Anti-f-actin antibodies

Abstract

Antibodies that specifically bind to actin are provided. In particular, the antibodies 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 anti-actin antibodies, medical uses thereof and methods of generating anti-F-actin antibodies.

Description

[0001] Anti-actin Antibodies Field of the Invention The present invention relates to antibodies that specifically bind to actin, for instance exhibiting preferential binding for F-actin over G-actin. The invention provides anti-actin antibodies, medical uses thereof and methods of generating anti-F-actin antibodies. Background Filamentous-actin (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 α / β 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). F-actin is an ATPase and it is this nucleotide hydrolysis that regulates the transition between F-actin and G-actin, resulting in highly dynamic actin filaments which enable cells to change shape. Antibodies that bind to actin are known. However, such antibodies typically cannot distinguish between the monomeric form of actin, G-actin, from filamentous F-actin and / or fail to bind with sufficient affinity or avidity to trigger immunologically useful outcomes such as phagocytosis, described herein. For example, Abcam antibody ab2051binds to G-actin as well as F-actin. The Novus Biologicals antibody NBP2- 616102also binds to both F-actin and G-actin. The Sigma Aldrich antibody AC-74 (A2228) is beta-actin specific. The Abcam antibody AC-40 (ab11003) is pan-actin binding. WO 2022 / 163809 (which is incorporated herein by reference in its entirety) discloses antigen-binding molecules which bind to a cellular component that is exposed following cell death, such as F-actin. However, the relative binding specificities of the antibodies postulated in WO 2022 / 163809 to F- and G-actin is not disclosed, nor is a way of achieving high specificity for F-actin suggested. The following anti-actin antibodies have been shown to bind to G-actin by western blotting: Cell Signaling Technology antibody #4967 (beta-actin specific); St John’s Laboratory antibody STJ91461 (pan-actin); Abcam antibody EPR16769 ab179467 (pan-actin). Even antibodies that are marketed as anti-F-actin antibodies have been shown to also bind to G-actin, such as the NH3 antibody (Dransfield, 1988), Abcam antibody 4E3.adl (ab130935) and antibody ABIN7314983. Yao et al, 2023 (incorporated herein by reference in its entirety) disclose plasma cells that are present in human pancreatic ductal adenocarcinomas, which purportedly secrete antibodies that bind to self- antigens such as F-actin. However, the F-actin binding antibody allegedly disclosed by Yao et al does not bind with very high affinity or avidity, showing an EC50 for F actin of 9.1 nM.1https: / / www.abcam.com / products / primary-antibodies / f-actin-antibody-nh3-ab205.html2https: / / www.novusbio.com / products / f-actin-antibody-nh3_nbp2-616103https: / / www.antibodies-online.com / antibody / 731498 / anti-F-Actin+AA+1-50+antibody / 008738577 2 The development of an antibody that is specific for F-actin would be of great utility because F-actin and G-actin have very distinct cellular functions. G-actin is maintained in a cytosolic pool which supplies the necessary building blocks for generating F-actin filaments (Dominguez & Homes, 2011, which is incorporated herein by reference in its entirety). In contrast, F-actin is a Damage-Associated Molecular Pattern (DAMP) that is recognised by DNGR-1, a receptor for dead cells (WO 2013 / 088136; Hanĉ, et al, 2015; Ahrens, 2012; Zhang, 2012; Sancho, 2009, each of which are incorporated by reference in their entireties). DNGR-1 (also known as CLEC9A) is expressed at high levels on type 1 conventional dendritic cells (cDC1). Upon binding to F-actin via its C-type lectin domain (CTLD), DNGR-1 triggers SYK signalling, which causes rupture of ligand-containing phagosomes, release of antigenic material into the cDC1 cytosol, and its entry into the endogenous MHC class I presentation pathway (Sancho, 2009; Canton, 2021; and WO2009 / 013484A1, each of which is incorporated herein by reference in its entirety), a process termed cross-presentation (XP). As a part of the cytoskeleton, F-actin is exposed upon damage to the cell membrane, such as during necrosis. F-actin is therefore a marker of necrotic cell death. Necrotic cell debris is a source of antigens for XP within tumours, and is avidly internalised by cDC1 (Galluzzi, 2017, which is incorporated herein by reference in its entirety). The present invention has been devised in light of the above considerations. Summary of the Invention This invention provides a way of obtaining antibodies that bind to F-actin with high affinity, avidity, and / or specificity. This invention therefore provides a class of antibodies that exhibit such properties. The inventors found that positively selecting members of an antibody population by F-actin binding activity, negatively selecting them with G-actin, and additionally using DNGR-1 as an antibody-eluting agent and / or trypsin (as described herein) can yield antibodies of this class. The inventors were motivated to try to obtain antibodies with these properties following their insight that actin binding antibodies might find utility in the treatment of cancers, such as solid tumours, and because the commercially available anti- actin antibodies lack the therapeutically desirable properties discussed herein. Although some previously known antibodies claim to be selective for F-actin, the inventors have found that they bind to G-actin to a similar degree (i.e., these known antibodies do not bind to F-actin specifically). In contrast, antibodies disclosed herein demonstrably exhibit specificity for F-actin over G- actin, as shown in the Examples below. This specificity means that the antibody can specifically target necrotic and damaged cells by binding to F-actin. As described above, F-actin is part of the cytoskeleton, whereas G-actin is not. Without wishing to be bound by theory, the inventors hypothesise that when the cell membrane becomes damaged or destroyed (for example during necrosis) G-actin will disperse, whereas F-actin will remain largely entangled with necrotic cell debris. When an antibody of the invention binds to exposed F-actin molecules, its Fc domain will bind to Fc receptors (FcRs) on the surface of antigen presenting cells (APCs). Preferably, the antibody has an Fc domain that preferentially binds to activating FcRs. Binding these FcRs promotes phagocytosis, and antigen presentation (including cross- presentation (XP)) of neoantigens. 008738577 3 In contrast, prior art antibodies bind F-actin with insufficient affinity / avidity, and / or also bind G-actin, which is not a DAMP and will therefore promote off-target effects and remove the antibody from necrotic foci. Thus, for certain applications, the antibody should comprise an Fc domain. The antibodies of the invention also find use as experimental tools. For example, the antibodies may find use in imaging applications. The antibodies may also find use as capture antibodies. For these applications, the antibody may not need an Fc domain. At its broadest, the invention provides actin-binding antibodies that exhibit one or more characteristics described herein. For instance, in a first aspect the invention provides an antibody, or antigen-binding fragment thereof, that specifically binds to filamentous actin (F-actin). Preferably the antibody has a higher affinity for F-actin than for globular actin (G-actin). Preferably, the antibody or antigen-binding fragment thereof has an EC50 value for F-actin of about 5 nM or less. Preferably, the antibody or antigen-binding fragment thereof has at least 5-fold higher affinity for F-actin than for G-actin. More preferably, the antibody or antigen-binding fragment thereof has at least 10-fold higher affinity for F-actin than for G-actin. In another aspect, the invention provides a human or humanised antibody, or antigen-binding fragment thereof, that specifically binds to filamentous actin (F-actin). Preferably the antibody is a monoclonal antibody. In another aspect, the invention provides an antibody, or antigen-binding fragment thereof, that specifically binds to filamentous actin (F-actin) and is of the human IgG1 isotype. Preferably, the antibody binds to an activating Fcγ receptor. In another aspect, the invention provides an antibody, or antigen-binding fragment thereof, that specifically binds to filamentous actin (F-actin) and can facilitate cross presentation (XP) of a dead cell- associated antigen to a CD8+ T cell. All of the individual features of the above aspects are combinable and are explicitly encompassed in the invention described herein. The embodiments described below are also combinable with any of the above aspects. Affinity of the antibody for F-actin and G-actin can be assessed by the following method. Immunoplates (MaxiSorp 96-well, Thermo Fisher Scientific) are coated with 5 µg / 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 mAbs or control reagent (DNGR-1-Fc fusion) starting at 300 nM are added to the ELISA plate for 1 hr. Actin-bound mAbs or control reagent are detected using a horseradish peroxidase (HRP)-conjugated mouse-specific anti- 008738577 4 IgG (Jackson ImmunoResearch) antibody (0.16 µg / 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 mAb are calculated by applying a nonlinear regression (curve fit) of a log (agonist) vs response - variable slope (four parameters). Preferably, the antibody can facilitate cross presentation (XP) of a dead cell associated antigen to a CD8+ T cell. In some embodiments, the XP is mediated by a cDC1. In other embodiments, the XP is mediated by a non-cDC1 cell that expresses an FcR, e.g. a cDC2 or a macrophage. Cross-presentation can be assessed by the following method. Tumour cells are irradiated to induce necrosis. 16 hours after irradiation, necrotic tumour cells are soaked for 1 hour at 37oC in 10 mg / ml albumin (OVA, Sigma Aldrich) in RPMI 1640 medium and then washed. The OVA-necrotic cells are then incubated with APCs (5 x 104cDC1 or 1 x 105non-cDC1) at a 2:1 ratio for 4 h in the presence of the candidate antibody (or control agent) in a U-bottomed well of a 96-well plate. An Fc-DNGR-1 construct can be used as a positive control and a non-binding Fc-DNGR-1 variant such as the “2WA” mutant can be used as a negative control. Then, pre-activated OT-I CD8+ T cells (which can be generated by culturing splenic single cell suspensions from OT-I mice in R10+ medium supplemented with 0.1 nM SIINFEKL peptide and / or 100 U / ml recombinant mouse IL-2 for 4-5 days) are added to the APC co- culture for 24 hours. Finally, ELISA is used to measure the concentration of T cell-derived IFNγ after the 24 hour co-culture with the pre-activated OT-I CD8+ T cells. This assay is repeated using five evenly distributed antibody concentrations between about 100 pM to 10 nM, e.g. at 200 pM, 400 pM, 800 pM, 1.6 nM and 3.2 nM (and with the same five concentrations of controls). The results can be expressed as the concentration of T cell-derived IFNγ (pg / mL) against the concentration of the antibody (see Figure 3 for example). One way to classify an antibody’s capacity to mediate XP is to take the average area under the curve (AUC) from the cross-presentation assay described above of 3 non-binding Fc-DNGR-1 variants, such as the 2WA mutant and define this value as 0% baseline (negative control). The average AUC from the cross-presentation assay of 3 WT Fc-DNGR-1 constructs is defined as 100% (positive control). The AUC from the cross-presentation assay of a candidate antibody is corrected to the baseline and converted to a %. The antibody is considered to have the capacity to mediate XP if this % is 5%, 10%, 12% or 15% or above. Alternatively, an antibody is considered to have the capacity to mediate XP if the IFNγ signal from the cross-presentation assay described above is at least 3 standard deviations above a negative control (e.g., 2WA or irrelevant antibody). In these embodiments, the IFNγ signal may be at least 4, at least 5, or at least 6 standard deviations above a negative control. Preferably, the antibody or antigen-binding fragment thereof exhibits specificity for binding to necrotic cells over live cells, as measured by FACS or flow cytometry. In some embodiments, the antibody or antigen-binding fragment thereof has at least 3-fold specificity for binding necrotic cells over live cells. In some embodiments, the antibody or antigen-binding fragment thereof has at least 4-fold, at least 5-fold, at 008738577 5 least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold specificity for binding necrotic cells over live cells. The antibody or antigen-binding fragment thereof may have at least 10% necrotic cell binding. The antibody or antigen-binding fragment thereof may have at least 20%, at least 30% or at least 40% necrotic cell binding. In some embodiments, the necrotic cell binding is not inhibited by the presence of G-actin. Necrotic cell binding can be assessed by the following method. Necrotic tumour cells (e.g. irradiated mouse BRAFV600Emutant 5555 melanoma cells or human HEK293T cells) are cultured overnight in R0 medium (lacking 10 % FCS) and then incubated with antibody (or control agents) in DPBS. An Fc- DNGR-1 construct can be used as a positive control and a non-binding Fc-DNGR-1 variant such as the “2WA” mutant can be used as a negative control. To assess whether binding to F-actin is inhibited by the presence of G-actin, soluble cytochalasin D-stabilised human platelet G-actin (Cytoskeleton) is added simultaneously. Samples are washed and incubated with secondary AF488-conjugated anti-mouse IgG2a (Thermo Fisher Scientific, 1:400) at 4oC for 30 min. Samples are again washed and resuspended in DPBS before acquisition on an LSRFortessa or FACSymphony (BD Biosciences). Preferably, the antibody can facilitate phagocytosis of necrotic cells. In some embodiments, the phagocytosis is mediated by a cDC1. In other embodiments, the phagocytosis is mediated by a non- cDC1 cell that expresses an FcR, e.g. a cDC2 or a macrophage. The phagocytosis may be mediated by a monocyte-derived dendritic cell (moDC), e.g. a human moDC or by a monocyte-derived macrophage (moMac), e.g. a human moMac. Necrotic cell phagocytosis can be assessed by the following method. Tumour cells are labelled with Cell Tracker-Deep Red (CT-DR) dye (Thermo Fisher Scientific; 1:1000 dilution) for 1 hour at 37oC prior to irradiation. CT-DR-labelled necrotic cells are then added to cDC at a 2:1 ratio for 4 hours in the presence of antibody (or control agents). An Fc-DNGR-1 construct can be used as a positive control and a non- binding Fc-DNGR-1 variant such as the “2WA” mutant can be used as a negative control. After 4 hours, samples are prepared for flow cytometry by surface staining with a combination of: XCR1 (Biolegend, ZET), CD172a (Biolegend, P84), I-A / I-E (Biolegend, M5 / 114.15.2), B220 (BD Biosciences, RA3-6B2), and CD11c (Biolegend, N418). DAPI or LIVE / DEAD Fixable Blue Dead Cell Dye (Thermo Fisher Scientific) is used to exclude non-internalised necrotic cell material. Samples are acquired on an LSRFortessa or FACSymphony (BD Biosciences). Phagocytic index (which integrates frequency and absolute quantity of phagocytosed material) is calculated using the formula (% CT-DR+ x CT-DR GMFI of CT-DR+) / 10,000. Preferably, this assay is repeated using at least five evenly-distributed antibody concentrations between about 30 pM to about 30 nM. An elevated phagocytic index versus the phagocytic index achieved by a negative control in this concentration range indicates that the antibody can facilitate phagocytosis of necrotic cells. Preferably, the antibody is a human or humanised antibody. Preferably, the antibody comprises an activating Fc domain (that preferentially binds activating FcRs), for instance wherein the antibody is of the human IgG1 isotype. 008738577 6 In some embodiments, the antibody competes with human DNGR-1 for binding to F-actin. In other embodiments, the antibody does not compete with human DNGR-1 for binding to F-actin. Preferably, the antibody binds to the same epitope, or overlaps with the same epitope, as human DNGR-1. In other embodiments, the antibody does not bind to the same epitope as human DNGR-1. In some embodiments, the antibody does not compete with the following antibodies for binding to F-actin: ab11003 (Abcam), ab205 (Abcam), ab130935 (Abcam), A2228 or A5316 (a.k.a. “Sigma-Aldrich), NBP2- 61610 (Novus Biologicals), MA1-80729 (ThermoFisher) and / or BS-1571R (ThermoFisher). Preferably, antibody binding to F-actin is not inhibited by the presence of G-actin. In some embodiments, the antibody or antigen binding fragment of the invention may be defined as specifically binding to the discontinuous, conformational epitope that is bound by human DNGR-1. This epitope 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 conformational epitope consists of three or more residues selected from: R196 and / or E237 of a first subunit of F-actin and A114, E117, Q121, A365, G366 and P367 of a second subunit of F-actin. In some embodiments, the conformational epitope 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. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibodies are polyclonal antibodies. Monoclonal and polyclonal antibodies produced by the methods of the invention are encompassed. 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: Heavy chain: CDR1 comprises GGTFRSY or a variant thereof comprising one or two amino acid substitutions with respect to GGTFRSY; CDR2 comprises NPIFDT or a variant thereof comprising one or two amino acid substitutions with respect to NPIFDT; and CDR3 comprises TVIGAFDS or a variant thereof comprising one or two amino acid substitutions with respect to TVIGAFDS; Light chain: CDR1 comprises TRTSGDIGGYNFVS or a variant thereof comprising one, two or three amino acid substitutions with respect to TRTSGDIGGYNFVS CDR2 comprises DVNSRPS or a variant thereof comprising one or two amino acid substitutions with respect to DVNSRPS; and 008738577 7 CDR3 comprises SSYTSRNTV or a variant thereof comprising one or two amino acid substitutions with respect to SSYTSRNTV, 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. 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. 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. 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. 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. 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. 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. In another instance, the antibody or antigen-binding fragment comprises the following heavy and light chain CDRs: Heavy chain: CDR1 comprises GYIFTSY or a variant thereof comprising one or two amino acid substitutions with respect to GYIFTSY; 008738577 8 CDR2 comprises SAYNGH or a variant thereof comprising one or two amino acid substitutions with respect to SAYNGH; and CDR3 comprises GKISSWFVLED or a variant thereof comprising one, two or three amino acid substitutions with respect to GKISSWFVLED; Light chain: CDR1 comprises SGGTSNIGKNYVS or a variant thereof comprising one, two or three amino acid substitutions with respect to SGGTSNIGKNYVS CDR2 comprises DNNMRPS or a variant thereof comprising one or two amino acid substitutions with respect to DNNMRPS; and CDR3 comprises GMWIRSLSRWV or a variant thereof comprising one, two or three amino acid substitutions with respect to GMWIRSLSRWV, 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. 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. 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. 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. 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. 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 008738577 9 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. In another instance, the antibody or antigen-binding fragment comprises the following heavy and light chain CDRs: Heavy chain: CDR1 comprises GFTFSAY or a variant thereof comprising one or two amino acid substitutions with respect to GFTFSAY; CDR2 comprises SYDGNN or a variant thereof comprising one or two amino acid substitutions with respect to SYDGNN; and CDR3 comprises DFRDYVWGTYPSAY or a variant thereof comprising one, two or three amino acid substitutions with respect to DFRDYVWGTYPSAY; Light chain: CDR1 comprises SGSSSNIGRRHVF or a variant thereof comprising one, two or three amino acid substitutions with respect to SGSSSNIGRRHVF CDR2 comprises RGDQRPS or a variant thereof comprising one or two amino acid substitutions with respect to RGDQRPS; and CDR3 comprises ATWDDGLSGYV or a variant thereof comprising one, two or three amino acid substitutions with respect to ATWDDGLSGYV, 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. 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. 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. 008738577 10 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. 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. 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. 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. In another instance, the antibody or antigen-binding fragment thereof comprises the following heavy and light chain CDRs: Heavy chain: CDR1 comprises GGAFRNS or a variant thereof comprising one or two amino acid substitutions with respect to GGAFRNS; CDR2 comprises IPMSGT or a variant thereof comprising one or two amino acid substitutions with respect to IPMSGT; and CDR3 comprises EKERTFGVVMRTSYYYVMEV or a variant thereof comprising one, two, three or four amino acid substitutions with respect to EKERTFGVVMRTSYYYVMEV; Light chain: CDR1 comprises RASQSISSYLN or a variant thereof comprising one or two amino acid substitutions with respect to RASQSISSYLN 008738577 11 CDR2 comprises AASSLQS or a variant thereof comprising one or two amino acid substitutions with respect to AASSLQS; and CDR3 comprises QQSYSTPYT or a variant thereof comprising one or two amino acid substitutions with respect to QQSYSTPYT, wherein complementarity determining regions (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. 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. 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. 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. 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. 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. 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 008738577 12 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. In another aspect, the invention provides antibodies and antigen-binding fragments thereof, as defined herein, for use in medicine. Relatedly, the antibodies and antigen-binding fragments of the invention may be used in methods of treating a subject, e.g. a cancer patient. In embodiments in which the antibody or antigen-binding fragment thereof is for use in treating a cancer in a patient, the antibody or antigen- binding fragment thereof preferably comprises an Fc region that preferentially binds an activating Fc receptor as defined herein. In other embodiments, the antibody or antigen-binding fragment thereof is for use in treating an autoimmune condition. In these embodiments, the antibody or antigen-binding fragment thereof preferably comprises an Fc region that preferentially binds an inhibitory Fc receptor as defined herein. In some embodiments where the antibody or antigen-binding fragment is for use in medicine, including for use in a method of treating cancer, the use comprises systemic administration of the antibody or antigen- binding fragment. In some embodiments, the invention provides an antibody or antigen-binding fragment which is competitive for binding to F-actin with the antibody or antigen-binding fragment as defined in whole or in part by their amino acid sequences as described herein above (which may be termed a reference antibody). The antibody or antigen-binding fragment may compete with the reference antibody by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. The antibody or antigen- binding fragment may be capable of neutralising the binding of the reference antibody to F-actin. In another aspect, the invention provides a method of providing and / or 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, and contacting the binding substrate with trypsin or with a competitor agent that comprises 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. In one embodiment, the binding substrate is contacted with trypsin. In another embodiment, the binding substrate is contacted with a competitor agent that comprises the CTLD of DNGR-1. In some embodiments, the antibody is selected because it has 10-fold specificity for F-actin over G-actin. In these embodiments, the method of selecting the antibody comprises screening said one or more lead antibodies for 10-fold specificity. In some embodiments, the antibody is selected because it has at least 3-fold specificity for binding necrotic cells binding over live cells. In these embodiments, the method of selecting the antibody comprises performing FACS or flow cytometry to screen said one or more lead antibodies for at least 3- fold specificity for binding necrotic cells binding over live cells. 008738577 13 In some embodiments, the antibody is selected because it has capacity to promote cross-presentation. In these embodiments, the method of selecting the antibody comprises screening said one or more lead antibodies for the capacity to promote cross-presentation. In some embodiments, the antibody is selected because it has capacity to promote phagocytosis. In these embodiments, the method of selecting the antibody comprises screening said one or more lead antibodies for the capacity to promote phagocytosis. In some embodiments, the method of selecting the antibody comprises an initial step of ‘panning’ the population of candidate antibodies before contacting the binding substrate with the population. The population may be panned against F-actin beads (positive screen) and G-actin beads (negative screen). In some embodiments, the antibody binds to the same epitope, or overlaps with the same epitope, on F- actin as DNGR-1. The epitope that DNGR-1 binds to was elucidated in Hanĉ et al 2015, which is hereby incorporated by reference in it entirety. Hanĉ et al show that DNGR-1 binds the interface of two actin protofilaments. The epitope comprises three actin subunits helically arranged in the actin filament, bridging over two protofilaments, as well as two neighbouring actin subunits along one protofilament. Thus, the F-actin antibody of the invention is specific for F-actin over G-actin because its epitope constitutes part of the fully formed tertiary structure of the F-actin filament (a conformational epitope). G- actin does not comprise this 3D structure. Hanĉ et al disclose that the most extensive interactions between DNGR-1 and F-actin are between the actin subunit 2. There were moderate interactions with subunit 1 and only weak interactions were seen with subunit 3. Thus, subunit 3 was deemed largely dispensable for binding. 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 at least 2-fold, at least 5-fold, at least 10-fold, at least 20- fold, or at least 50-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. In another aspect, the invention provides an antibody produced by the method of the invention. In some embodiments, the antibody is polyclonal. The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. 008738577 14 Sequences The following amino acid sequences relate to the antibodies of the invention. Antibody A, VH 1 QVQLVQSGAE VKKPGSSVKV SCKSSGGTFR SYIISWVRQA PGQGLEWMGG FNPIFDTTIY AQKFQGRVTF 71 TADESTSTAY MDLSSLRSED TAVYYCARTV IGAFDSWGQG TPVTVSS Antibody A, VL 1 QSALTQPASV SGSPGQSITI SCTRTSGDIG GYNFVSWYQQ HPGKAPKLMI YDVNSRPSGV SDRFSGSKSG 71 DTASLTISGL QAEDEADYYC SSYTSRNTVF GGGTKLTVL The FRs and CDRs of Antibody A can be defined as follows, using the Chothia system: FR-H1 QVQLVQSGAEVKKPGSSVKVSCKSS CDR-H1 GGTFRSY FR-H2 IISWVRQAPGQGLEWMGGF CDR-H2 NPIFDT FR-H3 TIYAQKFQGRVTFTADESTSTAYMDLSSLRSEDTAVYYCAR CDR-H3 TVIGAFDS FR-H4 WGQGTPVTVSS FR-L1 QSALTQPASVSGSPGQSITISC CDR-L1 TRTSGDIGGYNFVS FR-L2 WYQQHPGKAPKLMIY CDR-L2 DVNSRPS FR-L3 GVSDRFSGSKSGDTASLTISGLQAEDEADYYC CDR-L3 SSYTSRNTV FR-L4 FGGGTKLTVL The FRs and CDRs of Antibody A can be defined as follows, using the Kabat system: FR-H1 QVQLVQSGAEVKKPGSSVKVSCKSSGGTFR CDR-H1 SYIIS FR-H2 WVRQAPGQGLEWMG CDR-H2 GFNPIFDTTIYAQKFQG FR-H3 RVTFTADESTSTAYMDLSSLRSEDTAVYYCAR CDR-H3 TVIGAFDS FR-H4 WGQGTPVTVSS FR-L1 QSALTQPASVSGSPGQSITISC CDR-L1 TRTSGDIGGYNFVS 008738577 15 FR-L2 WYQQHPGKAPKLMIY CDR-L2 DVNSRPS FR-L3 GVSDRFSGSKSGDTASLTISGLQAEDEADYYC CDR-L3 SSYTSRNTV FR-L4 FGGGTKLTVL The FRs and CDRs of Antibody A can be defined as follows, using the IMGT system: FR-H1 QVQLVQSGAEVKKPGSSVKVSCKSS CDR-H1 GGTFRSYI FR-H2 ISWVRQAPGQGLEWMGG CDR-H2 FNPIFDTT FR-H3 IYAQKFQGRVTFTADESTSTAYMDLSSLRSEDTAVYYC CDR-H3 ARTVIGAFDS FR-H4 WGQGTPVTVSS FR-L1 QSALTQPASVSGSPGQSITISCTRT CDR-L1 SGDIGGYNF FR-L2 VSWYQQHPGKAPKLMIY CDR-L2 DVN FR-L3 SRPSGVSDRFSGSKSGDTASLTISGLQAEDEADYYC CDR-L3 SSYTSRNTV FR-L4 FGGGTKLTVL The FRs and CDRs of Antibody A can be defined as follows, using the Martin / Contact system: FR-H1 QVQLVQSGAEVKKPGSSVKVSCKSSGGTF CDR-H1 RSYIIS FR-H2 WVRQAPGQGLE CDR-H2 WMGGFNPIFDTTI FR-H3 YAQKFQGRVTFTADESTSTAYMDLSSLRSEDTAVYYC CDR-H3 ARTVIGAFD FR-H4 SWGQGTPVTVSS FR-L1 QSALTQPASVSGSPGQSITISCTRTSGD CDR-L1 IGGYNFVSWY FR-L2 QQHPGKAPK CDR-L2 LMIYDVNSRP FR-L3 SGVSDRFSGSKSGDTASLTISGLQAEDEADYYC 008738577 16 CDR-L3 SSYTSRNT FR-L4 VFGGGTKLTVL Antibody B, VH1 QMQLVQSGAE VKKPGASVKV SCKAPGYIFT SYGISWVRQA PGQGLEWMGR ISAYNGHTNY AQNLQDRVTL 71 TTDTSTSTAY MELRSLRYDD TAIYYCATGK ISSWFVLEDW GQGTQVTVSS Antibody B, VL 1 QSVVTQPPSV SAAPGQKVTI SCSGGTSNIG KNYVSWYQQL PGTAPRLLIY DNNMRPSGIP DRFSGSKSGT 71 SATLAITGLQ TGDEADYYCG MWIRSLSRWV FGGGTKLAVL The framework regions (FRs) and CDRs of Antibody B can be defined as follows, using the Chothia system: FR-H1 QMQLVQSGAEVKKPGASVKVSCKAP CDR-H1 GYIFTSY FR-H2 GISWVRQAPGQGLEWMGRI CDR-H2 SAYNGH FR-H3 TNYAQNLQDRVTLTTDTSTSTAYMELRSLRYDDTAIYYCAT CDR-H3 GKISSWFVLED FR-H4 WGQGTQVTVSS FR-L1 QSVVTQPPSVSAAPGQKVTISC CDR-L1 SGGTSNIGKNYVS FR-L2 WYQQLPGTAPRLLIY CDR-L2 DNNMRPS FR-L3 GIPDRFSGSKSGTSATLAITGLQTGDEADYYC CDR-L3 GMWIRSLSRWV FR-L4 FGGGTKLAVL The FRs and CDRs of Antibody B can be defined as follows, using the Kabat system: FR-H1 QMQLVQSGAEVKKPGASVKVSCKAPGYIFT CDR-H1 SYGIS FR-H2 WVRQAPGQGLEWMG CDR-H2 RISAYNGHTNYAQNLQD FR-H3 RVTLTTDTSTSTAYMELRSLRYDDTAIYYCAT CDR-H3 GKISSWFVLED FR-H4 WGQGTQVTVSS FR-L1 QSVVTQPPSVSAAPGQKVTISC 008738577 17 CDR-L1 SGGTSNIGKNYVS FR-L2 WYQQLPGTAPRLLIY CDR-L2 DNNMRPS FR-L3 GIPDRFSGSKSGTSATLAITGLQTGDEADYYC CDR-L3 GMWIRSLSRWV FR-L4 FGGGTKLAVL The FRs and CDRs of Antibody B can be defined as follows, using the IMGT system: FR-H1 QMQLVQSGAEVKKPGASVKVSCKAP CDR-H1 GYIFTSYG FR-H2 ISWVRQAPGQGLEWMGR CDR-H2 ISAYNGHT FR-H3 NYAQNLQDRVTLTTDTSTSTAYMELRSLRYDDTAIYYC CDR-H3 ATGKISSWFVLED FR-H4 WGQGTQVTVSS FR-L1 QSVVTQPPSVSAAPGQKVTISCSGG CDR-L1 TSNIGKNY FR-L2 VSWYQQLPGTAPRLLIY CDR-L2 DNN FR-L3 MRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYC CDR-L3 GMWIRSLSRWV FR-L4 FGGGTKLAVL The FRs and CDRs of Antibody B can be defined as follows, using the Martin / Contact system: FR-H1 QMQLVQSGAEVKKPGASVKVSCKAPGYIF CDR-H1 TSYGIS FR-H2 WVRQAPGQGLE CDR-H2 WMGRISAYNGHTN FR-H3 YAQNLQDRVTLTTDTSTSTAYMELRSLRYDDTAIYYC CDR-H3 ATGKISSWFVLE FR-H4 DWGQGTQVTVSS FR-L1 QSVVTQPPSVSAAPGQKVTISCSGGTSN CDR-L1 IGKNYVSWY FR-L2 QQLPGTAPR CDR-L2 LLIYDNNMRP 008738577 18 FR-L3 SGIPDRFSGSKSGTSATLAITGLQTGDEADYYC CDR-L3 GMWIRSLSRW FR-L4 VFGGGTKLAVL Antibody C, VH 1 QVQLVESGGG VVQPGRSLRL SCAASGFTFS AYAMHWVRQA PGKGLEWMAV ISYDGNNIHY ADSVKGRFTV 71 SRDNSKNTLF LQMDGLRTED TAVYYCARDF RDYVWGTYPS AYWGQGTLVT VSS Antibody C, VL 1 QSVLTQPPSV SGTPGQRVII SCSGSSSNIG RRHVFWYQQF PESAPKLLIY RGDQRPSGVP ERYSGSKSGT 71 SASLAISGLR SEDEADYYCA TWDDGLSGYV FGTGTRVTVL The FRs and CDRs of Antibody C can be defined as follows, using the Chothia system: FR-H1 QVQLVESGGGVVQPGRSLRLSCAAS CDR-H1 GFTFSAY FR-H2 AMHWVRQAPGKGLEWMAVI CDR-H2 SYDGNN FR-H3 IHYADSVKGRFTVSRDNSKNTLFLQMDGLRTEDTAVYYCAR CDR-H3 DFRDYVWGTYPSAY FR-H4 WGQGTLVTVSS FR-L1 QSVLTQPPSVSGTPGQRVIISC CDR-L1 SGSSSNIGRRHVF FR-L2 WYQQFPESAPKLLIY CDR-L2 RGDQRPS FR-L3 GVPERYSGSKSGTSASLAISGLRSEDEADYYC CDR-L3 ATWDDGLSGYV FR-L4 FGTGTRVTVL The FRs and CDRs of Antibody C can be defined as follows, using the Kabat system: FR-H1 QVQLVESGGGVVQPGRSLRLSCAASGFTFS CDR-H1 AYAMH FR-H2 WVRQAPGKGLEWMA CDR-H2 VISYDGNNIHYADSVKG FR-H3 RFTVSRDNSKNTLFLQMDGLRTEDTAVYYCAR CDR-H3 DFRDYVWGTYPSAY FR-H4 WGQGTLVTVSS FR-L1 QSVLTQPPSVSGTPGQRVIISC 008738577 19 CDR-L1 SGSSSNIGRRHVF FR-L2 WYQQFPESAPKLLIY CDR-L2 RGDQRPS FR-L3 GVPERYSGSKSGTSASLAISGLRSEDEADYYC CDR-L3 ATWDDGLSGYV FR-L4 FGTGTRVTVL The FRs and CDRs of Antibody C can be defined as follows, using the IMGT system: FR-H1 QVQLVESGGGVVQPGRSLRLSCAAS CDR-H1 GFTFSAYA FR-H2 MHWVRQAPGKGLEWMAV CDR-H2 ISYDGNNI FR-H3 HYADSVKGRFTVSRDNSKNTLFLQMDGLRTEDTAVYYC CDR-H3 ARDFRDYVWGTYPSAY FR-H4 WGQGTLVTVSS FR-L1 QSVLTQPPSVSGTPGQRVIISCSGS CDR-L1 SSNIGRRH FR-L2 VFWYQQFPESAPKLLIY CDR-L2 RGD FR-L3 QRPSGVPERYSGSKSGTSASLAISGLRSEDEADYYC CDR-L3 ATWDDGLSGYV FR-L4 FGTGTRVTVL FRs and CDRs of Antibody C can be defined as follows, using the Contact system: FR-H1 QVQLVESGGGVVQPGRSLRLSCAASGFTF CDR-H1 SAYAMH FR-H2 WVRQAPGKGLE CDR-H2 WMAVISYDGNNIH FR-H3 YADSVKGRFTVSRDNSKNTLFLQMDGLRTEDTAVYYC CDR-H3 ARDFRDYVWGTYPSA FR-H4 YWGQGTLVTVSS FR-L1 QSVLTQPPSVSGTPGQRVIISCSGSSSN CDR-L1 IGRRHVFWY FR-L2 QQFPESAPK CDR-L2 LLIYRGDQRP 008738577 20 FR-L3 SGVPERYSGSKSGTSASLAISGLRSEDEADYYC CDR-L3 ATWDDGLSGY FR-L4 VFGTGTRVTVL Antibody D, VH 1 QVQLVQSGAE VKKTGSSVKV SCKVSGGAFR NSAINWVRQA PGRGLEWMGV IIPMSGTTNY ARNFQGRVTI 71 SADESTSTAY MELSTLTSGD TADYYCAREK ERTFGVVMRT SYYYVMEVWG QGTTVTVSS Antibody D, VL 1 AIRMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD 71 FTLTISSLQP EDFATYYCQQ SYSTPYTFGQ GTKLEIK The FRs and CDRs of Antibody D can be defined as follows, using the Chothia system: FR-H1 QVQLVQSGAEVKKTGSSVKVSCKVS CDR-H1 GGAFRNS FR-H2 AINWVRQAPGRGLEWMGVI CDR-H2 IPMSGT FR-H3 TNYARNFQGRVTISADESTSTAYMELSTLTSGDTADYYCAR CDR-H3 EKERTFGVVMRTSYYYVMEV FR-H4 WGQGTTVTVSS FR-L1 IRMTQSPSSLSASVGDRVTITC CDR-L1 RASQSISSYLN FR-L2 WYQQKPGKAPKLLIY CDR-L2 AASSLQS FR-L3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC CDR-L3 QQSYSTPYT FR-L4 FGQGTKLEIK The FRs and CDRs of Antibody D can be defined as follows, using the Kabat system: FR-H1 QVQLVQSGAEVKKTGSSVKVSCKVSGGAFR CDR-H1 NSAIN FR-H2 WVRQAPGRGLEWMG CDR-H2 VIIPMSGTTNYARNFQG FR-H3 RVTISADESTSTAYMELSTLTSGDTADYYCAR CDR-H3 EKERTFGVVMRTSYYYVMEV FR-H4 WGQGTTVTVSS FR-L1 IRMTQSPSSLSASVGDRVTITC 008738577 21 CDR-L1 RASQSISSYLN FR-L2 WYQQKPGKAPKLLIY CDR-L2 AASSLQS FR-L3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC CDR-L3 QQSYSTPYT FR-L4 FGQGTKLEIK The FRs and CDRs of Antibody D can be defined as follows, using the IMGT system: FR-H1 QVQLVQSGAEVKKTGSSVKVSCKVS CDR-H1 GGAFRNSA FR-H2 INWVRQAPGRGLEWMGV CDR-H2 IIPMSGTT FR-H3 NYARNFQGRVTISADESTSTAYMELSTLTSGDTADYYC CDR-H3 AREKERTFGVVMRTSYYYVMEV FR-H4 WGQGTTVTVSS FR-L1 IRMTQSPSSLSASVGDRVTITCRAS CDR-L1 QSISSY FR-L2 LNWYQQKPGKAPKLLIY CDR-L2 AAS FR-L3 SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC CDR-L3 QQSYSTPYT FR-L4 FGQGTKLEIK The FRs and CDRs of Antibody D can be defined as follows, using the Martin / Contact system: FR-H1 QVQLVQSGAEVKKTGSSVKVSCKVSGGAF CDR-H1 RNSAIN FR-H2 WVRQAPGRGLE CDR-H2 WMGVIIPMSGTTN FR-H3 YARNFQGRVTISADESTSTAYMELSTLTSGDTADYYC CDR-H3 AREKERTFGVVMRTSYYYVME FR-H4 VWGQGTTVTVSS FR-L1 IRMTQSPSSLSASVGDRVTITCRASQSI CDR-L1 SSYLNWY FR-L2 QQKPGKAPK CDR-L2 LLIYAASSLQ 008738577 22 FR-L3 SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC CDR-L3 QQSYSTPY FR-L4 TFGQGTKLEIK Antibody E, VH 1 QVQLVQSGAE VKKTGSSVKV SCKVFGGAFR NSAINWVRQA PGRGLEWMGV LIPMSGTTNY ARNFQGRVTI 71 SADESTSTAY MELSTLTSGD TAVYYCAREK ERTFGVVMRT SYYYVMEVWG QGTTVTVSS Antibody E, VL 1 AIRMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD 71 FTLTISSLQP EDFATYYCQQ SYSTPYTFGQ GTKLEIK The FRs and CDRs of Antibody E can be defined as follows, using the Chothia system: FR-H1 QVQLVQSGAEVKKTGSSVKVSCKVF CDR-H1 GGAFRNS FR-H2 AINWVRQAPGRGLEWMGVL CDR-H2 IPMSGT FR-H3 TNYARNFQGRVTISADESTSTAYMELSTLTSGDTAVYYCAR CDR-H3 EKERTFGVVMRTSYYYVMEV FR-H4 WGQGTTVTVSS FR-L1 IRMTQSPSSLSASVGDRVTITC CDR-L1 RASQSISSYLN FR-L2 WYQQKPGKAPKLLIY CDR-L2 AASSLQS FR-L3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC CDR-L3 QQSYSTPYT FR-L4 FGQGTKLEIK The FRs and CDRs of Antibody E can be defined as follows, using the Kabat system: FR-H1 QVQLVQSGAEVKKTGSSVKVSCKVFGGAFR CDR-H1 NSAIN FR-H2 WVRQAPGRGLEWMG CDR-H2 VLIPMSGTTNYARNFQG FR-H3 RVTISADESTSTAYMELSTLTSGDTAVYYCAR CDR-H3 EKERTFGVVMRTSYYYVMEV FR-H4 WGQGTTVTVSS FR-L1 IRMTQSPSSLSASVGDRVTITC 008738577 23 CDR-L1 RASQSISSYLN FR-L2 WYQQKPGKAPKLLIY CDR-L2 AASSLQS FR-L3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC CDR-L3 QQSYSTPYT FR-L4 FGQGTKLEIK The FRs and CDRs of Antibody E can be defined as follows, using the IMGT system: FR-H1 QVQLVQSGAEVKKTGSSVKVSCKVF CDR-H1 GGAFRNSA FR-H2 INWVRQAPGRGLEWMGV CDR-H2 LIPMSGTT FR-H3 NYARNFQGRVTISADESTSTAYMELSTLTSGDTAVYYC CDR-H3 AREKERTFGVVMRTSYYYVMEV FR-H4 WGQGTTVTVSS FR-L1 IRMTQSPSSLSASVGDRVTITCRAS CDR-L1 QSISSY FR-L2 LNWYQQKPGKAPKLLIY CDR-L2 AAS FR-L3 SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC CDR-L3 QQSYSTPYT FR-L4 FGQGTKLEIK The FRs and CDRs of Antibody E can be defined as follows, using the Contact system: FR-H1 QVQLVQSGAEVKKTGSSVKVSCKVFGGAF CDR-H1 RNSAIN FR-H2 WVRQAPGRGLE CDR-H2 WMGVLIPMSGTTN FR-H3 YARNFQGRVTISADESTSTAYMELSTLTSGDTAVYYC CDR-H3 AREKERTFGVVMRTSYYYVME FR-H4 VWGQGTTVTVSS FR-L1 IRMTQSPSSLSASVGDRVTITCRASQSI CDR-L1 SSYLNWY FR-L2 QQKPGKAPK CDR-L2 LLIYAASSLQ 008738577 24 FR-L3 SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC CDR-L3 QQSYSTPY FR-L4 TFGQGTKLEIK The antibody of the invention can be defined as having six CDR sequences that correspond to the CDR sequences of one of the clones defined above. Summary of the Figures Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which: Figure 1. Phage selection funnel for F-actin monoclonal antibodies. Figure 2. Ranking of anti-F-actin IgG2a clones by ability to induce necrotic cell XP in cDC2. Hoxb8- CDP-derived cDC2 were incubated with OVA-soaked necrotic 5555 BrafV600Emelanoma cells with a dose titration (50, 10, 2, 0.4 nM) of 2WA Fc-DNGR-1 (black circles), WT Fc-DNGR-1 (white circles), or anti-F- actin IgG2a clones (white triangles) and OT-I CD8+ T cells. T cell-derived IFNγ in supernatants was measured by ELISA after 24 h. Area under the curve (AUC) was calculated for IFNγ production for each mAb and ranked in descending manner. Prioritised clones are identified by the grey box. Figure 3. Repeat XP of candidate anti-F-actin IgG2a clones in cDC2. Hoxb8-CDP-derived cDC2 were incubated with OVA-soaked necrotic 5555 BrafV600Emelanoma cells with a dose titration of 2WA Fc- DNGR-1 (black circles), WT Fc-DNGR-1 (white circles), or anti-F-actin IgG2a clones (white triangles) and OT-I CD8+ T cells. T cell-derived IFNγ in supernatants was measured by ELISA after 24 h. Mean + / - SEM is shown from duplicate measurements. Figure 4. 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. Figure 5. Necrotic cell binding of candidate anti-F-actin clones. Flow cytometric analysis of UV- irradiated necrotic mouse 5555 BrafV600Emelanoma cells (A) or necrotic human HEK293T cells (B) incubated with serial dilutions of anti-F-actin IgG2a 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. Figure 6. Necrotic cell phagocytosis by primary cDC in the presence of anti-F-actin IgG2a. Flow cytometric analysis of internalisation of CT-DR-labelled necrotic cell debris by IFN-I-primed primary FLT3L-cDC from murine bone marrow in the presence of F-actin binding reagents. cDC1 (A) and cDC2 (B) were analysed after 4 h of incubation and phagocytic index calculated using the following formula: (% of CT-DR positive cDC x GMFI of CT-DR positive cDC) / 10,000. Mean + / - SEM is shown from duplicate measurements. 008738577 25 Figure 7. Necrotic cell XP by primary cDC in the presence of anti-F-actin IgG2a. Primary IFN-I- primed bone marrow FLT3L-cDC cultures were separated into cDC1 and non-cDC1 fractions using XCR1-targeted MACS enrichment. These were then incubated with OVA-soaked UV-irradiated necrotic 5555 BrafV600Enecrotic cells and a serial dilution of WT Fc-DNGR-1, irrelevant IgG2a, or anti-F-actin IgG2a clones and OT-I CD8+ T cells. T cell-derived IFNγ was measured after 24 h for both cDC1 (A) and non-cDC1 (B). Mean + / - SEM is shown from duplicate measurements. Figure 8. Necrotic cell binding of candidate commercial anti-actin clones. Flow cytometric analysis of UV-irradiated necrotic mouse 5555 BrafV600Emelanoma cells incubated with serial dilutions of anti-actin IgG2a clones (AC-40 or AC-74) or Fc-DNGR-1 fusion proteins (2WA or WT) and stained with secondary AF488-conjugated anti-mouse IgG. Figure 9. Necrotic cell phagocytosis by primary cDC in the presence of commercial anti-actin IgG2a. Flow cytometric analysis of internalisation of CT-DR-labelled necrotic cell debris by IFN-I-primed primary FLT3L-cDC from murine bone marrow in the presence of anti-actin IgG2a clones (AC-40 and AC- 74) or Fc-DNGR-1 (2WA or WT). cDC1 (A) and cDC2 (B) were analysed after 4 h of incubation and phagocytic index calculated using the following formula: (% of CT-DR positive cDC x GMFI of CT-DR positive cDC) / 10,000. Mean + / - SEM is shown from duplicate measurements. Figure 10. Necrotic cell XP by primary cDC in the presence of commercial anti-F-actin IgG2a. Primary IFN-I-primed bone marrow FLT3L-cDC cultures were separated into cDC1 and non-cDC1 fractions using XCR1-targeted MACS enrichment. These were then incubated with OVA-soaked UV- irradiated necrotic 5555 BrafV600Enecrotic cells and a serial dilution of 2WA or WT Fc-DNGR-1 or commercial anti-actin IgG2a (AC-40 or AC-74) and OT-I CD8+ T cells. T cell-derived IFNγ was measured after 24 h for both cDC1 (A) and non-cDC1 (B). Mean + / - SEM is shown from duplicate measurements. Figure 11. Ranking anti-actin antibodies for potency and efficacy in XP. EC50 (potency) and maximum IFNγ signal (efficacy) for each antibody / Fc-DNGR-1 were calculated from XP results in Figure 10 between cDC1 (A) and non-cDC1 (B). Dotted line shows IFNγ signal in the absence of monoclonal antibodies. Figure 12. G-actin competition for necrotic cell binding by anti-actin antibodies. Flow cytometric analysis of UV-irradiated necrotic mouse 5555 BrafV600Emelanoma 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. Figure 13. Tumour control by anti-F-actin IgG2a antibodies. Anti-F-actin IgG2a clones or Fc-DNGR-1 (WT or 2WA) were administered intra-tumourally (100 μg for Fc-DNGR-1 or 150 μg for anti-F-actin IgG2a in 50 μl) to C57BL / 6 mice bearing MCA205 tumours. Agents were administered in combination with 1.25 mg / kg doxorubicin on day 7 to induce immunogenic cell death. A second dose of the antibodies / Fc- DNGR-1 was administered on day 11. N = 10 per group. Mean + / - SEM is plotted. (A) Schematic of experimental set-up. (B) Tumour volumes measured with Fc-DNGR-1 control and antibodies A, B and E. 008738577 26 Figure 14. 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. 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 μg) or antibody A (p.t.150 μg) 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 IgG2a (400 μg i.p.). Antibody A was detected using either anti-mouse IgG2a (top row) or anti-CH1-IgG (bottom row). Mean ± SEM is plotted. (D) Tumour growth profiles from MC38- implanted C57BL / 6 mice treated systemically with anti-F-actin or isotype IgG2a (400 μg 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. (F) Schematics and standard curves of anti-DNGR-1 (top) and anti-human CH1 (bottom) ELISAs for PK analysis. 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. Figure 16. Necrotic cells localise close to FcγR+APC in tumours. (A) Confocal microscopy of 20 μm sections of BrafV600Emelanoma tumour spheroids cultured in vitro with intracellular binding reagents. (B) Representative confocal images of co-localisation of actin cytoskeleton- and DNA-binding reagents from A. (C) Example filamentous staining pattern of Fc-DNGR-1 on necrotic cells within tumour spheroids, prepared as in A. (D) Top: Confocal microscopy of murine MCA205 LifeAct (LA)-OVA tumours (day 8 post-implantation into C57BL / 6 mice) after local injection with WT or 2WA Fc-DNGR-1 (n = 9 independent tumours). Bottom: Data pooled from multiple experiments and mean ± SEM is plotted. (E) Top: Confocal microscopy to identify 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 μm distance to necrotic core analysed. Bottom: Mean is plotted. (F) Confocal microscopy showing staining of MCA205 LA-OVA-mCherry tumours with anti-mouse FcγRI. Arrows indicate cells co- staining for MHC-II and FcγRI. Data are representative of two (E), three (A-C) or more (D) 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. Figure 17. Commercial anti-actin antibody binding to F-actin. ELISA assessment of commercial anti- actin and AFA binding to F-actin-coated plates. 008738577 27 Figure 18. Human IgG1 AFAs compared to 8-3. (A) ELISA analysis of human IgG1 AFA and 8-3 antibody binding to F-actin-coated plates. (B) Flow cytometric analysis of human IgG1 AFA and 8-3 binding to UV-irradiated necrotic mouse 5555V600Etumour cells. Figure 19. Fc receptor expression on human tumour cDC2 and moDCs. (A) Human intra-tumoural DC2 FCGR expression levels, assessed from single cell RNA sequencing (scRNAseq) data derived by interrogation of the human pan-cancer myeloid cell atlas portal (http: / / panmyeloid.cancer-pku.cn). (B) Flow cytometric analysis of Fc receptor expression on monocyte-derived DCs (GM-CSF / IL-4) generated from healthy human peripheral blood. Figure 20. Antibody A promotes necrophagy by human monocyte-derived DCs. (A) Confocal microscopy analysis of necrophagy in human moDCs incubated with UV-irradiated necrotic cells and WT and mutant AFA A IgG1. Uptake expressed as necrotic cells internalized per DC, with each dot representing a field of view (left) or the average uptake observed across different donors (right). Mean ± SEM is plotted. LALA-PG = Fc-null antibody; WT = conventional human IgG1 Fc; GASDALIE = Fc- enhanced antibody. (B) Confocal microscopy of necrophagy as in A in the presence of a Fc receptor- blocking antibody or a SYK inhibitor. Each dot represents a field of view. Data are representative of two or more independent experiments. Data was analysed using a one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Figure 21. Antibody A promotes necrophagy by human monocyte-derived macrophages. (A) Flow cytometric analysis of Fc receptor expression in human monocyte-derived macrophages (moMacs, M- CSF-derived). (B) Confocal microscopy analysis of necrophagy by human moMacs, as per moDCs in Figure 19. Data are representative of two or more independent experiments. Data was analysed using a one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Detailed Description of the Invention 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. F-actin 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. 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. 008738577 28 In general, alpha and gamma2 isotypes are found in muscle (alpha-skeletal, alpha-aortic smooth, alpha- cardiac and gamma2-enteric smooth) while beta and gamma1 isotypes are found in non-muscle cells (beta-cytoplasmic and gamma1-cytoplasmic). 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. 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”. 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. 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. F-actin can be stabilised by various molecules which bind to it and inhibit depolymerisation, such as phalloidin, jasplakinolide or tropomyosin. 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. 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). Exemplary human actin sequences include: Alpha Actin (alpha skeletal muscle) MCDEDETTALVCDNGSGLVKAGEFAGDDAPRAVEPSIVCGRPRHOGVMVGMGQKDSYVGDEAQSKRG ILTLKYPIEHGIITNWDDMEKIWHHTFYNELRVAPEEHPTLLTEAPLNPKANREKMIQIMEETENVPAMYVAI QAVLSLYASGRTTGIVLDSGDGVTHNVPIYEGYALPHAIMRLDLAGRDLTDYLMKILTERGYSEVITAEREI VRDIKEKLCYVALDFENEMATAASSSSLEKSYELPDGQVITIGNERFRCPETLEFQPSEFIGMESAGIHETT YNSIMKCDIDIRKDLYANNVMSGGTTMYPGIADRMOKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLST EFQOMWITKOEYDEAGPSIVHRKCE 008738577 29 Beta Actin (cytoplasmic 1) MDDDIAALVVDNGSGMCKAGEFAGDDAPRAVEPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTL KYPIEHGIVINWDDMEKIWHHTEYNELRVAPEEHPVLLTEAPLNPKANREKMTQIMFETENTPAMYVAIQA VLSLYASGRTTGIVMDSGDGVTHIVPIYEGYALPHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVRD IKEKLCYVALDFEQEMATAASSSSLEKSYBELPDGOVITIGNERFRCPEALFQPSEFLGMESCGIHETTEN SIMKCDVDIRKDLYANTVLSGGTTMYPGIADRMOKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTEQ OMWISKQEYDESGPSIVHRKCE Gamma Actin (cytoplasmic 2) MEEEIAALVIDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHQOGVMVGMGQOKDSYVGDEAQSKRGILTI LKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANREKMTQIMFETFNTPAMYVAIQ AVLSLYASGRTTGIVMDSGDGVIHIVPIYEGYALPHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVR DIKEKLCYVALDFEQEMATAASSSSLEKSYELPDGQVITIGNERFRCPEALFQPSFLGMESCGIHETTFNSI MKCDVDIRKDLYANTVLSGGITMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQO MWISKQEYDESGPSIVHRKCE Actin is a highly conserved protein, with 90% identity between yeast and humans (Srinivasan, 2016). Each monomer of actin is a 375-amino acid polypeptide which folds into two major α / β 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). 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. Anti-F-actin antibodies and antigen binding fragments 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 008738577 30 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). 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. Fragments of antibodies, such as Fab and Fab2 fragments may also be used, as can genetically engineered antibodies and antibody fragments. As used herein, an “antigen binding fragment” of an antibody is a fragment that comprises the antigen binding portion of the VH / VL 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). 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). 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. 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. 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 008738577 31 "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). An “antibody that binds to the same epitope” as DNGR-1 may refer to an antibody that blocks binding of DNGR-1 to its antigen in a competition assay by 50% or more, and conversely, DNGR-1 blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein. An “antibody that binds to the same epitope” as a reference antibody may refer to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein. As used herein, the term “AFA” refers to an “anti-F-actin antibody”. Preparation of anti-F-actin antibodies 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 Köhler, 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 "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). 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 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 Fcy receptor-mediated activity. Fc domains can be mutated to 008738577 32 alter the binding characteristics with their Fc receptors. Preferably, the antibody of the invention has an Fc domain. Fcγ receptors Preferably, the Fc domain of the antibody binds to an activating Fcγ receptor. Human activating Fcγ receptors include FcγRI, FcγRIIA, FcγRIIC and FcγRIIIB, whilst FcγRIIB is an inhibitory FcγR. Mouse activating Fcγ receptors include FcγRI, FcγRIII, and FcγRIV. Mouse FcγRIIB is an inhibitory FcγR. Both mice and human FcγRs display different affinities for different IgG Fc domains. Table 2 displays the binding affinities of various human FcγRs for different human IgG Fc domains and Table 3 displays the binding affinities of various mouse FcγRs for different mouse IgG Fc domains. Activatory Inhibitory IgG FcγRI FcγRIIA FcγRIIC FcγRIIIA FcγRIIIB FcγRIIB Variants - R / H131 - F / V158 - - IgG1 6x1073 / 5x1061x1051 / 2x1052x1051x105IgG2 - 1 / 4x1052 x1043 / 7x104- 2x104IgG3 6x1079x1052x1050.8 / 1x1071x1062x105IgG4 3x1072x1052x1052x105- 2x105Table 2: Affinities of human FcγR for human IgG Fc domains, indicated as KA (M-1). Adapted from Castro- Dopico, 2019 (which is hereby incorporated by reference in its entirety). Activatory Inhibitory IgG FcγRI FcγRIII FcγRIV FcγRIIB IgG1 - 3x105- 3x106IgG2a 3x1077x1053x1074x105IgG2b 1x1056x1052x1072x106IgG3 (+) - - - Table 3: Affinities of mouse FcγR for mouse IgG Fc domains, indicated as KA(M-1). Adapted from Castro- Dopico, 2019. 008738577 33 Substitutions and sequence identity 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. 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, Gln, Ser, Thr and Tyr; and non-polar amino acids are Ala, Gly, Val, Leu, Ile, Pro, Phe, Met, Trp and Cys. 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 Gln; basic amino acids are His, Arg and Lys; small hydrophobic amino acids are Met, Ile, Leu and Val; and aromatic amino acids are Phe, Tyr and Trp. Substitutions which score positive in the BLOSUM62 matrix are as follows: Original Residue C S T P A G N D E Q H R K M I L V F Y W Substitution - T S - S - S N D E N Q E I M M M Y H F A D E Q R Y K Q L L I I W F Y N H K K R V V V L W 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. 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. 008738577 34 Professional / non-professional antigen presenting cells 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. By binding to the Fc domain of the antibodies of the invention, professional APCs that do not specialise in XP of cell-associated antigens (cDC2, monocyte-derived cells) and potentially non-professional cells expressing FcγR (neutrophils) can be enabled to cross- present necrotic cell-associated antigens on MHC-I for activation of CD8+T cells in the context of necrotic cells. Pharmaceutical compositions 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. 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. 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. 008738577 35 Routes of Administration 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. 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. Combinations with other Anticancer treatments As described herein, the medical methods, medical uses and pharmaceutical compositions of the invention may involve the antibody in combination with another anticancer treatment. In some embodiments, the anticancer treatment is an additional immunotherapy. Currently, the most common cancer immunotherapies are checkpoint inhibitors. The antibody of the invention may be used in combination with a checkpoint inhibitor. Checkpoint inhibitors suitable for use in combination with the antibody 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-TGFβ, transforming growth factor-β; anti-TIM3, T cell membrane protein 3; or anti-CD27. Other immunotherapies, such as T cell therapy, can be used in conjunction with the antibodies disclosed herein. T cell therapies include administration of autologous or allogeneic T cells. In some embodiments, the antibody is administered in combination with a CAR-T cell (a T cell that expresses a chimeric antigen receptor). In some embodiments, the anticancer treatment is a cytotoxic chemotherapeutic, meaning that the antibody 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 antibody to bind to the tumour cells. Cytotoxic chemotherapeutic agents 008738577 36 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. In some embodiments, the anticancer treatment is radiotherapy. In some embodiments, the anticancer treatment is surgery. 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). Cancers 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. 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). 008738577 37 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. The cancer that is the subject of the treatments and medical uses of the present invention may be selected from the lists provided above. The epitope of an antibody may be determined using methods apparent to persons skilled in the art. For example, the antibody may be determined by X-ray crystallography, and / or by electron cryomicroscopy (Cryo-EM) (Hanĉ, et al, 2015). In another aspect, competition assays may be used to identify an antibody that competes with an anti-F- actin antibody as described herein for binding to F-actin. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope). Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol.66 (Humana Press, Totowa, NJ) . In an exemplary competition assay, immobilized antigen is incubated in a solution comprising a first labelled antibody that binds to the antigen and a second unlabelled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen. The second antibody may be present in a hybridoma supernatant. As a control, immobilized antigen is incubated in a solution comprising the first labelled antibody but not the second antibody. After incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed, and the amount of label associated with immobilized antigen is measured. If the amount of label associated with immobilized antigen is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). *** 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. 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 008738577 38 be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. 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. Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. 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. 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%. Examples MATERIALS & METHODS Mice C57BL / 6Jax and OT-I / Rag1- / -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. Murine primary cells and cell lines RPMI 1640 supplemented with 2 mM glutamine, 100 units / ml penicillin, 100 μg / ml streptomycin, non- essential amino acids, 10 mM HEPES, 50 μM 2-mercaptoethanol (all from Gibco) and 10 % heat- inactivated fetal calf serum (FCS) (R10+ medium) was used for all cell culture. BrafV600E5555 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 μg / ml streptomycin, and 10 % heat-inactivated FCS. 008738577 39 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 cDC cultures were additionally primed with 200 ng / ml IFNα (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. For Hoxb8-cDC2 generation, CDP were immortalized using the ER-Hoxb8 system. Briefly, bone marrow CDP (Lin-CD117-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 µM E2 (β-oestradiol, Sigma Aldrich). Immortalised CDP were separated from congenic bone marrow progenitors via FACS. To differentiate cDC 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. For pre-activated effector OT-I cultures, single cell suspensions were generated from spleen and lymph nodes of OT-I / Rag1- / -mice 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 (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. Human tumour Fc receptor expression Data from the human pan-cancer myeloid cell atlas was analysed at http: / / panmyeloid.cancer-pku.cn. All tumour types were analysed for tumour FCGR gene expression in cDC2. The following tumour types were analysed: kidney, colorectal cancer (CRC), breast cancer (BRCA), ovarian or fallopian tube carcinoma (OV-FTC), thyroid carcinoma (THCA), pancreatic adenocarcinoma (PAAD), melanoma (MEL), lung cancer, uterine corpus endometrial carcinoma (UCEC), hepatocellular carcinoma (HCC), stomach cancer (STAD), oesophageal carcinoma (ESCA), and nasopharyngeal cancer (NPC). Human primary cells Peripheral blood was obtained from healthy volunteers at the Francis Crick Institute with prior ethical approval from the local ethics committee. Monocytes were enriched using biotinylated anti-human CD14 IgG (clone HCD14, Biolegend) and anti-biotin microbeads (Miltenyi) as per the manufacturer’s instructions, followed by incubation with R10+ medium supplemented with 100 ng / ml GM-CSF (Peprotech) and 40 ng / ml IL-4 (Peprotech) for DCs or 50 ng / ml M-CSF (Peprotech) for macrophages. For the latter, medium was refreshed on day 3, and both DCs and macrophages were harvested on day 6-8. 008738577 40 Fc receptor expression profiling Human moDCs and moMacs were harvested with PBS / EDTA or 1X TrypLE (Thermo Fisher Scientific), washed 2X in DPBS, and incubated with anti-human antibodies (1:200 unless stated) against the following proteins for 45 min at 4oC: CD14 (clone HCD14, Biolegend, HLA-DR (clone L243, Biolegend), CD11c (clone, Biolegend), FcγRI (clone 10.1, BD Biosciences, 1:100), FcγRIIA (clone IV.3, StemCELL Technologies, 1:50), FcγRIIB (clone 2B6, Biolegend, 1:100), FcγRIIIA / B (clone 3G8, BD Biosciences, 1:100). LIVE / DEAD Fixable Blue Dead Cell Dye (Thermo Fisher Scientific) was used to exclude dead cells and samples were acquired on an LSRFortessa or FACSymphony (BD Biosciences). Data were analysed using FlowJo software version 10. 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 NheI 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. 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 NaCl (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 95ºC 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 (Bio- rad) and analysed on ImageQuant 800 (Amersham) imaging system. Large-scale production of Fc- DNGR-1 was contracted commercially (ImmunoPrecise (Netherlands) and FairJourney Biologics (Portugal)). 008738577 41 De novo anti-F-actin IgG generation Anti-F-actin IgG monoclonal antibody selection was performed using phage display using a naïve human Fab library, and was contracted commercially (FairJourney Biologics, Portugal). After three rounds of positive selection for F-actin binding and negative selection for G-actin binding, a total of 736 clones were identified. For each round of screening, F-actin-bound phages 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 (see below).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 flow cytometric analysis on live and necrotic HEK293 (human) and NIH3T3 (mouse) cells. This led to 68 clones with ≥3- fold dead cell selectivity. These clones were reformatted to murine IgG2a 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.45 clones met this threshold. Large-scale production of anti-F-actin mouse IgG2a, human CH1 domain-containing IgG2a, human IgG1 monoclonal antibodies, as well as Fc-mutant antibodies, was contracted commercially (ImmunoPrecise, Netherlands or Biointron, China). Commercial & previously described anti-actin antibodies Purified commercial anti-actin antibodies of the mouse IgG2a isotype that bound to either alpha, beta- actin or pan-actin were identified. Pan-actin-binding AC-40 was obtained from Abcam (ab11003). Beta- actin-specific AC-74 was obtained from Sigma Aldrich (A2228). Alpha-actin-specific C4 was obtained from Merck Millipore (MAB1501). Anti-F-actin antibody 8-3 was previously identified as an expanded clone in pancreatic cancer patients (Yao et al., 2023). Synthesis of this antibody as a human IgG1 was contracted commercially to Biointron (China) based on publicly available sequences enclosed within the original manuscript. Bio-F-actin: Mix 20 µl Bio-G-actin (1 mg / ml, i.e.20 µM, Cytoskeleton Inc.) with 20 µl non-biotinylated G-actin (1 mg / ml, i.e.20 µM, Cytoskeleton Inc.), 5 µl F-buffer (10 x polymerisation buffer, Cytoskeleton Inc) and 5 µl phalloidin (200 µM, 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 µM) and store at 4°C (do not freeze) for up to 4 weeks. Bio-G-actin: 008738577 42 Mix 2 x 20µl Bio-G-actin (1 mg / ml, i.e.20 µM, Cytoskeleton Inc.) with 5 µl G-buffer (General actin buffer, Cytoskeleton Inc) and 5 µl Cytochalasin D (200 µM, 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 µM), and store at 4°C (do not freeze) for up to 4 weeks. F-actin / myosin-II (FM) complexes were prepared as previously described (Canton, 2021). Lyophilized nonbiotinylated G-actin and myosin II (Cytoskeleton) were reconstituted in sterile water at the final concentration of 10 mg / ml and stored at −80°C. Before use, the G-actin aliquots were diluted into G-actin buffer to final concentration 1 mg / ml and incubated for at least 30 min on ice. To generate biotinylated F- actin, nonbiotinylated G-actin was mixed in a 1:1 molar ratio with biotinylated G-actin (Cytoskeleton), which was always freshly reconstituted. Then, 20 μg of each G-actin preparation was mixed with F-actin buffer and incubated for 1 h at RT. To complex the biotinylated F-actin with myosin II, F-actin was mixed in a 1:1 molar ratio with myosin II and incubated for 1 h at RT. For the coating of microbeads, OVA was biotinylated using the DSB-X biotinylation kit (Thermo Fisher Scientific). The concentration of biotinylated OVA was adjusted to 2 mg / ml. Streptavidin coated microbeads with a diameter of 2.0 μm (Polysciences) were used in nonfluorescent or yellow / green- fluorescent form and were labelled with biotinylated OVA (1:1000) for 1 h at 4ºC. The OVA bead preparations were washed with 1 % BSA in DPBS for 3 min at 10,000 x g. The OVA beads were then subjected to labelling with F-actin / myosin II. In vitro polymerized biotinylated F-actin / myosin II was added to OVA beads and incubated for at least 1 h on ice. Quantitation of monoclonal antibody (mAb) binding to F-actin via ELISA 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 µg / 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 or AP- conjugated anti-mouse or anti-human IgG (Jackson ImmunoResearch) antibody (0.16 µg / ml) and developed with an ELISA substrate for HRP- or AP-based detection (TMB, eBioscience or SIGMAFAST p-Nitrophenyl substrate solution, Sigma 008738577 43 Aldrich). 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). Necrotic cell binding assay 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 IgG2a, anti-F-actin IgG1 or commercially available / previously described anti-actin IgG 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 IgG2a (Thermo Fisher Scientific, 1:400) or AF568-conjugated anti-human IgG1 (Thermo Fisher Scientific, 1:400) at 4oC 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. Prior to irradiation, tumour cells were labelled with Cell Tracker-Deep Red (CT-DR) dye (Thermo Fisher Scientific; 1:1000 dilution) for 1 h at 37oC. CT-DR-labelled necrotic cells were added to cDC at a 2:1 ratio for 4 h in the presence of equimolar quantities of F-actin binding reagents. After 4 h, samples were surface stained for flow cytometry with a combination of the following: XCR1 (Biolegend, ZET), CD172a (Biolegend, P84), I-A / I-E (Biolegend, M5 / 114.15.2), B220 (BD Biosciences, RA3-6B2), and CD11c (Biolegend, N418). DAPI or LIVE / DEAD Fixable Blue Dead Cell Dye (Thermo Fisher Scientific) was used to exclude non-internalised necrotic cell material. Samples were acquired on an LSRFortessa or FACSymphony (BD Biosciences). Data were analysed using FlowJo software version 10. Phagocytic index integrates frequency and absolute quantity of phagocytosed material and was calculated using the following formula: (% CT-DR+ x CT-DR GMFI of CT-DR+) / 10,000. 5 x 104cDC1 or 1 x 105non-cDC1 were plated per well in U-bottomed 96-well plates.16 h post-UV irradiation, necrotic tumour cells were harvested from 10 cm tissue culture plates and soaked for 1 h at 37oC in 10 mg / ml albumin from chicken egg white (OVA, Sigma Aldrich) in RPMI 1640 medium before washing 3X in DPBS.1 ml OVA solution was used per 10 cm tissue culture plate. OVA-necrotic cells were incubated with cDC at a 2:1 ratio for 4 h in the presence of equimolar quantities of Fc-DNGR-1, anti- F-actin IgG2a, or commercial anti-actin IgG2a. 008738577 44 Pre-activated OT-I CD8+ T cells were generated by culturing splenic single cell suspensions in R10+ medium supplemented with 0.1 nM SIINFEKL peptide and / or 100 U / ml recombinant mouse IL-2 (Peprotech) for 4-5 days. Fresh medium containing 100 U / ml IL-2 was added every day and cells used on day 5 / 6.2:1 pre-activated OT-I were added to cDC cultures for 24 h and T cell-derived IFNγ release was measured by in-house ELISA. Briefly, 96-well high-affinity Nunc MaxiSorp plates (Thermo Fisher Scientific) were coated overnight with rat anti-mouse IFNγ IgG (clone R4-6A2, BD Biosciences, 8 μg / 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 T cell culture supernatants and recombinant IFNγ (Peprotech) standard curve samples for 2 h. After washing, plates were incubated with biotin rat anti-mouse IFNγ IgG (clone XMG1.2, BD Biosciences, 1 μg / ml in blocking buffer) for 2 h, then ExtrAvidin-Alkaline Phosphatase (Sigma Aldrich, 1:5,000 in blocking buffer) for 30 min, before developing with SIGMAFAST p-Nitrophenyl substrate solution (Sigma Aldrich), as per the manufacturer’s instructions. Absorbances at 405 nm (IFNγ signal) and 540 nm (background) were measured after 20-30 min on a Spark plate reader (Tecan) and absolute concentrations determined using the standard curve. Area under the curve, EC50 (potency), and maximum IFNγ signals (efficacy) were calculated in GraphPad Prism. 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 µg / 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. 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 Prism10. 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 μl) and injected s.c. in the shaved right flank of each recipient C57BL / 6Jax mouse. Tumour growth was monitored every 1 to 3 days, 008738577 45 and the longest tumour diameter (l) 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. Fc- DNGR-1 or anti-F-actin IgG2a was administered via intratumoural injection (100 μg in 50 μl PBS for Fc- DNGR-1; 150 μg in 50 μl PBS for anti-F-actin IgG2a) on day 7 and 11. On day 7, mice were additionally injected with 1.25 mg / kg doxorubicin (Merck Life Science UK). Necrotic cell phagocytosis assay - Microscopy For cDC / moDC / moMac imaging, glass coverslips (18 mm) were coated with a 0.1 % solution of poly-l- lysine (Sigma Aldrich) for 30 min at RT. The excess poly-l-lysine was washed 3X with DPBS. The coverslips were incubated in a 2.5% solution of glutaraldehyde (Sigma Aldrich) for 15 min at RT. After washing 3X with DPBS, coverslips were incubated in a solution of 1 μg / ml anti-mouse MHC class II (clone M5 / 114.15.2, Thermo Fisher Scientific) or anti-human HLA-DR (clone L243, Biolegend) in DPBS for 30 min at RT. Coverslips were washed again with DPBS and incubated overnight in 0.2 M glycine in DPBS at 4ºC. On the day of the experiment, the coverslips were washed 3X with fresh DPBS.2.5-5 x 105murine FLT3L cDC1s, non-cDC1s or Hoxb8-cDC2s, or human moDCs or moMacs were added to individual coverslips and allowed to attach for 1 h at 37ºC. Cells were then challenged with CT-DR / green- labelled necrotic cells ± anti-F-actin mAbs for 3-4 h. In some instances, human cells were pre-incubated for 30 min with 10 μg / ml anti-human FcγRIIA IgG (Biotechne) or 0.5 μM SYK inhibitor IV (Merck Millipore) prior to addition of necrotic cells. Cells were fixed in 4% paraformaldehyde, washed 3X with DPBS, and stained with rhodamine-conjugated wheat germ agglutinin (1:10,000) in blocking buffer for 30 min at RT. Washed coverslips were mounted onto glass slides using Prolong Diamond Antifade Mountant (Fisher Scientific). Samples were imaged on a Zeiss LSM880 inverted confocal microscope. Image processing and analysis was performed using Fiji / ImageJ or Imaris v9.1.2. Tumour microscopy For tumouroids, 5 x 103BrafV600Emelanoma 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 μg / ml Fc-DNGR-1, Alexa Fluor 555 phalloidin (1:400, Fisher Scientific), or 2 μg / 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. For tumours, 2.5 x 106MCA205-LA-OVA-mCherry tumour cells were injected into the shaved flanks of C57BL / 6 mice. After 7 days, Fc-DNGR-1 was injected peri-tumourally (50-200 μg) in DPBS and tumours harvested after 6-24 h. Alternatively, MCA205 tumours were harvested on day 21 post-injection (0.5 x 106cells) and 3 days after injection of isotype or anti-F-actin IgG2a (400 μg, i.p.). Tumours were fixed in 4% PFA overnight at 4ºC, followed incubation in 30% sucrose overnight at 4ºC. Tumouroids and tumours were embedded in O.C.T. (Tissue-Tek), sectioned (10-30 μm) using a cryostat (Leica), mounted onto SuperFrost Plus glass slides (Thermo Fisher Scientific), and stored at -80ºC. 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). 008738577 46 Sections were stained (all at 1:400 in blocking buffer, unless otherwise stated) with a combination of goat anti-mouse IgG2a-AF647 (Thermo Fisher Scientific), AF555-conjugated phalloidin (Fisher Scientific), DAPI, Hoechst (Thermo Fisher Scientific), and anti-mouse MHC class II FITC or AF594 (clone M5 / 114.15.2, 1:100, Thermo Fisher Scientific or Biolegend) for 1 h at RT. For certain tumour samples, sections were also incubated with goat anti-mouse CD103 (AF1990, 1:100, R&D Systems), rabbit polyclonal anti-RFP (600-401-379, 1:500, Rockland Inc.), and / or rabbit anti-mouse CD64 / FcγRI (clone 027, 1:1000, Sino) overnight at 4ºC before secondary staining with donkey anti-goat IgG (H+L) AF488 (A32814, Thermo Fisher Scientific), donkey anti-rabbit IgG AF405 (A48258, Thermo Fisher Scientific), and / or donkey anti-rabbit IgG (H+L) AF594 (A21207, Thermo Fisher Scientific) for 1 h at RT. Slides were washed and mounted using Prolong Diamond Antifade Mountant (Fisher Scientific), then imaged and analysed as above. For confocal microscopy of actin cytoskeleton binding, HeLa cells were fixed in 4% PFA for 20 min at RT, washed 3X DPBS, and permeabilised / blocked with 2% BSA with 0.3% Triton-X in DPBS for 15 min. Cells were stained with Hoechst (2.5 μg / ml), phalloidin-A647 (1:400), and anti-F-actin IgG2a (32 nM) for 1 h in 500 μl 2% BSA in DPBS. Samples were washed 3X, stained with anti-mouse IgG2a-AF488 for 1 h in 2% BSA in DPBS, washed and mounted using Prolong Diamond Antifade Mountant (Fisher Scientific), then imaged as above. Fc-DNGR-1 and anti-F-actin antibody pharmacokinetics analysis C57BL / 6 mice were injected i.p. with 100 μg 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 μg / 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 μg / ml) for 2 h, before developing with alkaline phosphatase, as above. Antibody A titre was calculated similarly but using CaptureSelect™ biotin anti-IgG-CH1 (Thermo Fisher Scientific) on ELISA plates coated with ExtrAvidin (Merck Life Science, 5 μg / ml) overnight, and goat anti-mouse IgG2a-alkaline phosphatase detection (SouthernBiotech). EXAMPLE 1 – Generation of candidate antibodies A phage selection funnel was used to select the lead antibody candidates (Figure 1). A total of 736 clones were generated by screening a naïve 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 008738577 47 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 IgG2a 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 4. F / G actin Dead cell / viable cell Dead cell / viable cell Cross presentation Ab ID binding binding (human) binding (murine) (AUC) A 12.4 389.4 237.3 2265 B 18.7 313.9 313.2 1861 C 12.8 361.9 186.4 1107 D 30.8 126.4 129.5 803.9 E 23.7 109.2 187.7 929.2 1 13.4 296.2 331.6 442 2 18.3 84.6 303.6 492.5 3 11.2 358 571.4 246.4 4 16.2 195.1 317.8 51.38 5 648 315.4 301.1 77.67 6 19.1 265 440.6 680.7 7 13.9 151.6 106.3 206.1 8 13.5 228.4 227.9 354.3 9 45.7 138.8 154.1 224 10 12.2 82.6 326.1 280.9 11 20.4 293.8 287.8 439.5 12 25.5 552.1 287.9 255.9 13 18.2 303.5 230.2 84.29 14 11.9 459.5 348.2 008738577 48 F / G actin Dead cell / viable cell Dead cell / viable cell Cross presentation Ab ID binding binding (human) binding (murine) (AUC) 15 15.7 375.8 322.1 296.4 16 15.7 279.7 246.3 383.3 17 29 483.4 444.4 234 18 42.8 171.8 231 304.4 19 6.6 333.5 188.2 406.5 20 24.2 73.3 181.7 256.4 21 35.1 805.8 359.2 201.2 22 32.8 536.1 366.8 215.4 23 34.1 343 289.6 24 41.9 81.7 271.3 509.7 25 240 314.1 209.8 225.5 26 49.1 162.8 308.7 256.7 27 21.3 177 247.5 252.7 28 29.4 313.4 284.5 425.5 29 24.4 751.3 308.8 212 30 32.8 452.1 184.1 447.5 31 30.3 541.6 218.6 278.6 32 62.1 380.1 175 170.4 33 32.4 488.9 184.2 509.8 34 19 149.5 51.1 200.4 35 29.1 631.6 183.9 361.2 36 541 394.8 218.9 227.5 37 41.2 505 261.4 316.6 38 40.4 197.5 211.2 331.4 39 28.9 212.6 150.9 130.9 40 28.6 522.7 137.9 124.7 41 13 173.6 196.6 150.1 008738577 49 Table 4: Values for: F / G actin binding; Dead cell / viable cell binding (human); Dead cell / viable cell binding (murine); and Cross presentation (AUC) for all 46 IgG2a clones All 46 mAbs were assayed for their ability to induce necrotic cell XP in cDC2 cells (Figure 2). Hoxb8- CDP-derived cDC2 were incubated with OVA-soaked necrotic 5555 BrafV600E melanoma cells with a dose titration (50, 10, 2, 0.4 nM) of 2WA Fc-DNGR-1 (black circles), WT Fc-DNGR-1 (white circles), or anti-F-actin IgG2a clones (white triangles) and OT-I CD8+ T cells. T cell-derived IFNγ in supernatants was measured by ELISA after 24 h. Area under the curve (AUC) was calculated for IFNγ production for each mAb. They were then ranked in descending order (Figure 2) and the top 5 antibodies (A to E) that were able to augment XP by cDC2 were selected for further experiments. All 5 of these antibodies happened to come from the DNGR-1 elution fraction, as shown in Table 5 which also includes the 6thranked antibody (Ab ID: 7). Ab ID Cross-presentation (% WT) Eluted with Fc-DNGR-1 or Trypsin A 70.0 Fc-DNGR-1 B 55.0 Fc-DNGR-1 C 27.1 Fc-DNGR-1 D 16.1 Fc-DNGR-1 E 20.4 Fc-DNGR-1 7 11.1 Trypsin Table 5: Cross-presentation (% WT) values and elution information for the top 6 antibodies ranked by ability to augment XP by cDC2. EXAMPLE 2 – Further characterisation of lead antibodies The 5 lead antibodies were then characterised further. The XP assays in Hoxb8-CDP-derived cDC2 were repeated as described above and the results are shown in Figure 3. The Fc-DNGR-1 conjugate (white circles) was used as a positive control. The 2WA mutant Fc-DNGR-1 (black circles) is used as a negative control throughout the examples. This mutant was generated by Francis Crick Institute, and contains 2 W to A mutations in the CTLD DNGR-1 domain which abolishes binding to F-actin (Canton, 2021). XP was measured by T cell-derived IFNγ. All 5 antibodies were able to trigger XP at a level above control. An ELISA was performed to assay the F-actin binding properties of all 5 antibodies (Figure 4, 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 008738577 50 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. Antibody EC50 (nM) A 0.03 B 0.47 C 4.55 D 0.89 E 1.94 Fc-DNGR-1 0.04 Irrelevant N.D. Table 6: EC50 (nM) values for binding to F-actin. N.D. = not determined. EXAMPLE 3 – Functional analysis of lead antibodies To confirm that the antibodies were able to bind to F-actin on dead cells, UV-irradiated necrotic mouse 5555 cells (Figure 5A) or necrotic human HEK293T cells (Figure 5B) were incubated with serial dilutions of anti-F-actin IgG2a 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 5). The ability of the antibodies to induce phagocytosis was assessed in primary cDC1 cells (Figure 6A) and primary cDC2 cells (Figure 6B) by flow cytometric analysis of internalisation of CT-DR-labelled necrotic cell debris by IFN-I-primed primary FLT3L-cDC from murine bone marrow in the presence of F-actin binding reagents. The cDC cells were analysed after 4 h of incubation and phagocytic index calculated using the following formula: (% of CT-DR positive cDC x GMFI of CT-DR positive cDC) / 10,000. Figure 6 shows that all 5 antibodies boosted uptake of CT-DR-labelled necrotic cell debris by IFN-I-primed primary FLT3L-cDC, and had distinctly different phagocytic index profiles. The ability of the antibodies to trigger XP in a cDC2 cell line was established in Examples 1 and 2 above and shown in Figure 3. The ability to trigger XP in primary cDC1 and non-cDC1 cells was then investigated. Primary IFN-I-primed bone marrow FLT3L-cDC cultures were separated into cDC1 and non-cDC1 fractions using XCR1-targeted MACS enrichment. These were then incubated with OVA- soaked UV-irradiated necrotic 5555 BrafV600E necrotic cells and a serial dilution of WT Fc-DNGR-1, irrelevant IgG2a, or anti-F-actin IgG2a clones and OT-I CD8+ T cells. T cell-derived IFNγ was measured after 24 h as a readout of XP. All 5 antibodies triggered XP in both cDC1 (Figure 7A) and non-cDC1 (Figure 7B) cells. The ability of each antibody to trigger XP was in a similar proportion to their ability to trigger phagocytosis in Figure 6. 008738577 51 These functional experiments provide confirmation that all 5 antibodies are able to bind F-actin on dead cells, trigger phagocytosis and subsequent XP of the phagocytosed material. EXAMPLE 4 – Testing of commercial antibodies and comparison to lead antibodies A, B and E 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-β-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 IgG2a clones (AC-40 or AC- 74) or Fc-DNGR-1 fusion proteins (2WA or WT) and stained with secondary AF488-conjugated anti- mouse 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 8). After establishing the binding profiles of AC-40 and AC-74 to dead cells, the ability of these antibodies to trigger necrotic cell phagocytosis was determined by flow cytometric analysis of internalisation of CT-DR- labelled necrotic cell debris by IFN-I-primed primary FLT3L-cDC from murine bone marrow in the presence of anti-actin IgG2a clones (AC-40 and AC-74) or Fc-DNGR-1 (2WA or WT). cDC1 (Figure 9A) and cDC2 (Figure 9B) were analysed after 4 h of incubation and phagocytic index was calculated using the following formula: (% of CT-DR positive cDC x GMFI of CT-DR positive cDC) / 10,000. As expected based on the dead cell binding profiles, AC-74 leads to phagocytosis of necrotic cell debris by primary FLT3L-cDC, but AC-40 does not (Figure 9). The ability of AC-40 and AC-74 to trigger XP in primary cDC1 and cDC2 was measured as described above for the lead antibodies. Unsurprisingly, AC-40 was not able to trigger XP. AC-74 was able to induce XP by non-cDC1 (Figure 10B), but poorly by cDC1 (Figure 10A). The lead antibodies A, B and E were then directly compared to the commercial antibodies AC-40 and AC- 74 by ranking the EC50 (potency) and maximum IFNγ signal (efficacy) for each antibody / Fc-DNGR-1, calculated from XP results in Figure 10 between cDC1 (Figure 11A) and non-cDC1 (Figure 11B). As shown, antibodies A and B consistently induce XP comparably to Fc-DNGR-1 by both cDC1 and non- cDC1. In contrast, the commercially available antibodies have poor potency and efficacy in cDC1. In non- cDC1, only AC-74 has any efficacy with AC-40 having poor efficacy and potency. 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 12A and 12B, 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. 008738577 52 EXAMPLE 5 - Anti-F-actin antibodies promote tumour control with chemotherapy Tumour control by the lead anti-F-actin antibodies A, B and E was assessed (Figure 13). Anti-F-actin IgG2a clones or Fc-DNGR-1 (WT or 2WA) were administered intra-tumourally (100 μg for Fc-DNGR-1 or 150 μg for anti-F-actin IgG2a in 50 μl) to C57BL / 6 mice bearing MCA205 tumours. Agents were administered in combination with 1.25 mg / kg doxorubicin on day 7 to induce immunogenic cell death. A second dose of the antibodies / Fc-DNGR-1 was administered on day 11. As shown in Figure 13B, antibodies A, B and E exhibited tumour control that was greater relative to the negative control. 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 14). Antibody E shows minimal competition. None of the antibodies matched the ability of DNGR-1 to compete with itself. 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 death- inducing 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). 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 serum- compatible 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). 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 IgG2a or human CH1 (Figure 15C). 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. Notably, systemically administered antibody A alone was sufficient to promote control of MC38 tumours compared to isotype- matched control antibody 008738577 53 (Figure 15D). Mice did not exhibit any outward signs of systemic inflammation and continued to accumulate weight as expected (Figure 15E). EXAMPLE 8 - Fc-DNGR-1 reveals tumour necrotic cell localisation in vivo. 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. Three-dimensional tumour spheroids (tumouroids) were generated in vitro and incubated with Fc-DNGR-1 variants. WT Fc-DNGR-1, but not 2WA Fc-DNGR-1, was able to highlight cells within the tumouroid core (data not shown), an area with likely restricted nutrient exchange and significant cleaved caspase-3 staining. Fc-DNGR-1 staining reflected a general loss of tumour cell membrane integrity as necrotic cells could also be visualised by culturing tumouroids with other reagents that bind F-actin or DNA, such as phalloidin or DAPI, respectively (Figure 16A-B). Consistent with binding to the actin cytoskeleton, several necrotic cells exhibited discrete filament-like Fc-DNGR-1 staining patterns (Figure 16C). To identify areas of tumour necrosis in vivo, Fc-DNGR-1 was administered peri-tumourally to mice bearing a fibrosarcoma generated by transplantation of an MCA205-derived tumour line modified to express a fluorescent marker, mCherry (Giampazolias, 2021). Consistent with the tumouroid data, WT Fc-DNGR-1 effectively labelled necrotic cells within the tumour core (Figure 16D), while no staining was observed with 2WA Fc-DNGR-1 (Figure 16D). Bright Fc-DNGR-1+cells were observed at the necrotic core border, with diffuse Fc-DNGR-1 staining within the centre. This more diffuse staining pattern overlapped with a drop in nuclear DNA staining intensity, consistent with cellular disintegration and release of intracellular debris. Next, to assess the proximity of different APC populations to dead cells within the tumour, the inventors co-stained for MHC-II and CD103 to identify CD103+cDC1s and CD103- non-cDC1 APCs (e.g., cDC2s, MCs) (Figure 16E). 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 FcγRI (Figure 16F), suggesting that they are optimally positioned to acquire necrotic cell antigens in vivo in the presence of Fc-DNGR-1. This data provides proof-of-principle that the necrotic core of tumours can be targeted via binding F-actin and that FcγR-expressing cells reside in close proximity to these regions. EXAMPLE 9 – Testing of commercial antibodies binding to F-actin and comparison to lead antibody A To further compare the binding and F-actin specificity of lead antibody A 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. C4 is an alpha-actin specific antibody. The ability of these 008738577 54 antibodies to bind to F-actin was investigated by ELISA using immobilised F-actin. The inventors were able to show that neither AC-40 nor C4 is able to efficiently bind to F-actin, whereas antibody A can bind efficiently to F-actin (Figure 17). EXAMPLE 10 – Testing of humanised commercial antibody and comparison to humanised lead antibodies A, B and E Humanised IgG1 antibodies of lead antibodies A, B and E were prepared. To compare the efficacy and potency of these antibodies to the previously described 8-3 antibody, which is reported in Yao et. al to bind to F-actin, was generated on a human IgG1 backbone. The ability of these antibodies to bind to F- actin was determined by ELISA (Figure 18A). While all antibodies exhibited binding to F-actin-coated plates, Antibodies A, B, and E all exhibited much higher potency (EC50) and efficacy (maximum binding) compared to 8.3. Next, the ability of these antibodies to bind to F-actin on necrotic cells was investigated by flow cytometry. Once more, maximal binding (efficacy) and potency (EC50) were significantly higher for Antibodies A, B, and E relative to 8-3 (Figure 18B). For example, antibody A had an EC50 of 2.759 nM compared to an EC50 of 15.10 nM for antibody 8-3. Therefore, human IgG1 antibodies A, B, and E bind strongly to F-actin and necrotic cells, whereas the described antibody 8-3 does not. Thus, despite what is discussed in the literature, the data shows that not all available antibodies bind to F- actin with sufficient strength to achieve the therapeutically desirable properties discussed herein. After confirming that the humanised antibodies were able to bind to F-actin and necrotic cells, the ability of Antibody A to trigger phagocytosis of necrotic cells (necrophagy) by various human cells was investigated. cDC2s are a major target of CTT in the tumour microenvironment and express activating FcγRIIA and inhibitory FcγRIIB (Figure 19A). To model these cells, we generated DC-like cells from human peripheral blood monocytes using GM-CSF / IL-4 (termed moDCs for simplicity), which were found to have a very similar Fc receptor expression pattern by flow cytometry (Figure 19B). We assessed the ability of antibody A to promote necrophagy by moDCs by confocal microscopy. WT antibody A was able to promote necrophagy by human moDCs compared to an antibody deficient in Fc receptor binding (LALA-PG mutant) (Figure 20A). This activity was further augmented by the introduction of mutations in the IgG1 Fc domain that enhance affinity for Fc receptors (GASDALIE). Therefore, AFA A promotes necrophagy via Fc receptors and this can be further enhanced by Fc engineering. Uptake was consistently observed across moDCs derived from different donors (Figure 20A). Furthermore, the uptake was shown to be dependent on FcγRIIa and SYK (Figure 20B), consistent with the previously- defined Fc receptor expression pattern (Figure 19B). 008738577 55 Macrophages express high levels of Fc receptors and represent a major tumour myeloid cell subset. To assess AFA function in these cells, peripheral blood monocytes were differentiated into macrophages with M-CSF (termed moMacs). As expected, moMacs expressed high levels of all Fc receptors (Figure 21A). As observed for moDCs, AFA A promoted necrophagy in moMacs, as determined by confocal microscopy (Figure 21B). Activity was abolished (LALA-PG) or enhanced (GASDALIE) with mutations that reduced or increased the affinity of IgG1 Fc for human Fc receptors, respectively. Therefore, AFA A promotes necrophagy by various human myeloid cells via Fc receptor engagement, and this activity can be further tuned by mutations within the Fc domain that alter Fc receptor affinity. The data described above show the generation of a class of antibodies that bind to F-actin with high affinity, avidity, and / or specificity (Figure 11). These antibodies are able to specifically bind to F-actin on necrotic cells (Figure 5), trigger phagocytosis of necrotic cell debris (Figure 6) and induce XP by both cDC1 and non-cDC1 cells (Figure 7). These functional characteristics, as well as the specificity for F- actin, highlights these antibodies as therapeutically useful, for example in the treatment of cancers. 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 12). Whilst one of the antibodies (AC-74) was able to trigger XP, its inability to distinguish between F-actin and G-actin excludes it as a viable therapeutic due to the risk of off-target effects. The antibodies of the present invention are also shown to display improved pharmacokinetics in circulation relative to Fc-DNGR-1, accumulate in necrotic sites within tumours, and promote tumour control upon systemic administration.

[0002] 008738577 56 References 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. Ahrens, S. et al. F-actin is an evolutionarily conserved damage-associated molecular pattern recognized by DNGR-1, a receptor for dead cells. Immunity 36, 635-645 (2012) Canton, J., et al., The receptor DNGR-1 signals for phagosomal rupture to promote cross-presentation of dead-cell-associated antigens. Nat Immunol, 2021.22(2): p.140-153. Carmi, Y., et al., Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity. Nature, 2015: p.1-6. Carmi, Y. et al. Tumor-binding antibodies induce potent dendritic cell-mediated tumor immunity. Oncoimmunology 8(10), e1078063 (2019) Castro-Dopico, T. & Clatworthy, M. IgG and Fcγ Receptors in Intestinal Immunity and Inflammation. Frontiers in Immunology 10:805 (2019) Dominguez, R. & Holmes, K. C., Actin Structure and Function. Annu Rev Biophys 40:169-186 (2011) Dransfield, I. et al., Initial characterization of an anti-actin monoclonal antibody (NH3). Biochemical Society Transactions, 1988, vol.16 p.163-164 Galluzzi, L., et al., Immunogenic cell death in cancer and infectious disease. Nature Reviews Immunology, 2017.17(2): p.97-111. Giampazolias, E., et al., Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunity. Cell, 2021.184(15): p.4016-4031.e22 Guilliams, M., et al., The function of Fcy receptors in dendritic cells and macrophages. Nature reviews Immunology, 2014.14(2): p.94-108. Hanĉ, 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) Mylvaganam, S., Freeman, S., & Grinstein, S. (2021). The cytoskeleton in phagocytosis and macropinocytosis. Current Biology, 31(10), R619-R632. https: / / doi.org / 10.1016 / j.cub.2021.01.036 Regnault, A., et al., Fcgamma receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. The Journal of experimental medicine, 1999.189(2): p.371-80 Sancho, D. et al. Identification of a dendritic cell receptor that couples sensing of necrosis to immunity. Nature 458, 899-903 (2009) Srinivasan, N. et al., Actin is an evolutionarily-conserved damage-associated molecular pattern that signals tissue injury in Drosophila melanogaster. eLife, 5:e19662 (2016) 008738577 57 WO 2009 / 013484 WO 2013 / 088136 WO 2022 / 163809 Min Yao, Jonathan Preall, Johannes T.-H. Yeh, Darryl Pappin, Paolo Cifani, Yixin Zhao, Sophia Shen, Philip Moresco, Brian He, Hardik Patel, Amber N. Habowski, Daniel A. King, Kara Raphael, Arvind Rishi, Divyesh Sejpal, Matthew J. Weiss, David Tuveson, and Douglas T. Fearon. Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies against self-antigens. JCI Insight, September 26, 2023 Zhang, J.-G. et al. The dendritic cell receptor Clec9A binds damaged cells via exposed actin filaments. Immunity 36, 646-657 (2012) For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual.3 ed.2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

Claims

008738577 58 Claims:

1. An antibody, or antigen-binding fragment thereof, that specifically binds to filamentous actin (F- actin), wherein the antibody has an EC50 value for F-actin of about 5 nM or less, and / or wherein the antibody has at least 5-fold higher affinity for F-actin than for globular actin (G-actin).

2. The antibody or antigen-binding fragment according to claim 1, wherein the antibody can facilitate cross presentation (XP) of a dead cell-associated antigen to a CD8+ T cell.

3. The antibody or antigen-binding fragment according to claim 2, wherein the XP is mediated by a cDC1.

4. The antibody or antigen-binding fragment according to claim 2, wherein the XP is mediated by a non-cDC1 cell that expresses an FcR, e.g. a cDC2 or a macrophage.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, having at least 3-fold specificity for binding necrotic cells over live cells, as measured by FACS or flow cytometry.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the necrotic cell binding is not inhibited by the presence of G-actin.

7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody can facilitate phagocytosis of necrotic cells.

8. The antibody or antigen-binding fragment according to claim 7, wherein the phagocytosis is mediated by a cDC1.

9. The antibody or antigen-binding fragment according to claim 7, wherein the phagocytosis is mediated by a non-cDC1 cell that expresses an FcR, e.g. a cDC2 or a macrophage.

10. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody is a human or humanised antibody.

11. The antibody or antigen-binding fragment according to claim 10, wherein the antibody is of the human IgG1 isotype.

12. The antibody or antigen-binding fragment according to any preceding claim, wherein the antibody does not compete for binding to F-actin with any of the antibodies selected from the group consisting of: ab11003 (Abcam), ab205 (Abcam), ab130935 (Abcam), A2228 or A5316 (Sigma-Aldrich), NBP2-61610 (Novus Biologicals), MA1-80729 (ThermoFisher) and BS-1571R (ThermoFisher).008738577 59 13. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody competes with human DNGR-1 for binding to F-actin.

14. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody binds to the same epitope or overlaps with the same epitope as human DNGR-1.

15. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein antibody binding to F-actin is not inhibited by the presence of G-actin.

16. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen binding fragment 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.

17. The antibody or antigen-binding fragment according to claim 16, wherein the antigen binding portion 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.

18. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: a) a CDR1 having a sequence GGTFRSY or a variant thereof comprising one or two amino acid substitutions with respect to GGTFRSY; b) a CDR2 having a sequence NPIFDT or a variant thereof comprising one or two amino acid substitutions with respect to NPIFDT; and c) a CDR3 having a sequence TVIGAFDS or a variant thereof comprising one or two amino acid substitutions with respect to TVIGAFDS; and wherein the light chain comprises: d) a CDR1 having a sequence TRTSGDIGGYNFVS or a variant thereof comprising one, two or three amino acid substitutions with respect to TRTSGDIGGYNFVS e) a CDR2 having a sequence DVNSRPS or a variant thereof comprising one or two amino acid substitutions with respect to DVNSRPS; and f) a CDR3 having a sequence SSYTSRNTV or a variant thereof comprising one or two amino acid substitutions with respect to SSYTSRNTV, wherein complementarity determining regions (CDRs) are identified using Chothia numbering.

19. The antibody or antigen-binding fragment according to claim 18, 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, wherein008738577 60 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.

20. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: a) a CDR1 having a sequence GYIFTSY or a variant thereof comprising one or two amino acid substitutions with respect to GYIFTSY; b) a CDR2 having a sequence SAYNGH or a variant thereof comprising one or two amino acid substitutions with respect to SAYNGH; and c) a CDR3 having a sequence GKISSWFVLED or a variant thereof comprising one, two or three amino acid substitutions with respect to GKISSWFVLED; and wherein the light chain comprises: d) a CDR1 having a sequence SGGTSNIGKNYVS or a variant thereof comprising one or two or three amino acid substitutions with respect to SGGTSNIGKNYVS008738577 61 e) a CDR2 having a sequence DNNMRPS or a variant thereof comprising one or two amino acid substitutions with respect to DNNMRPS; and f) a CDR3 having a sequence GMWIRSLSRWV or a variant thereof comprising one, two or three amino acid substitutions with respect to GMWIRSLSRWV, wherein complementarity determining regions (CDRs) are identified using Chothia numbering.

21. The antibody or antigen-binding fragment according to claim 20, 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.

22. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises:008738577 62 a) a CDR1 having a sequence GFTFSAY or a variant thereof comprising one or two amino acid substitutions with respect to GFTFSAY; b) a CDR2 having a sequence SYDGNN or a variant thereof comprising one or two amino acid substitutions with respect to SYDGNN; and c) a CDR3 having a sequence DFRDYVWGTYPSAY or a variant thereof comprising one, two or three amino acid substitutions with respect to DFRDYVWGTYPSAY; and wherein the light chain comprises: d) a CDR1 having a sequence SGSSSNIGRRHVF or a variant thereof comprising one, two or three amino acid substitutions with respect to SGSSSNIGRRHVF e) a CDR2 having a sequence RGDQRPS or a variant thereof comprising one or two amino acid substitutions with respect to RGDQRPS; and f) a CDR3 having a sequence ATWDDGLSGYV or a variant thereof comprising one, two or three amino acid substitutions with respect to ATWDDGLSGYV, wherein complementarity determining regions (CDRs) are identified using Chothia numbering.

23. The antibody or antigen-binding fragment according to claim 22, 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;008738577 63 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.

24. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: a) a CDR1 having a sequence GGAFRNS or a variant thereof comprising one or two amino acid substitutions with respect to GGAFRNS; b) a CDR2 having a sequence IPMSGT or a variant thereof comprising one or two amino acid substitutions with respect to IPMSGT; and c) a CDR3 having a sequence EKERTFGVVMRTSYYYVMEV or a variant thereof comprising one, two, three or four amino acid substitutions with respect to EKERTFGVVMRTSYYYVMEV; and wherein the light chain comprises: d) a CDR1 having a sequence RASQSISSYLN or a variant thereof comprising one or two amino acid substitutions with respect to RASQSISSYLN e) a CDR2 having a sequence AASSLQS or a variant thereof comprising one or two amino acid substitutions with respect to AASSLQS; and f) a CDR3 having a sequence QQSYSTPYT or a variant thereof comprising one or two amino acid substitutions with respect to QQSYSTPYT, wherein complementarity determining regions (CDRs) are identified using Chothia numbering.

25. The antibody or antigen-binding fragment thereof according to claim 24, 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 the008738577 64 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.

26. The antibody or antigen-binding fragment according to claim 18, 20, 22 or 24, wherein said one or more substitutions are conservative substitutions.

27. The antibody or antigen-binding fragment according to any one of the preceding claims, for use in medicine.

28. The antibody or antigen-binding fragment according to any one of claims 1 to 26, for use in a method of treating cancer.

29. The antibody or antigen-binding fragment for the use according to claim 28, wherein the cancer comprises a solid tumour.

30. The antibody or antigen-binding fragment for the use of any one of claims 27-29, wherein the use comprises systemic administration of the antibody or antigen-binding fragment.

31. The antibody or antigen-binding fragment according to any one of claims 1 to 17, wherein the antibody or antigen-binding fragment is competitive for binding to F-actin with the antibody or antigen- binding fragment according to any one of claims 18 to 26.008738577 65 32. A method of selecting an antibody or antigen-binding fragment thereof that specifically binds to F- actin, the method comprising: 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, and then selecting an antibody from said one or more lead antibodies.

33. The method according to claim 32, 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.

34. The method according to claim 32 or claim 33, 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.

35. The method according to any one of claims 32 to 34, wherein the method further comprises screening said one or more lead antibodies for capacity to promote cross-presentation, wherein the selected antibody has said capacity.

36. The method according to any one of claims 32 to 35, wherein the method further comprises screening said one or more lead antibodies for capacity to promote phagocytosis, wherein the selected antibody has said capacity.

37. An antibody produced by the method according to any one of claims 32 to 36.

38. The antibody according to claim 37, wherein the antibody is polyclonal.

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