Anti-f-actin antibodies

Abstract

Antibodies that specifically bind to actin are provided. In particular, the antibodies of the invention exhibit preferential binding to F-actin over G-actin, which enables their use in medical and research settings. The invention provides anti-actin antibodies, their medical uses, and methods of producing anti-F-actin antibodies.

Description

Technical Field

[0001] This invention relates to antibodies that specifically bind to actin, for example, exhibiting preferential binding to F-actin relative to G-actin. The invention provides anti-actin antibodies, their medical uses, and methods for generating anti-F-actin antibodies. Background Technology

[0002] Filamentous actin (F-actin) is a polymer of monomeric globular actin (G-actin). Each actin monomer is a 375-amino acid polypeptide that folds into two major α / β domains separated by deep clefts and consists of subdomains 1-4. F-actin assembles into two protofilaments that intertwine to form a right-handed double-stranded long helix, in which approximately 13 molecules repeat every 6 turns at an axial distance of 35.9 nm (Dominguez and Holmes, 2011, which is incorporated herein by reference in its entirety).

[0003] F-actin is an ATPase, and it is this nucleotide hydrolysis that regulates the transition between F-actin and G-actin, producing highly dynamic actin filaments that enable cells to change shape.

[0004] Antibodies that bind to actin are known. However, such antibodies typically cannot distinguish between the monomeric form of actin, G-actin, and the filamentous form, F-actin, and / or cannot bind with sufficient affinity to trigger immunologically useful outcomes, such as phagocytosis, as described herein. For example, Abcam antibody ab205 (https: / / www.abcam.com / products / primary-antibodies / f-actin-antibody-nh3-ab205.html) binds to both G-actin and F-actin. Novus Biologicals antibody NBP2-61610 (https: / / www.novusbio.com / products / f-actin-antibody-nh3_nbp2-61610) also binds to both F-actin and G-actin. Sigma Aldrich antibody AC-74 (A2228) is β-actin specific. Abcam antibody AC-40 (ab11003) is pan-actin-binding. WO 2022 / 163809 (incorporated herein by reference in its entirety) discloses antigen-binding molecules that bind to cellular components exposed after cell death, such as F-actin. However, it does not disclose the relative binding specificity of the antibodies envisioned in WO 2022 / 163809 for F-actin and G-actin, nor does it propose methods for achieving high specificity for F-actin. The following anti-actin antibodies have been shown by Western blotting to bind to G-actin: Cell Signaling Technology antibody #4967 (β-actin specific); St. John's Laboratory antibody STJ91461 (pan-actin-binding); and Abcam antibody EPR16769 ab179467 (pan-actin-binding). Even antibodies marketed as anti-F-actin antibodies have shown binding to G-actin, such as NH3 antibody (Dransfield, 1988), Abcam antibody 4E3.adl (ab130935), and antibody ABIN731498 (https: / / www.antibodies-online.com / antibody / 731498 / anti-F-Actin+AA+1-50+antibody / ).

[0005] Yao et al., 2023 (incorporated herein by reference in its entirety), disclosed plasma cells present in human pancreatic ductal adenocarcinoma that reportedly secrete antibodies that bind to self-antigens such as F-actin. However, the F-actin-binding antibodies reported by Yao et al. reportedly do not bind with very high affinity or binding strength, indicating ECG resistance to F-actin. 50 It is 9.1 nM.

[0006] Developing antibodies specific to F-actin would be highly practical, as F-actin and G-actin have very different cellular functions. G-actin is maintained in a cytosol pool that provides the necessary structural units for the production of F-actin filaments (Dominguez and Homes, 2011, which is incorporated herein by reference in its entirety). In contrast, F-actin is a damage-associated molecular pattern (DAMP) recognized by the receptor DNGR-1 of dead cells (WO 2013 / 088136; Hanĉ et al., 2015; Ahrens, 2012; Zhang, 2012; Sancho, 2009, each of which is incorporated herein by reference in its entirety). DNGR-1 (also known as CLEC9A) is expressed at high levels on type 1 conventional dendritic cells (cDC1). DNGR-1 triggers SYK signaling upon binding to F-actin via its C-type lectin domain (CTLD). This leads to the rupture of the ligand-containing phagosome, releasing antigenic material into the cDC1 cytosol and into the endogenous MHC class I presentation pathway (Sancho, 2009; Canton, 2021; and WO 2009 / 013484A1, each of which is incorporated herein by reference in its entirety), a process known as cross-presentation (XP). As part of the cytoskeleton, F-actin is exposed when the cell membrane is damaged, such as during necrosis. Therefore, F-actin is a marker of necrotic cell death. Necrotic cell debris is a source of antigens for XP within the tumor and is efficiently internalized by cDC1 (Galluzzi, 2017, which is incorporated herein by reference in its entirety).

[0007] The present invention was designed in view of the above considerations. Summary of the Invention

[0008] This invention provides a method for obtaining antibodies that bind to F-actin with high affinity, cohesion, and / or specificity. Therefore, this invention provides a class of antibodies exhibiting such properties. The inventors have discovered that this class of antibodies can be generated by positively selecting antibody population members through F-actin binding activity, negatively selecting them with G-actin, and additionally using DNGR-1 as an antibody eluent and / or trypsin (as described herein). Recognizing the potential utility of actin-binding antibodies in the treatment of cancers (such as solid tumors), and because commercially available anti-actin antibodies lack the therapeutically desirable properties discussed herein, the inventors were motivated to attempt to obtain antibodies possessing these properties.

[0009] While some previously known antibodies claim selectivity for F-actin, the inventors have found that they bind to G-actin to a similar degree (i.e., these known antibodies do not specifically bind to F-actin). In contrast, the antibodies disclosed herein exhibit explicit specificity for F-actin relative to G-actin, as illustrated in the following examples. This specificity means that the antibodies can specifically target necrotic and damaged cells by binding to F-actin. As mentioned above, F-actin is part of the cytoskeleton, while G-actin is not. Not wishing to be bound by theory, the inventors hypothesize that when the cell membrane is damaged or disrupted (e.g., during necrosis), G-actin will disperse, while F-actin will remain largely entangled with necrotic cell debris. When the antibody of the present invention binds to exposed F-actin molecules, its Fc domain will bind to the Fc receptor (FcR) on the surface of the antigen-presenting cell (APC). Preferably, the antibody has an Fc domain that preferentially binds to activating FcRs. These FcRs combine to promote phagocytosis and antigen presentation of new antigens (including cross-presentation (XP)).

[0010] In contrast, antibodies from existing technologies bind to F-actin with insufficient affinity / cohesion, and / or also bind to G-actin, which is not a DAMP, and therefore will promote off-target effects and remove the antibody from the necrotic foci.

[0011] Therefore, for certain applications, antibodies should contain an Fc domain.

[0012] The antibodies of this invention can also be used as experimental tools. For example, antibodies can be used in imaging applications. Antibodies can also be used as capture antibodies. For these applications, the antibody may not require an Fc domain.

[0013] Most broadly, the present invention provides actin-binding antibodies exhibiting one or more of the features described herein.

[0014] For example, in a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to filamentous actin (F-actin). Preferably, the antibody has a higher affinity for F-actin compared to globular actin (G-actin). Preferably, the EC50 value of the antibody or its antigen-binding fragment for F-actin is about 5 nM or less. Preferably, the affinity of the antibody or its antigen-binding fragment for F-actin is at least 5 times that for G-actin. More preferably, the affinity of the antibody or its antigen-binding fragment for F-actin is at least 10 times that for G-actin.

[0015] In another aspect, the present invention provides a human or humanized antibody or an antigen-binding fragment thereof, said human or humanized antibody or antigen-binding fragment thereof specifically binding to filamentous actin (F-actin). Preferably, the antibody is a monoclonal antibody.

[0016] In another aspect, the present 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.

[0017] In another aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to filamentous actin (F-actin) and can promote the cross-presentation (XP) of dead cell-associated antigens to CD8+ T cells.

[0018] All the individual features of the above aspects are composable and are explicitly covered in the invention described herein. The embodiments described below can also be combined with any of the above aspects.

[0019] The affinity of antibodies for F-actin and G-actin can be assessed using the following method. Immunoplates (MaxiSorp 96-well, Thermo Fisher Scientific) were coated overnight at 4°C with 5 µg / ml NeutrAvidin™ (Thermo Fisher Scientific) in PBS, washed, blocked for 2 h at room temperature with 2.5% BSA in PBS, and stored overnight at 4°C after adding biotinylated F-actin or G-actin (2 nM). Capture of biotinylated F-actin and G-actin was validated using a commercially available actin-specific mAb (AC-40, Sigma) that identifies both F-actin and G-actin. To prevent depolymerization of F-actin or polymerization of G-actin during assay, F-actin and G-actin were stabilized during actin stock solution preparation by adding phalloidin or pinocembrin D, respectively. After washing to remove unbound actin, a three-fold serial dilution of either mAb or control reagent (DNGR-1-Fc fusion) starting at 300 nM was added to the ELISA plate and allowed to stand for 1 h. Actin-bound mAbs or control reagents were detected using a horseradish peroxidase (HRP)-conjugated mouse-specific anti-IgG (Jackson ImmunoResearch) antibody (0.16 µg / ml), and color development was performed using an ELISA substrate for HRP-based detection (TMB, eBioscience). Substrate development was stopped by adding sulfuric acid (H2SO4, Fisher). Titration curves were plotted using a GraphPad Prism 7, and the EC50 value for each mAb was calculated by applying a nonlinear regression (curve fitting) of log(agonist) against response-variable slope (four parameters).

[0020] Preferably, the antibody can promote the cross-presentation (XP) of dead cell-associated antigens to CD8+ T cells. In some embodiments, XP is mediated by cDC1. In other embodiments, XP is mediated by non-cDC1 cells expressing FcR (e.g., cDC2 or macrophages).

[0021] Cross-presentation can be evaluated using the following method: Irradiate tumor cells to induce necrosis. Sixteen hours after irradiation, necrotic tumor cells are immersed for 1 hour at 37ºC in RPMI 1640 medium with 10 mg / ml albumin (OVA, Sigma Aldrich), followed by washing. Then, in the presence of a candidate antibody (or control), the OVA-treated necrotic cells are crossed with APC (5 x 10⁻⁶ cells / mL). 4 One cDC1 or 1 x 10 5Fc-DNGR-1 constructs were co-incubated with pre-activated OT-I CD8+ T cells at a 2:1 ratio in U-bottom wells of a 96-well plate for 4 h. The Fc-DNGR-1 construct could be used as a positive control, and non-binding Fc-DNGR-1 variants (such as the “2WA” mutant) could be used as a negative control. Pre-activated OT-I CD8+ T cells (which can be generated by culturing a single-cell suspension of spleen cells 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) were then added to the APC co-culture and incubated for 24 h. Finally, after 24 hours of co-culturing with pre-activated OT-I CD8+ T cells, the concentration of T cell-derived IFNγ was measured using ELISA. This assay was repeated using five uniformly distributed antibody concentrations between approximately 100 pM and 10 nM, for example, 200 pM, 400 pM, 800 pM, 1.6 nM, and 3.2 nM (and control concentrations of the same five concentrations). The results can be expressed as a relationship between the concentration of T-cell-derived IFNγ (pg / mL) and the antibody concentration (see, for example, [reference needed]). Figure 3 ).

[0022] One approach to classifying the ability of antibodies to mediate XP is to obtain the mean area under the curve (AUC) from cross-presentation assays of the three non-binding Fc-DNGR-1 variants (such as the 2WA mutant) described above, and define this value as 0% baseline (negative control). The mean AUC from cross-presentation assays of the three WT Fc-DNGR-1 constructs is defined as 100% (positive control). The AUC from the cross-presentation assays of the candidate antibody is corrected relative to baseline and converted to a %. If this % is 5%, 10%, 12%, or 15% or higher, the antibody is considered to have the ability to mediate XP.

[0023] Alternatively, an antibody is considered to have the ability to mediate XP if the IFNγ signal from the cross-presentation assay described above is at least 3 standard deviations higher than the negative control (e.g., 2WA or an irrelevant antibody). In these embodiments, the IFNγ signal may be at least 4, at least 5, or at least 6 standard deviations higher than the negative control.

[0024] Preferably, the antibody or its antigen-binding fragment exhibits specificity for binding to necrotic cells relative to living cells, as measured by FACS or flow cytometry. In some embodiments, the antibody or its antigen-binding fragment has at least 3 times the specificity for binding to necrotic cells relative to living cells. In some embodiments, the antibody or its antigen-binding fragment has at least 4, 5, 6, 7, 8, 9, or 10 times the specificity for binding to necrotic cells relative to living cells. The antibody or its antigen-binding fragment may have at least 10% necrotic cell binding. The antibody or its antigen-binding fragment may have at least 20%, 30%, or 40% necrotic cell binding. In some embodiments, necrotic cell binding is not inhibited by the presence of G-actin.

[0025] The binding of necrotic cells can be assessed using the following methods. Necrotic tumor cells (e.g., irradiated mouse BRAF) are then tested. V600E Mutant 5555 melanoma cells or human HEK293T cells were cultured overnight in R0 medium (lacking 10% FCS) and then incubated in DPBS with an antibody (or control). The Fc-DNGR-1 construct could be used as a positive control, and a non-binding Fc-DNGR-1 variant (such as the “2WA” mutant) could be used as a negative control. To assess whether binding to F-actin was inhibited by the presence of G-actin, soluble pinocembrin D-stabilized human platelet G-actin (Cytoskeleton) was added. Samples were washed and incubated with AF488-conjugated anti-mouse IgG2a secondary antibody (Thermo Fisher Scientific, 1:400) at 4ºC for 30 min. Samples were washed again and resuspended in DPBS, then collected on an LSR Tortessa or FACSymphony (BD Biosciences).

[0026] Preferably, the antibody can promote phagocytosis of necrotic cells. In some embodiments, phagocytosis is mediated by cDC1. In other embodiments, phagocytosis is mediated by non-cDC1 cells expressing FcR (e.g., cDC2 or macrophages). Phagocytosis can be mediated by monocyte-derived dendritic cells (moDCs) (e.g., human moDCs) or monocyte-derived macrophages (moMacs) (e.g., human moMacs).

[0027] The following method can be used to assess necrotic cell phagocytosis. Tumor cells were labeled with Cell Tracker-DeepRed (CT-DR) dye (Thermo Fisher Scientific; 1:1000 dilution) at 37ºC for 1 hour and then irradiated. CT-DR-labeled necrotic cells were then added to cDCs at a 2:1 ratio for 4 hours in the presence of an antibody (or control). The Fc-DNGR-1 construct can be used as a positive control, and non-binding Fc-DNGR-1 variants (such as the “2WA” mutant) can be used as a negative control. After 4 hours, samples were prepared for flow cytometry by surface staining with the following combination: XCR1 (Biolegend, ZET), CD172a (Biolegend, P84), IA / IE (Biolegend, M5 / 114.15.2), B220 (BD Biosciences, RA3-6B2), and CD11c (Biolegend, N418). Non-internalized necrotic cell material was excluded using DAPI or LIVE / DEAD fixed blue dead cell dye (Thermo Fisher Scientific). Samples were collected on an LSR Tortessa or FACSymphony (BD Biosciences). The phagocytic index (which integrates the frequency and absolute amount of phagocytosed material) was calculated using the formula (% CT-DR+ x CT-DR+ of CT-DR GMFI) / 10,000. Preferably, this assay was repeated using at least five uniformly distributed antibody concentrations between about 30 pM and about 30 nM. Within this concentration range, an increased phagocytic index compared to the negative control indicates that the antibody promotes the phagocytosis of necrotic cells.

[0028] Preferably, the antibody is a human or humanized antibody. Preferably, the antibody contains an activating Fc domain (which preferentially binds to an activating FcR), for example, wherein the antibody is a human IgG1 isotype.

[0029] 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 as human DNGR-1, or the epitope bound by the antibody overlaps with the epitope bound by human DNGR-1. In other embodiments, the antibody does not bind to the same epitope as human DNGR-1.

[0030] 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 (also known as "Sigma-Aldrich"), NBP2-61610 (Novus Biologicals), MA1-80729 (ThermoFisher), and / or BS-1571R (ThermoFisher). Preferably, the binding of the antibody to F-actin is not inhibited by the presence of G-actin.

[0031] In some embodiments, the antibody or antigen-binding fragment of the present invention can be defined as specifically binding to a discontinuous conformational epitope that binds to human DNGR-1. This epitope comprises amino acid residues present on at least two subunits of F-actin, wherein the discontinuous conformational epitope comprises three or more residues selected from: R196 and / or E237 of the first subunit of F-actin on the first filament, and A114, E117, Q121, A365, G366, and P367 of the second subunit of F-actin on an F-actin filament different from the filament containing the first subunit. In some embodiments, the conformational epitope consists of three or more residues selected from: R196 and / or E237 of the first subunit of F-actin and A114, E117, Q121, A365, G366, and P367 of the second subunit of F-actin. In some embodiments, the conformational epitope further comprises residue D80 of the third subunit of F-actin, which is part of the same filament as the first subunit and is adjacent to the first subunit.

[0032] In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody. Both monoclonal and polyclonal antibodies produced by the methods of the present invention are covered.

[0033] An antibody or its antigen-binding fragment can be defined wholly or partially by its amino acid sequence. For example, an antibody or antigen-binding fragment may contain the following heavy chain CDRs and light chain CDRs:

[0034] Heavy chain:

[0035] CDR1 contains GGTFRSY or its variants containing one or two amino acid substitutions relative to GGTFRSY;

[0036] CDR2 contains NPIFDT or its variants containing one or two amino acid substitutions relative to NPIFDT; and

[0037] CDR3 contains TVIGAFDS or its variants containing one or two amino acid substitutions relative to TVIGAFDS;

[0038] Light chain:

[0039] CDR1 contains TRTSGDIGGYNFVS or its variants containing one, two, or three amino acid substitutions relative to TRTSGDIGGYNFVS;

[0040] CDR2 contains DVNSRPS or its variants containing one or two amino acid substitutions relative to DVNSRPS; and

[0041] CDR3 contains SSYTSRNTV or its variants containing one or two amino acid substitutions relative to SSYTSRNTV.

[0042] CDRs are identified using Chothia numbers.

[0043] Preferably, one or more substitutions are conservative substitutions, i.e. substitutions within an amino acid class and / or substitutions that obtain a positive score in the BLOSUM62 matrix.

[0044] In some implementations, G at position 2 of CDRH1 is replaced by F or Y; T at position 3 of CDRH1 is replaced by I or A; R at position 5 of CDRH1 is replaced by S or T; S at position 6 of CDRH1 is replaced by A or N; and / or Y at position 7 of CDRH1 is replaced by S.

[0045] In some implementations, N at position 1 of CDRH2 is replaced by I; P at position 2 of CDRH2 is replaced by S; I at position 3 of CDRH2 is replaced by A, S or M; F at position 4 of CDRH2 is replaced by Y or S; D at position 5 of CDRH2 is replaced by N or G; and / or T at position 6 of CDRH2 is replaced by G.

[0046] In some implementations, T at position 1 of CDRH3 is replaced by G or Y; I at position 3 of CDRH3 is replaced by W or V; G at position 4 of CDRH3 is replaced by M; A at position 5 of CDRH3 is replaced by R, T or K; F at position 6 of CDRH3 is replaced by T, Y or I; D at position 7 of CDRH3 is replaced by P or S; and / or S at position 8 of CDRH3 is replaced by Y.

[0047] In some implementations, T at position 1 of CDRL1 is replaced by S; R at position 2 of CDRL1 is replaced by G; T at position 3 of CDRL1 is replaced by A, S, or G; S at position 4 of CDRL1 is replaced by T; G ​​at position 5 of CDRL1 is replaced by Q or S; D at position 6 of CDRL1 is replaced by S or N; G at position 8 of CDRL1 is replaced by S; G at position 9 of CDRL1 is replaced by S; Y at position 10 of CDRL1 is replaced by R or K; N at position 11 of CDRL1 is replaced by R or L; F at position 12 of CDRL1 is replaced by Y, H, or N; and / or S at position 14 of CDRL1 is replaced by F.

[0048] In some implementations, D at position 1 of CDRL2 is replaced by A or R; V at position 2 of CDRL2 is replaced by A, N or G; N at position 3 of CDRL2 is replaced by S or D; S at position 4 of CDRL2 is replaced by M or Q; R at position 5 of CDRL2 is replaced by L; and / or P at position 6 of CDRL2 is replaced by Q.

[0049] In some implementations, S at position 1 of CDRL3 is replaced by Q; S at position 2 of CDRL3 is replaced by R or G; Y at position 3 of CDRL3 is replaced by W; T at position 4 of CDRL3 is replaced by S or V; S at position 5 of CDRL3 is replaced by T; R at position 6 of CDRL3 is replaced by P; and / or N at position 7 of CDRL3 is replaced by Y.

[0050] In another case, the antibody or antigen-binding fragment contains the following heavy chain CDR and light chain CDR:

[0051] Heavy chain:

[0052] CDR1 contains GYIFTSY or its variants containing one or two amino acid substitutions relative to GYIFTSY;

[0053] CDR2 contains SAYNGH or its variants containing one or two amino acid substitutions relative to SAYNGH; and

[0054] CDR3 contains GKISSWFVLED or its variants containing one, two or three amino acid substitutions relative to GKISSWFVLED;

[0055] Light chain:

[0056] CDR1 contains SGGTSNIGKNYVS or its variants containing one, two, or three amino acid substitutions relative to SGGTSNIGKNYVS;

[0057] CDR2 contains DNNMRPS or its variants containing one or two amino acid substitutions relative to DNNMRPS; and

[0058] CDR3 contains GMWIRSLSRWV or its variants containing one, two, or three amino acid substitutions relative to GMWIRSLSRWV.

[0059] CDRs are identified using Chothia numbers.

[0060] Preferably, one or more substitutions are conservative substitutions, i.e. substitutions within an amino acid class and / or substitutions that obtain a positive score in the BLOSUM62 matrix.

[0061] In some implementations, Y at position 2 of CDRH1 is replaced by F or G; I at position 3 of CDRH1 is replaced by T or A; T at position 5 of CDRH1 is replaced by S or R; S at position 6 of CDRH1 is replaced by A or N; and / or Y at position 7 of CDRH1 is replaced by S.

[0062] In some implementations, S at position 1 of CDRH2 is replaced by P; A at position 2 of CDRH2 is replaced by S, I, or M; Y at position 3 of CDRH2 is replaced by F or S; N at position 4 of CDRH2 is replaced by D or G; G at position 5 of CDRH2 is replaced by T; and / or H at position 6 of CDRH2 is replaced by N.

[0063] In some implementations, G at position 1 of CDRH3 is replaced by M; K at position 2 of CDRH3 is replaced by R, T, or A; I at position 3 of CDRH3 is replaced by T, Y, or F; S at position 4 of CDRH3 is replaced by P or D; S at position 5 of CDRH3 is replaced by Y; W at position 6 of CDRH3 is replaced by Y or A; F at position 7 of CDRH3 is replaced by Y; L at position 9 of CDRH3 is replaced by M; and / or D at position 11 of CDRH3 is replaced by V.

[0064] In some implementations, the S at position 1 of CDRL1 is replaced by T; the G at position 2 of CDRL1 is replaced by R; the G at position 3 of CDRL1 is replaced by A, S, or T; the T at position 4 of CDRL1 is replaced by S; the S at position 5 of CDRL1 is replaced by Q or G; the N at position 6 of CDRL1 is replaced by S or D; the G at position 8 of CDRL1 is replaced by S; the K at position 9 of CDRL1 is replaced by R or Y; the N at position 10 of CDRL1 is replaced by R or L; the Y at position 11 of CDRL1 is replaced by H, F, or N; and / or the S at position 13 of CDRL1 is replaced by F.

[0065] In some implementations, D at position 1 of CDRL2 is replaced by A or R; N at position 2 of CDRL2 is replaced by A, V or G; N at position 3 of CDRL2 is replaced by S, N or D; M at position 4 of CDRL2 is replaced by S or Q; R at position 5 of CDRL2 is replaced by L; and / or P at position 6 of CDRL2 is replaced by Q.

[0066] In some implementations, G at position 1 of CDRL3 is replaced by A; M at position 2 of CDRL3 is replaced by T; I at position 4 of CDRL3 is replaced by D; R at position 5 of CDRL3 is replaced by D; S at position 6 of CDRL3 is replaced by G; L at position 7 of CDRL3 is replaced by Q; S at position 8 of CDRL3 is replaced by Q; R at position 9 of CDRL3 is replaced by S or G; W at position 10 of CDRL3 is replaced by Y; and / or V at position 11 of CDRL3 is replaced by S or T.

[0067] In another case, the antibody or antigen-binding fragment contains the following heavy chain CDR and light chain CDR:

[0068] Heavy chain:

[0069] CDR1 contains GFTFSAY or its variants containing one or two amino acid substitutions relative to GFTFSAY;

[0070] CDR2 contains SYDGNN or its variants containing one or two amino acid substitutions relative to SYDGNN; and

[0071] CDR3 contains DFRDYVWGTYPSAY or its variants containing one, two, or three amino acid substitutions relative to DFRDYVWGTYPSAY;

[0072] Light chain:

[0073] CDR1 contains SGSSSNIGRRHVF or its variants containing one, two, or three amino acid substitutions relative to SGSSSNIGRRHVF;

[0074] CDR2 contains RGDQRPS or its variants containing one or two amino acid substitutions relative to RGDQRPS; and

[0075] CDR3 contains ATWDDGLSGYV or its variants containing one, two, or three amino acid substitutions relative to ATWDDGLSGYV.

[0076] CDRs are identified using Chothia numbers.

[0077] Preferably, one or more substitutions are conservative substitutions, i.e. substitutions within an amino acid class and / or substitutions that obtain a positive score in the BLOSUM62 matrix.

[0078] In some implementations, F at position 2 of CDRH1 is replaced by G or Y; T at position 3 of CDRH1 is replaced by I or A; S at position 5 of CDRH1 is replaced by R or T; A at position 6 of CDRH1 is replaced by S or N; and / or Y at position 7 of CDRH1 is replaced by S.

[0079] In some implementations, S at position 1 of CDRH2 is replaced by A, I, or M; Y at position 2 of CDRH2 is replaced by F or S; D at position 3 of CDRH2 is replaced by N or G; G at position 4 of CDRH2 is replaced by T; and / or N at position 5 of CDRH2 is replaced by H.

[0080] In some implementations, D at position 1 of CDRH3 is replaced by K; F at position 2 of CDRH3 is replaced by E; D at position 4 of CDRH3 is replaced by T; Y at position 5 of CDRH3 is replaced by G or T; W at position 7 of CDRH3 is replaced by I or V; G at position 8 of CDRH3 is replaced by M; T at position 9 of CDRH3 is replaced by R, A, or K; Y at position 10 of CDRH3 is replaced by T, F, or I; P at position 11 of CDRH3 is replaced by D or S; S at position 12 of CDRH3 is replaced by Y; A at position 13 of CDRH3 is replaced by Y or W; and / or Y at position 14 of CDRH3 is replaced by F.

[0081] In some implementations, the S at position 1 of CDRL1 is replaced by T; the G at position 2 of CDRL1 is replaced by R; the S at position 3 of CDRL1 is replaced by A, T, or G; the S at position 4 of CDRL1 is replaced by T; the S at position 5 of CDRL1 is replaced by Q or G; the N at position 6 of CDRL1 is replaced by S or D; the G at position 8 of CDRL1 is replaced by S; the R at position 9 of CDRL1 is replaced by Y or K; the R at position 10 of CDRL1 is replaced by L or N; the H at position 11 of CDRL1 is replaced by F, N, or Y; and / or the F at position 13 of CDRL1 is replaced by S.

[0082] In some implementations, R at position 1 of CDRL2 is replaced by A or D; G at position 2 of CDRL2 is replaced by A, N or V; D at position 3 of CDRL2 is replaced by S or N; Q at position 4 of CDRL2 is replaced by S or M; R at position 5 of CDRL2 is replaced by L; and / or P at position 6 of CDRL2 is replaced by Q.

[0083] In some implementations, A at position 1 of CDRL3 is replaced by G; T at position 2 of CDRL3 is replaced by M; D at position 4 of CDRL3 is replaced by I; D at position 5 of CDRL3 is replaced by R; G at position 6 of CDRL3 is replaced by S; L at position 7 of CDRL3 is replaced by Q; S at position 8 of CDRL3 is replaced by Q; G at position 9 of CDRL3 is replaced by S or R; Y at position 10 of CDRL3 is replaced by W; and / or V at position 11 of CDRL3 is replaced by S or T.

[0084] In another case, the antibody or its antigen-binding fragment contains the following heavy chain CDR and light chain CDR:

[0085] Heavy chain:

[0086] CDR1 contains GGAFRNS or its variants containing one or two amino acid substitutions relative to GGAFRNS;

[0087] CDR2 contains IPMSGT or its variants containing one or two amino acid substitutions relative to IPMSGT; and

[0088] CDR3 contains EKERTFGVVMRTSYYYVMEV or its variants containing one, two, three, or four amino acid substitutions relative to EKERTFGVVMRTSYYYVMEV;

[0089] Light chain:

[0090] CDR1 contains RASQSISSYLN or its variants containing one or two amino acid substitutions relative to RASQSISSYLN;

[0091] CDR2 contains AASSLQS or its variants containing one or two amino acid substitutions relative to AASSLQS; and

[0092] CDR3 contains QQSYSTPYT or its variants containing one or two amino acid substitutions relative to QQSYSTPYT.

[0093] The Complementary Decision Region (CDR) is identified using Chothia numbers.

[0094] Preferably, one or more substitutions are conservative substitutions, i.e. substitutions within an amino acid class and / or substitutions that obtain a positive score in the BLOSUM62 matrix.

[0095] In some implementations, G at position 2 of CDRH1 is replaced by F or Y; A at position 3 of CDRH1 is replaced by T or I; R at position 5 of CDRH1 is replaced by S or T; N at position 6 of CDRH1 is replaced by A or S; and / or S at position 7 of CDRH1 is replaced by Y.

[0096] In some implementations, I at position 1 of CDRH2 is replaced by N; P at position 2 of CDRH2 is replaced by S; M at position 3 of CDRH2 is replaced by I, A, or S; S at position 4 of CDRH2 is replaced by F or Y; G at position 5 of CDRH2 is replaced by D or N; and / or T at position 6 of CDRH2 is replaced by G.

[0097] In some implementations, K at position 2 of CDRH3 is replaced by D; E at position 3 of CDRH3 is replaced by F; T at position 5 of CDRH3 is replaced by D; G at position 7 of CDRH3 is replaced by Y or T; V at position 9 of CDRH3 is replaced by W or I; M at position 10 of CDRH3 is replaced by G; R at position 11 of CDRH3 is replaced by A, T, or K; T at position 12 of CDRH3 is replaced by F, Y, or I; S at position 13 of CDRH3 is replaced by P or D; Y at position 14 of CDRH3 is replaced by S; Y at position 15 of CDRH3 is replaced by W or A; Y at position 16 of CDRH3 is replaced by F; M at position 18 of CDRH3 is replaced by L; and / or V at position 20 of CDRH3 is replaced by D.

[0098] In some implementations, R at position 1 of CDRL1 is replaced by G; A at position 2 of CDRL1 is replaced by S, T, or G; S at position 3 of CDRL1 is replaced by T; Q at position 4 of CDRL1 is replaced by S or G; S at position 5 of CDRL1 is replaced by D or N; S at position 7 of CDRL1 is replaced by G; S at position 8 of CDRL1 is replaced by G; Y at position 9 of CDRL1 is replaced by R or K; L at position 10 of CDRL1 is replaced by N or R; and / or N at position 11 of CDRL1 is replaced by F, H, or Y.

[0099] In some implementations, A at position 1 of CDRL2 is replaced by D or R; A at position 2 of CDRL2 is replaced by V, N or G; S at position 3 of CDRL2 is replaced by N or D; S at position 4 of CDRL2 is replaced by M or Q; L at position 5 of CDRL2 is replaced by R; and / or Q at position 6 of CDRL2 is replaced by P.

[0100] In some implementations, Q at position 1 of CDRL3 is replaced by L; Q at position 2 of CDRL3 is replaced by S; S at position 3 of CDRL3 is replaced by R or G; Y at position 4 of CDRL3 is replaced by W; S at position 5 of CDRL3 is replaced by T or V; T at position 6 of CDRL3 is replaced by S; P at position 7 of CDRL3 is replaced by R; and / or Y at position 8 of CDRL3 is replaced by N.

[0101] In some implementations, the six CDR sequences of the antibody correspond to the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 sequences annotated herein.

[0102] In another aspect, the present invention provides antibodies and antigen-binding fragments thereof as defined herein for medical use. Accordingly, the antibodies and antigen-binding fragments of the present invention can be used in methods of treating subjects (e.g., cancer patients). In embodiments where the antibody or antigen-binding fragment thereof is used in treating a patient's cancer, the antibody or antigen-binding fragment thereof preferably comprises an Fc region that preferentially binds to an activating Fc receptor as defined herein.

[0103] In other embodiments, the antibody or its antigen-binding fragment is used for the treatment of autoimmune diseases. In these embodiments, the antibody or its antigen-binding fragment preferably comprises an Fc region that preferentially binds to an inhibitory Fc receptor as defined herein.

[0104] In some embodiments where the antibody or antigen-binding fragment is used in medicine (including in methods of treating cancer), the use includes systemic administration of the antibody or antigen-binding fragment.

[0105] In some embodiments, the present invention provides an antibody or antigen-binding fragment that competitively binds to F-actin with an antibody (which may be referred to as a reference antibody) or antigen-binding fragment as defined in whole or in part by its amino acid sequence as described above. The antibody or antigen-binding fragment may compete with the reference antibody for 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 neutralizing the binding of the reference antibody to F-actin.

[0106] In another aspect, the present invention provides a method for providing and / or selecting an antibody or antigen-binding fragment thereof that specifically binds to F-actin. The method includes 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 a competitive agent of a CTLD comprising DNGR-1 to displace one or more lead antibodies from the binding substrate. An 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 competitive agent of a CTLD comprising DNGR-1.

[0107] In some embodiments, antibodies are selected because they exhibit 10-fold specificity against F-actin relative to G-actin. In these embodiments, the method for selecting antibodies includes screening one or more lead antibodies for 10-fold specificity.

[0108] In some embodiments, antibodies are selected because they exhibit at least three times the specificity relative to living cells for binding necrotic cells. In these embodiments, the method for selecting antibodies includes performing FACS or flow cytometry to screen for one or more lead antibodies targeting at least three times the specificity relative to living cells for binding necrotic cells.

[0109] In some embodiments, antibodies are selected because of their ability to promote cross-presentation. In these embodiments, the method of selecting antibodies includes screening one or more lead antibodies for their ability to promote cross-presentation.

[0110] In some embodiments, antibodies are selected because of their ability to promote phagocytosis. In these embodiments, the method of selecting antibodies includes screening one or more lead antibodies for their ability to promote phagocytosis.

[0111] In some implementations, the method for selecting antibodies includes an initial step of “panning” a population of candidate antibodies before exposing the binding substrate to the population. The population can be panned against F-actin beads (positive screening) and G-actin beads (negative screening).

[0112] In some embodiments, the antibody binds to an F-actin epitope that is identical to the epitope on F-actin to which DNGR-1 binds, or the F-actin epitope to which the antibody binds overlaps with the F-actin epitope to which DNGR-1 binds. The epitope to which DNGR-1 binds is elucidated in Hanĉ et al. 2015 (which is hereby incorporated by reference in its entirety). Hanĉ et al. demonstrated that DNGR-1 binds to the interface of two actin protofilaments. The epitope comprises three actin subunits helically arranged within the actin filament (bridging the two protofilaments) and two adjacent actin subunits along one protofilament. Therefore, the F-actin antibody of the present invention is specific to F-actin relative to G-actin because its epitope constitutes part of the fully formed tertiary structure of the F-actin filament (conformational epitope). G-actin does not contain this 3D structure. Hanĉ et al. disclosed that the most extensive interaction between DNGR-1 and F-actin is between actin subunits 2. Moderate interaction with subunit 1 was observed, and only weak interaction with subunit 3 was observed. Therefore, subunit 3 is considered to be largely non-essential for binding.

[0113] In some embodiments, the specificity for F-actin is between 2 and 100 times that for G-actin. In some embodiments, the specificity for F-actin is at least 2, at least 5, at least 10, at least 20, or at least 50 times that for G-actin. In some embodiments, the specificity for F-actin is at least 2 times that for G-actin. In some embodiments, the specificity for F-actin is at least 5 times that for G-actin. In some embodiments, the specificity for F-actin is at least 10 times that for G-actin. In some embodiments, the specificity for F-actin is at least 100 times that for G-actin. Specificity is explicitly intended to encompass affinity and / or cohesion.

[0114] In another aspect, the present invention provides an antibody produced by the method of the present invention. In some embodiments, the antibody is polyclonal.

[0115] The present invention includes combinations of the described aspects and preferred features, except where such combinations are clearly not permitted or explicitly avoided.

[0116] sequence

[0117] The following amino acid sequence is associated with the antibody of this invention.

[0118] Antibodies A and V H

[0119] 1 QVQLVQSGAE VKKPGSSVKV SCKSSGGTFR SYIISWVRQA PGQGLEWMGGFNPIFDTTIY AQKFQGRVTF

[0120] 71 TADESTSTAY MDLSSLRSED TAVYYCARTV IGAFDSWGQG TPVTVSS

[0121] Antibody A, V L

[0122] 1 QSALTQPASV SGSPGQSITI SCTRTSGDIG GYNFVSWYQQ HPGKAPKLMIYDVNSRPSGV SDRFSGSKSG

[0123] 71 DTASLTISGL QAEDEADYYC SSYTSRNTVF GGGTKLTVL

[0124] Using the Chothia system, the FRs and CDRs of Antibody A can be defined as follows:

[0125] FR-H1QVQLVQSGAEVKKPGSSVKVSCKSS

[0126] CDR-H1GGTFRSY

[0127] FR-H2IISWVRQAPGQGLEWMGGF

[0128] CDR-H2NPIFDT

[0129] FR-H3TIYAQKFQGRVTFTADESTSTAYMDLSSLRSEDTAVYYCAR

[0130] CDR-H3TVIGAFDS

[0131] FR-H4WGQGTPVTVSS

[0132] FR-L1QSALTQPASVSGSPGQSITISC

[0133] CDR-L1TRTSGDIGGYNFVS

[0134] FR-L2WYQQHPGKAPKLMIY

[0135] CDR-L2DVNSRPS

[0136] FR-L3GVSDRFSGSKSGDTASLTISGLQAEDEADYYC

[0137] CDR-L3SSYTSRNTV

[0138] FR-L4FGGGTKLTVL

[0139] Using the Kabat system, the FR and CDR of antibody A can be defined as follows:

[0140] FR-H1QVQLVQSGAEVKKPGSSVKVSCKSSGGTFR

[0141] CDR-H1SYIIS

[0142] FR-H2WVRQAPGQGLEWMG

[0143] CDR-H2GFNPIFDTTIYAQKFQG

[0144] FR-H3RVTFTADESTSTAYMDLSSLRSEDTAVYYCAR

[0145] CDR-H3TVIGAFDS

[0146] FR-H4WGQGTPVTVSS

[0147] FR-L1QSALTQPASVSGSPGQSITISC

[0148] CDR-L1TRTSGDIGGYNFVS

[0149] FR-L2WYQQHPGKAPKLMIY

[0150] CDR-L2DVNSRPS

[0151] FR-L3GVSDRFSGSKSGDTASLTISGLQAEDEADYYC

[0152] CDR-L3SSYTSRNTV

[0153] FR-L4FGGGTKLTVL

[0154] Using the IMGT system, the FR and CDR of antibody A can be defined as follows:

[0155] FR-H1QVQLVQSGAEVKKPGSSVKVSCKSS

[0156] CDR-H1GGTFRSYI

[0157] FR-H2ISWVRQAPGQGLEWMGG

[0158] CDR-H2FNPIFDTT

[0159] FR-H3IYAQKFQGRVTFTADESTSTAYMDLSSLRSEDTAVYYC

[0160] CDR-H3ARTVIGAFDS

[0161] FR-H4WGQGTPVTVSS

[0162] FR-L1QSALTQPASVSGSPGQSITISCTRT

[0163] CDR-L1SGDIGGYNF

[0164] FR-L2VSWYQQHPGKAPKLMIY

[0165] CDR-L2DVN

[0166] FR-L3SRPSGVSDRFSGSKSGDTASLTISGLQAEDEADYYC

[0167] CDR-L3SSYTSRNTV

[0168] FR-L4FGGGTKLTVL

[0169] Using the Martin / Contact system, the FRs and CDRs of Antibody A can be defined as follows:

[0170] FR-H1QVQLVQSGAEVKKPGSSVKVSCKSSGGTF

[0171] CDR-H1RSYIIS

[0172] FR-H2WVRQAPGQGLE

[0173] CDR-H2WMGGFNPIFDTTI

[0174] FR-H3YAQKFQGRVTFTADESTSTAYMDLSSLRSEDTAVYYC

[0175] CDR-H3ARTVIGAFD

[0176] FR-H4SWGQGTPVTVSS

[0177] FR-L1QSALTQPASVSGSPGQSITISCTRTSGD

[0178] CDR-L1IGGYNFVSWY

[0179] FR-L2QQHPGKAPK

[0180] CDR-L2LMIYDVNSRP

[0181] FR-L3SGVSDRFSGSKSGDTASLTISGLQAEDEADYYC

[0182] CDR-L3SSYTSRNT

[0183] FR-L4VFGGGTKLTVL

[0184] Antibodies B and V H

[0185] 1 QMQLVQSGAE VKKPGASVKV SCKAPGYIFT SYGISWVRQA PGQGLEWMGRISAYNGHTNY AQNLQDRVTL

[0186] 71 TTTDSTSTAY MELRSLRYDD TAIYYCATGK ISSWFVLEDW GQGTQVTVSS

[0187] Antibodies B and V L

[0188] 1 QSVVTQPPSV SAAPGQKVTI SCSGGTSNIG KNYVSWYQQL PGTAPRLLIYDNNMRPSGIP DRFSGSKSGT

[0189] 71 SATLAITGLQ TGDEADYYCG MWIRSLSRWV FGGTKLAVL

[0190] Using the Chothia system, the frame region (FR) and CDR of antibody B can be defined as follows:

[0191] FR-H1QMQLVQSGAEVKKPGASVKVSCKAP

[0192] CDR-H1GYIFTSY

[0193] FR-H2GISWVRQAPGQGLEWMGRI

[0194] CDR-H2SAYNGH

[0195] FR-H3TNYAQNLQDRVTLTTDTSTSTAYMELRSLRYDDTAIYYCAT

[0196] CDR-H3GKISSWFVLED

[0197] FR-H4WGQGTQVTVSS

[0198] FR-L1QSVVTQPPSVSAAPGQKVTISC

[0199] CDR-L1SGGTSNIGKNYVS

[0200] FR-L2WYQQLPGTAPRLLIY

[0201] CDR-L2DNNMRPS

[0202] FR-L3GIPDRFSGSKSGTSATLAITGLQTGDEADYYC

[0203] CDR-L3GMWIRSLSRWV

[0204] FR-L4FGGGTKLAVL

[0205] Using the Kabat system, the FRs and CDRs of Antibody B can be defined as follows:

[0206] FR-H1QMQLVQSGAEVKKPGASVKVSCKAPGYIFT

[0207] CDR-H1SYGIS

[0208] FR-H2WVRQAPGQGLEWMG

[0209] CDR-H2RISAYNGHTNYAQNLQD

[0210] FR-H3RVTLTTDTSTSTAYMELRSLRYDDTAIYYCAT

[0211] CDR-H3GKISSWFVLED

[0212] FR-H4WGQGTQVTVSS

[0213] FR-L1QSVVTQPPSVSAAPGQKVTISC

[0214] CDR-L1SGGTSNIGKNYVS

[0215] FR-L2WYQQLPGTAPRLLIY

[0216] CDR-L2DNNMRPS

[0217] FR-L3GIPDRFSGSKSGTSATLAITGLQTGDEADYYC

[0218] CDR-L3GMWIRSLSRWV

[0219] FR-L4FGGGTKLAVL

[0220] Using the IMGT system, the FR and CDR of antibody B can be defined as follows:

[0221] FR-H1QMQLVQSGAEVKKPGASVKVSCKAP

[0222] CDR-H1GYIFTSYG

[0223] FR-H2ISWVRQAPGQGLEWMGR

[0224] CDR-H2ISAYNGHT

[0225] FR-H3NYAQNLQDRVTLTTDTSSTAYMELRSLRYDDTAIYYC

[0226] CDR-H3ATGKISSWFVLED

[0227] FR-H4WGQGTQVTVSS

[0228] FR-L1QSVVTQPPSVSAAPGQKVTISCSGG

[0229] CDR-L1TSNIGKNY

[0230] FR-L2VSWYQQLPGTAPRLLIY

[0231] CDR-L2DNN

[0232] FR-L3MRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYC

[0233] CDR-L3GMWIRSLSRWV

[0234] FR-L4FGGGTKLAVL

[0235] Using the Martin / Contact system, the FR and CDR of antibody B can be defined as follows:

[0236] FR-H1QMQLVQSGAEVKKPGASVKVSCKAPGYIF

[0237] CDR-H1TSYGIS

[0238] FR-H2WVRQAPGQGLE

[0239] CDR-H2WMGRISAYNGHTN

[0240] FR-H3YAQNLQDRVTLTTDTSTSTAYMELRSLRYDDTAIYYC

[0241] CDR-H3ATGKISSWFVLE

[0242] FR-H4DWGQGTQVTVSS

[0243] FR-L1QSVVTQPPSVSAAPGQKVTISCSGGTSN

[0244] CDR-L1IGKNYVSWY

[0245] FR-L2QQLPGTAPR

[0246] CDR-L2LLIYDNNMRP

[0247] FR-L3SGIPDRFSGSKSGTSATLAITGLQTGDEADYYC

[0248] CDR-L3GMWIRSLSRW

[0249] FR-L4VFGGGTKLAVL

[0250] Antibody C, V H

[0251] 1 QVQLVESGGG VVQPGRSLRL SCAASGFTFS AYAMHWVRQA PGKGLEWMAVISYDGNNIHY ADSVKGRFTV

[0252] 71 SRDNSKNTLF LQMDGLRTED TAVYYCARDF RDYVWGTYPS AYWGQGTLVT VSS

[0253] Antibody C, V L

[0254] 1 QSVLTQPPSV SGTPGQRVII SCSGSSSNIG RRHVFWYQQF PESAPKLLIYRGDQRPSGVP ERYSGSKSGT

[0255] 71 SASLAISGLR SEDEADYYCA TWDDGLSGYV FGTGTRVTVL

[0256] Using the Chothia system, the FR and CDR of antibody C can be defined as follows:

[0257] FR-H1QVQLVESGGGVVQPGRSLRLSCAAS

[0258] CDR-H1GFTFSAY

[0259] FR-H2AMHWVRQAPGKGLEWMAVI

[0260] CDR-H2SYDGNN

[0261] FR-H3IHYADSVKGRFTVSRDNSKNTLFLQMDGLRTEDTAVYYCAR

[0262] CDR-H3DFRDYVWGTYPSAY

[0263] FR-H4WGQGTLVTVSS

[0264] FR-L1QSVLTQPPSVSGTPGQRVIISC

[0265] CDR-L1SGSSSNIGRRHVF

[0266] FR-L2WYQQFPESAPKLLIY

[0267] CDR-L2RGDQRPS

[0268] FR-L3GVPERYSGSKSGTSASLAISGLRSEDEADYYC

[0269] CDR-L3ATWDDGLSGYV

[0270] FR-L4FGTGTRVTVL

[0271] Using the Kabat system, the FR and CDR of antibody C can be defined as follows:

[0272] FR-H1QVQLVESGGGVVQPGRSLRLSCAASGFTFS

[0273] CDR-H1AYAMH

[0274] FR-H2WVRQAPGKGLEWMA

[0275] CDR-H2VISYDGNNIHYADSVKG

[0276] FR-H3RFTVSRDNSKNTLFLQMDGLRTEDTAVYYCAR

[0277] CDR-H3DFRDYVWGTYPSAY

[0278] FR-H4WGQGTLVTVSS

[0279] FR-L1QSVLTQPPSVSGTPGQRVIISC

[0280] CDR-L1SGSSSNIGRRHVF

[0281] FR-L2WYQQFPESAPKLLIY

[0282] CDR-L2RGDQRPS

[0283] FR-L3GVPERYSGSKSGTSASLAISGLRSEDEADYYC

[0284] CDR-L3ATWDDGLSGYV

[0285] FR-L4FGTGTRVTVL

[0286] Using the IMGT system, the FRs and CDRs of antibody C can be defined as follows:

[0287] FR-H1QVQLVESGGGVVQPGRSLRLSCAAS

[0288] CDR-H1GFTFSAYA

[0289] FR-H2MHWVRQAPGKGLEWMAV

[0290] CDR-H2ISYDGNNI

[0291] FR-H3HYADSVKGRFTVSRDNSKNTLFLQMDGLRTEDTAVYYC

[0292] CDR-H3ARDFRDYVWGTYPSAY

[0293] FR-H4WGQGTLVTVSS

[0294] FR-L1QSVLTQPPSVSGTPGQRVIISCSGS

[0295] CDR-L1SSNIGRRH

[0296] FR-L2VFWYQQFPESAPKLLIY

[0297] CDR-L2RGD

[0298] FR-L3QRPSGVPERYSGSKSGTSASLAISGLRSEDEADYYC

[0299] CDR-L3ATWDDGLSGYV

[0300] FR-L4FGTGTRVTVL

[0301] Using the Contact system, the FRs and CDRs of antibody C can be defined as follows:

[0302] FR-H1QVQLVESGGGVVQPGRSLRLSCAASGFTF

[0303] CDR-H1SAYAMH

[0304] FR-H2WVRQAPGKGLE

[0305] CDR-H2WMAVISYDGNNIH

[0306] FR-H3YADSVKGRFTVSRDNSKNTLFLQMDGLRTEDTAVYYC

[0307] CDR-H3ARDFRDYVWGTYPSA

[0308] FR-H4YWGQGTLVTVSS

[0309] FR-L1QSVLTQPPSVSGTPGQRVIISCSGSSSN

[0310] CDR-L1IGRRHVFWY

[0311] FR-L2QQFPESAPK

[0312] CDR-L2LLIYRGDQRP

[0313] FR-L3SGVPERYSGSKSGTSASLAISGLRSEDEADYYC

[0314] CDR-L3 ATWDDGLSGY

[0315] FR-L4 VFGTGTRVTVL

[0316] Antibody D, V H

[0317] 1 QVQLVQSGAE VKKTGSSVKV SCKVSGGAFR NSAINWVRQA PGRGLEWMG VIIPMSGTTNY ARNFQGRVTI

[0318] 71 SADESTSTAY MELSTLTSGD TADYYCAREK ERTFGVVMRT SYYYVMEVWGQGTTVTVSS

[0319] Antibody D, V L

[0320] 1 AIRMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIY AASSLQSGVPS RFSGSGSGTD

[0321] 71 FTLTISSLQP EDFATYYCQQ SYSTPYTFGQ GTKLEIK

[0322] Using the Chothia system, the FRs and CDRs of Antibody D can be defined as follows:

[0323] FR-H1 QVQLVQSGAEVKKTGSSVKVSCKVS

[0324] CDR-H1 GGAFRNS

[0325] FR-H2 AINWVRQAPGRGLEWMGVI

[0326] CDR-H2 IPMSGT

[0327] FR-H3 TNYARNFQGRVTISADESTSTAYMELSTLTSGDTADYYCAR

[0328] CDR-H3 EKERTFGVVMRTSYYYVMEV

[0329] FR-H4 WGQGTTVTVSS

[0330] FR-L1 IRMTQSPSSLSASVGDRVTITC

[0331] CDR-L1RASQSISSYLN

[0332] FR-L2WYQQKPGKAPKLLIY

[0333] CDR-L2AASSLQS

[0334] FR-L3GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

[0335] CDR-L3QQSYSTPYT

[0336] FR-L4FGQGTKLEIK

[0337] Using the Kabat system, the FRs and CDRs of Antibody D can be defined as follows:

[0338] FR-H1QVQLVQSGAEVKKTGSSVKVSCKVSGGAFR

[0339] CDR-H1NSAIN

[0340] FR-H2WVRQAPGRGLEWMG

[0341] CDR-H2VIIPMSGTTNYARNFQG

[0342] FR-H3RVTISADESTSTAYMELSTLTSGDTADYYCAR

[0343] CDR-H3EKERTFGVVMRTSYYYVMEV

[0344] FR-H4WGQGTTVTVSS

[0345] FR-L1IRMTQSPSSLSASVGDRVTITC

[0346] CDR-L1RASQSISSYLN

[0347] FR-L2WYQQKPGKAPKLLIY

[0348] CDR-L2AASSLQS

[0349] FR-L3GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

[0350] CDR-L3QQSYSTPYT

[0351] FR-L4FGQGTKLEIK

[0352] Using the IMGT system, the FR and CDR of antibody D can be defined as follows:

[0353] FR-H1QVQLVQSGAEVKKTGSSVKVSCKVS

[0354] CDR-H1GGAFRNSA

[0355] FR-H2INWVRQAPGRGLEWMGV

[0356] CDR-H2IIPMSGTT

[0357] FR-H3NYARNFQGRVTISADESTSTAYMELSTLTSGDTADYYC

[0358] CDR-H3AREKERTFGVVMRTSYYYVMEV

[0359] FR-H4WGQGTTVTVSS

[0360] FR-L1IRMTQSPSSSLSASVGDRVTITCRAS

[0361] CDR-L1QSISSY

[0362] FR-L2LNWYQQKPGKAPKLLIY

[0363] CDR-L2AAS

[0364] FR-L3SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

[0365] CDR-L3QQSYSTPYT

[0366] FR-L4FGQGTKLEIK

[0367] Using the Martin / Contact system, the FR and CDR of antibody D can be defined as follows:

[0368] FR-H1QVQLVQSGAEVKKTGSSVKVSCKVSGGAF

[0369] CDR-H1RNSAIN

[0370] FR-H2WVRQAPGRGLE

[0371] CDR-H2WMGVIIPMSGTTN

[0372] FR-H3YARNFQGRVTISADESTSTAYMELSTLTSGDTADYYC

[0373] CDR-H3AREKERTFGVVMRTSYYYVME

[0374] FR-H4VWGQGTTVTVSS

[0375] FR-L1IRMTQSPSSLSASVGDRVTITCRASQSI

[0376] CDR-L1SSYLNWY

[0377] FR-L2QQKPGKAPK

[0378] CDR-L2LLIYAASSLQ

[0379] FR-L3SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

[0380] CDR-L3QQSYSTPY

[0381] FR-L4TFGQGTKLEIK

[0382] Antibody E, V H

[0383] 1 QVQLVQSGAE VKKTGSSVKV SCKVFGGAFR NSAINWVRQA PGRGLEWMGVLIPMSGTTNY ARNFQGRVTI

[0384] 71 SADESTSTAY MELSTLTSGD TAVYYCAREK ERTFGVVMRT SYYYVMEVWGQGTTVTVSS

[0385] Antibody E, V L

[0386] 1 AIRMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYAASSLQSGVPS RFSGSGSGTD

[0387] 71 FTLTISSLQP EDFATYYCQQ SYSTPYTFGQ GTKLEIK

[0388] Using the Chothia system, the FRs and CDRs of Antibody E can be defined as follows:

[0389] FR-H1QVQLVQSGAEVKKTGSSVKVSCKVF

[0390] CDR-H1GGAFRNS

[0391] FR-H2AINWVRQAPGRGLEWMGVL

[0392] CDR-H2IPMSGT

[0393] FR-H3TNYARNFQGRVTISADESTSTAYMELSTLTSGDTAVYYCAR

[0394] CDR-H3EKERTFGVVMRTSYYYVMEV

[0395] FR-H4WGQGTTVTVSS

[0396] FR-L1IRMTQSPSSLSASVGDRVTITC

[0397] CDR-L1RASQSISSYLN

[0398] FR-L2WYQQKPGKAPKLLIY

[0399] CDR-L2AASSLQS

[0400] FR-L3GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

[0401] CDR-L3QQSYSTPYT

[0402] FR-L4FGQGTKLEIK

[0403] Using the Kabat system, the FRs and CDRs of Antibody E can be defined as follows:

[0404] FR-H1QVQLVQSGAEVKKTGSSVKVSCKVFGGAFR

[0405] CDR-H1NSAIN

[0406] FR-H2WVRQAPGRGLEWMG

[0407] CDR-H2VLIPMSGTTNYARNFQG

[0408] FR-H3RVTISADESTSTAYMELSTLTSGDTAVYYCAR

[0409] CDR-H3EKERTFGVVMRTSYYYVMEV

[0410] FR-H4WGQGTTVTVSS

[0411] FR-L1IRMTQSPSSLSASVGDRVTITC

[0412] CDR-L1RASQSISSYLN

[0413] FR-L2WYQQKPGKAPKLLIY

[0414] CDR-L2AASSLQS

[0415] FR-L3GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

[0416] CDR-L3QQSYSTPYT

[0417] FR-L4FGQGTKLEIK

[0418] Using the IMGT system, the FRs and CDRs of antibody E can be defined as follows:

[0419] FR-H1QVQLVQSGAEVKKTGSSVKVSCKVF

[0420] CDR-H1GGAFRNSA

[0421] FR-H2INWVRQAPGRGLEWMGV

[0422] CDR-H2LIPMSGTT

[0423] FR-H3NYARNFQGRVTISADESTSTAYMELSTLTSGDTAVYYC

[0424] CDR-H3AREKERTFGVVMRTSYYYVMEV

[0425] FR-H4WGQGTTVTVSS

[0426] FR-L1IRMTQSPSSLSASVGDRVTITCRAS

[0427] CDR-L1QSISSY

[0428] FR-L2LNWYQQKPGKAPKLLIY

[0429] CDR-L2AAS

[0430] FR-L3SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

[0431] CDR-L3QQSYSTPYT

[0432] FR-L4FGQGTKLEIK

[0433] Using the Contact system, the FR and CDR of antibody E can be defined as follows:

[0434] FR-H1QVQLVQSGAEVKKTGSSVKVSCKVFGGAF

[0435] CDR-H1RNSAIN

[0436] FR-H2WVRQAPGRGLE

[0437] CDR-H2WMGVLIPMSGTTN

[0438] FR-H3YARNFQGRVTISADESTSTAYMELSTLTSGDTAVYYC

[0439] CDR-H3AREKERTFGVVMRTSYYYVME

[0440] FR-H4VWGQGTTVTVSS

[0441] FR-L1IRMTQSPSSSLSASVGDRVTITCRASQSI

[0442] CDR-L1SSYLNWY

[0443] FR-L2QQKPGKAPK

[0444] CDR-L2LLIYAASSLQ

[0445] FR-L3SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

[0446] CDR-L3QQSYSTPY

[0447] FR-L4TFGQGTKLEIK

[0448] The antibody of the present invention can be defined as having six CDR sequences having a CDR sequence corresponding to one of the clones defined above. Attached Figure Description

[0449] The embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying drawings, in which:

[0450] Figure 1 Phage selection funnel for .F-actin monoclonal antibody.

[0451] Figure 2 Ranking of anti-F-actin IgG2a clones based on their ability to induce XP in necrotic cells in cDC2. Hoxb8-CDP-derived cDC2 cells were soaked in OVA with necrotic 5555 Braf. V600E Melanoma cells were incubated with dose-adjusted (50 nM, 10 nM, 2 nM, 0.4 nM) 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. After 24 h, T cell-derived IFNγ in the supernatant was measured by ELISA. The area under the curve (AUC) of IFNγ production for each mAb was calculated and ranked in descending order. Preferred clones were marked with gray boxes.

[0452] Figure 3 XP replications of candidate anti-F-actin IgG2a clones in cDC2. Hoxb8-CDP-derived cDC2 was soaked in OVA-treated necrotic 5555 Braf. V600E Melanoma cells were incubated with dose-adjusted 2WA Fc-DNGR-1 (black circles), WTFc-DNGR-1 (white circles), or anti-F-actin IgG2a clones (white triangles) and OT-I CD8+ T cells. T cell-derived IFNγ in the supernatant was measured by ELISA after 24 h. Mean + / - SEM values ​​from repeated measurements are shown.

[0453] Figure 4 ELISA of F-actin versus G-actin in candidate anti-F-actin clones. Binding of selected mAbs and F-actin specificity were measured by ELISA using immobilized F-actin and G-actin.

[0454] Figure 5 Necrotic cell binding of candidate anti-F-actin clones. Necrotic mice 5555 Braf irradiated with UV light. V600EFlow cytometry analysis of melanoma cells (A) or necrotic human HEK293T cells (B) was performed by incubating both cell types with serially diluted anti-F-actin IgG2a clones or Fc-DNGR-1 fusion protein (2WA [negative control] or WT [positive control]) and staining with AF488-conjugated anti-mouse IgG secondary antibody. Mean + / - SEM results from repeated measurements are shown.

[0455] Figure 6 Phagocytosis of necrotic cells in primary cDCs in the presence of anti-F-actin IgG2a. Flow cytometry analysis of internalization of CT-DR-labeled necrotic cell debris by primary FLT3L-cDCs from mouse bone marrow pretreated with IFN-I in the presence of an F-actin binding reagent. cDC1 (A) and cDC2 (B) were analyzed after 4 h of incubation, and the phagocytic index was calculated using the following formula: (% of CT-DR-positive cDCs x GMFI of CT-DR-positive cDCs) / 10,000. Mean + / - SEM values ​​from repeated measurements are shown.

[0456] Figure 7 Necrotic primary cDC cells (XP) in the presence of anti-F-actin IgG2a. Primary bone marrow FLT3L-cDC cultures pretreated with IFN-I were separated into cDC1 and non-cDC1 fractions using MACS enrichment targeting XCR1. These were then compared with OVA-soaked UV-irradiated necrotic cells. V600E Cells were incubated with serially diluted WT Fc-DNGR-1, unrelated IgG2a or anti-F-actin IgG2a clones, and OT-I CD8+ T cells. IFNγ derived from T cells was measured after 24 h for both cDC1 (A) and non-cDC1 (B). Mean + / - SEM values ​​from repeated measurements are shown.

[0457] Figure 8 Necrotic cell binding of candidate commercial anti-actin clones. Necrotic mice 5555Braf irradiated with UV light. V600E Flow cytometry analysis of melanoma cells involved incubating the cells with serially diluted anti-actin IgG2a clones (AC-40 or AC-74) or Fc-DNGR-1 fusion protein (2WA or WT) and staining with AF488-conjugated anti-mouse IgG secondary antibody.

[0458] Figure 9Phagocytosis of necrotic cells in primary cDCs in the presence of commercially available anti-actin IgG2a. Flow cytometry analysis of internalization of CT-DR-labeled necrotic cell debris by primary FLT3L-cDCs from mouse bone marrow pretreated with IFN-I 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 analyzed after 4 h of incubation, and the phagocytic index was calculated using the following formula: (% of CT-DR-positive cDCs x GMFI of CT-DR-positive cDCs) / 10,000. Mean + / - SEM results from repeated measurements are shown.

[0459] Figure 10 Necrotic primary cDC cells (XP) were extracted in the presence of commercially available anti-F-actin IgG2a. Primary bone marrow FLT3L-cDC cultures pretreated with IFN-I were separated into cDC1 and non-cDC1 fractions using MACS enrichment targeting XCR1. These were then compared with OVA-soaked UV-irradiated necrotic cells (5555 Braf). V600E Cells were incubated with serially diluted 2WA or WT Fc-DNGR-1 or commercial anti-actin IgG2a (AC-40 or AC-74) and OT-I CD8+ T cells. IFNγ derived from both cDC1 (A) and non-cDC1 (B) T cells was measured after 24 h. Mean + / - SEM values ​​from repeated measurements are shown.

[0460] Figure 11 Ranked according to their potency and efficacy against actin antibodies in XP. Figure 10 XP results between cDC1(A) and non-cDC1(B) were used to calculate the EC50 (potency) and maximum IFNγ signal (efficacy) for each antibody / Fc-DNGR-1. The dashed line indicates the IFNγ signal in the absence of a monoclonal antibody.

[0461] Figure 12 G-actin competes with anti-actin antibodies for binding to necrotic cells. In the presence of escalating doses of soluble pinocembrin D-stabilized human platelet G-actin, anti-actin antibodies (10 nM) compared with UV-irradiated necrotic mice (Braf 5555). V600E Flow cytometry analysis of melanoma cell binding. (A) Solid black line = no G-actin; dashed black line = 256 nM G-actin. (B) Plotting individual points for each condition.

[0462] Figure 13Tumor control with anti-F-actin IgG2a antibody. Anti-F-actin IgG2a clones or Fc-DNGR-1 (WT or 2WA) were administered intratumorally (100 μg for Fc-DNGR-1 or 150 μg for anti-F-actin IgG2a in 50 μl) to C57BL / 6 mice carrying MCA205 tumors. On day 7, the drug was administered in combination with 1.25 mg / kg doxorubicin to induce immunogenic cell death. A second dose of antibody / Fc-DNGR-1 was administered on day 11. N = 10 / group. Mean + / - SEM plotted. (A) Schematic diagram of the experimental setup. (B) Tumor volume measured in the Fc-DNGR-1 control and in the cases of antibodies A, B, and E.

[0463] Figure 14 Anti-F-actin mAbs differ in their ability to inhibit the binding of DNGR-1 to F-actin. A comparison of the ability of three anti-F-actin mAbs to competitively bind to F-actin against the extracellular domain (ECD) of DNGR-1 is presented. Anti-F-actin mAbs were mixed with 10 nM FLAG-labeled DNGR-1 ECD at different doses as indicated and added to immobilized F-actin. Inhibition curves for each competitor are shown in the figure. OD was measured according to the method and materials section. 405 The value is used to calculate the percentage of inhibition.

[0464] Figure 15 Systemic anti-F-actin antibody promotes therapeutic tumor control. (A) Implantation of MCA205 cells (0.5 x 10⁻⁶) 6(a) Schematic diagram and tumor growth curve of C57BL / 6 mice (n = 10 / group) treated with WT or 2WA Fc-DNGR-1 (100 μg per tumor cycle) or antibody A (150 μg per tumor cycle) in combination with doxorubicin (2.5 mg / kg intratumorally, day 7). Plot mean ± SEM. (B) ELISA of serum antibody A or Fc-DNGR-1 levels after systemic administration (n = 2 / time point). Plot mean ± SEM. (C) Confocal microscopy analysis of MCA205 tumors treated with doxorubicin three days after systemic injection of antibody A or isotype IgG2a (400 μg intraperitoneally). Antibody A was detected using anti-mouse IgG2a (top row) or anti-CH1-IgG (bottom row). Plot mean ± SEM. (D) Tumor growth curves (n = 10) of C57BL / 6 mice implanted with MC38, treated systemically with anti-F-actin or allotype IgG2a (400 μg intraperitoneally, every two weeks starting from day 4). Plotted as mean ± SEM. (E) Relative body weight changes in mice treated in D. Plotted as mean ± SEM. (F) Schematic diagram and standard curves of anti-DNGR-1 (top) and anti-human CH1 (bottom) ELISAs used for PK analysis. Data represent two independent experiments. Data (A, D) were analyzed using Bonferroni-corrected two-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

[0465] Figure 16 Necrotic cells are located in the FcγR region of tumors. + Near APC. (A) Braf cells cultured in vitro with intracellular binding reagent. V600E (B) Confocal microscopy analysis of 20 μm sections of melanoma tumor spheroids. (C) Representative confocal images of colocalization of actin cytoskeleton binding reagent and DNA binding reagent from A. (D) Exemplary filamentous staining pattern of Fc-DNGR-1 on necrotic cells within tumor spheroids prepared as in A. (E) Top: Confocal microscopy analysis of mouse MCA205 LifeAct (LA)-OVA tumors (day 8 post-implantation in C57BL / 6 mice) after local injection of WT or 2WA Fc-DNGR-1 (n = 9 independent tumors). Bottom: Data pooled from multiple experiments, plotted as mean ± SEM. (E) Top: Identification of Fc-DNGR-1 in tumors from D. + Necrotic cells and MHC-II + APC group (CD103) + cDC1 and CD103 -Confocal microscopy analysis of non-cDC1 cells. Each point represents a single cell within a tumor, and only APCs with a distance < 500 μm to the necrotic core were analyzed. Bottom: Plotted mean. (F) Confocal microscopy analysis of staining MCA205 LA-OVA-mCherry tumors with anti-mouse FcγRI is shown. Arrows indicate cell co-staining with MHC-II and FcγRI. Data represent two (E), three (AC), or more (D) independent experiments. Data were analyzed using unpaired Student's t-test (DE). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

[0466] Figure 17 Binding of commercially available anti-actin antibodies to F-actin. ELISA assessment of the binding of commercially available anti-actin and AFA to F-actin-coated plates.

[0467] Figure 18 Comparison of human IgG1 AFA and 8-3. (A) ELISA analysis of the binding of human IgG1 AFA and 8-3 antibodies to F-actin-coated plates. (B) Human IgG1 AFA and 8-3 with UV-irradiated necrotic mice. V600E Flow cytometry analysis of tumor cell binding.

[0468] Figure 19 Fc receptor expression on human tumor cDC2 and moDCs. (A) DC2 FCGR expression levels in human tumors, assessed using single-cell RNA sequencing (scRNAseq) data obtained from the Human Pan-Cancer Myeloid Atlas Portal (http: / / panmyeloid.cancer-pku.cn). (B) Flow cytometry analysis of Fc receptor expression on DCs (GM-CSF / IL-4) derived from monocytes produced from peripheral blood of healthy individuals.

[0469] Figure 20Antibody A promotes necrophagy in human mononuclear cell-derived dendritic cells (DCs). (A) Confocal microscopy analysis of necrophagy in human moDCs incubated with UV-irradiated necrotic cells, WT, and mutant AFA A IgG1. Uptake is represented as necrotic cells internalized per DC, where each point represents a field of view (left) or the mean uptake observed in different donors (right). Plotted mean ± SEM. LALA-PG = Fc null antibody; WT = standard human IgG1 Fc; GASDALIE = Fc enhancer antibody. (B) Confocal microscopy analysis of necrophagy as in A in the presence of Fc receptor blocking antibodies or SYK inhibitors. Each point represents a field of view. Data represent two or more independent experiments. Data were analyzed using one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

[0470] Figure 21 Antibody A promotes necrotizing phagocytosis in macrophages derived from human monocytes. (A) Flow cytometry analysis of Fc receptor expression in human monocyte-derived macrophages (moMac, M-CSF-derived). (B) According to... Figure 19 Confocal microscopy analysis of necrotic phagocytosis in moDC (human moMac). Data represent two or more independent experiments. One-way ANOVA was used to analyze the data. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Detailed Implementation

[0471] Aspects and embodiments of the invention will now be discussed with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All references to this document are incorporated herein by reference.

[0472] F-actin

[0473] 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 muscle cell contraction apparatus.

[0474] Higher eukaryotes possess several different actin variants, each classified as α, β, or γ. Any given organism can have more than one variant of any particular class. For example, mammals express at least six different actin variants, each encoded by a separate gene, while lower eukaryotes may have fewer variants. For instance, most yeasts possess only one.

[0475] Typically, the α and γ2 isoforms are found in muscles (α-skeletal muscle type, α-aortic smooth muscle type, α-cardiac muscle type, and γ2-intestinal smooth muscle type), while the β and γ1 isoforms are found in non-muscle cells (β-cytoplasmic type and γ1-cytoplasmic type).

[0476] Actin molecules possess ATPase activity. They have deep nucleotide-binding cleavages that can bind ATP or ADP and hydrolyze ATP into ADP.

[0477] The monomeric (globose) form of any variant actin molecule is usually named "G-actin". G-actin can polymerize into chains, and the polymerized form is named "F-actin".

[0478] Filament actin (F-actin) consists of two parallel actin chains (filaments) in a helical configuration, with a diameter of approximately 7 nm and a pitch (i.e., the distance along the axis of a complete turn) of approximately 37 nm.

[0479] G-actin polymerization into F-actin can be achieved by incubating the monomeric form in a suitable buffer containing physiological saline and ATP. Furthermore, F-actin filaments of a defined length (approximately 100 subunits) can be prepared by subjecting F-actin to actin shearing and the action of capping proteins (such as gelling proteins).

[0480] F-actin can be stabilized by binding to and inhibiting the depolymerization of various molecules, such as phalloidin, jasplakinolide, or tropomyosin.

[0481] For the purposes of this specification, the term "F-actin" may be considered to refer to any substance containing two or more actin subunits, said actin subunits being conformally covalently or non-covalently associated in a conformation mimicking that of actin subunits in physiological F-actin chains or filaments. The substance may contain any suitable number of associating monomers, such as 5 or more, 10 or more, 20 or more, 50 or more, or 100 or more monomer units. For example, it may contain between 2 and 100 monomer units, such as 2-10 monomer units, 10-50 monomer units, or 50-100 monomer units.

[0482] F-actin can be composed of a single F-actin isoform or a mixture of different F-actin isoforms. For example, it can be composed entirely of α, β, or γ subunits, or it can be a mixture of them. It can be composed entirely of non-muscle (NM) isoforms (NM F-actin) or entirely of muscle isoforms (muscle actin).

[0483] An exemplary human actin sequence includes:

[0484] α-actin (α-skeletal muscle type)

[0485] MCDEDETTALVCDNGSGLVKAGEFAGDDAPRAVEPSIVCGRPRHOGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIITNWDDMEKIWHHTFYNELRVAPEEHPTLLTEAPLNPKANREKMIQIMEETENVPAMYVAIQAVLSLYASGRTTGIVLDSGDGVTHNVPIYEGYALPHAIMRLDLAGRDLTD YLMKILTERGYSEVITAEREIVRDIKEKLCYVALDFENEMATAASSSSLEKSYELPDGQVITIGNERFRCPETLEFQPSEFIGMESAGIHETTYNSIMKCDIDIRKDLYANNVMSGGTTMYPGIADRMOKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTEFQOMWITKOEYDEAGPSIVHRKCE

[0486] β-actin (cytoplasmic type 1)

[0487] MDDDIAALVVDNGSGMCKAGEFAGDDAPRAVEPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIVINWDDMEKIWHHTEYNELRVAPEEHPVLLTEAPLNPKANREKMTQIMFETENTPAMYVAIQAVLSLYASGRTTGIVMDSGDGVTHIVPIYEGYALPHAILRLDLAGRDLTDY LMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQEMATAASSSSLEKSYBELPDGOVITIGNERFRCPEALFQPSEFLGMESCGIHETTENSIMKCDVDIRKDLYANTVLSGGTTMYPGIADRMOKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTEQOMWISKQEYDESGPSIVHRKCE

[0488] γ-actin (cytoplasmic type 2)

[0489] MEEEIAALVIDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHQOGVMVGMGQOKDSYVGDEAQSKRGILTILKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANREKMTQIMFETFNTPAMYVAIQAVLSLYASGRTTGIVMDSGDGVIHIVPIYEGYALPHAILRLDLAGRDLT DYLMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQEMATAASSSSLEKSYELPDGQVITIGNERFRCPEALFQPSFLGMESCGIHETTFNSIMKCDVDIRKDLYANTVLSGGITMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQOMWISKQEYDESGPSIVHRKCE

[0490] Actin is a highly conserved protein, sharing 90% identity between yeast and humans (Srinivasan, 2016). Each actin monomer is a 375-amino acid polypeptide that folds into two main 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 inserted into domains 1 and 3, respectively (Dominguez and Holmes, 2011, which is hereby incorporated by reference in its entirety).

[0491] F-actin assembles into two protofilaments that intertwine to form a right-handed double-stranded long helix, in which approximately 13 molecules repeat every 6 turns at an axial distance of 35.9 nm (Dominguez and Holmes, 2011).

[0492] F-actin refers to the filamentous form of actin. Therefore, the term "F-actin" refers to heterogeneous molecules of different lengths.

[0493] Anti-F-actin antibody and antigen-binding fragment

[0494] Monoclonal antibodies (mAbs) can be used in the methods of the present invention and are a homogeneous population of antibodies that specifically target a single epitope on an antigen. Suitable monoclonal antibodies can be prepared using methods well known in the art (see, for example, 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 Supplement A: S106-12; Helen E. Chadd and Steven M. Chamow; "Therapeutic antibody expression technology," Current Opinion in Biotechnology 12, Vol. 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", JGR Hurrell (CRC Press, 1982). Neuberger et al. (1988, 8th International Congress on Biotechnology, Part 2, 792-799) discussed chimeric antibodies, each of which is incorporated herein by reference in its entirety.

[0495] Polyclonal antibodies can be used in some methods of the present invention. However, monospecific polyclonal antibodies are preferred. Suitable polyclonal antibodies can be prepared using methods well known in the art.

[0496] Antibody fragments (such as Fab and Fab2 fragments) can also be used, as can genetically engineered antibodies and antibody fragments.

[0497] As used in this article, the "antigen-binding fragment" of an antibody is a fragment containing V. H / V L The fragment is an antigen-binding portion of the antibody, allowing it to retain essentially the same specific binding activity as the full-length antibody. (One or more amino acid residues present in the full-length antibody have been removed from the fragment, but this does not significantly affect the antigen-binding activity of the fragment.)

[0498] The fact that the variable heavy chain (VH) and variable light chain (VL) domains of antibodies are involved in antigen recognition was first recognized through early protease digestion experiments. This was further confirmed through the "humanization" of rodent antibodies. The rodent-derived variable domains can be fused with human-derived constant domains, so that the resulting antibodies retain the antigen specificity of the rodent parent antibodies (Morrison et al. (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855, which is incorporated herein by reference in its entirety).

[0499] Experiments involving bacterial expression of antibody fragments (all containing one or more variable domains) have shown that antigen specificity is conferred by the variable domains and is independent of the constant 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 in which the VH and VL paired domains are linked via flexible oligopeptides (Bird et al. (1988) Science 242, 423; Huston et al. (1988) Proc. Natl. Acad. Sd. USA 85, 5879); and single-domain antibodies (sdAbs) containing a separated V domain (Ward et al. (1989) Nature 341, 544). A general overview of the techniques involved in the synthesis of antibody fragments that retain their specific binding sites can be found in Winter and Milstein (1991) Nature 349, 293-299.

[0500] "ScFv molecule" refers to a molecule in which VH and VL paired domains are covalently linked, for example, directly, via peptide, or via flexible oligopeptides. Fab, Fv, ScFv, and sdAb antibody fragments can all be expressed in and secreted from *E. coli*, allowing for the easy production of large quantities of said fragments.

[0501] The complete antibody and the F(ab')2 fragment are "bivalent." "Bivalent" means that the antibody and the F(ab')2 fragment have two antigen-binding sites. In contrast, the Fab, Fv, ScFv, and sdAb fragments are monovalent, having only one antigen-binding site. Synthetic antibodies binding to the targets discussed herein can also be prepared using phage display techniques well-known in the art (see, for example, "Phage display: a molecular tool for the generation of antibodies--a review". Placenta. 21 Supplement A: 106-12. Helen E. Chadd and Steven M. Chamow; "Phageantibodies: filamentous phage displaying antibody variable domains". Nature 348 (6301): 552-554).

[0502] "Antibody that binds to the same epitope as that bound by DNGR-1" can refer to an antibody that blocks the binding of DNGR-1 to its antigen by 50% or more in a competitive assay, or conversely, an antibody that blocks the binding of its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0503] "Antibody that binds to the same epitope as the reference antibody" can refer to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, or conversely, a reference antibody that blocks the binding of an antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0504] As used in this article, the term "AFA" refers to "anti-F-actin antibody".

[0505] Preparation of anti-F-actin antibody

[0506] The antibodies of this invention can be prepared using any suitable method known in the art. These include hybridoma, phage display, and panning. These descriptions appear in, for example, 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 Supplement A: S106-12; Helen E. Chadd and Steven M. Chamow; “Therapeutic antibody expression technology,” Current Opinion in Biotechnology 12, Vol. 2 (April 1, 2001): 188-194; McCafferty, J.; Griffiths, A.; Winter, G.; Chiswell, D. (1990). "Phage antibodies: Fibrous phage displaying antibody variabledomains. Nature 348 (6301): 552-554; "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Applications", JGR Hurrell (CRC Press, 1982), each of which is incorporated in its entirety by reference. Chimeric antibodies were discussed by Neuberger et al. (1988, 8th International Congress on Biotechnology, Part 2, 792-799).

[0507] For any of the above methods, a standard elution buffer containing trypsin (e.g., at 1 mg / ml) or elution can be performed competitively with Fc-DNGR1 (e.g., at 1000 nM).

[0508] Fc structural domain

[0509] The Fc domain (crystallizable fragment region) is a region in 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 the antibody to activate the immune system. The IgG Fc domain contains a highly conserved N-glycosylation site, which is essential for Fcγ receptor-mediated activity. The Fc domain can be mutated to alter its binding characteristics to its Fc receptor. Preferably, the antibody of the present invention has an Fc domain.

[0510] Fcγ receptor

[0511] Preferably, the Fc domain of the antibody binds to an activating Fcγ receptor. Human activating Fcγ receptors include FcγRI, FcγRIIA, FcγRIIC, and FcγRIIIB, while FcγRIIB is an inhibitory FcγR. Mouse activating Fcγ receptors include FcγRI, FcγRIII, and FcγRIV. Mouse FcγRIIB is an inhibitory FcγR. Both mouse and human FcγRs exhibit different affinities for different IgG Fc domains. Table 2 shows the binding affinity of various human FcγRs for different human IgG Fc domains, and Table 3 shows the binding affinity of various mouse FcγRs for different mouse IgG Fc domains.

[0512]

[0513] Table 2: Affinity of human FcγR to the Fc domain of human IgG, expressed as K. A (M) -1 Adapted from Castro-Dopico, 2019 (which is hereby incorporated in its entirety by reference).

[0514]

[0515] Table 3: Affinity of mouse FcγR to mouse IgG Fc domain, expressed as K A (M) -1 Adapted from Castro-Dopico, 2019.

[0516] Substitution and Sequence Identity

[0517] Conservative substitution can be defined as substitution within an amino acid class and / or substitution that scores positive in the BLOSUM62 matrix.

[0518] According to one classification, amino acids are categorized as acidic, basic, uncharged polar, and nonpolar, with acidic amino acids being Asp and Glu; basic amino acids being Arg, Lys, and His; uncharged polar amino acids being Asn, Gln, Ser, Thr, and Tyr; and nonpolar amino acids being Ala, Gly, Val, Leu, Ile, Pro, Phe, Met, Trp, and Cys.

[0519] According to another classification, amino acids are categorized as slightly hydrophilic, acid / acidic amide / hydrophilic, basic, slightly hydrophobic, and aromatic. Slightly hydrophilic amino acids are Ser, Thr, Pro, Ala, and Gly; acid / acidic amide / hydrophilic amino acids are Asn, Asp, Glu, and Gln; basic amino acids are His, Arg, and Lys; slightly hydrophobic amino acids are Met, Ile, Leu, and Val; and aromatic amino acids...

[0520] The acids are Phe, Tyr, and Trp.

[0521] The replacements that score positive in the BLOSUM62 matrix are as follows:

[0522]

[0523] The percentage of amino acid sequence identity (%) relative to a reference sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference sequence after aligning the candidate sequence with the reference sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. The identity % value can be determined using 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 default values. Adjustable parameter settings include: overlap span = 1, overlap score = 0.125, and word threshold (T) = 11. The amino acid sequence identity % value is determined by dividing the number of identical matching residues as determined by WU-BLAST-2 by the total number of residues in the reference sequence (vacancies introduced into the reference sequence by WU-BLAST-2 to maximize the alignment score are ignored) and then multiplying by 100.

[0524] Alternatively, a specific pairwise alignment procedure can be used. A suitable example is "lalign" with the default parameters (implementing the algorithm of Huang and Miller; Adv. Appl. Math. (1991) 12:337-357).

[0525] Professional antigen-presenting cells / Non-professional antigen-presenting cells

[0526] Certain immune cells, such as dendritic cells and specific macrophage populations, are considered “professional” antigen-presenting cells (professional APCs). While most cell types can present antigens via MHC class I molecules (especially when the antigen has already been synthesized intracellularly (endogenous antigens)), professional APCs can additionally process and present exogenous antigens via MHC class II molecules and / or cross-present exogenous antigens via MHC class I molecules. Whether professional APCs participate in antigen presentation via MHC-II or MHC-I is also influenced by their cell type and the nature of the antigen. Importantly, cDC1 (a type of dendritic cell) is particularly adept at XP of cell-associated antigens (such as tumor antigens) (partly due to its unique receptor expression pattern) and plays a crucial role in antitumor immunity. By binding to the Fc domain of the antibody of this invention, professional APCs (cDC2, monocyte-derived cells) that are not specifically trained to present cell-associated antigens (CPAs) and potentially non-professional cells expressing FcγR (neutrophils) can cross-present CPAs via MHC-I in the context of necrotic cells to activate CD8. + T cells.

[0527] Pharmaceutical Composition

[0528] Pharmaceutical compositions can be prepared using a pharmaceutically acceptable “carrier” composed of materials considered safe and effective. “Pharmaceutically acceptable” means that the molecular entity and composition are “generally considered safe” when administered to humans, for example, physiologically tolerable and typically do not produce allergic reactions or similar adverse reactions, such as gastrointestinal upset. In some embodiments, this term refers to molecular entities and compositions approved by U.S. federal or state regulatory agencies for use in animals and more particularly for use in humans, such as the GRAS lists under Sections 204(s) and 409 of the Federal Food, Drug, and Cosmetic Act (which are subject to premarket review and approval by the FDA), or similar lists, the United States Pharmacopeia, or another recognized pharmacopoeia.

[0529] The term "carrier" refers to diluents, binders, lubricants, and disintegrants. Those skilled in the art are familiar with such drug carriers and methods of formulating drug compositions using such carriers.

[0530] The pharmaceutical compositions provided herein may contain one or more excipients, such as solvents, solubility enhancers, suspending agents, buffers, isotonic agents, antioxidants, or antimicrobial preservatives. When used, the excipients in the composition will not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient (i.e., carrier, cell, and / or chimeric receptor) used in the composition. Therefore, those skilled in the art will understand that such compositions are provided in which there is no incompatibility between any components in the dosage form. Excipients may be selected from buffers, solubilizers, sizing agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

[0531] Application route

[0532] Pharmaceutical agents and compositions according to aspects of the invention can be formulated for administration via a variety of routes, including but not limited to systemic, parenteral, intravenous, intra-arterial, intramuscular, intratumoral, oral, and nasal administration. The agents and compositions can be formulated into fluid or solid forms. Fluid formulations can be formulated for administration by injection to selected areas of the human or animal body.

[0533] Preferred dosage is the "therapeuticly effective dose," which is sufficient to demonstrate benefit to the individual. The actual dosage, as well as the rate and timing of administration, will depend on the nature and severity of the disease being treated. Prescribing treatment (e.g., decisions regarding dosage, etc.) is within the purview of general practitioners and other physicians, and typically takes into account the barrier to be treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th edition, 2000, pub. Lippincott, Williams & Wilkins.

[0534] Combination with other anticancer treatments

[0535] As described herein, the medical methods, medical uses, and pharmaceutical compositions of the present invention may involve combinations of antibodies with another anticancer therapy. In some embodiments, the anticancer therapy is an additional immunotherapy.

[0536] Currently, the most common cancer immunotherapy is checkpoint inhibitors. The antibodies of this invention can be used in combination with checkpoint inhibitors. Checkpoint inhibitors suitable for use in combination with the antibodies of this invention include checkpoint inhibitors that inhibit CTLA4 (cytotoxic T-lymphocyte-associated antigen 4), such as 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-associated protein 1); and anti-BTLA (B and T lymphocyte attenuation factor); anti-GAL9 (galactagogue 9); anti-HVEM (herpesvirus entry mediator); anti-ICOS (inducible T cell co-stimulatory molecule); anti-IL (interleukin); anti-KIR (cytotoxic cell immunoglobulin-like receptor); anti-LAG3 (lymphocyte activation gene 3); anti-VISTA (T cell activation V domain Ig repressor); anti-B7-H3; anti-B7-H4; anti-TGFβ (transforming growth factor β); anti-TIM3 (T cell membrane protein 3); or anti-CD27.

[0537] Other immunotherapies, such as T-cell therapy, can be used in combination with the antibodies disclosed herein. T-cell therapy involves the administration of autologous or allogeneic T cells. In some embodiments, antibodies are administered in combination with CAR-T cells (T cells expressing chimeric antigen receptors).

[0538] In some embodiments, the anticancer treatment is a cytotoxic chemotherapeutic agent, meaning that the antibodies of the present invention can be used in combination with cytotoxic chemotherapeutic agents. Combination with cytotoxic chemotherapeutic agents has the advantage of causing necrosis and exposure of F-actin on tumor cells, thereby allowing the antibody to bind to the tumor cells. Cytotoxic chemotherapeutic agents non-exclusively refer to alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors, antitumor drugs, and arsenic trioxide, carmustine, fludarabine, IDA ara-C, myalotang, GO, nitrogen mustard (mustalgen), cyclophosphamide, gemcitabine, bendamustine, total body irradiation, cytarabine, etoposide, melphalan, pentostatin, and radiation.

[0539] In some implementations, cancer treatment is radiation therapy. In other implementations, cancer treatment is surgery.

[0540] Subjects

[0541] The subject to treatment can be any animal or human. The subject is preferably a mammal, more preferably a human. The subject can be a non-human mammal, but more preferably a human. The subject can be male or female. The subject can be a patient. The therapeutic use can be for human or animal (veterinary use).

[0542] cancer

[0543] "Cancer" can include any one or more of the following: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, female reproductive system cancer, male reproductive system cancer, central nervous system lymphoma, cervical cancer, rhabdomyosarcoma in children, sarcoma in children, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colorectal cancer, colon cancer, endometrial cancer, endometrial sarcoma, esophageal cancer, eye cancer, gallbladder cancer, and gastric cancer. Cancer), gastrointestinal cancer, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, 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 cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary adenoma, plasma cell tumor, primary central nervous system lymphoma, prostate cancer, rectal cancer, respiratory system cancer, retinoblastoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, stomach cancer. Cancers include testicular cancer, thyroid cancer, urinary tract cancer, uterine sarcoma, vaginal cancer, vascular cancer, Waldenström macroglobulinemia, and nephroblastoma. In some implementations, the cancer is not prostate cancer.

[0544] Cancer can be of specific types. Examples of cancer types include astrocytoma, carcinoma (e.g., adenocarcinoma, hepatocellular carcinoma, medullary carcinoma, papillary carcinoma, squamous cell carcinoma), glioma, lymphoma, medulloblastoma, melanoma, myeloma, meningioma, neuroblastoma, and sarcoma (e.g., angiosarcoma, chondrosarcoma, osteosarcoma).

[0545] Some cancers cause solid tumors. These solid tumors can be located in any tissue, such as the pancreas, lungs, breast, uterus, stomach, kidneys, or testes. In contrast, blood cancers (such as leukemia) may not cause solid tumors and can be called liquid tumors.

[0546] Cancers that are the object of the therapeutic and medical uses of this invention can be selected from the list provided above.

[0547] Epitope determination

[0548] The epitopes of antibodies can be determined using methods known to those skilled in the art. For example, antibodies can be identified by X-ray crystallography and / or by cryo-electron microscopy (Cryo-EM) (Hanĉ et al., 2015).

[0549] Competition Measurement

[0550] On the other hand, competitive assays can be used to identify antibodies that compete with anti-F-actin antibodies as described herein for binding to F-actin. In some embodiments, such competing antibodies bind to the same epitope (e.g., a linear epitope or a conformational epitope). Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996), “Epitope Mapping Protocols,” Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0551] In an exemplary competitive assay, the immobilized antigen is incubated in a solution containing a first labeled antibody and a second unlabeled antibody that binds to the antigen, and the ability of the second unlabeled antibody to competitively bind to the antigen against the first antibody is tested. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution containing the first labeled antibody but not the second antibody. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed, and the amount of label associated with the immobilized antigen is measured. If the amount of label associated with the immobilized antigen in the test sample is significantly reduced relative to the control sample, it indicates that the second antibody competes with the first antibody for antigen binding. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (Cold SpringHarbor Laboratory, Cold Spring Harbor, NY).

[0552] ***

[0553] Features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their particular form or according to the means for performing the disclosed function, or the method or process for obtaining the disclosed result (as the case may be), may be used alone or in any combination of these features to implement the invention in its various forms.

[0554] While the invention has been described in conjunction with the exemplary embodiments set forth above, many equivalent modifications and variations will be apparent to those skilled in the art when this disclosure is given. Therefore, the exemplary embodiments of the invention set forth above are to be considered illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention.

[0555] To avoid any doubt, any theoretical explanations provided in this article are intended to enhance the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0556] Any chapter headings used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0557] Throughout this specification, including the following claims, unless the context otherwise requires, the words “comprise” and “include”, as well as variations such as “comprises”, “comprising”, and “including”, shall be understood to imply inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0558] It must be noted that, as used in the specification and appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. A range herein may be expressed as “about” a particular value and / or “about” another particular value. When such a range is expressed, another embodiment includes a range from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it will be understood that a particular value forms another embodiment. The term “about” in relation to numerical values ​​is optional and means, for example, + / - 10%.

[0559] Example

[0560] Materials and Methods

[0561] mice

[0562] C57BL / 6Jax and OT-I / Rag1 - / -Mice were housed at the Francis Crick Institute under specific pathogen-free conditions. In vivo tumor experiments were conducted using female mice aged 6–8 weeks. All animal experiments were conducted in accordance with national and institutional animal care guidelines and were approved by the Francis Crick Institute Biological Resources Facility Strategic Oversight Committee (an animal welfare and ethics review body) and the UK Home Office.

[0563] Mouse primary cells and cell lines

[0564] All cell cultures were performed using RPMI 1640 (R10+ medium) 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 bovine serum (FCS). V600E The 5555 melanoma cell line was a friendly gift from George Kassiotis. The MCA205 fibrosarcoma cell line and the human HEK293T cell line were obtained from the Cell Services Science and Technology Platform at the Francis Crick Institute. The HEK293T cells were maintained in DMEM containing 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, and 10% heat-inactivated FCS.

[0565] For FLT3L-cDC, bone marrow was extracted from the hind limbs of mice, and erythrocytes were lysed (ThermoFisher Scientific). Cells were then cultured in R10 reactors containing 150 ng / ml recombinant mouse FLT3L (R&D systems). + Cultured in medium for 9 days. On day 8, FTL3L cDC cultures were additionally pretreated 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 (all from Miltenyi) according to the manufacturer's instructions.

[0566] To generate Hoxb8-cDC2, CDPs were immortalized using the ER-Hoxb8 system. In short, bone marrow CDPs (Lin-CD117-CD115+FLT3+DNGR-1+) were isolated via FACS and compared with homologous bone marrow cells in R10 buffer containing 20 ng / ml IL-3, IL-6, and SCF.+ Cells were co-cultured in culture media. On the second day, cells were transduced with a retrovirus containing MSCV-Neo-HA-ER-Hoxb8 (obtained from David Sykes, Harvard University, Cambridge, Massachusetts) and selectively expanded with 1 mg / ml G418 in R10+ supplemented with conditioned medium (final concentration 75 ng / ml FLT3L) and 0.5 µM E2 (β-estradiol, Sigma Aldrich). Immortalized CDPs were separated from homologous bone marrow progenitor cells via FACS. To induce cDC differentiation in vitro, Hoxb8 CDPs were washed twice with Dulbecco's PBS (DPBS) and cultured for 5–7 days in R10+ containing 75 ng / ml recombinant mouse FLT3L (R&D systems) to produce cDC2.

[0567] For pre-activated effector OT-I cultures, from OT-I / Rag1 - / - Single-cell suspensions were produced from the spleen and lymph nodes of mice, and erythrocytes were lysed from them. Cells were then subjected to R10 buffer supplemented with 100 U / ml IL-2 (Peprotech) and 0.1 nMSIINFEKL (produced at the Francis Crick Institute). + Cultured in medium for 3 days. On days 3 and 4, cells were aliquoted at a ratio of 1:2 and the medium was completely replaced with R10+ medium containing 100 U / ml IL-2. OT-I culture was introduced on day 5.

[0568] Human tumor Fc receptor expression

[0569] Data from the Human Pan-Cancer Myeloid Atlas were analyzed at http: / / panmyeloid.cancer-pku.cn. Tumor FCGR gene expression in cDC2 was analyzed for all tumor types. The following tumor types were analyzed: renal cell carcinoma, colorectal cancer (CRC), breast cancer (BRCA), ovarian or fallopian tube cancer (OV-FTC), thyroid cancer (THCA), pancreatic adenocarcinoma (PAAD), melanoma (MEL), lung cancer, endometrial cancer of the uterine corpus (UCEC), hepatocellular carcinoma (HCC), gastric cancer (STAD), esophageal cancer (ESCA), and nasopharyngeal carcinoma (NPC).

[0570] Human primary cells

[0571] With prior ethical approval from the local ethics committee, peripheral blood was obtained from healthy volunteers at the Francis Crick Institute. Monocytes were enriched with biotinylated anti-human CD14 IgG (clone HCD14, Biolegend) and anti-biotin microbeads (Miltenyi) according to the manufacturer's instructions, and then incubated 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, the medium was changed on day 3, and both DCs and macrophages were harvested on days 6–8.

[0572] Fc receptor expression profile analysis

[0573] Human moDCs and moMacs were harvested with PBS / EDTA or 1X TrypLE (Thermo Fisher Scientific), washed twice in DPBS, and incubated at 4ºC for 45 min with anti-human antibodies against the following proteins (1:200 unless otherwise specified): 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), and FcγRIIIA / B (clone 3G8, BD Biosciences, 1:100). Dead cells were excluded using LIVE / DEAD fixative blue dead cell dye (Thermo Fisher Scientific), and samples were collected on LSL Fortessa or FACSymphony (BD Biosciences). Data were analyzed using FlowJo software version 10.

[0574] Fc-DNGR-1 fusion protein

[0575] The Fc-DNGR-1 variant fusion protein was produced at the Francis Crick Institute (small-scale) or commissioned to a commercial facility (large-scale, at ImmunoPrecise, Netherlands). Briefly, primers designed for infusion (SigmaAldrich) were used to amplify the long ECD DNA sequence of WT and 2WA mouse DNGR-1 allotype 4 from the existing pFB neo plasmid. The amplified pFUSEN-mG2aFc plasmid (InvivoGen) was linearized using NheI and EcoRV restriction enzymes, gel purified, and infused with the amplified 2WA or WT mouse DNGR-1 ECD DNA sequence according to the manufacturer's instructions (Takara). The plasmid was transformed into Stellar competent cells and overnight selected on gemcitabine agar plates. Single colonies were cloned, sequenced, and used for downstream expression. The plasmid was transformed into XL10 Gold bacteria (Agilent Technologies) and grown overnight on LB agar plates. Single colonies were then grown in LB broth containing genomiconine. Plasmid DNA was extracted using the QIAprep SpinMiniprep kit (QIAGEN) and sequenced.

[0576] The validated plasmid with the amplified sequence was transiently transfected into Expi293F cells, and the supernatant was harvested within a few days. The Fc-DNGR-1 fusion protein was purified using Protein A beads (Generon; M1300-5) according to the Pierce Gentle Ag / Ab Binding and Elution Buffer Kit (Thermo Fisher Scientific, 21030). The protein was then dialyzed in endotoxin-free 25 mM Tris (pH 7.2) and 150 mM NaCl (BupH Tris buffer, Thermo Fisher Scientific). Endotoxin levels were tested using the Pierce Chromatographic Endotoxin Quantification Kit-60 reaction (Thermo Fisher Scientific) and confirmed to be < 0.05 EU / ml. The purified protein was analyzed by non-reducing SDS-PAGE on a 7.5% mini-PROTEAN TGX pre-prepared gel (Bio-rad) containing Precision Plus dual-color standards, heated to 95ºC for 5 min in Laemmli buffer. The gel was developed using Coomassie Brilliant Blue R-250 staining solution (Bio-rad) and analyzed on an ImageQuant 800 (Amersham) imaging system. Large-scale production of Fc-DNGR-1 was outsourced to commercial companies (ImmunoPrecise (Netherlands) and FairJourney Biologics (Portugal)).

[0577] De novo anti-F-actin IgG production

[0578] Selection of anti-F-actin IgG monoclonal antibodies was performed using phage display on a natural human Fab library, commissioned to a commercial organization (FairJourney Biologics, Portugal). A total of 736 clones were identified after three rounds of positive selection for F-actin binding and negative selection for G-actin binding. For each round of screening, F-actin-binding phages were eluted using standard elution buffer containing trypsin (1 mg / mL) (Group 1) or via competition with Fc-DNGR-1 (at 1000 nM) (Group 2). These clones were then subjected to ELISA to determine binding to both F-actin and G-actin (see below). 497 clones met the threshold of ≥3-fold selectivity for F-actin binding relative to G-actin binding. Following Fab sequencing and sequence analysis (including VH and VL gene diversity and sequence liability), 78 unique clones were further evaluated. These clones were then filtered using necrotic cell binding by flow cytometry analysis of live and necrotic HEK293 (human) and NIH3T3 (mouse) cells. This yielded 68 clones with ≥ 3-fold necrotic cell selectivity. These clones were reformatted to mouse IgG2a and filtered for the following: ≥ 5-fold F-actin binding relative to G-actin binding; ≥ 5-fold F-actin binding relative to neutral avidin binding; ≥ 10% necrotic cell binding; and ≥ 3-fold necrotic cell MFI relative to background MFI. 45 clones met this threshold.

[0579] The large-scale production of anti-F-actin mouse IgG2a, IgG2a containing the human CH1 domain, human IgG1 monoclonal antibody, and Fc mutant antibody was outsourced to commercial organizations (ImmunoPrecise, Netherlands; or Biointron, China).

[0580] Commercial and previously described anti-actin antibodies

[0581] Purified commercially available anti-actin antibodies binding to mouse IgG2a isotypes, including α-actin, β-actin, and pan-actin, were identified. Pan-actin-binding AC-40 was obtained from Abcam (ab11003). β-actin-specific AC-74 was obtained from Sigma Aldrich (A2228). α-actin-specific C4 was obtained from Merck Millipore (MAB1501). Anti-F-actin antibody 8-3 was previously identified as an amplified clone in pancreatic cancer patients (Yao et al., 2023). Based on the publicly available sequence accompanying the original manuscript, the synthesis of this antibody as human IgG1 was commissioned to the commercial company Biointron (China).

[0582] Preparation of biotinylated and stable F-actin and G-actin

[0583] Biotinylated F-actin:

[0584] 20 µl of biotinylated G-actin (1 mg / ml, 20 µM, Cytoskeleton Inc.) was mixed with 20 µl of non-biotinylated G-actin (1 mg / ml, 20 µM, Cytoskeleton Inc.), 5 µl of F-buffer (10x polymerization buffer, Cytoskeleton Inc.), and 5 µl of phalloidin (200 µM, Thermo Fisher Scientific), and the mixture was incubated at room temperature for at least 1 h. The mixture was diluted with 0.75 ml PBS, and the polymerized F-actin was precipitated by ultracentrifugation at 150,000 g for 1 h at 4ºC. The supernatant was discarded, and the precipitate was resuspended in 0.8 ml PBS to produce stock solution of F-actin (1 µM), which was stored at 4ºC (do not freeze) for up to 4 weeks.

[0585] Biotinylated G-actin:

[0586] Mix 2 x 20 µl of biotinylated G-actin (1 mg / ml, i.e., 20 µM, Cytoskeleton Inc.) with 5 µl of G-buffer (universal actin buffer, Cytoskeleton Inc.) and 5 µl of pinocembrin D (200 µM, Thermo Fisher Scientific). Immediately dilute the mixture with 0.75 ml of G-buffer and remove contaminating F-actin by ultracentrifuging the solution at 150,000 g for 1 h at 4ºC. Carefully remove the supernatant (≤ 0.8 ml) (which represents the stock solution of G-actin (1 µM)) and store at 4ºC (do not freeze) for up to 4 weeks.

[0587] Preparation of F-actin / myosin-II (FM) beads

[0588] The F-actin / myosin-II (FM) complex was prepared as previously described (Canton, 2021). Lyophilized, non-biotinylated G-actin and myosin II (Cytoskeleton) were reconstituted in sterile water to a final concentration of 10 mg / ml and stored at -80ºC. Prior to use, aliquots of G-actin were diluted to a final concentration of 1 mg / ml in G-actin buffer and incubated on ice for at least 30 min. To generate biotinylated F-actin, non-biotinylated G-actin was mixed with freshly reconstituted biotinylated G-actin (Cytoskeleton) at a 1:1 molar ratio. Then, 20 μg of each G-actin formulation was mixed with F-actin buffer and incubated at room temperature for 1 h. To enable biotinylated F-actin to complex with myosin II, F-actin was mixed with myosin II at a 1:1 molar ratio and incubated at room temperature for 1 h.

[0589] To coat the 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 (Polysciences) with a diameter of 2.0 μm were used in a non-fluorescent or yellow-green fluorescent form and labeled with biotinylated OVA (1:1000) at 4ºC for 1 h. The OVA bead formulation was washed with 1% BSA in DPBS at 10,000 xg for 3 min. The OVA beads were then labeled with F-actin / myosin II. In vitro polymerized biotinylated F-actin / myosin II was added to the OVA beads and incubated on ice for at least 1 h.

[0590] Quantitative analysis of the binding of monoclonal antibodies (mAbs) to F-actin via ELISA

[0591] Binding to selected mAbs and F-actin specificity were measured by ELISA using immobilized F-actin and G-actin. Immunosorbent plates (MaxiSorp 96-well, Thermo Fisher Scientific) were coated overnight at 4°C with 5 µg / ml NeutrAvidin™ (Thermo Fisher Scientific) in PBS, washed, blocked for 2 h at room temperature with 2.5% BSA in PBS, and stored overnight at 4°C after the addition of biotinylated F-actin or G-actin (2 nM). Capture of biotinylated F-actin and G-actin was validated using a commercially available actin-specific mAb (AC-40, Sigma) that identifies both F-actin and G-actin. To prevent depolymerization of F-actin or polymerization of G-actin during assay, F-actin and G-actin were stabilized during actin stock solution preparation by the addition of phalloidin or pinocembrin D, respectively. After washing to remove unbound actin, a three-fold serial dilution of mAb or control reagent (DNGR-1-Fc fusion) starting at 300 nM (unless otherwise specified) was added to the ELISA plate and allowed to stand for 1 h. Actin-bound mAbs or control reagents were detected using horseradish peroxidase (HRP)-conjugated or AP-conjugated anti-mouse or anti-human IgG (Jackson Immuno Research) antibodies (0.16 µg / ml), and color development was performed using ELISA substrates for HRP- or AP-based detections (TMB, eBioscience; or SIGMAFAST p-nitrophenyl substrate solution, Sigma Aldrich). Substrate color development was stopped by adding sulfuric acid (H2SO4, Fisher). Titration curves were plotted using a GraphPad Prism 7, and the EC50 value for each mAb was calculated by applying a nonlinear regression (curve fitting) of log(agonist) against response-variable slope (four parameters).

[0592] Necrotic cell binding assay

[0593] By using 240 mJ / cm in DPBS 2 UVC irradiation of mice with BRAF V600EMutant 5555 melanoma cells or human HEK293T cells were cultured overnight in R0 medium (lacking 10% FCS) to generate necrotic cells. The necrotic cells were incubated in DPBS for 0.5–1 h with molar equivalents of Fc-DNGR-1, anti-F-actin IgG2a, anti-F-actin IgG1, or commercially available / previously described anti-actin IgG antibodies. In some cases, soluble pinocembrin D-stabilized human platelet G-actin (Cytoskeleton) was added simultaneously to assess binding inhibition of non-F-actin-specific antibodies. Samples were washed twice in DPBS and incubated for 30 min at 4ºC with AF488-conjugated anti-mouse IgG2a secondary antibody (Thermo Fisher Scientific, 1:400) or AF568-conjugated anti-human IgG1 secondary antibody (Thermo Fisher Scientific, 1:400). Samples were washed and resuspended in DPBS, and then collected on an LSR Tortessa or FACSymphony (BD Biosciences). Data were analyzed using FlowJo software version 10.

[0594] Assay for phagocytosis of necrotic cells - Flow cytometry

[0595] Prior to irradiation, tumor cells were labeled with Cell Tracker-Deep Red (CT-DR) dye (ThermoFisher Scientific; 1:1000 dilution) at 37ºC for 1 h. CT-DR-labeled necrotic cells were added to cDCs at a 2:1 ratio for 4 h in the presence of an equimolar amount of F-actin binding reagent. After 4 h, the samples were surface-stained for flow cytometry using the following combinations: XCR1 (Biolegend, ZET), CD172a (Biolegend, P84), IA / IE (Biolegend, M5 / 114.15.2), B220 (BD Biosciences, RA3-6B2), and CD11c (Biolegend, N418). Non-internalized necrotic cell material was excluded using DAPI or LIVE / DEAD fixed blue dead cell dye (ThermoFisher Scientific). Samples were collected on LSR Fretessa or FACSymphony (BD Biosciences). Data were analyzed using FlowJo software version 10. The phagocytosis index integrates the frequency and absolute amount of phagocytosed material and was calculated using the following formula: (% CT-DR+ x CT-DR+ of CT-DR GMFI) / 10,000.

[0596] Cross-presentation determination

[0597] Fill each hole of the U-shaped bottom 96-hole plate with 5 x 10 mm paving stones. 4 One cDC1 or 1 x 10 5 Non-cDC1 cells were harvested from 10 cm tissue culture plates 16 h after UV irradiation and incubated for 1 h at 37ºC in 10 mg / ml albumin derived from egg white (OVA, Sigma Aldrich) in RPMI 1640 medium, followed by three washes in DPBS. 1 ml of OVA solution was used per 10 cm tissue culture plate. OVA-necrotic cells were incubated with cDCs at a 2:1 ratio for 4 h in the presence of equimolar amounts of Fc-DNGR-1, anti-F-actin IgG2a, or commercially available anti-actin IgG2a.

[0598] Preactivated OT-I CD8+ T cells were generated by culturing spleen 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 daily, and cells were used on days 5 and 6. Preactivated OT-I was added to cDC cultures at a 2:1 ratio for 24 h, and T cell-derived IFNγ release was measured by internal 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) and then thoroughly washed in 0.05% Tween-20 in DPBS. The plate was blocked with 3% FCS (blocking buffer) for 1 h, washed once, and incubated for 2 h with T cell culture supernatant and recombinant IFNγ (Peprotech) standard curve samples. After washing, the plate was incubated with biotinylated rat anti-mouse IFNγ IgG (clone XMG1.2, BD Biosciences, 1 μg / ml, in blocking buffer) for 2 h, then with ExtrAvidin-alkaline phosphatase (Sigma Aldrich, 1:5,000 in blocking buffer) for 30 min. The nitrophenyl substrate solution (Sigma Aldrich) was then developed with SIGMAFAST according to the manufacturer's instructions. After 20–30 min, the absorbance at 405 nm (IFNγ signal) and 540 nm (background) was measured on a Spark plate reader (Tecan), and the absolute concentration was determined using the standard curve. The area under the curve, EC50 (potency), and maximum IFNγ signal (efficacy) were calculated in GraphPad Prism.

[0599] Competing with ELISA - competing with DNGR-1

[0600] The ability of selected anti-F-actin mAbs to competitively bind F-actin to the soluble DNGR-1 receptor was measured by ELISA using immobilized F-actin. In short, immunoassay plates (MaxiSorp 96-well, Thermo Fisher Scientific) were coated overnight at 4°C with 5 µg / ml NeutrAvidin™ (Thermo Fisher Scientific) in PBS, washed, blocked for 2 h at room temperature with 2.5% BSA in PBS, and stored overnight at 4°C after the addition of biotinylated F-actin (100 nM). To prevent depolymerization of F-actin during assay, F-actin was stabilized by the addition of phalloidin during the preparation of the F-actin stock solution. After washing to remove unbound F-actin, a 10-fold serial dilution of anti-F-actin mAb (starting at 100 nM), an irrelevant antibody (negative control), and DNGR-1-Fc fusion (positive control) was mixed with 10 nM FLAG-labeled DNGR-1 (DNGR-1-FLAG) and added to an ELISA plate for 1 h. Binding of DNGR-1-FLAG was detected using an alkaline phosphatase (AP)-conjugated FLAG-specific antibody (Sigma, clone M2), and colorimetric analysis was performed using an ELISA substrate for AP-based detection (pNitrophosphate, Sigma). The plate was read at 405 nm using an ELISA reader to determine the optical density (OD) of each sample. The OD was calculated according to the formula (OD...). max -OD 竞争剂 ) / (OD max - OD min ) x 100% to calculate the inhibition curve, where OD max and OD min The values ​​were defined as the OD value of DNGR-1-FLAG binding in the absence of a competing agent and the determination background in the absence of DNGR-1-FLAG, respectively, and plotted using GraphPad Prism10.

[0601] In vivo tumor experiments

[0602] MCA205 tumor cells were dissociated with trypsin (0.25%) and washed three times in PBS. The final cell pellet was resuspended and diluted in endotoxin-free PBS (0.5 x 10⁻⁶ μL per 100 μl). 6(1 cell), and subcutaneously injected into the shaved right abdomen of each recipient C57BL / 6Jax mouse. Tumor growth was monitored every 1 to 3 days, and the longest tumor diameter (l) and vertical width (w) were measured using digital calipers; tumor volume was calculated using the following formula: length x width 2 / 2, and represented as mm 3 Fc-DNGR-1 or anti-F-actin IgG2a was administered via intratumoral injection on days 7 and 11 (100 μg for Fc-DNGR-1 in 50 μl PBS; 150 μg for anti-F-actin IgG2a in 50 μl PBS). On day 7, mice were additionally injected with 1.25 mg / kg doxorubicin (Merck Life Science UK).

[0603] Assay for phagocytosis of necrotic cells - microscopy

[0604] For imaging cDC / moDC / moMac, glass coverslips (18 mm) were coated with 0.1% poly-l-lysine solution (Sigma Aldrich) for 30 min at room temperature. Excess poly-l-lysine was washed three times with DPBS. The coverslips were incubated in 2.5% glutaraldehyde solution (Sigma Aldrich) for 15 min at room temperature. After washing three times with DPBS, the coverslips were incubated for 30 min at room temperature 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. The coverslips were washed again with DPBS and incubated overnight at 4ºC in 0.2 M glycine in DPBS. On the day of the experiment, the coverslips were washed three times with fresh DPBS. 2.5–5 x 10 5Add individual mouse FLT3L cDC1, non-cDC1, or Hoxb8-cDC2, or human moDC or moMac cells to a single coverslip and allow them to adhere at 37ºC for 1 h. Then, challenge the cells with CT-DR / green-labeled necrotic cells ± anti-F-actin mAb for 3–4 h. In some cases, pre-incubate human cells for 30 min with 10 μg / ml anti-human FcγRIIA IgG (Biotechne) or 0.5 μM SYK inhibitor IV (Merck Millipore) before adding necrotic cells. Fix the cells in 4% paraformaldehyde, wash three times with DPBS, and stain for 30 min at room temperature with rhodamine-conjugated wheat germ lectin (1:10,000) in blocking buffer. Mount the washed coverslips onto glass slides using Prolong Diamond Antifade (Fisher Scientific). Image the sample on a Zeiss LSM880 inverted confocal microscope. Perform image processing and analysis using Fiji / ImageJ or Imaris v9.1.2.

[0605] Tumor microsurgery

[0606] For tumor-like structures, 5 x 10 3 Braf V600E Melanoma cells were inoculated into R10 + The tumor-like cells were cultured in Corning matrix basement membrane matrix (VWR) with continuous shaking for 5 days. The tumor-like cells were dissociated from the matrix using Corning cell recovery solution (Fisher Scientific) and then reacted with 10 μg / ml Fc-DNGR-1, AlexaFluor 555 phalloidin (1:400, Fisher Scientific), or 2 μg / ml DAPI in R10. + Incubate together in the culture medium for 24 h. Wash the tumor-like cells in DPBS for 1 h, fix them in 4% PFA for 1 h, and then incubate overnight in 30% sucrose. For tumors, use 2.5 x 10⁻⁶... 6 MCA205-LA-OVA-mCherry tumor cells were injected into the shaved flanks of C57BL / 6 mice. Seven days later, the tumor cells were peri-injected with Fc-DNGR-1 (50-200 μg) in DPBS, and tumors were harvested after 6-24 h. Alternatively, 0.5 x 10⁻⁶ mCherry tumor cells were injected into the flanks of C57BL / 6 mice. 6MCA205 tumors were harvested on day 21 after administration of the cytokine (cells) and on day 3 after administration of isotype or anti-F-actin IgG2a (400 μg, intraperitoneal). Tumors were fixed overnight in 4% PFA at 4ºC, followed by overnight incubation in 30% sucrose at 4ºC. Tumor-like structures and tumors were embedded in OCT (Tissue-Tek), sectioned (10–30 μm) using a Leica cryostat, mounted on SuperFrost Plus glass slides (Thermo Fisher Scientific), and stored at -80ºC. Sections were thawed to room temperature, rehydrated in DPBS for 10 min, and blocked for 1 h in DPBS (blocking buffer) containing 0.3% Triton-X and 2%–5% normal goat serum or 3% BSA.

[0607] Slides were stained at room temperature 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) at 1 h (unless otherwise specified, all in blocking buffer at 1:400). For certain tumor samples, sections were incubated overnight at 4ºC 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), followed by secondary staining at room temperature for 1 h 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). The slides were washed and mounted with Prolong Diamond Antifade (Fisher Scientific) before imaging and analysis as described above.

[0608] To perform confocal microscopy analysis of actin cytoskeleton binding, HeLa cells were fixed in 4% PFA at room temperature for 20 min, washed three times with DPBS, and permeabilized / blocked for 15 min with 2% BSA and 0.3% Triton-X in DPBS. Cells were stained for 1 h with Hoechst (2.5 μg / ml), phalloidin-A647 (1:400), and anti-F-actin IgG2a (32 nM) in 500 μl of 2% BSA in DPBS. The samples were washed three times, stained with anti-mouse IgG2a-AF488 in 2% BSA in DPBS for 1 h, washed, and mounted with Prolong Diamond Antifade (FisherScientific) before imaging as described above.

[0609] Pharmacokinetic analysis of Fc-DNGR-1 and anti-F-actin antibody

[0610] 100 μg of antibody A or Fc-DNGR-1 (5 mice / group) was injected intraperitoneally into C57BL / 6 mice. Blood was harvested between 0 and 6 days post-injection, and serum was separated using a serum Z-gel tube (Sarstedt). Fc-DNGR-1 titers were calculated using a serum-compatible anti-DNGR-1 ELISA. In short, 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), and then thoroughly washed in 0.05% Tween-20 in DPBS. The plates were blocked with 3% FCS (blocking buffer) for 1 h, washed once more, and incubated for 2 h with serum diluted in DPBS (1:100 and 1:1000) or an Fc-DNGR-1 standard curve. After washing, the plates were incubated with biotinylated rat anti-mouse DNGR-1 IgG (clone 7H11, 1 μg / ml) for 2 h, followed by color development with alkaline phosphatase, as described above. Antibody A titers were calculated similarly, but captured using CaptureSelect™ biotinylated anti-IgG-CH1 (Thermo Fisher Scientific) on ELISA plates coated overnight with ExtrAvidin (Merck Life Science, 5 μg / ml), and detected with goat anti-mouse IgG2a-alkaline phosphatase (SouthernBiotech).

[0611] Example 1 - Generation of Candidate Antibodies

[0612] Use a phage selection funnel to select lead antibody candidates. Figure 1 A total of 736 clones were generated by screening native phage display libraries. These were produced through four rounds of positive selection based on F-actin binding and negative selection based on G-actin binding. Two parallel groups were performed: for each round of screening, F-actin-bound phages were eluted using standard elution buffer containing trypsin (1 mg / mL) (Group 1) or via competition with Fc-DNGR-1 (at 1000 nM) (Group 2). These clones were then subjected to ELISA to determine binding to both F-actin and G-actin. 497 clones met the threshold of ≥ 3-fold selectivity for F-actin binding relative to G-actin binding. Following Fab sequencing and sequence analysis (including VH and VL gene diversity and sequence adversities), 78 unique clones were further evaluated. These clones were then filtered using necrotic cell binding by FACS analysis on untreated and necrotic HEK293 and NIH3T3 cells. This yielded 68 clones with ≥ 3-fold dead cell selectivity. These clones were reformatted to mouse IgG2a and filtered for the following: ≥ 5-fold F-actin binding relative to G-actin binding; ≥ 5-fold F-actin binding relative to neutral avidin binding; ≥ 10% necrotic cell binding; and ≥ 3-fold necrotic cell MFI relative to background MFI. 46 clones met this threshold, and the data are shown in Table 4.

[0613]

[0614]

[0615] Table 4: The following values ​​for all 46 IgG2a clones: F / G actin binding; dead cell / live cell binding (human); dead cell / live cell binding (mouse); and cross-presentation (AUC).

[0616] The ability of all 46 mAbs to induce necrotic XP cells in cDC2 cells was determined. Figure 2 Hoxb8-CDP-derived cDC2 was incubated with OVA-soaked necrotic 5555 BrafV600E melanoma cells and dose-adjusted (50 nM, 10 nM, 2 nM, 0.4 nM) 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. After 24 h, T cell-derived IFNγ in the supernatant was measured by ELISA. The area under the curve (AUC) of IFNγ production for each mAb was calculated. They were then ranked in descending order. Figure 2 Furthermore, the top 5 antibodies (A through E) that could enhance XP via cDC2 were selected for further experiments. All 5 antibodies were derived from the DNGR-1 elution fraction, as shown in Table 5, which also includes the 6th ranked antibody (Ab ID: 7).

[0617]

[0618] Table 5: Cross-presentation (% WT) values ​​and elution information of the top 6 antibodies ranked by their ability to enhance XP via cDC2.

[0619] Example 2 - Further Characterization of the Lead Antibody

[0620] The five lead antibodies were then further characterized. XP assays in cDC2 derived from Hoxb8-CDP were repeated as described above, and the results are shown in [the table / image / etc.]. Figure 3 In this study, the Fc-DNGR-1 conjugate (white circle) was used as a positive control. The 2WA mutant Fc-DNGR-1 (black circle) was used as a negative control throughout the study. This mutant was generated by the Francis Crick Institute and contains two W-to-A mutations in the CTLD DNGR-1 domain, which eliminates binding to F-actin (Canton, 2021). XP was measured by T-cell-derived IFNγ. All five antibodies were able to trigger XP at levels higher than the control.

[0621] ELISA was performed to determine the F-actin binding properties of all five antibodies. Figure 4 (Solid circles). Binding to G-actin was also measured (hollow circles). As shown, all five antibodies exhibited only minimal binding to G-actin and are therefore specific for F-actin. The EC50 (nM) values ​​for binding to F-actin are shown in Table 6. All antibodies are in the nanomolar range and therefore exhibit excellent potency. In particular, antibodies A, B, and D have potency below 1 nM.

[0622]

[0623] Table 6: EC50 (nM) values ​​for binding to F-actin. ND = Undetermined.

[0624] Example 3 - Functional Analysis of Lead Antibody

[0625] To confirm that the antibody could bind to F-actin on dead cells, necrotic mouse 5555 cells irradiated with UV light were used. Figure 5 A) or necrotic human HEK293T cells ( Figure 5B) Incubate with serially diluted anti-F-actin IgG2a clone or Fc-DNGR-1 fusion protein (2WA as a negative control or WT as a positive control), and detect binding with AF488-conjugated anti-mouse IgG secondary antibody. All five antibodies were able to bind to both mouse and human necrotic cells, exhibiting similar curves and affinities. Figure 5 ).

[0626] Flow cytometry analysis was performed on the internalization of CT-DR-labeled necrotic cell debris in primary FLT3L-cDCs from mouse bone marrow pretreated with IFN-I in the presence of an F-actin binding reagent. The results were obtained in primary cDC1 cells (…). Figure 6 A) and primary cDC2 cells ( Figure 6 In B), the ability of the antibody to induce phagocytosis was assessed. cDCs were analyzed after 4 h of incubation, and the phagocytic index was calculated using the following formula: (% of CT-DR positive cDCs x GMFI of CT-DR positive cDCs) / 10,000. Figure 6 The results showed that all five antibodies promoted the uptake of CT-DR-labeled necrotic cell debris by primary FLT3L-cDCs pretreated with IFN-I, and exhibited significantly different phagocytic index curves.

[0627] The ability of the antibody to trigger XP in the cDC2 cell line was determined in Examples 1 and 2 above, and is shown in... Figure 3 The ability to trigger XP in primary cDC1 and non-cDC1 cells was then investigated. Primary bone marrow FLT3L-cDC cultures pretreated with IFN-I were separated into cDC1 and non-cDC1 fractions using MACS enrichment targeting XCR1. These were then incubated with OVA-soaked, UV-irradiated necrotic 5555 BrafV600E cells and serially diluted 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 an outcome of XP. In cDC1 ( Figure 7 A) and non-cDC1 ( Figure 7 In both B) cells, all five antibodies triggered XP. The ability of each antibody to trigger XP is related to its... Figure 6 The ability to trigger a phagocytic process is in a similar proportion.

[0628] These functional experiments confirmed that all five antibodies were able to bind to F-actin on dead cells, triggering phagocytosis and subsequent XP of the phagocytosed material.

[0629] Example 4 - Testing of commercially available antibodies and comparison with lead antibodies A, B, and E

[0630] To compare the specificity and function of the lead antibody with 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 necrotic mouse 5555 BrafV600E melanoma cells irradiated with UV light was investigated by flow cytometry analysis. The cells were incubated with serially diluted anti-actin IgG2a clones (AC-40 or AC-74) or Fc-DNGR-1 fusion protein (2WA or WT) and stained with AF488-conjugated anti-mouse IgG secondary antibody. The inventors demonstrated that AC-40 did not bind effectively to necrotic cells, while AC-74 could bind effectively. Figure 8 ).

[0631] After establishing binding curves of AC-40 and AC-74 to dead cells, the ability of these antibodies to trigger necrotic cell phagocytosis was determined by flow cytometry analysis of the internalization of CT-DR-labeled necrotic cell debris by primary FLT3L-cDCs from mouse bone marrow pretreated with IFN-I in the presence of anti-actin IgG2a clones (AC-40 and AC-74) or Fc-DNGR-1 (2WA or WT). cDC1 was analyzed after 4 h of incubation. Figure 9 A) and cDC2 ( Figure 9 B), and the phagocytic index was calculated using the following formula: (% of CT-DR positive cDCs x GMFI of CT-DR positive cDCs) / 10,000. As expected based on the dead cell binding curve, AC-74 caused primary FLT3L-cDCs to phagocytose necrotic cell debris, but AC-40 did not. Figure 9 ).

[0632] As described above regarding the lead antibodies, the ability of AC-40 and AC-74 to trigger XP in primary cDC1 and cDC2 was measured. Unsurprisingly, AC-40 failed to trigger XP. AC-74 was able to induce XP via non-cDC1 pathways. Figure 10 B), but the induction via cDC1 is poor ( Figure 10 A).

[0633] Then, by measuring the EC50 (potency) and maximum IFNγ signal (efficacy) of each antibody / Fc-DNGR-1 (according to... Figure 10 cDC1 ( Figure 11 A) and non-cDC1 ( Figure 11The XP results between B) were ranked, directly comparing lead antibodies A, B, and E to commercially available antibodies AC-40 and AC-74. As shown, antibodies A and B consistently induced XP comparable to Fc-DNGR-1 in both cDC1 and non-cDC1. In contrast, the commercially available antibodies showed poorer potency and efficacy in cDC1. In non-cDC1, only AC-74 showed some efficacy, while AC-40 showed poor potency and efficacy.

[0634] Then, the binding of AC-74 and antibodies A, B, and E to necrotic cells in the presence of G-actin was measured. Figure 12 A and Figure 12 As shown in Figure B, antibodies A, B, and E exhibit competitive resistance to G-actin and maintain binding to necrotic cells via F-actin. In contrast, AC-74 does not exhibit competitive resistance to G-actin. This data indicates that the antibodies of the present invention are specific to F-actin compared to G-actin, while AC-74 is not.

[0635] Example 5 - Anti-F-actin antibody promotes tumor control with chemotherapy

[0636] Evaluation of tumor control of lead anti-F-actin antibodies A, B, and E ( Figure 13 The anti-F-actin IgG2a clone or Fc-DNGR-1 (WT or 2WA) was administered intratumorally to C57BL / 6 mice carrying MCA205 tumors (100 μg for Fc-DNGR-1 or 150 μg for anti-F-actin IgG2a in 50 μl). On day 7, the drug was combined with 1.25 mg / kg doxorubicin to induce immunogenic cell death. A second dose of antibody / Fc-DNGR-1 was administered on day 11. Figure 13 As shown in B, antibodies A, B, and E exhibited greater tumor control compared to the negative control.

[0637] Example 6 - Anti-F-actin antibody competitively binds to F-actin with DNGR-1

[0638] The degree of competition among antibodies A, B, and E was assessed. The ability of these antibodies to compete with DNGR-1 varied considerably, with antibody B showing the highest level of competition with DNGR-1 for binding to F-actin. Figure 14 Antibody E showed minimal competition. No antibody could match DNGR-1's ability to compete with itself.

[0639] Example 7 - Systemic administration of anti-F-actin antibody promotes tumor control

[0640] To further evaluate the in vivo therapeutic efficacy of the anti-F-actin antibody, antibody A was compared with equimolar concentrations of Fc-DNGR-1 in promoting tumor control when administered peritumorally. Consistent with the results shown in Example 5, in MCA205 fibrosarcoma tumors treated with the cell-death-inducing chemotherapeutic agent doxorubicin, both Fc-DNGR-1 and antibody A promoted therapeutic tumor control at equivalent levels compared to mice receiving 2WA of Fc-DNGR-1. Figure 15 A).

[0641] To determine whether systemic administration is feasible, the pharmacokinetics of the anti-F-actin antibody compared to DNGR-1 were evaluated. To this end, the inventors developed two mouse serum compatibility ELISAs for reagent detection: an Fc-DNGR-1 ELISA using two monoclonal anti-mouse DNGR-1 antibodies targeting different epitopes in the ECD, and an anti-F-actin antibody ELISA utilizing the detection of the human CH1 domain incorporated into the antibody backbone. Figure 15 Using this method, the inventors demonstrated that antibody A exhibits a significantly enhanced serum half-life compared to Fc-DNGR-1. Figure 15 B). Consistent with improved pharmacokinetics, antibody A, compared with an isotype-matched control antibody, accumulated in necrotic areas within doxorubicin-treated MCA205 tumors after systemic administration, as detected by mouse IgG2a or human CH1. Figure 15 C).

[0642] Consistent with its short half-life, systemically administered Fc-DNGR-1 (different from the agent administered peritumorally) did not stain tumors or demonstrate therapeutic efficacy in various models (data not shown). Given the excellent pharmacokinetic characteristics of the anti-F-actin antibody, the inventors evaluated the efficacy of systemically administered antibody A. Single-agent activity of peritumorally administered Fc-DNGR-1 has been demonstrated in the MC38 tumor model. Notably, systemically administered antibody A alone was sufficient to promote control of MC38 tumors compared to an isotype-matched control antibody. Figure 15 D). The mice did not exhibit any external signs of systemic inflammation and, as expected, continued to gain weight. Figure 15 E).

[0643] Example 8 - Fc-DNGR-1 reveals the localization of tumor necrosis cells in vivo.

[0644] For cancer immunotherapy, Fc-DNGR-1 requires the close juxtaposition of intratumoral APCs with necrotic cancer cells. However, little is known about the spatiotemporal dynamics of immune cells sensing necrotic cells in vivo. To address this issue, the inventors used Fc-DNGR-1 to microscopically highlight necrotic regions within tumors. Three-dimensional tumor spheroids (tumor-like structures) were generated in vitro and incubated with a variant of Fc-DNGR-1. WT Fc-DNGR-1 (instead of 2WA Fc-DNGR-1) was able to highlight cells within the tumor-like core (a region potentially with restricted nutrient exchange and significantly cleaved caspase-3 staining) (data not shown). Fc-DNGR-1 staining reflected the pervasive loss of tumor cell membrane integrity, as necrotic cells can also be visualized by culturing the tumor-like structures separately with other agents binding F-actin or DNA, such as phalloidin or DAPI. Figure 16 A- Figure 16 B). Consistent with the binding of the actin cytoskeleton, several necrotic cells exhibited a discrete filamentous Fc-DNGR-1 staining pattern (B). Figure 16 C).

[0645] To identify tumor necrosis areas in vivo, Fc-DNGR-1 was administered peritumorally to mice carrying fibrosarcoma, which was generated by transplantation of an MCA205-derived tumor line modified to express the fluorescent marker mCherry (Giampazolias, 2021). Consistent with tumor-like data, WT Fc-DNGR-1 effectively labeled necrotic cells within the tumor core. Figure 16 D), while no staining was observed in the 2WA Fc-DNGR-1 case (D), Figure 16 D). Bright Fc-DNGR-1 was observed at the boundary of the necrotic core. + Cells exhibited diffuse Fc-DNGR-1 staining at the center. This more diffuse staining pattern coincided with a decrease in nuclear DNA staining intensity, consistent with cell rupture and the release of intracellular debris. Next, to assess the proximity of different APC populations to tumor-derived dead cells, the inventors co-stained MHC-II and CD103 to identify CD103. + cDC1 and CD103 - Non-cDC1 APCs (e.g., cDC2, MC) Figure 16 E). Notably, cDC1 is located peripherally to the tumor, far from necrotic sites, suggesting that their ability to acquire dead cellular material in vivo may be somewhat limited. In contrast, MHC-II... + CD103 - APCs were located throughout the tumor, including at the boundary of the necrotic core and within the core. Further confirmation of these CD103s...- APC expression of FcγRI ( Figure 16 F), indicating that in the presence of Fc-DNGR-1, they are located in the optimal position for acquiring necrotic cell antigens in vivo.

[0646] This data provides proof of principle that the necrotic core of tumors can be targeted by binding to F-actin, and that cells expressing FcγR reside closely in these areas.

[0647] Example 9 - Testing of a commercially available antibody that binds to F-actin and comparison with lead antibody A

[0648] To further compare the binding of lead antibody A with currently available antibodies and its F-actin specificity, 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 α-actin-specific antibody. The binding ability of these antibodies to F-actin was studied by ELISA using immobilized F-actin. The inventors demonstrated that neither AC-40 nor C4 effectively binds to F-actin, while antibody A effectively binds to F-actin. Figure 17 ).

[0649] Example 10 - Testing of humanized commercial antibodies and comparison with humanized lead antibodies A, B, and E

[0650] Humanized IgG1 antibodies, including lead antibodies A, B, and E, were prepared. To compare the efficacy and potency of these antibodies with the previously described 8-3 antibody (which, according to Yao et al., binds to F-actin), 8-3 antibodies were generated on the human IgG1 backbone. The ability of these antibodies to bind to F-actin was determined by ELISA. Figure 18 A). Although all antibodies showed binding to F-actin-coated plates, antibodies A, B, and E all exhibited significantly 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. Again, compared to 8.3, antibodies A, B, and E showed significantly higher maximum binding (potency) and potency (EC50). Figure 18 (B) For example, the EC50 of antibody A is 2.759 nM, compared to 15.10 nM for antibody 8-3. Therefore, human IgG1 antibodies A, B, and E bind strongly to F-actin and necrotic cells, while antibody 8-3, as described, does not.

[0651] Therefore, despite the discussion in the literature, data show that not all available antibodies bind to F-actin with sufficient strength to achieve the therapeutically desired properties discussed in this article.

[0652] Example 11 - Functional Analysis of Humanized Antibody A

[0653] After confirming that the humanized antibody can bind to F-actin and necrotic cells, the ability of antibody A to trigger phagocytosis of necrotic cells by various human cells (necrophagocytosis) was investigated.

[0654] cDC2 is a major target of CTT in the tumor microenvironment and expresses activating FcγRIIA and inhibitory FcγRIIB ( Figure 19 A). To model these cells, we generated DC-like cells (referred to as moDCs for simplicity) from human peripheral blood mononuclear cells using GM-CSF / IL-4. Flow cytometry revealed that these cells exhibited very similar Fc receptor expression patterns. Figure 19 B).

[0655] We evaluated the ability of antibody A to promote necrophagocytosis of human moDCs using confocal microscopy. Compared with antibodies defective in Fc receptor binding (LALA-PG mutant), WT antibody A was able to promote necrophagocytosis of human moDCs. Figure 20 A). This activity was further enhanced by introducing a mutation (GASDALIE) into the IgG1 Fc domain that enhances affinity for the Fc receptor. Therefore, AFA A promotes necrophagocytosis via the Fc receptor, and this can be further enhanced by Fc engineering. Uptake was consistently observed in moDCs derived from different donors. Figure 20 A). Furthermore, it was shown that uptake depends on FcγRIIa and SYK ( Figure 20 B), compared to the previously defined Fc receptor expression pattern ( Figure 19 B) Consistent.

[0656] Macrophages express high levels of Fc receptors and represent a major subset of tumor myeloid cells. To assess AFA function in these cells, peripheral blood monocytes were differentiated into macrophages (moMacs) using M-CSF. As expected, moMacs expressed high levels of all Fc receptors (…). Figure 21 A). As observed in moDC, AFA promotes necrotic phagocytosis in moMac, as determined by confocal microscopy ( Figure 21 B). Activity was either eliminated by mutations that decreased or increased the affinity of IgG1 Fc for the human Fc receptor (LALA-PG) or enhanced by mutations that increased the affinity (GASDALIE). Thus, AFA A promotes necrotic phagocytosis of various human myeloid cells via Fc receptor binding, and this activity can be further modulated by mutations that alter Fc receptor affinity within the Fc domain.

[0657] Summarize

[0658] The data described above have generated a class of antibodies that bind to F-actin with high affinity, cohesion, and / or specificity. Figure 11 These antibodies can specifically bind to F-actin on necrotic cells. Figure 5 ), triggering the phagocytosis of necrotic cell debris ( Figure 6 ), and XP is induced by both cDC1 and non-cDC1 cells ( Figure 7 These functional characteristics, along with their specificity for F-actin, highlight the therapeutic potential of these antibodies, such as in the treatment of cancer.

[0659] The inventors also demonstrated that the commercially available antibodies selected were not specific to F-actin and bound to G-actin to a similar degree. Figure 12 While one of the antibodies (AC-74) can trigger XP, it cannot distinguish between F-actin and G-actin, which excludes it as a viable therapeutic agent due to the risk of off-target effects. The antibody of the present invention also exhibits improved pharmacokinetics in circulation relative to Fc-DNGR-1 after systemic administration, accumulates at necrotic sites within the tumor, and promotes tumor control.

[0660] References

[0661] Numerous publications have been cited above to provide a more comprehensive description and disclosure of the present invention and the current state of the art to which it pertains. Full citations of these references are provided below. The entire contents of each of these references are incorporated herein by reference.

[0662] 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)

[0663] Canton, J., et al., The receptor DNGR-1 signals for phagosomalrupture to promote cross-presentation of dead-cell-associated antigens. NatImmunol, 2021. 22(2): p. 140-153.

[0664] Carmi, Y., et al., Allogeneic IgG combined with dendritic cellstimuli induce antitumour T-cell immunity. Nature, 2015: p. 1-6.

[0665] Carmi, Y. et al. Tumor-binding antibodies induce potent dendriticcell-mediated tumor immunity. Oncoimmunology 8(10), e1078063 (2019)

[0666] Castro-Dopico, T. & Clatworthy, M. IgG and Fcγ Receptors inIntestinal Immunity and Inflammation. Frontiers in Immunology 10:805 (2019)

[0667] Dominguez, R. & Holmes, K. C., Actin Structure and Function. Annu RevBiophys 40:169-186 (2011)

[0668] Dransfield, I. et al., Initial characterization of an anti-actinmonoclonal antibody (NH3). Biochemical Society Transactions, 1988, vol.16 p.163-164

[0669] Galluzzi, L., et al., Immunogenic cell death in cancer and infectiousdisease. Nature Reviews Immunology, 2017. 17(2): p. 97-111.

[0670] Giampazolias, E., et al., Secreted gelsolin inhibits DNGR-1-dependentcross-presentation and cancer immunity. Cell, 2021. 184(15): p. 4016-4031.e22

[0671] Guilliams, M., et al., The function of Fcy receptors in dendriticcells and macrophages. Nature reviews Immunology, 2014. 14(2): p. 94-108.

[0672] 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 deadcell-associated antigens. Immunity 42(5): 839-849 (2015)

[0673] Mylvaganam, S., Freeman, S., & Grinstein, S. (2021). The cytoskeletonin phagocytosis and macropinocytosis. Current Biology, 31(10), R619-R632.https: / / doi.org / 10.1016 / j.cub.2021.01.036

[0674] Regnault, A., et al., Fcgamma receptor-mediated induction ofdendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. TheJournal of experimental medicine, 1999. 189(2): p. 371-80

[0675] Sancho, D. et al. Identification of a dendritic cell receptor thatcouples sensing of necrosis to immunity. Nature 458, 899-903 (2009)

[0676] Srinivasan, N. et al., Actin is an evolutionarily-conserved damage-associated molecular pattern that signals tissue injury in Drosophilamelanogaster. eLife, 5:e19662 (2016)

[0677] WO 2009 / 013484

[0678] WO 2013 / 088136

[0679] WO 2022 / 163809

[0680] Min Yao, Jonathan Preall, John T.-H. Yeh, Darryl Pappin, PaoloCifani, Yixin Zhao, Sophia Shen, Philip Moresco, Brian He, Hardik Patel, Amber N. Habowski, Daniel A. King, Kara Raphael, Arvind Rishi, DivyeshSejpal, Matthew J. Weiss, David Tuveson, and Douglas T. Fearon. Human pancreatic ductal adenocarcinoma plasma cells secrete antibodies against self-antigens. JCI Insight, September 26,

[0681] Zhang, J.-G. et al. The dendritic cell receptor Clec9A binds damagedcells via exposed actin filaments. Immunity 36, 646–657 (2012).

[0682] References Sambrook, J., Russel, DW MolecularCloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: ColdSpring Harbor Laboratory Press

Claims

1. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment specifically binding to filamentous actin (F-actin), wherein said antibody is effective against the EC50 of F-actin. 50 The value is about 5 nM or less, and / or the affinity of the antibody for F-actin is at least 5 times that for globular actin (G-actin).

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody is capable of promoting cross-presentation (XP) of dead cell-associated antigens to CD8+ T cells.

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

4. The antibody or antigen-binding fragment of claim 2, wherein the XP is mediated by non-cDC1 cells expressing FcR, such as cDC2 or macrophages.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof has at least 3 times the specificity for binding necrotic cells relative to living 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 is capable of promoting phagocytosis of necrotic cells.

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

9. The antibody or antigen-binding fragment of claim 7, wherein the phagocytosis is mediated by non-cDC1 cells expressing FcR, such as cDC2 or macrophages.

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

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

12. An antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody does not compete with any antibody selected from the following for binding to F-actin: ab11003 (Abcam), ab205 (Abcam), ab130935 (Abcam), A2228 or A5316 (Sigma-Aldrich), NBP2-61610 (Novus Biologicals), MA1-80729 (ThermoFisher), and BS-1571R (ThermoFisher).

13. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody competitively binds to F-actin with human DNGR-1.

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

15. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the binding of the antibody 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, the discontinuous conformational epitope comprising amino acid residues present on at least two subunits of F-actin, wherein the discontinuous conformational epitope comprises three or more residues selected from: R196 and / or E237 of the first subunit of F-actin on the first filament and A114, E117, Q121, A365, G366 and P367 of the second subunit of F-actin on an F-actin filament different from the filament containing the first subunit.

17. The antibody or antigen-binding fragment of claim 16, wherein the antigen-binding portion binds to a conformational epitope, the conformational epitope further comprising residue D80 of a third subunit of F-actin, the third subunit being 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 comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: a) CDR1, wherein CDR1 has the sequence GGTFRSY or its variants contain one or two amino acid substitutions relative to GGTFRSY; b) CDR2, wherein CDR2 has the sequence NPIFDT or a variant thereof containing one or two amino acid substitutions relative to NPIFDT; and c) CDR3, wherein the CDR3 has the sequence TVIGAFDS or its variants contain one or two amino acid substitutions relative to TVIGAFDS; And said light chain comprises: d) CDR1, wherein the CDR1 has the sequence TRTSGDIGGYNFVS or its variants contain one, two or three amino acid substitutions relative to TRTSGDIGGYNFVS; e) CDR2, wherein the CDR2 has the sequence DVNSRPS or its variants contain one or two amino acid substitutions relative to DVNSRPS; and f) CDR3, wherein CDR3 has the sequence SSYTSRNTV or its variants contain one or two amino acid substitutions relative to SSYTSRNTV. The Complementary Decision Region (CDR) is identified using Chothia numbers.

19. The antibody or antigen-binding fragment according to claim 18, wherein the G at position 2 of CDRH1 is replaced by F or Y; wherein the T at position 3 of CDRH1 is replaced by I or A; wherein the R at position 5 of CDRH1 is replaced by S or T; wherein the S at position 6 of CDRH1 is replaced by A or N; and / or wherein the Y at position 7 of CDRH1 is replaced by S; and / or Wherein, N at position 1 of CDRH2 is replaced by I; where P at position 2 of CDRH2 is replaced by S; where I at position 3 of CDRH2 is replaced by A, S, or M; where F at position 4 of CDRH2 is replaced by Y or S; where D at position 5 of CDRH2 is replaced by N or G; and / or where T at position 6 of CDRH2 is replaced by G; and / or Wherein, T at position 1 of CDRH3 is replaced by G or Y; I at position 3 of CDRH3 is replaced by W or V; G at position 4 of CDRH3 is replaced by M; A at position 5 of CDRH3 is replaced by R, T, or K; F at position 6 of CDRH3 is replaced by T, Y, or I; D at position 7 of CDRH3 is replaced by P or S; and / or S at position 8 of CDRH3 is replaced by Y; and / or Wherein, T at position 1 of CDRL1 is replaced by S; R at position 2 of CDRL1 is replaced by G; T at position 3 of CDRL1 is replaced by A, S, or G; S at position 4 of CDRL1 is replaced by T; G ​​at position 5 of CDRL1 is replaced by Q or S; D at position 6 of CDRL1 is replaced by S or N; G at position 8 of CDRL1 is replaced by S; G at position 9 of CDRL1 is replaced by S; Y at position 10 of CDRL1 is replaced by R or K; N at position 11 of CDRL1 is replaced by R or L; F at position 12 of CDRL1 is replaced by Y, H, or N; and / or S at position 14 of CDRL1 is replaced by F; and / or Wherein, D at position 1 of CDRL2 is replaced by A or R; V at position 2 of CDRL2 is replaced by A, N, or G; N at position 3 of CDRL2 is replaced by S or D; S at position 4 of CDRL2 is replaced by M or Q; R at position 5 of CDRL2 is replaced by L; and / or P at position 6 of CDRL2 is replaced by Q; and / or Wherein, S at position 1 of CDRL3 is replaced by Q; where S at position 2 of CDRL3 is replaced by R or G; where Y at position 3 of CDRL3 is replaced by W; where T at position 4 of CDRL3 is replaced by S or V; where S at position 5 of CDRL3 is replaced by T; where R at position 6 of CDRL3 is replaced by P; and / or where N at position 7 of CDRL3 is replaced by Y.

20. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: a) CDR1, wherein CDR1 has the sequence GYIFTSY or its variants contain one or two amino acid substitutions relative to GYIFTSY; b) CDR2, wherein CDR2 has the sequence SAYNGH or a variant thereof containing one or two amino acid substitutions relative to SAYNGH; and c) CDR3, wherein the CDR3 has the sequence GKISSWFVLED or its variants contain one, two or three amino acid substitutions relative to GKISSWFVLED; And said light chain comprises: d) CDR1, wherein the CDR1 has the sequence SGGTSNIGKNYVS or its variants contain one, two or three amino acid substitutions relative to SGGTSNIGKNYVS; e) CDR2, wherein CDR2 has the sequence DNNMRPS or its variants contain one or two amino acid substitutions relative to DNNMRPS; and f) CDR3, wherein the CDR3 has the sequence GMWIRSLSRWV or its variants contain one, two, or three amino acid substitutions relative to GMWIRSLSRWV. The Complementary Decision Region (CDR) is identified using Chothia numbers.

21. The antibody or antigen-binding fragment of claim 20, wherein the Y at position 2 of CDRH1 is replaced by F or G; wherein the I at position 3 of CDRH1 is replaced by T or A; wherein the T at position 5 of CDRH1 is replaced by S or R; wherein the S at position 6 of CDRH1 is replaced by A or N; and / or wherein the Y at position 7 of CDRH1 is replaced by S; Wherein, S at position 1 of CDRH2 is replaced by P; where A at position 2 of CDRH2 is replaced by S, I or M; where Y at position 3 of CDRH2 is replaced by F or S; where N at position 4 of CDRH2 is replaced by D or G; where G at position 5 of CDRH2 is replaced by T; and / or where H at position 6 of CDRH2 is replaced by N; Wherein, G at position 1 of CDRH3 is replaced by M; K at position 2 of CDRH3 is replaced by R, T, or A; I at position 3 of CDRH3 is replaced by T, Y, or F; S at position 4 of CDRH3 is replaced by P or D; S at position 5 of CDRH3 is replaced by Y; W at position 6 of CDRH3 is replaced by Y or A; F at position 7 of CDRH3 is replaced by Y; L at position 9 of CDRH3 is replaced by M; and / or D at position 11 of CDRH3 is replaced by V; Wherein, S at position 1 of CDRL1 is replaced by T; G ​​at position 2 of CDRL1 is replaced by R; G at position 3 of CDRL1 is replaced by A, S, or T; T at position 4 of CDRL1 is replaced by S; S at position 5 of CDRL1 is replaced by Q or G; N at position 6 of CDRL1 is replaced by S or D; G at position 8 of CDRL1 is replaced by S; K at position 9 of CDRL1 is replaced by R or Y; N at position 10 of CDRL1 is replaced by R or L; Y at position 11 of CDRL1 is replaced by H, F, or N; and / or S at position 13 of CDRL1 is replaced by F. Wherein, D at position 1 of CDRL2 is replaced by A or R; N at position 2 of CDRL2 is replaced by A, V, or G; N at position 3 of CDRL2 is replaced by S or D; M at position 4 of CDRL2 is replaced by S or Q; R at position 5 of CDRL2 is replaced by L; and / or P at position 6 of CDRL2 is replaced by Q; and / or Wherein, G at position 1 of CDRL3 is replaced by A; M at position 2 of CDRL3 is replaced by T; I at position 4 of CDRL3 is replaced by D; R at position 5 of CDRL3 is replaced by D; S at position 6 of CDRL3 is replaced by G; L at position 7 of CDRL3 is replaced by Q; S at position 8 of CDRL3 is replaced by Q; R at position 9 of CDRL3 is replaced by S or G; W at position 10 of CDRL3 is replaced by Y; and / or V at position 11 of CDRL3 is replaced by 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 comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: a) CDR1, wherein CDR1 has the sequence GFTFSAY or a variant thereof containing one or two amino acid substitutions relative to GFTFSAY; b) CDR2, wherein CDR2 has the sequence SYDGNN or a variant thereof containing one or two amino acid substitutions relative to SYDGNN; and c) CDR3, wherein the CDR3 has the sequence DFRDYVWGTYPSAY or its variants contain one, two or three amino acid substitutions relative to DFRDYVWGTYPSAY; And said light chain comprises: d) CDR1, wherein the CDR1 has the sequence SGSSSNIGRRHVF or its variants contain one, two or three amino acid substitutions relative to SGSSSNIGRRHVF; e) CDR2, wherein the CDR2 has the sequence RGDQRPS or a variant thereof containing one or two amino acid substitutions relative to RGDQRPS; and f) CDR3, wherein the CDR3 has the sequence ATWDDGLSGYV or a variant thereof containing one, two, or three amino acid substitutions relative to ATWDDGLSGYV. The Complementary Decision Region (CDR) is identified using Chothia numbers.

23. The antibody or antigen-binding fragment according to claim 22, wherein F at position 2 of CDRH1 is replaced by G or Y; wherein T at position 3 of CDRH1 is replaced by I or A; wherein S at position 5 of CDRH1 is replaced by R or T; wherein A at position 6 of CDRH1 is replaced by S or N; and / or wherein Y at position 7 of CDRH1 is replaced by S; and / or Wherein, S at position 1 of CDRH2 is replaced by A, I or M; where Y at position 2 of CDRH2 is replaced by F or S; where D at position 3 of CDRH2 is replaced by N or G; where G at position 4 of CDRH2 is replaced by T; and / or where N at position 5 of CDRH2 is replaced by H; Wherein, D at position 1 of CDRH3 is replaced by K; F at position 2 of CDRH3 is replaced by E; D at position 4 of CDRH3 is replaced by T; Y at position 5 of CDRH3 is replaced by G or T; W at position 7 of CDRH3 is replaced by I or V; G at position 8 of CDRH3 is replaced by M; T at position 9 of CDRH3 is replaced by R, A, or K; Y at position 10 of CDRH3 is replaced by T, F, or I; P at position 11 of CDRH3 is replaced by D or S; S at position 12 of CDRH3 is replaced by Y; A at position 13 of CDRH3 is replaced by Y or W; and / or Y at position 14 of CDRH3 is replaced by F; and / or Wherein, the S at position 1 of CDRL1 is replaced by T; the G at position 2 of CDRL1 is replaced by R; the S at position 3 of CDRL1 is replaced by A, T, or G; the S at position 4 of CDRL1 is replaced by T; the S at position 5 of CDRL1 is replaced by Q or G; the N at position 6 of CDRL1 is replaced by S or D; the G at position 8 of CDRL1 is replaced by S; the R at position 9 of CDRL1 is replaced by Y or K; the R at position 10 of CDRL1 is replaced by L or N; the H at position 11 of CDRL1 is replaced by F, N, or Y; and / or the F at position 13 of CDRL1 is replaced by S; and / or Wherein, R at position 1 of CDRL2 is replaced by A or D; G at position 2 of CDRL2 is replaced by A, N, or V; D at position 3 of CDRL2 is replaced by S or N; Q at position 4 of CDRL2 is replaced by S or M; R at position 5 of CDRL2 is replaced by L; and / or P at position 6 of CDRL2 is replaced by Q; and / or Wherein, A at position 1 of CDRL3 is replaced by G; T at position 2 of CDRL3 is replaced by M; D at position 4 of CDRL3 is replaced by I; D at position 5 of CDRL3 is replaced by R; G at position 6 of CDRL3 is replaced by S; L at position 7 of CDRL3 is replaced by Q; S at position 8 of CDRL3 is replaced by Q; G at position 9 of CDRL3 is replaced by S or R; Y at position 10 of CDRL3 is replaced by W; and / or V at position 11 of CDRL3 is replaced by 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 comprises a heavy chain and a light chain variable sequence, wherein the heavy chain comprises: a) CDR1, wherein the CDR1 has the sequence GGAFRNS or a variant thereof containing one or two amino acid substitutions relative to GGAFRNS; b) CDR2, wherein CDR2 has the sequence IPMSGT or a variant thereof containing one or two amino acid substitutions relative to IPMSGT; and c) CDR3, wherein the CDR3 has the sequence EKERTFGVVMRTSYYYVMEV or its variants contain one, two, three or four amino acid substitutions relative to EKERTFGVVMRTSYYYVMEV; And said light chain comprises: d) CDR1, wherein CDR1 has the sequence RASQSISSYLN or a variant thereof containing one or two amino acid substitutions relative to RASQSISSYLN; e) CDR2, wherein CDR2 has the sequence AASSLQS or a variant thereof containing one or two amino acid substitutions relative to AASSLQS; and f) CDR3, wherein CDR3 has the sequence QQSYSTPYT or a variant thereof containing one or two amino acid substitutions relative to QQSYSTPYT. The Complementary Decision Region (CDR) is identified using Chothia numbers.

25. The antibody or antigen-binding fragment thereof according to claim 24, wherein the G at position 2 of CDRH1 is replaced by F or Y; wherein the A at position 3 of CDRH1 is replaced by T or I; wherein the R at position 5 of CDRH1 is replaced by S or T; wherein the N at position 6 of CDRH1 is replaced by A or S; and / or wherein the S at position 7 of CDRH1 is replaced by Y; and / or Wherein, I at position 1 of CDRH2 is replaced by N; where P at position 2 of CDRH2 is replaced by S; where M at position 3 of CDRH2 is replaced by I, A, or S; where S at position 4 of CDRH2 is replaced by F or Y; where G at position 5 of CDRH2 is replaced by D or N; and / or where T at position 6 of CDRH2 is replaced by G; and / or Wherein, K at position 2 of CDRH3 is replaced by D; E at position 3 of CDRH3 is replaced by F; T at position 5 of CDRH3 is replaced by D; G at position 7 of CDRH3 is replaced by Y or T; V at position 9 of CDRH3 is replaced by W or I; M at position 10 of CDRH3 is replaced by G; R at position 11 of CDRH3 is replaced by A, T, or K; T at position 12 of CDRH3 is replaced by F, Y, or I; S at position 13 of CDRH3 is replaced by P or D; Y at position 14 of CDRH3 is replaced by S; Y at position 15 of CDRH3 is replaced by W or A; Y at position 16 of CDRH3 is replaced by F; M at position 18 of CDRH3 is replaced by L; and / or V at position 20 of CDRH3 is replaced by D; and / or Wherein, R at position 1 of CDRL1 is replaced by G; A at position 2 of CDRL1 is replaced by S, T, or G; S at position 3 of CDRL1 is replaced by T; Q at position 4 of CDRL1 is replaced by S or G; S at position 5 of CDRL1 is replaced by D or N; S at position 7 of CDRL1 is replaced by G; S at position 8 of CDRL1 is replaced by G; Y at position 9 of CDRL1 is replaced by R or K; L at position 10 of CDRL1 is replaced by N or R; and / or N at position 11 of CDRL1 is replaced by F, H, or Y; and / or Wherein, A at position 1 of CDRL2 is replaced by D or R; where A at position 2 of CDRL2 is replaced by V, N, or G; where S at position 3 of CDRL2 is replaced by N or D; where S at position 4 of CDRL2 is replaced by M or Q; where L at position 5 of CDRL2 is replaced by R; and / or where Q at position 6 of CDRL2 is replaced by P; and / or Where Q at position 1 of CDRL3 is replaced by L; Q at position 2 of CDRL3 is replaced by S; S at position 3 of CDRL3 is replaced by R or G; Y at position 4 of CDRL3 is replaced by W; S at position 5 of CDRL3 is replaced by T or V; T at position 6 of CDRL3 is replaced by S; P at position 7 of CDRL3 is replaced by R; and / or Y at position 8 of CDRL3 is replaced by N.

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

27. An 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 purpose of claim 28, wherein the cancer includes solid tumors.

30. The antibody or antigen-binding fragment for the purpose according to any one of claims 27 to 29, wherein the purpose includes 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 competes with the antibody or antigen-binding fragment according to any one of claims 18 to 26 for binding to F-actin.

32. A method for selecting an antibody or antigen-binding fragment thereof that specifically binds to F-actin, the method comprising: Provide candidate antibody populations, The binding substrate containing F-actin is contacted with the candidate antibody population. The binding substrate is contacted with trypsin or a competitive agent of a CTLD containing DNGR-1 to displace one or more lead antibodies from the binding substrate, and then... Choose an antibody from one or more of the lead antibodies.

33. The method of claim 32, wherein the method comprises screening the one or more lead antibodies for 10-fold specificity relative to G-actin against F-actin, wherein the selected antibody has the 10-fold specificity.

34. The method of claim 32 or claim 33, wherein the method comprises screening the one or more lead antibodies for at least 3 times specificity relative to live cells for binding necrotic cells, as measured by FACS or flow cytometry, wherein the selected antibody has said 3 times specificity.

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

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

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

38. The antibody of claim 37, wherein the antibody is polyclonal.

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