is noted that the antibodies of the present invention are in no way limited to the antibodies described below. MATERIALS & METHODS

[0853] Mice

[0854] C57BL / 6Jax and OT-\ / RagT!- mice were bred at the Francis Crick Institute under specific-pathogen-free conditions. Female mice were used at 6-8 weeks of age for in vivo tumour experiments. All animal experiments were performed in accordance with national and institutional guidelines for animal care and were approved by the Francis Crick Institute Biological Resources Facility Strategic Oversight Committee (incorporating the Animal Welfare and Ethical Review Body) and by the Home Office, United Kingdom.

[0855] Primary cells and cell lines

[0856] RPMI 1640 supplemented with 2 mM glutamine, 100 units / ml penicillin, 100 pg / ml streptomycin, non- essential amino acids, 10 mM HEPES, 50 pM 2-mercaptoethanol (all from Gibco) and 10 % heat- inactivated fetal calf serum (FCS) (R10+ medium) was used for all cell culture. Braf'7600E5555 melanoma was a kind gift from George Kassiotis. MCA205 fibrosarcoma cell line and human HEK293T cell line were obtained from the Francis Crick Institute Cell Services Science Technology Platform. HEK293T cells were maintained in DMEM containing 2 mM glutamine, 100 U / ml penicillin, 100 pg / ml streptomycin, and 10 % heat-inactivated FCS.

[0857] For FLT3L-cDCs, bone marrow was extracted from hind legs of mice and subjected to red blood cell lysis (Thermo Fisher Scientific). Cells were cultured for 9 days in R10+ medium containing 150 ng / ml recombinant mouse FLT3L (R&D systems). On day 8, FTL3L eDC cultures were additionally primed with 200 ng / ml IFNa (R&D systems). cDC1 and non-cDC1 were separated using biotinylated anti-mouse XCR- 1 IgG (Biolegend, clone ZET), Anti-Biotin MicroBeads, and LS Columns (both Miltenyi) according to manufacturer’s instructions.

[0858] For Hoxb8-cDC2 generation, CDP were immortalized using the ER-Hoxb8 system. Briefly, bone marrow CDP (Lin-CD1 17-CD115+FLT3+DNGR-1 +) were isolated by FACS and co-cultured with congenic bone marrow cells in R10+ medium with 20 ng / ml IL-3, IL-6, and SCF. The cells were transduced the following day with retrovirus containing MSCV-Neo-HA-ER-Hoxb8 (obtained from David Sykes, Harvard, Cambridge, MA) and selectively expanded with 1 mg / ml G418 in R10+ supplemented with conditioned media generated from CHO-FLT3L-producing cells (final concentration 75 ng / ml FLT3L) and 0.5 pM E2 (p-oestradiol, Sigma Aldrich). Immortalised CDP were separated from congenic bone marrow progenitors via FACS. To differentiate eDC in vitro, Hoxb8 CDP were washed two times with Dulbecco’s PBS (DPBS) and cultured in R10+ with 75 ng / ml recombinant mouse FLT3L (R&D systems) for 5-7 days to generate cDC2.

[0859] For pre-activated effector OT-I cultures, single cell suspensions were generated from spleen and lymph nodes of OT-l / RagTAmice and subjected to red blood cell lysis. Cells were cultured in R10+ medium supplemented with 100 U / ml IL-2 (Peprotech) and 0.1 nM SIINFEKL (SEQ ID NO:190) (generated at the Francis Crick Institute) for 3 days. On days 3 and 4, cells were split 1 :2 and culture medium completely replaced with R10+ medium containing 100 U / ml IL-2. OT-I cultures were used on day 5. Fc-DNGR-1 fusion

[0860] Fc-DNGR-1 variant fusion proteins were generated at the Francis Crick Institute (small scale) or contracted commercially (large scale at ImmunoPrecise, Netherlands). Briefly, WT and 2WA mouse DNGR-1 isoform 4 (long) ECD DNA sequences were amplified from existing pFB neo plasmids using Infusion-designed primers (Sigma Aldrich). Amplified pFUSEN-mG2aFc plasmid (InvivoGen) was linearised using Nhel and EcoRV restriction enzymes, gel purified, and subjected to Infusion reaction (Takara) with amplified 2WA or WT mouse DNGR-1 ECD DNA sequences according to manufacturer’s instructions. Plasmids were transformed into Stellar competent cells and subjected to overnight selection on zeocin agar plates. Single colonies were cloned, sequenced, and used for downstream expression. Plasmids were transformed into XL10 Gold bacteria (Agilent Technologies) and grown overnight on LB agar plates, single colonies grown in zeocin-containing LB broth, plasmid DNA extracted using QIAprep Spin Miniprep Kit (QIAGEN), and sequenced.

[0861] Amplified sequence-verified plasmids were transiently transfected into Expi293F cells and supernatants harvested over several days. Fc-DNGR-1 fusion proteins were purified using protein A beads (Generon; M1300-5) following Pierce gentle Ag / Ab Binding and Elution Buffer Kit (Thermo Fisher Scientific, 21030) protocol. Proteins were then dialysed in endotoxin-free 25mM Tris pH7.2, 150mM NaCI (BupH Tris Buffered Saline Packs, Thermo Fisher Scientific). Samples were tested for endotoxin (Pierce Chromogenic Endotoxin Quant Kit-60 reactions, Thermo Fisher Scientific) and confirmed to be < 0.05 EU / ml. Non-reducing SDS-PAGE was performed on purified proteins by heating to 95°C for 5 min in Laemmli buffer and running on 7.5 % mini-PROTEAN TGX pre-cast gels (Bio-rad) with Precision Plus Dual Color Standards. Gels were developed with Coomassie Brilliant Blue R-250 Staining Solution (Biorad) and analysed on ImageQuant 800 (Amersham) imaging system. Large-scale production of Fc- DNGR-1 was contracted commercially (ImmunoPrecise (Netherlands) and FairJourney Biologies (Portugal)).

[0862] Binding and F-actin specificity of selected mAbs was measured by ELISA using immobilised F- and G- actin. Immunoplates (MaxiSorp 96-well, Thermo Fisher Scientific) were coated with 5 pg / ml NeutrAvidinTM (Thermo Fisher Scientific) in PBS overnight at 4°C, washed, blocked with 2.5% BSA in PBS for 2h at room temperature and stored overnight at 4°C after addition of biotinylated F- or G-actin (2 nM). Capture of biotinylated F- and G-actin was verified using a commercial actin-specific mAb (AC-40, Sigma) that recognises both, F- and G-actin. To prevent the depolymerisation of F-actin or the polymerisation of G-actin during the assay, F- and G-actin were stabilised by adding phalloidin or cytochalasin D, respectively, during the preparation of the actin stocks. After washing to remove unbound actin, three-fold serial dilutions of mAbs or control reagent (DNGR-1 -Fc fusion) starting at 300nM (unless stated otherwise) were added to the ELISA plate for 1 hr. Actin-bound mAbs or control reagent were detected using a horseradish peroxidase (HRP)-conjugated mouse-specific anti-IgG (Jackson ImmunoResearch) antibody (0.16 pg / ml) and developed with an ELISA substrate for HRP based detection (TMB, eBioscience). Substrate development was stopped by adding sulphuric acid (H2SO4, Fisher). Titration curves were plotted using GraphPad Prism7 and EC50 values for each mAb were calculated by applying a nonlinear regression (curve fit) of a log (agonist) vs response - variable slope (four parameters).

[0863] Bio-F-actin:

[0864] Mix 20 pl Bio-G-actin (1 mg / ml, i.e. 20 pM, Cytoskeleton Inc.) with 20 pl non-biotinylated G-actin (1 mg / ml, i.e. 20 pM, Cytoskeleton Inc.), 5 pl F-buffer (10 x polymerisation buffer, Cytoskeleton Inc) and 5 pl phalloidin (200 pM, Thermo Fisher Scientific) and incubate mixture for at least 1 hr at room temperature. Dilute mixture with 0.75 ml PBS and pellet polymerised F-actin by ultra-centrifugation at 150.000g for 1 hr at 4°C. Discard supernatant, resuspend pellet in 0.8 ml PBS to generate F-actin stock (1 pM) and store at 4°C (do not freeze) for up to 4 weeks.

[0865] Bio-G-actin:

[0866] Mix 2 x 20pl Bio-G-actin (1 mg / ml, i.e. 20 pM, Cytoskeleton Inc.) with 5 pl G-buffer (General actin buffer buffer, Cytoskeleton Inc) and 5 pl Cytochalasin D (200 pM, Thermo Fisher Scientific). Dilute mixture immediately with 0.75 ml G-buffer and remove contaminating F-actin by ultra-centrifuging the solution at 150.000g for 1 hr at 4°C. Carefully remove the supernatant (< 0.8 ml), which represents the G-actin stock (1 pM), and store at 4°C (do not freeze) for up to 4 weeks.

[0867] Commercial anti-actin antibodies

[0868] Purified commercial anti-actin antibodies of the mouse lgG2a isotype that bound to either beta-actin or pan-actin were identified. Pan-actin-binding AC-40 was obtained from Abeam (ab11003). Beta-actin- specific AC-74 was obtained from Sigma Aldrich (A2228).

[0869] Necrotic cells were generated by irradiating mouse BRAFV600Emutant 5555 melanoma cells or human HEK293T cells with 240 mJ / cm2UVC in DPBS followed by culture overnight in R0 medium (lacking 10 % FCS). Necrotic cells were incubated with molar equivalent concentrations of Fc-DNGR-1 , anti-F-actin lgG2a, or commercial anti-actin lgG2a antibodies for 0.5-1 h in DPBS. In some instances, soluble cytochalasin D-stabilised human platelet G-actin (Cytoskeleton) was added simultaneously to assess binding inhibition of non-F-actin-specific antibodies. Samples were washed 2X in DPBS and incubated with secondary AF488-conjugated anti-mouse lgG2a (Thermo Fisher Scientific, 1 :400) at 4°C for 30 min. Samples were washed and resuspended in DPBS before acquisition on an LSRFortessa or FACSymphony (BD Biosciences). Data was analysed using FlowJo software version 10. ELISA

[0870] The ability of selected anti-F-actin mAbs to compete with soluble DNGR-1 receptor for binding to F-actin was measured by ELISA using immobilised F-actin. Briefly, Immunoplates (MaxiSorp 96-well, Thermo Fisher Scientific) were coated with 5 pg / ml NeutrAvidin™ (Thermo Fisher Scientific) in PBS overnight at 4°C, washed, blocked with 2.5% BSA in PBS for 2h at room temperature and stored overnight at 4°C after addition of biotinylated F-actin (100 nM). To prevent the depolymerisation of F-actin during the assay, F-actin was stabilised by adding phalloidin during the preparation of the F-actin stock. After washing to remove unbound F-actin, ten-fold serial dilutions of anti-F-actin mAbs, irrelevant antibody (negative control) and DNGR-1 -Fc fusion (positive control) starting at 100nM were mixed with 10nM FLAG-tagged DNGR-1 (DNGR-1-FLAG) and added to the ELISA plate for 1 hr. Binding of DNGR-1-FLAG was detected using an alkaline phosphatase (AP)-conjugated FLAG-specific antibody (Sigma, clone M2) and developed with an ELISA substrate for AP based detection (pNitrophosphate, Sigma). Plates were read using an ELISA plate reader at 405nm to determine optical densities (OD) for each sample.

[0871] Inhibition curves were calculated according to the formula (ODmax - ODcompetitor) / (ODmax - ODmin) x100%, where ODmax and ODmin are defined as OD values of DNGR-1 -FLAG binding in the absence of competitor and assay background in the absence of DNGR-1 -FLAG, respectively, and plotted using GraphPad PrismI O.

[0872] In vivo tumour experiments

[0873] MCA205 tumour cells were dissociated with trypsin (0.25 %) and washed 3X in PBS. The final cell pellet was resuspended and diluted in endotoxin-free PBS (0.5 x 106cells per 100 pl) and injected s.c. in the shaved right flank of each recipient C57BL / 6Jax mouse. Tumour growth was monitored every 1 to 3 days, and the longest tumour diameter (I) and perpendicular width (w) were measured using digital Vernier callipers; tumour volume was calculated using the formula: length x width2 / 2 and expressed as mm3. Anti- F-actin lgG2a was administered via intraperitoneal injection (400 pg in 200 pl PBS for irrelevant and anti- F-actin lgG2a) on day 0, 3, 6, 11 , 14 and 18 (Figure 5). On day 6, mice were additionally injected with 1.25 mg / kg doxorubicin via intratumoural injection (Merck Life Science UK).

[0874] F-actin binding ELISA

[0875] The ability of selected anti-F-actin mAbs as well as soluble DNGR-1 receptor to bind to F-actin was measured by ELISA using immobilised F-actin. Briefly, Immunoplates (MaxiSorp 96-well, Thermo Fisher Scientific) were coated with 5 pg / ml ExtrAvidin®(Merck Life Science) in PBS overnight at 4°C. The following day, plates were washed, blocked with 2.5% BSA in PBS for 2 h at room temperature and stored overnight at 4°C after addition of biotinylated F-actin (100 nM), which had been prepared the same or the previous day. To prevent the depolymerisation of F-actin during the assay, F-actin was stabilised by adding phalloidin during the preparation of the F-actin stock. Ten-fold serial dilutions of anti-F-actin mAbs and Fc-DNGR-1 fusion protein (positive control) starting at 100 nM were prepared in neat mouse serum or PBS containing 10Opg / ml hGSN (human GSN) or 1 % BSA and added to the ELISA plate for 1 h. Binding of anti-F-actin mAbs and mFc-mDNGR-1 was detected using an alkaline phosphatase (AP)- conjugated mslgG2a-specific antibody (Southern Biotech) and developed with an ELISA substrate for AP based detection (pNitrophosphate, Sigma). Plates were read using an ELISA plate reader at 405 nm to determine optical densities (OD) for each sample and plotted using GraphPad PrismIO.

[0876] Necrotic tumour cells were incubated with 10 nM Fc-DNGR-1 or anti-F-actin lgG2a at RT for 1 h in DPBS in the presence of increasing concentrations of normal mouse serum. Samples were washed 2X and incubated with secondary AF488-conjugated anti-mouse IgG (Thermo Fisher Scientific, 1 :400) at 4°C for 30 min. Samples were washed and resuspended in DPBS before acquisition on an LSRFortessa (BD Biosciences). Data was analysed using FlowJo software version 10.

[0877] Fc-DNGR-1 and anti-F-actin antibody pharmacokinetics analysis

[0878] C57BL / 6 mice were injected i.p. with 100 pg of antibody A or Fc-DNGR-1 (5 mice per group), blood was harvested between days 0-6 post-injection, and serum isolated using serum Z-Gel tubes (Sarstedt). Fc- DNGR-1 titre was calculating using a serum-compatible anti-DNGR-1 ELISA. Briefly, 96-well high-affinity Nunc MaxiSorp plates (Thermo Fisher Scientific) were coated overnight with rat anti-mouse DNGR-1 IgG (clone 42D2, 5 pg / ml in 0.1 M sodium bicarbonate buffer) before extensive washing in 0.05% Tween-20 in DPBS. Plates were blocked for 1 h with 3% FCS (blocking buffer), washed once more, and incubated with DPBS-diluted serum (1 :100 and 1 :1000) or Fc-DNGR-1 standard curve for 2 h. After washing, plates were incubated with biotin rat anti-mouse DNGR-1 IgG (clone 7H11 , 1 pg / ml) for 2 h, before developing with alkaline phosphatase, as above. Antibody A titre was calculated similarly but using CaptureSelect™ biotin anti-lgG-CH1 (Thermo Fisher Scientific) on ELISA plates coated with ExtrAvidin (Merck Life Science, 5 pg / ml) overnight, and goat anti-mouse lgG2a-alkaline phosphatase detection (SouthernBiotech).

[0879] EXAMPLE 4 - Generation of candidate antibodies

[0880] A phage selection funnel was used to select the lead antibody candidates (Figure 6). A total of 736 clones were generated by screening a naive phage display library. These were generated by 4 rounds of positive selection based on F-actin binding and negative selection based on G-actin binding. Two parallel arms were performed: for each round of screening, the F-actin-bound phage were eluted using either standard elution buffer containing Trypsin (1 mg / mL) (Arm 1) or via competition with Fc-DNGR-1 (at 1000 nM) (Arm 2). An ELISA was then performed on these clones to determine binding to F-actin and G-actin. 497 clones met the threshold of >3-fold F-actin binding selectivity over G-actin. Following Fab sequencing and sequence analysis, including VH and VL gene diversity and sequence liabilities, 78 unique clones were taken forward for further evaluation. Necrotic binding was then used to filter these clones, by FACS analysis on untreated and necrotic HEK293 and NIH3T3 cells. This led to 68 clones with >3-fold dead cell selectivity. These clones were reformatted to murine lgG2a and filtered for: >5-fold F-actin over G-actin binding; >5-fold F-actin over neutravidin binding; >10% necrotic cell binding; and >3-fold necrotic cell MFI over background MFI. 46 clones met this threshold, and the data is displayed in Table 5.

[0881] Table 5: Values for: F / G actin binding; Dead cell / viable cell binding (human); Dead cell / viable cell binding

[0882] (murine) for all 46 lgG2a clones

[0883] EXAMPLE 5 - Further characterisation of lead antibodies 5 lead antibodies (Antibodies A-E in Table 5) were then characterised further. An ELISA was performed to assay the F-actin binding properties of all 5 antibodies (Figure 7, closed circles). The binding to G-actin was also measured (open circles). As shown, all 5 antibodies displayed only minimal binding to G-actin and are therefore specific for F-actin. The EC50 (nM) values for binding to F-actin are shown in Table 6. All antibodies are in the nanomolar range and thus have excellent potency. In particular, antibodies A, B and D have potencies below 1 nM.

[0884]

[0885] Table 6: EC50 (nM) values for binding to F-actin. N.D. = not determined.

[0886] EXAMPLE 6 - Functional analysis of lead antibodies

[0887] To confirm that the antibodies were able to bind to F-actin on dead cells, UV-irradiated necrotic mouse 5555 cells (Figure 8A) or necrotic human HEK293T cells (Figure 8B) were incubated with serial dilutions of anti-F-actin lgG2a clones or Fc-DNGR-1 fusion proteins (2WA as negative control or WT as positive control) and binding was detected with secondary AF488-conjugated anti-mouse IgG. All 5 antibodies were capable of binding to both mouse and human necrotic cells, with similar profiles and affinity (Figure 8).

[0888] These functional experiments provide confirmation that all 5 antibodies are able to bind F-actin on dead cells.

[0889] EXAMPLE 7 - Testing of commercial antibodies and comparison to lead antibodies A, B and E

[0890] To compare the specificity and function of the lead antibodies to currently available antibodies, two commercially available anti-actin antibodies were obtained. AC-40 is a pan anti-actin monoclonal antibody that binds to both F-actin and G-actin. AC-74 is an anti-p-actin antibody. The ability of these antibodies to bind to F-actin on dead cells was investigated by flow cytometric analysis of UV-irradiated necrotic mouse 5555 BrafV600E melanoma cells incubated with serial dilutions of anti-actin lgG2a clones (AC-40 or AC- 74) or Fc-DNGR-1 fusion proteins (2WA or WT) and stained with secondary AF488-conjugated antimouse IgG. The inventors were able to show that AC-40 is not able to efficiently bind to dead cells, whereas AC-74 can bind to dead cells (Figure 9).

[0891] Necrotic cell binding in the presence of G-actin was then measured for AC-74 and Antibodies A, B and E. As shown in Figure 10A and 10B, antibodies A, B and E are resistant to G-actin competition and maintain binding to necrotic cells via F-actin. In contrast, AC-74 is not resistant to G-actin competition. This data shows that the antibodies of the invention are specific for F-actin over G-actin, whereas AC-74 is not. EXAMPLE 8 - Competition of Anti-F-actin antibodies with DNGR-1 binding to F-actin

[0892] The extent of competition of antibodies A, B and E was assessed. The ability to compete with DNGR-1 varied greatly between these antibodies, with Antibody B showing the highest level of competition with DNGR-1 for binding F-actin (Figure 11). Antibody E shows minimal competition. None of the antibodies matched the ability of DNGR-1 to compete with itself.

[0893] EXAMPLE 9 - sGSN inhibition of Anti-F-actin antibodies

[0894] To further probe the binding activity of the antibodies, the effect of sGSN on binding of mAbs to immobilised F-actin was measured, using 1 % BSA as a negative control (Figure 12A). When mouse serum was added, binding of all antibodies tested (and Fc-DNGR-1) was inhibited (Figure 12B). Human GSN (hGSN) also led to the inhibition of all antibodies tested and Fc-DNGR-1 (Figure 12C).

[0895] The inhibition of sGSN on necrotic tumour cells was also measured. Mouse serum (which contains high levels of sGSN, Piktel 2018) was added to the cells and was shown to reduce binding of all antibodies tested and Fc-DNGR-1 (Figure 13).

[0896] EXAMPLE 10 - Systemic administration of anti-F-actin antibody promotes tumour control

[0897] To further assess the in vivo therapeutic efficacy of the anti-F-actin antibodies, antibody A was compared to equimolar concentrations of Fc-DNGR-1 in promoting tumour control when given peri-tumourally. Consistent with the results shown in Example 5, in MCA205 fibrosarcoma tumours treated with cell deathinducing chemotherapeutic doxorubicin, both Fc-DNGR-1 and antibody A promoted therapeutic tumour control to equivalent levels compared to mice receiving 2WA Fc-DNGR-1 (Figure 15A).

[0898] To determine whether systemic administration would be feasible, the pharmacokinetics of anti-F-actin antibodies compared to DNGR-1 was assessed. To do this, the inventors developed two mouse serumcompatible ELISAs for reagent detection: an Fc-DNGR-1 ELISA using two monoclonal anti-mouse DNGR-1 antibodies that target distinct epitopes in the ECD and an anti-F-actin antibody ELISA that leveraged detection of a human CH1 domain incorporated into the antibody backbone (Figure 15F).

[0899] Using this approach, the inventors demonstrated that antibody A exhibited much enhanced serum half-life compared to Fc-DNGR-1 (Figure 15B). In line with its improved pharmacokinetics, following systemic administration, antibody A accumulated at necrotic areas within doxorubicin-treated MCA205 tumours, compared to an isotype-matched control antibody, either via detection of mouse lgG2a or human CH1 (Figure 15C).

[0900] Consistent with its short half-life, systemically-administered Fc-DNGR-1 , unlike reagent given peri- tumourally, did not stain tumours or demonstrate therapeutic efficacy in various models (data not shown). Given the superior pharmacokinetic profile of anti-F-actin antibodies, the inventors therefore assessed the efficacy of systemically administered antibody A. The MC38 tumour model in which single agent activity with Fc-DNGR-1 given peri-tumourally has been shown previously. Notably, systemically administered antibody A alone was sufficient to promote control of MC38 tumours compared to isotype- matched control antibody (Figure 15D). Mice did not exhibit any outward signs of systemic inflammation and continued to accumulate weight as expected (Figure 15E).

[0901] EXAMPLE 11 - Fc-DNGR-1 reveals tumour necrotic cell localisation in vivo.

[0902] For cancer immunotherapy, Fc-DNGR-1 would require close apposition of APCs and necrotic cancer cells within tumours. However, the spatiotemporal dynamics of necrotic cell sensing by immune cells in vivo is poorly understood. To address this, the inventors used Fc-DNGR-1 to highlight areas of necrosis within tumours by microscopy. Following on from Examples 1-3 showing that Fc-DNGR-1 can penetrate tumour spheroids and tumours in vivo, further experiments were performed.

[0903] To assess the proximity of different APC populations to dead cells within the tumour, the inventors costained for MHC-II and CD103 to identify CD103+cDC1s and CD103- non-cDC1 APCs (e.g., cDC2s, MCs) (Figure 16A). Remarkably, cDC1s were located towards the tumour periphery, away from necrotic sites, revealing that they may be somewhat limited in their ability to acquire dead cell material in vivo. In contrast, MHC-II+CD103- APCs were located throughout the tumour, including at the boundary of and within the necrotic core. These CD103- APCs were further confirmed to express FcyRI (Figure 16B), suggesting that they are optimally positioned to acquire necrotic cell antigens in vivo in the presence of Fc-DNGR-1.

[0904] This data provides further proof-of-principle that the necrotic core of tumours can be targeted via binding F-actin.

[0905] The data described above show the generation of a class of antibodies that bind to F-actin with high affinity, avidity, and / or specificity, and are inhibited by sGSN. These antibodies are able to specifically bind to F-actin on necrotic cells (Figure 8). These functional characteristics, as well as the specificity for F-actin, highlights these antibodies as therapeutically useful, for example in the treatment of cancers.

[0906] The inventors have also shown that a selection of commercially available antibodies are not specific for F- actin, and bind to G-actin to a similar degree (Figure 10).

[0907] The data also shows that this class of antibodies display improved pharmacokinetics in circulation relative to Fc-DNGR-1 , accumulate in necrotic sites within tumours, and promote tumour control upon systemic administration. Anti-F-actin affimers Examples

[0908] The following examples demonstrate that anti-F-actin affimers are able to bind to F-actin with high affinity, avidity, and / or specificity. It is noted that the affimers of the present invention are in no way limited to the affimers described below.

[0909] MATERIALS & METHODS

[0910] Affimer-Fc fusion proteins

[0911] Affimer-Fc fusion proteins were generated by FairJourney Biologies. Affimers 6, 14, and 24 bind to F-actin and Control Affimer does not bind to F-actin (negative control). All Affimers used in the examples are conjugated to mouse lgG2a Fc (mFc), optionally via a linker, at their N-terminus. sGS / V inhibition

[0912] For sGSN inhibition assays, recombinant mouse sGSN was generated by FairJourney Biologies. sGSN was incubated in PBS containing 100 pM CaCh with UV-treated 5555 dead cells for 1 h. Dead cells were then incubated with 10 nM Affimer-Fc for 30 min before detecting binding by staining with anti-mouse IgG A647.

[0913] DNGR-1 cross-blocking assay

[0914] UV-treated 5555 dead tumour cells were incubated with equimolar amounts of Fc-fusion proteins for 1 h in PBS. Blocking of DNGR-1 binding was the assessed by incubating dead cells with FLAG-tagged mC9 for 30 min at 1 pg / ml for 30 min. Binding of Fc-fusion proteins and Affimer-Fc was detected using antimouse IgG and anti-FLAG IgG antibodies.

[0915] EXAMPLE 12 - Affimer-Fc binding properties

[0916] This example shows that binding agents that bind to F-actin with an F-actin affimer are able to specifically bind F-actin.

[0917] 3 different affimers specific for F-actin were conjugated to mouse lgG2a Fc (mFc) at their N-terminus. The affimer-Fc constructs used are termed Affimer 6, Affimer 14 and Affimer 24. To investigate the ability of the affimer-Fc constructs to bind to F-actin, the constructs were incubated with UV-treated 555 dead tumour cells for 1 hour. A control affimer that does not bind F-actin was included as a negative control. Figure 14A shows that Affimers 14 and 24 were able to bind to F-actin. Binding was then detected using anti-mouse lgG-A647 (GMFI) (Figure 14B). sGSN inhibition of affimer-Fc binding to dead cells was investigated by incubating UV-5555 dead cells with 10 pg / ml sGSN for 1 h at 4°C, followed by incubation with 10 nM Affimer-Fc. sGSN inhibited the binding of all 3 affimer-Fc constructs to dead cells (Figure 14C & 14D).

[0918] These preliminary experiments provide confirmation that constructs which employ an affimer as the F- actin binding moiety are physically able to bind dead or dying cells and the affimer-Fc binding to dead cells is sensitive to inhibition by sGSN. Figure 17A shows a schematic of Fc fusions using anti-F-actin affimer proteins. Figure 17B confirms that anti-actin affimer-Fc exhibited preferential binding to F- versus G-actin beads, and Figure 17C shows that anti-actin affimer-Fc stained necrotic tumour cell debris with similar potency and effectiveness to Fc- DNGR-1 (top line is Fc-DNGR-1 , middle line is anti-actin affimer and bottom line is control affimer). Anti- actin affimer-Fc bound to a distinct F-actin epitope from one engaged by DNGR-1 ECD, as assessed by competition with DNGR-1 -FLAG (Figure 17D - in the bottom left hand panel, the top line shows anti-actin affimer, the middle line shows WT Fc-DNGR-1 and the bottom line shows the 2WA Fc-DNGR-1 mutant; in the bottom right hand panel, the top line shows the 2WA Fc-DNGR-1 mutant, the open white circles show the control affimer-Fc, the third line from the top shows the anti-actin affimer-Fc and the bottom line shows the WT-Fc-DNGR-1). Notably, like Fc-DNGR-1 , anti-actin affimer-Fc, but not control affimer-Fc, boosted necrotic cell XP by both cDC1s and non-cDC1s (Figure 17E), once more normalising the ability of both cell types to activate CTLs in response to dead cell-associated antigen.

[0919] References

[0920] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[0921] Ashman, N. et al., Non-internalising antibody-drug conjugates. Chem Soc Rev, 2022 51 , 9182

[0922] Bargh, J.D. et al. Cleavable linkers in antibody-drug conjugates. Chem. Soc. Rev., 2019, 48, 4361-4374

[0923] Castro-Dopico, T. & Clatworthy, M. IgG and Fey Receptors in Intestinal Immunity and Inflammation. Frontiers in Immunology 10:805 (2019)

[0924] Chomet, M. et al., State of the Art in Radiolabeling of Antibodies with Common and Uncommon Radiometals for Preclinical and Clinical Immuno-PET. Bioconjugate Chemistry, 2021. 32:1315-1330

[0925] Dominguez, R. & Holmes, K. C., Actin Structure and Function. Annu Rev Biophys 40:169-186 (2011)

[0926] Eckerman, K. F. & Enzo, A. MIRD: Radionuclide Data and Decay Schemes, 2nd edn. (Society of Nuclear Medicine, 2008)

[0927] Giampazolias, E., et al., Secreted gelsolin inhibits DNGR-1 -dependent cross-presentation and cancer immunity. Cell, 2021. 184(15): p. 4016-4031 ,e22.

[0928] Hanc, P. et al., Structure of the complex of F-actin and DNGR-1 , a C-type lectin receptor involved in dendritic cell crosspresentation of dead cell-associated antigens. Immunity 42(5): 839-849 (2015)

[0929] Holik, H. A. et al., The Chemical Scaffold of Theranostic Radiopharmaceuticals: Radionuclide, Bifunctional Chelator, and Pharmacokinetics Modifying Linker. Molecules, 2022, 27, 3062

[0930] Ivanova, J. R. et al., Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells. ACS Nano 2024, 18, 8919-8933

[0931] Khongorzul, P. et al., Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res (2020) 18 (1): 3-19.

[0932] Lopata, A. et al., Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells. Scientific Reports (2018) 8:6572

[0933] Piktel, E. et al., Plasma Gelsolin: Indicator of Inflammation and Its Potential as a Diagnostic Tool and Therapeutic Target. Int. J. Mol. Sci. 2018, 19, 2516

[0934] Sgouros, G. et al., Radiopharmaceutical therapy in cancer: clinical advantages and challenges. Nature Reviews Drug Discovery, 2020 19:589-608

[0935] Staudacher, A. H. et al., Therapeutic targeting of tumor hypoxia and necrosis with antibody a- radioconjugates. Antibody Therapeutics, 2018, 1 (2):55-63

[0936] WO 2023 / 203 / 198

[0937] WO 2024 / 018062 For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A

[0938] Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

Claims

1. Claims:1 . A radiolabelled binding agent comprising: a. an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and b. a radionuclide.

2. The radiolabelled binding agent according to claim 1 , wherein the radiolabelled binding agent has at least 5-fold higher affinity for F-actin than for globular actin (G-actin).

3. The radiolabelled binding agent according to any preceding claim, wherein binding of the radiolabelled binding agent to F-actin is inhibited by secreted gelsolin (sGSN).

4. The radiolabelled binding agent according to any preceding claim, wherein the radionuclide is a radioisotope of a metal.

5. The radiolabelled binding agent according to any preceding claim, wherein the radionuclide is an alpha (a)-emitting radionuclide, optionally wherein the alpha(a)-emitting radionuclide is selected from the group consisting of:227Th,223Ra,212Pb,212Bi,211At,225Ac,213Bi ,230U,226Th,224Ra,213Po, and149Tb.

6. The radiolabelled binding agent according to any preceding claim, wherein the radionuclide is a beta (p-)-emitting radionuclide, optionally wherein the beta (p-)-emitting radionuclide is selected from the group consisting of:32P,47Sc,64Cu,67Cu,153Sm,177Lu,90Y,131l,199Au,166Ho,186Re,188Re,194lr,159Gd, 149Pm,142Pr,109Pd,225Ra,213Bi ,212Bi,161Tb,89Sr,77As and149Tb.

7. The radiolabelled binding agent according to any preceding claim, wherein the radionuclide is a gamma (y)-emitting radionuclide, optionally wherein the gamma (y)-emitting radionuclide is selected from the group consisting of:123l,1311 ,203Pb,205Bi ,99mTc,133Xe,201TI,51Cr,67Ga,166Ho,61Cu,64Cu, 110mAg, "Y,188Re,166Ho,159Gd, and137Cs.

8. The radiolabelled binding agent according to any preceding claim, wherein the radionuclide is a high energy beta (p+)-emitting radionuclide, optionally wherein the high energy beta (p+)-emitting radionuclide is selected from the group consisting of:18F-AI,44Sc,52Mn,76Br,77Br,86Y, "Y,89Zr,124l, 66Ga,68Ga,18F,11C,82Rb,13N,61Cu, and64Cu.

9. The radiolabelled binding agent according to any preceding claim, wherein the radionuclide decays by electron capture, optionally wherein the radionuclide is selected from the group consisting of:1111n, 18F,67Ga,123l,125l,61Cu,186Re,77As and211At.

10. The radiolabelled binding agent according to any preceding claim, wherein the radionuclide decays by isomeric transition, optionally wherein the radionuclide is117mSn.

11. The radiolabelled binding agent according to any preceding claim, wherein the radionuclide is an Auger electron-emitting radionuclide, optionally wherein the Auger electron-emitting radionuclide is selected from the group consisting of:67Ga,77Br and103Pd.

12. The radiolabelled binding agent according to any preceding claim, wherein the radiolabelled binding agent further comprises a chelator that is co-ordinated to the radionuclide.

13. The radiolabelled binding agent according to claim 12, wherein the chelator is selected from the group of chelators disclosed in Table 1 , and salts or functional variants or derivatives thereof capable of chelating the metal.

14. The radiolabelled binding agent according to any preceding claim, wherein the radiolabelled binding agent further comprises a linker to couple the radionuclide to the F-actin binding moiety.

15. The radiolabelled binding agent according to claim 14, wherein the linker is selected from the group comprising: aminohexanoic acid, ethylene glycol bis(succinimidyl succinate) (EGS), disuccinimidyl subera (DSS), EMCS-Bz, MESS-Bz, MIH, 6-carboxy-1 ,4,8,11 -tetraazaundecane (N4), p- aminomethylaniline-diglycolic acid, PEG, NCS, maleimide, N-suc-TFP-ester, maleimide-monoamide, NHS, and MMA.

16. The radiolabelled binding agent according to any preceding claim, wherein the F-actin binding moiety is selected from the group consisting of: an antibody or antigen-binding fragment thereof, an affimer, an aptamer, a DarPin, the CTLD of DNGR-1 , LifeAct peptide and phalloidin.

17. The radiolabelled binding agent according to claim 16, wherein the F-actin binding moiety is an antigen-binding fragment of an antibody.

18. The radiolabelled binding agent according to claim 17, wherein the antigen-binding fragment of an antibody is a Fab fragment, a single chain Fc fragment (ScFv), a single domain antibody (sdAb), a diabody or a variable domain (Fv).

19. The radiolabelled binding agent of claim 16, wherein the F-actin binding moiety is an antibody.

20. The radiolabelled binding agent according to claim 19, wherein the antibody is a human or humanised antibody.

21. The radiolabelled binding agent according to claim 20, wherein the antibody is of the human IgG 1 isotype.

22. The radiolabelled binding agent according to any one of claims 15 to 21 , wherein the antibody or antigen-binding fragment thereof does not comprise an Fc domain.

23. The radiolabelled binding agent according to any one of claims 15 to 21 , wherein the antibody or antigen-binding fragment thereof comprises an Fc domain.

24. The radiolabelled binding agent according to claim 23, wherein the Fc domain comprises an amino acid sequence that has been mutated from that of a wild type Fc domain such that the mutated Fc domain binds to an Fc receptor with lower affinity and / or avidity than the affinity and / or avidity of the wild type Fc domain for the Fc receptor.

25. The radiolabelled binding agent according to claim 24, wherein the mutated Fc domain does not bind to an Fc receptor.

26. The radiolabelled binding agent according to any one of claims 23 to 25, wherein the Fc domain comprises an amino acid sequence that has been mutated from that of a wild type Fc domain such that the mutated Fc domain binds to a neonatal Fc receptor (FcRn) with higher affinity and / or avidity than the affinity and / or avidity of the wild type Fc domain for the Fc receptor.

27. The radiolabelled binding agent according to any one of the preceding claims, for use in medicine.

28. The radiolabelled binding agent according to any one of claims 1 to 26, for use in a method of treating cancer, the method comprising administering the radiolabelled binding agent to a subject that has cancer.

29. The radiolabelled binding agent for the use according to claim 28, wherein the cancer comprises a solid tumour.

30. The radiolabelled binding agent for the use according to any one of claims 28 or 29, wherein the cancer is characterised by F-actin presentation on necrotic cancer cells.

31. The radiolabelled binding agent for the use according to any one of claims 28 to 30, wherein the method of treating cancer comprises killing cancer cells via necrosis.

32. The radiolabelled binding agent for the use according to claim 31 , wherein the method comprises contacting the necrotic cancer cells with the radiolabelled binding agent.

33. The radiolabelled binding agent for the use according to any one of claims 28 to 32, wherein the method of treating cancer comprises (i) administering a therapy to the subject to cause necrosis of the cancer cells, and then (ii) administering the radiolabelled binding agent to the subject and thereby contacting the necrotic cancer cells with the radiolabelled binding agent.

34. The radiolabelled binding agent for the use according to claim 33, wherein the therapy administered to the subject in step (i) is selected from the group comprising: an F-actin binding agent, a chemotherapeutic agent or a radiotherapy.

35. The radiolabelled binding agent for the use according to any one of 28 to 34, wherein the use comprises administration of the radiolabelled binding agent in combination with an additional anticancer therapy.

36. The radiolabelled binding agent for the use according to claim 35, wherein the additional anticancer therapy is an immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor is an anti-PD1 monoclonal antibody.

37. The radiolabelled binding agent for the use according to any one of claims 35 or 36, wherein the additional anticancer therapy is a radiotherapy.

38. The radiolabelled binding agent for the use according to any one of claims 27 to 37, wherein the use comprises administration of the radiolabelled binding agent in combination with a chemotherapeutic agent.

39. The radiolabelled binding agent for the use according to any one of claims 27 to 38, wherein the use comprises co-administration of the radiolabelled binding agent with an anti-F-actin antibody that is not radiolabelled.

40. The radiolabelled binding agent for the use according to any one of claims 27 to 39, wherein the use comprises systemic administration of the antibody or antigen-binding fragment.

41. The radiolabelled binding agent according to any one of claims 1 to 26, for use in a method of radioimaging.

42. The radiolabelled binding agent for the use according to claim 41 , wherein the radionuclide comprises a non-ionising isotope.

43. The radiolabelled binding agent for the use according to any one of claims 41 or 42, wherein the radioimaging comprises imaging a tumour or a cancer.

44. The radiolabelled binding agent for the use according to claim 43, wherein the cancer is characterised by F-actin presentation on necrotic cancer cells.

45. A method of radio-immuno imaging, comprising administering the radiolabelled binding agent according to any one of claims 1 to 26 to a subject and detecting the radionuclide.

46. The method of claim 45, wherein the method of radio-immuno imaging is a positron-emission tomography (PET) method, and wherein the radionuclide is a high energy beta (p+)-emitting radionuclide.

47. The radiolabelled binding agent according to any one of claims 1 to 26, for use in an in vivo method of diagnosis of cancer.

48. A method of producing a radiolabelled binding agent according to any one of claims 1 to 26, the method comprising: a. obtaining an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and b. labelling the F-actin binding moiety with a radionuclide.

49. The method according to claim 48, wherein the radionuclide is selected from the group consisting of: 227Th,223Ra,212Pb,212Bi ,211At,225Ac,213Bi ,230U,226Th,224Ra,213Po,149Tb,32P,47Sc,64Cu,67Cu,153Sm, 177Lu,"Y,131l,199Au,166Ho,186Re,188Re,194lr,159Gd,149Pm,142Pr,109Pd,225Ra,213Bi,212Bi,161Tb,89Sr, 77As,149Tb,123l ,1311 ,203Pb,205Bi ,99mTc,133Xe,201TI,51Cr,67Ga,166Ho,61Cu,64Cu,110mAg, "Y,188Re, 166Ho,159Gd,137Cs,18F-AI,44Sc,52Mn,76Br,77Br,86Y, "Y,89Zr,124l ,66Ga,68Ga,18F,11C,82Rb,13N,61Cu, 64Cu,111ln,18F,67Ga,123l ,125l ,61Cu,186Re,77As,211At,1 17mSn,67Ga,77Br and103Pd.

50. A kit comprising: a. an F-actin binding moiety that specifically binds to filamentous actin (F-actin); and b. a radionuclide.

51. A kit according to claim 50, further comprising instructions for radiolabelling the F-actin binding moiety that specifically binds to filamentous actin (F-actin) with the radionuclide.

Images