Identification of agonist combinations that stimulate macrophages to induce anti-tumor T cells and overcome immunosuppression

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Igney, Daniela Reiss, John E. Park This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6195356/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Macrophages are the major immune cell population in most human solid cancers. Therefore, we hypothesized that appropriate activation of macrophages in tumors could convert an immunosuppressive into a proinflammatory tumor microenvironment and, thus, enable T cell-mediated killing of tumor cells. To identify appropriate activating receptors, we selected putative agonists from literature and used them for stimulation of primary monocyte-derived human macrophages as single agents or in combination. Stimulated macrophages were co-cultured with autologous T cells to test their ability to reactivate antigen-specific T cells. To mimic tumor-specific clustering, we expressed selected agonistic antibodies on the cell surface of tumor cells and used them in coculture assays to determine macrophage stimulation and T cell activation. For the most promising agonists and combinations, we also tested their ability to repolarize M2 to M1 macrophages and determined the induced cytokine and chemokine profile. The most effective agonist combinations were: CD40 + TLR4, CD40 + TLR7/8, CD40 + Clec5a, CD40 + CSF1R, CD40 + CSF2RB and TLR4 + TLR7/8. Macrophages that were stimulated with these combinations activated autologous antigen-specific T cells that were cytotoxic towards tumor cells. These combinations were also able to overcome TGF-beta-mediated immunosuppression in vitro. Tumor-specific activation of macrophages with these agonist combinations may represent a promising approach to enhance the anti-tumor immune response and increase survival of patients with macrophage-rich solid tumors. Biological sciences/Cancer/Tumour immunology Biological sciences/Immunology/Immunotherapy Biological sciences/Immunology/Immunotherapy/Immunosuppression Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages cancer immunotherapy tumor-associated macrophages T cell activation tumor microenvironment CD40 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Tumor-infiltrating macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) represent the major immune population in most human solid cancers and can reach up to 50% of tumor mass 1 , 2 . High numbers of these cells are associated with poor prognosis and response to therapy in many tumor types 3 . Therefore, different strategies to therapeutically target this abundant myeloid population are under investigation with the aim to induce immune-mediated destruction of tumors or to restore sensitivity to immune-checkpoint therapy. An early proof of this concept was achieved by the clinical success of the Toll-like receptor (TLR) 4 agonist Bacillus Calmette − Guerin (BCG), which is an approved therapy for bladder cancer 4 . Similarly, imiquimod is a TLR7 agonist that has been approved for the topical treatment of basal cell carcinoma and genital warts 5 , 6 . TLRs are pattern recognition receptors that activate innate immune cells, such as macrophages and dendritic cells, and induce inflammatory responses. Colony-stimulating factor 1 receptor (CSF1R) is a key regulator of macrophage survival and differentiation. CSF1R inhibitors have reached late-stage development in a variety of solid tumor types, but several have recently been discontinued due to disappointing efficacy 7 , 8 . The small molecule CSF1R inhibitor pexidartinib (PLX3397) has been approved for the treatment of tenosynovial giant cell tumors, a rare proliferative tumor caused by an overproduction of colony-stimulating factor 1. CD40 is a cell surface receptor that is mainly expressed on macrophages, dendritic cells, B cells and other non-immune cells 9 . Upon clustering by the ligand CD40L (CD154), antigen-presenting cells secrete proinflammatory cytokines and up-regulate costimulatory molecules such as CD80 and CD86 that are required for costimulation of T cells. Agonistic anti-CD40 antibodies have shown promising preclinical results and initial encouraging clinical data 9 – 11 . However, only few myeloid-cell targeting concepts have made it into clinical practice for cancer therapy. Many of these agonists suffer from lack of convincing efficacy or from high systemic toxicity. Toxicity can also restrict the use of agonists to suboptimal doses that result in insufficient immune activation and anti-tumor efficacy. An approach to overcome systemic toxicity is to specifically target an agonist to the tumor. Intratumoral administration of an anti-CD40 antibody into superficial lesions was well tolerated at clinically relevant doses and associated with pharmacodynamic responses 12 , 13 . However, this route of administration is limited to accessible lesions and a low frequency of injections. To reduce the likelihood of systemic toxicities, bispecific molecules have been developed that target both an activating receptor and a tumor antigen to achieve maximal activity in the presence of the tumor antigen and minimal activity in normal tissues with little antigen expression. A bispecific tumor-targeted anti-CD40 agonistic antibody, which simultaneously bound to CD40 and the tumor antigen carcinoembryonic antigen (CEA), enabled potent in vitro dendritic cell (DC) activation and consecutive T cell cross-priming in a CEA-specific manner 14 . The bispecific molecule ABBV-428 that targeted CD40 and mesothelin demonstrated enhanced activation of antigen-presenting cells and T cells upon binding to cell-surface mesothelin, and inhibition of tumor cell growth. In a phase I study, ABBV-428 monotherapy displayed a good tolerability profile, but had only minimal clinical activity 15 , 16 . The bispecific fibroblast activation protein (FAP)-targeted CD40 agonist RO7300490 was well tolerated up to the highest dose tested in a phase I study. It achieved a strong and sustained target engagement and immunomodulation in tumor tissue, but no objective responses 17 , 18 . SBT6050 is a therapeutic comprised of a TLR8 agonist payload conjugated to a HER2-directed monoclonal antibody. It demonstrated single agent efficacy in multiple mouse tumor models without peripheral cytokine production. Preliminary phase I data indicated a manageable safety profile and evidence of myeloid, NK and T cell activation 19 , 20 . To reduce CD40-related toxicities, also bispecific antibodies have been studied that targeted CD40 activation preferentially to dendritic cells, by coupling the CD40 agonist arm with a CLEC9A-targeting arm. This bispecific reagent demonstrated a superior safety profile compared to the parental anti-CD40 monospecific antibody while triggering potent antitumor activity in mice 21 . To increase anti-tumor efficacy and maximize the effects of myeloid cell-targeted therapies, combinations of anti-CD40 with other myeloid agonists have been studied in mouse models. Inhibition of CSF1R signaling sensitized TAMs to rapid reprogramming in the presence of a CD40 agonist before their depletion. Combination of anti-CSF1R and anti-CD40 was sufficient to create a proinflammatory tumor milieu that reinvigorated an effective T cell response in transplanted tumors 22 . In a recent study, combinations of anti-CD40 with various pattern-recognition receptor agonists were tested in a mouse model of pancreatic ductal adenocarcinoma 23 . Systemic administration of a TLR7/8, STING, NOD2, or Dectin-1 agonist in combination with agonistic anti-CD40 led to high rates of tumor cures and triggered immunological memory. This study demonstrated that cancer immune surveillance in pancreatic tumors that were resistant to checkpoint inhibition could be invoked by coactivation of complementary myeloid signaling pathways. We hypothesized that combining coactivation of two receptors with tumor targeting could overcome both challenges of systemic toxicity and low efficacy. Here, we aimed to identify the best combinations of activating receptors on macrophages to convert an immunosuppressive into a proinflammatory tumor microenvironment. We screened a range of agonists on primary human macrophages. Stimulated macrophages were co-cultured with autologous T cells to test their ability to activate antigen-specific T cells. The best combinations were able to overcome TGF-b-mediated immunosuppression in vitro and induced T cells that were cytotoxic towards tumor cells. These investigations will serve as a basis to develop multispecific antibody constructs for tumor-specific activation of macrophages. Methods Human primary cell isolation, macrophage differentiation and polarization Buffy coats or leukopaks of healthy blood donors were received from the Institute of Clinical Transfusion Medicine, Ulm, Germany. All experiments were approved by the Ethics Committee of Landesärztekammer Baden-Württemberg, and informed consent was obtained from all subjects. All methods were carried out in accordance with relevant guidelines and regulations. Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll gradient centrifugation. Monocytes were isolated by positive selection using CD14 MicroBeads (Miltenyi) according to the manufacturer's protocol. Remaining CD14-negative cells were cryo-preserved for later isolation of autologous T cells. Monocytes were differentiated to macrophages in RPMI-1640 (Gibco, ATCC modifications) with 5 % human serum (Sigma), 1 % Penicillin-Streptomycin (Sigma) and 50 ng/ml M-CSF (BioTechne) in UpCell plates (Thermo Scientific) for 5-7 d at 37 °C and 5% CO 2 in an incubator. If not indicated otherwise, these macrophages were used for all assays (M0 macrophages). For polarization to M2a macrophages, human IL-4 and IL-13 (20 ng/ml each; R&D Systems) were added for another 24 h. Autologous T cells were isolated from the CD14-negative fraction of PBMCs using the human Pan T Cell Isolation Kit (Miltenyi). Macrophage stimulation and autologous T cell activation Human monocyte-derived macrophages were removed from UpCell plates by temperature shift and loaded with peptides (1 µg/ml) for 1 h at 37 °C in cell repellent plates (Greiner-bio-one). The following peptide mixes were used (all from JPT Peptide Technologies): CEFX (CEFX Ultra SuperStim Pool), MHC-I (CEFX Ultra SuperStim Pool MHC-I Subset), CMV (Antigen Peptide CMV pp65 - HLA-A*0201 (NLVPMVATV)), Flu (Influenza A MP (58-66) Peptide (GILGFVFTL)), or SARS (PepMix SARS-CoV-2 (Spike Glycoprotein)). Autologous T cells were isolated from the CD14-negative PBMC fraction of the same donor. 2x10 4 macrophages and 10 5 T cells were cocultured in 96-well plates. For stimulation of macrophages, soluble agonists were added to the cocultures ( Supplementary Table 1 ); agonistic antibodies ( Supplementary Table 2 ) were coated (10 mg/ml) to high-binding plates (Costar) before addition of cells. Supernatants of cocultures were harvested after 5 d for analysis of IFNg or granzyme B by ELISA (R&D Systems). For stimulation with tumor-anchored agonists, 2x10 3 scFab-expressing HCT116 cells were added to the coculture, and supernatants were analysed after 3 d. Each condition was performed in triplicates. For normalization, the IFNg concentration of a specific sample was divided by the concentration of the indicated control (usually sample with CD40L stimulation). To investigate the effect of TGFb, the indicated concentrations of TGFb1 (R&D Systems) were added to the T cell:macrophage coculture. Generation of scFab-expressing HCT116 cells Fab sequences from antibodies ( Supplementary Table 3 ) were cloned into pOptiVEC as single chains with a GS linker, followed by a GPI anchor sequence. To enable co-expression, two sets of vectors were generated: one set contained sequences for V5 and FLAG tag in the linker sequence and a hygromycin resistance gene; the other set contained sequences for HA and Myc tag in the linker sequence and a puromycin resistance gene. DNA was synthesized at GeneArt (Thermo Fisher Scientific). HCT116 cells (ATCC) were transfected using Neon Transfection System (Invitrogen) and kept under the respective selection pressure to generate stable cell lines. For co-expression, a vector of the other set was co-transfected into a stable cell line and the second selection pressure added. Expression was analysed by conventional flow cytometry with antibodies against HA (clone 16B12, BV421, Biolegend), and/or Flag tag (clone L5, Alexa-647, Biolegend). CD40 and Clec5a activity assay For determining CD40 activity, 5x10 4 HEK-Blue CD40L cells (Invivogen) were seeded in DMEM with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin. The indicated number of scFab-expressing HCT116 cells were added and incubated for 24 h. CD40L (0.5 mg/ml) was used as positive control. Supernatants were harvested, and QuantiBlue solution (Invivogen) was added. SEAP reporter activity was measured at OD635. To determine Clec5a agonistic activity, U937 cells were differentiated with 10 nM phorbol 12-myristate 13-acetate (PMA; Sigma) for 3 d in 100 mm UpCell plates. 10 5 cells were seeded in 96-well plates (Costar) in medium without PMA, and indicated numbers of HCT116 cells expressing anti-Clec5a constructs were added. After 24 h CCL3 concentrations in supernatants were determined by ELISA (R&D Systems). Plate-coated anti-Clec5a (clone 283834) or isotype control was used as control. Macrophage chemokine/cytokine secretion and repolarization To determine chemokine and cytokine secretion, macrophages were stimulated either with the plate-bound and/or soluble agonists or cocultured with agonist-expressing HCT116 tumor cells for 24 h. Supernatants were harvested and cytokines and chemokines determined by LegendPlex Human Inflammation Panel 1 and LegendPlex Human Proinflammatory Chemokine Panel (Biolegend). Each condition for each donor was performed in triplicates. To determine repolarization, M2a-polarized macrophages were stimulated with the indicated plate-bound and/or soluble agonists for 48 h. Expression of MHC-II, CD80, CD83, CD163 and CD206 on live cells was analysed by multicolor conventional flow cytometry (all antibodies from BD Biosciences). T cell cytotoxicity PBMCs were tested for HLA-A2 expression and for their reactivity to CMV or Flu peptide stimulation. Only HLA-A2-positive donors that were reactive to CMV or flu peptide stimulation were used. Monocyte-derived macrophages were loaded with the respective peptide, activated with combinations of plate-bound antibodies or soluble agonists, and cocultured with autologous T cells as above. After 5 d, T cells were separated from these cocultures by gently aspirating the T cells in suspension from the adherent macrophages. Replicates were combined to obtain sufficient numbers of T cells as effector cells. As target cells, HCT116 cells stably expressing Renilla luciferase were loaded with the respective peptide. Alternatively, HCT116 cells were used that stably expressed a fusion peptide consisting of the CMV pp65 peptide and the M1 domain of influenza virus. 10 5 T cells were incubated with 10 4 target cells for 48 h (effector:target ratio 10:1). The number of living tumor cells was determined either by Renilla-Glo Luciferase Assay (Promega) or CellTiter-Glo Luminescent Cell Viability Assay (Promega). The signal of T cells in CellTiter-Glo assay was neglectable compared to tumor cell signal at the effector:target ratio used. Values were normalized to a control sample with target cells only. A final concentration of 0.5 % Triton X-100 was added 1 h before readout as control for maximal lysis. Results CD40L-stimulated macrophages activate autologous antigen-specific T cells. We hypothesized that modulation of macrophages in tumors could influence the function of cytotoxic T cells. Therefore, we tested whether human macrophages could directly reactivate antigen-experienced T cells. As model system, we used human monocyte-derived macrophages, and loaded them with a pool of peptides that were derived from common infectious agents and that were known to bind to MHC-I of a broad range of HLA subtypes. Macrophages were cocultured with autologous T cells, and T cell reactivation was quantified by measuring the release of IFNγ ( Figure 1A ). In the absence of peptides, IFNγ release was minimal. Peptide-loaded macrophages induced IFNγ release, indicating the reactivation of pre-existing T cells with specificity to the peptide pool of infectious agents. As CD40L is known to stimulate macrophages, we added recombinant CD40L to the cocultures. CD40L stimulation increased IFNγ release, confirming that macrophage modulation could influence T cell reactivation. Donor variability was high, and overall levels of IFNγ were low. Therefore, we switched to a related peptide pool containing MHC-I and MHC-II binding peptides, which resulted in a better assay window ( Figure 1B ). Measurement of granzyme B release confirmed the activation of cytotoxic T cells ( Figure 1C ). Stimulation of macrophages by agonist combinations can enhance T cell activation. To identify the most effective stimulation of macrophages, we selected a range of receptors from literature. Criteria for selection were expression on macrophages in tumor tissue, an activating function and availability of putative tool agonists ( Supplementary Table 1&2 ). First, we tested anti-receptor antibodies coated to high-binding plates for cross-linking as single agonists or in combination ( Figure 2A ). Peptide-loaded macrophages were cocultured with autologous T cells on these plates. CD40L in solution was used as control. Due to the high donor variability, values were normalized to CD40L stimulation for each donor. Several combinations of antibodies with CD40L induced higher IFNγ release than CD40L alone. Next, we screened a range of putative soluble agonists ( Figure 2B ). The TLR7/8 agonist resiquimod was more efficacious than CD40L as single agent, and strongly induced IFNγ when used in combinations. Other effective combinations included CD40L, IL-7, IL-17A and others. As some of the receptors, such as IL-7RA or IL-17RA, are not only expressed on macrophages, but also on T cells, a direct effect of agonists on T cells could not be excluded at this stage. Finally, we also tested combinations of plate-coated antibodies and soluble agonists that had shown some activity in the previous experiments ( Figure 2C ). Mainly combinations with anti-TLR4 showed enhanced activity. Confirmation and additional characterization of most effective agonist combinations. From the screens described above, we selected the most promising agonist combinations for further investigations, mainly based on strength and robustness of response. The majority of hits could be confirmed with additional donors ( Figure 3A&B ). With dual stimulation, T cell reactivation remained antigen-dependent, as there was no activation in absence of peptides ( Figure 3A ). To further confirm antigen-specific activation, we also analysed the response to a single antigenic peptide. As model antigen, we chose the pp65 antigen from cytomegalovirus (CMV) due to its relatively high prevalence in blood donors, and influenza A matrix protein due to wide-spread influenza vaccination. Donors were pre-tested for HLA-A2 expression and their reactivity to a specific peptide from cytomegalovirus (CMV) or influenza virus (flu). Macrophages from positive donors were loaded with the respective peptide, stimulated with the most promising agonist combinations and used for T cell activation ( Figure 3C ). IFNγ responses showed a similar pattern as in previous experiments. Expectedly, the strength of response to these antigens was different as compared to the previously used peptide mix. In tumors, macrophages are more polarized towards an M2 phenotype 3 . Therefore, we compared the ability of M0 and M2a macrophages to activate T cells ( Figure 3D ). For the agonists tested, stimulation of M2a macrophages enhanced T cell activation in a similar way as M0 macrophages, indicating that the concept could also work for TAMs. Expression of scFab on tumor cells as model for tumor-specific clustering of agonists. Next, we investigated whether the concept also worked when agonists were clustered on the cell surface of tumor cells. To circumvent any additional complexity with the characteristics of a specific tumor antigen and respective binders in a multi-specific construct, we expressed scFab fragments directly on the cell surface of the human tumor cell line HCT116 using a glycosylphosphatidylinositol (GPI) anchor. Fab constructs were derived from the sequences of published antibodies ( Supplementary Table 3 ). To enable co-expression of two different scFab constructs on the same cell, we generated two sets of vectors containing two different selection markers and four different tags ( Figure 4A ). Expression was determined by flow cytometry staining of the respective tags ( Figure 4B ). Due to the use of standardized constructs, IRES (internal ribosome entry site)-related correlation of Fab expression and selection marker as well as strong selection pressure, expression levels of different scFabs were comparable. To demonstrate functional activity of tumor-anchored scFabs, we performed a CD40 reporter gene assay. Tumor cells expressing anti-CD40 induced a strong reporter signal in a similar way as the positive control CD40L. Tumor cells that did not express anti-CD40 did not induce any reporter signal ( Figure 4C ). Titration of tumor cells expressing anti-CD40 and a second construct confirmed comparable activity of different transfected tumor cells ( Figure 4D ). We also tested agonistic activity of two anti-Clec5a scFabs. It has been shown that stimulation of differentiated U937 cells with a monoclonal anti-Clec5a antibody induced CCL3 release 24 . Therefore, we cocultured scFab-expressing HCT116 with differentiated U937 and confirmed that both anti-Clec5a scFabs were agonistic, whereas anti-CD40 alone did not induce CCL3 in this assay ( Figure 4E ). We did not directly test the function of other scFabs apart from anti-CD40 or anti-Clec5a, due to the wide range of receptors and pathways involved. Stimulation of macrophages by tumor-anchored scFab leads to T cell activation. To mimic tumor-specific clustering of agonists, we stimulated macrophages with HCT116 cell lines that expressed combinations of tumor-anchored anti-receptor scFabs. These macrophages were used to activate autologous antigen-specific T cells in triple cocultures ( Figure 5A&B ). Tumor-anchored anti-CD40 induced T cell reactivation as determined by IFNγ release. Similar to the experiments with plate-bound agonists above, several combinations induced higher IFNγ release than CD40-stimulation alone. Mainly combinations with anti-CD40 were effective. For some combinations (CD40+Clec5a), we also measured granzyme B release, which was in line with IFNγ release, confirming the activation of cytotoxic T cells ( Figure 5C ). We also tested reactivation of T cells to an unrelated model antigen, namely the spike glycoprotein from SARS-CoV-2 ( Figure 5D ). We hypothesized that due to the pandemic and related vaccinations most donors would be reactive to specific stimulation, so that pre-testing of donors would be unnecessary. However, we observed a very high donor variability. From 6 donors used, we excluded one with no response and one with an extreme response even in absence of additional stimulation. Similar to experiments above, anti-CD40 and anti-TLR4 induced IFNγ release, and several combinations with anti-CD40 resulted in an enhanced response. Agonist combinations can induce inflammatory chemokines and cytokines and repolarize M2a macrophages. To characterize the direct effect of stimulation on macrophages, we stimulated macrophages in the absence of T cells with the most promising agonists combinations. After stimulation either with plate-bound and/or soluble agonists or by coculture with scFab-expressing tumor cells, the secretion of a panel of inflammatory cytokines and chemokines was analysed ( Figure 6A ). CD40 stimulation alone resulted only in modest changes in cytokine/chemokine secretion. In general, strong increases were observed with combinations that were also effective in the T cell activation assays, such as CD40+TLR4, CD40+TLR7/8, CD40+Clec5a, and others. Because macrophages in tumors are polarized towards an M2 phenotype, we also investigated whether stimulation could repolarize M2a macrophages to a more proinflammatory M1 phenotype. After stimulation with plate-bound and/or soluble agonists, we analysed classical M1/M2 markers by flow cytometry ( Figure 6B ). While CD40 stimulation increased MHC-II expression, this effect was not observed with most of the other stimuli or combinations. TLR7/8 stimulation as well as several combinations enhanced the level of costimulatory molecules (CD80, CD83). Changes in the expression of M2 markers (CD163, CD206) were less pronounced. T cells activated by macrophages are cytotoxic towards tumor cells. To demonstrate that appropriate stimulation of macrophages could enable T cell-mediated killing of tumor cells, we analysed the cytotoxicity of activated T cells. Macrophages were loaded with CMV or flu peptide, activated with combinations of plate-bound antibodies or soluble agonists, and cocultured with autologous T cells to induce activation. T cells were separated from these cocultures and their cytotoxicity towards peptide-presenting HCT116 cells was determined ( Figure 7A ). Strongest cytotoxicity was observed after stimulation of TLR7/8, TLR4+TLR7/8, and several CD40 combinations ( Figure 7B ). Cytotoxicity of activated T cells was dependent on peptide-loading and stimulation of macrophages ( Figure 7C&D ). Stimulation of macrophages can overcome immunosuppression by TGF b . The tumor microenvironment, especially in tumors that are refractory to checkpoint inhibitor treatment, is characterized by immunosuppression 25 . One major contributing factor is TGFβ. Therefore, we tested the effect of TGFβ on activation of T cells by CD40-stimulated macrophages ( Figure 8A ). IFNγ release was almost completely reduced to baseline by 50 pg/ml TGFβ and completely blocked by higher concentrations. To investigate whether appropriate stimulation of macrophages could overcome TGFβ immunosuppression in vitro, we stimulated macrophages with different agonist combinations and used them for T cell stimulation in the absence or presence of TGFβ ( Figure 8B&C ). Although IFNγ release was reduced by TGFβ for all stimulation conditions, IFNγ secretion could be detected even in the presence of TGFβ. With the best agonist combinations, IFNγ levels were reached that were higher than with CD40-stimulation in the absence of TGFβ. Discussion In the tumor microenvironment, TAMs represent the largest proportion of immune cells. Therefore, we aimed to identify the best combinations of receptor agonists to activate macrophages in such a way that they promote T cell-mediated killing and immune-mediated destruction of tumor cells. We screened a range of putative agonists and characterized the most effective combinations for their ability to induce cytokine and chemokine release, repolarization, T cell activation, and cytotoxicity. Regarding all assays performed, the combinations that were most effective and fitted best to the concept of tumor-specific macrophage stimulation were: CD40 + TLR4, CD40 + TLR7/8, CD40 + Clec5a, CD40 + CSF1R, CD40 + CSF2RB and TLR4 + TLR7/8. Although combinations with IL-7 or IL-17a showed strong effects in the initial screens, we did not follow them up, because their receptors are not only expressed on macrophages, but also on T cells. Therefore, a direct effect of agonists on T cells could not be excluded, so that other concepts or constructs may be required. So far, efficacy of myeloid-targeted therapy in the clinic was rather limited, and treatment responses were potentially determined by patient-specific microenvironmental regulators, as has been shown for anti-CD40 26 . In our experiments, dual stimulation was more effective than single agonists, and T cell activation was also achieved in donors with low response to CD40L only. Importantly, dual stimulation was also able to overcome immunosuppression by TGFb (Fig. 8 B &C ). TGFb completely suppressed T cell activation by macrophages that were stimulated via CD40 only. However, stimulation of macrophages with combinations, such as CD40 + TLR7/8, CD40 + TLR4, or TLR4 + TLR7/8, resulted in stronger activation of T cells than with CD40-stimulation in the absence of TGFb. These data suggest that appropriate macrophage stimulation may restore anti-tumor T cell responses and sensitivity to checkpoint inhibitors in tumors that are characterized by an immunosuppressive microenvironment with high macrophage infiltration and high TGFb levels. While TGFb is a major factor, other molecules, such as prostaglandin E2, can also contribute to immunosuppression in the tumor microenvironment 25 , 27 . Therefore, further experiments are needed to demonstrate the value of the approach for patients, e.g. using dissociated patient samples ex vivo. Systemic stimulation of activating myeloid receptors can lead to severe toxicity 13 , 28 . Therefore, we hypothesized that combining coactivation of two receptors with tumor targeting could result in stronger efficacy and a better safety profile. To mimic tumor-specific clustering of agonists, we expressed scFab fragments directly on the cell surface of a human tumor cell line. Stimulation of macrophages by these tumor-anchored scFabs also led to T cell activation, demonstrating general feasibility of this concept (Fig. 5 ). Potential drugs could be envisaged as trispecific constructs: In case of two agonistic antibodies, such as anti-CD40 and anti-Clec5a, the construct could be composed of a trispecific antibody with CD40- and Clec5a-binding moieties and a tumor-specific binder, such as anti-FAP. The exact construct geometry and stoichiometry remains to be determined. In case of an agonistic antibody and a soluble small-molecule agonist, such as anti-CD40 and resiquimod, the drug construct may be a bispecific antibody-drug conjugate with a CD40 and a tumor-specific binder and a linked small molecule agonist. Proteinaceous agonists, such as flagellin, offer additional opportunities 29 . In addition, the characteristics of the tumor-specific antigen need to be considered, such as specificity and density of expression as well as propensity for internalization and clustering. For the most effective agonist combinations, we have observed synergistic effects on macrophage stimulation and subsequent T cell activation. For example, anti-Clec5a stimulation alone did not lead to T cell activation, but strongly enhanced the effect of CD40 stimulation (Fig. 3 & 5 ). T cell reactivation remained antigen-specific, as there was no activation in the absence of the antigenic peptide. It will be interesting to investigate the underlying signalling pathways and molecular mechanisms. While we have observed upregulation of costimulatory molecules (CD80, CD83) and induction of chemokines and cytokines (Fig. 6 ), a clear mechanism cannot be derived from these data. Further insight may be gained by transcriptomic analysis of stimulated macrophages and pathway analysis. The therapeutic effect of anti-CD40 has mainly been attributed to stimulation of DCs. To increase the therapeutic use of CD40 stimulation, bispecific antibodies have been designed that targeted CD40 activation preferentially to DCs by coupling the CD40 agonist arm with a CLEC9A-targeting arm 21 . DCs were also required for the anti-tumor activity of combinations of anti-CD40 with various pattern-recognition receptor agonists in a mouse model of pancreatic ductal adenocarcinoma 23 . Stimulation of DCs in a tumor by the agonist combinations presented here is likely and may contribute to a therapeutic effect. However, because macrophages are by far more abundant than DCs, the concept of the present paper is to exploit macrophage activation to enhance the anti-tumor immune response. Our experiments demonstrated that T cells could be activated by macrophages in the absence of DCs. In one experiment we used peptides from SARS-CoV-2 spike protein as alternative model antigens (Fig. 5 D). We hypothesized that due to the pandemic and related vaccinations most donors would be reactive to specific stimulation. Indeed, 5 out of 6 donors responded to stimulation with a large peptide pool derived from spike protein. However, we observed a very high donor variability, potentially caused by the individual patient history of vaccinations and infections. Therefore, like for CMV or flu peptide stimulation, pre-testing of donors may be necessary for use in such experiments. In summary, we have identified and characterized agonist combinations that stimulated macrophages to reactivate cytotoxic T cells and could overcome immunosuppression. These investigations will serve as a basis to develop multispecific antibody constructs for tumor-specific activation of macrophages that may represent a promising approach to enhance the anti-tumor immune response and increase survival of patients with macrophage-rich solid tumors. Declarations Acknowledgements The authors would like to thank Madlen Hahn, Daniel Hösch and Janine Schiele for expert technical assistance. Disclosure Statement The authors are employees of Boehringer Ingelheim. Data Availability Statement The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. 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A Bispecific Molecule Targeting CD40 and Tumor Antigen Mesothelin Enhances Tumor-Specific Immunity. Cancer Immunol Res 7 , 1864–1875 (2019). Luke, J. J. et al. Phase I study of ABBV-428, a mesothelin-CD40 bispecific, in patients with advanced solid tumors. J. Immunother. Cancer 9 , e002015 (2021). Melero, I. et al. 161P Fibroblast activation protein (FAP)-CD40 (RO7300490) mediates intratumoral DC maturation and modulation of the tumor microenvironment. Annals of Oncology 35 , S279–S280 (2024). Melero, I. et al. 617 A Phase I study of a tumor-targeted, fibroblast activation protein (FAP)-CD40 agonist (RO7300490) in patients with advanced solid tumors. J Immunother Cancer 11 , A703 (2023). Metz, H. et al. SBT6050, a HER2-directed TLR8 therapeutic, as a systemically administered, tumor-targeted human myeloid cell agonist. JCO 38 , 3110–3110 (2020). Klempner, S. J. et al. 209P Interim results of a phase I/Ib study of SBT6050 monotherapy and pembrolizumab combination in patients with advanced HER2-expressing or amplified solid tumors. Annals of Oncology 32 , S450 (2021). Salomon, R. et al. Bispecific antibodies increase the therapeutic window of CD40 agonists through selective dendritic cell targeting. Nat. Cancer 3 , 287–302 (2022). Hoves, S. et al. Rapid activation of tumor-associated macrophages boosts preexisting tumor immunity. J. Exp. Med. 215 , 859–876 (2018). Wattenberg, M. M. et al. Cancer immunotherapy via synergistic coactivation of myeloid receptors CD40 and Dectin-1. Sci. Immunol. 8 , eadj5097 (2023). Tosiek, M. J. et al. Activation of the Innate Immune Checkpoint CLEC5A on Myeloid Cells in the Absence of Danger Signals Modulates Macrophages’ Function but Does Not Trigger the Adaptive T Cell Immune Response. J. Immunol. Res. 2022 , 9926305 (2022). Combes, A. J., Samad, B. & Krummel, M. F. Defining and using immune archetypes to classify and treat cancer. Nat. Rev. Cancer 23 , 491–505 (2023). Murgaski, A. et al. Efficacy of CD40 agonists is mediated by distinct cDC subsets and subverted by suppressive macrophages. Cancer Res. 82 , 3785–3801 (2022). Mellman, I., Chen, D. S., Powles, T. & Turley, S. J. The cancer-immunity cycle: Indication, genotype, and immunotype. Immunity 56 , 2188–2205 (2023). Cheung, R. et al. Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal shock in mice. J Clin Invest 121 , 4446–4461 (2011). Clasen, S. J. et al. Silent recognition of flagellins from human gut commensal bacteria by Toll-like receptor 5. bioRxiv 2022.04.12.488020 (2022) doi:10.1101/2022.04.12.488020. YONGKE, Z., GUO-LIANG, Y. & WEIMIN, Z. ANTI-CD40 ANTIBODIES AND METHODS OF USE. (2021). Kosco-Vilbois, M., Graaf, K. L. D., Berney, T., Giovannoni, L. S. & Bosco, D. Anti-TLR4 antibodies and methods of use thereof. (2014). Elson, G. & LEGER, O. Neutralizing antibodies and methods of use thereof. (2008). BIGLER, M., G, H. P., H, P. J. & G, P. L. Anti-MDL-1 antibodies. (2012). SHIE-LIANG, H., CHI-HUEY, W., TSUI-LING, H. & SZU-TING, C. Compositions and methods for identifying response targets and treating flavivirus infection responses. (2013). JUSTIN, W. & MAXIMILIANO, V. ANTIBODIES THAT BIND CSF1R. (2011). CATHERINE, O. et al. CD131 BINDING PROTEINS AND USES THEREOF. (2017). LIN, P., YAN, W. & XIAOFEN, Y. ANTI-AXL ANTIBODIES AND METHODS OF USE. (2013). YIWEN, L., DAN, L., DAVID, S. & R, T. J. ANTI-FLT3 ANTIBODIES. (2009). CHIA-YANG, L., LI-FEN, L. & WENWU, Z. ANTAGONIST ANTI-IL-7 RECEPTOR ANTIBODIES AND METHODS. (2011). ADAM, M., XIZI, H. & BRIAN, H. TARGETING IL17 SIGNALING TO TREAT CANCER AND TO PREVENT AND TREAT ICI INDUCED IMMUNE RELATED ADVERSE EVENTS (IRAES). (2024). Additional Declarations Competing interest reported. The authors are employees of Boehringer Ingelheim. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6195356","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":441117048,"identity":"7d0d95dc-cef9-42fc-ae99-005d24b83c8c","order_by":0,"name":"Frederik H. Igney","email":"data:image/png;base64,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","orcid":"","institution":"Boehringer Ingelheim Pharma GmbH \u0026 Co. KG Discovery Research","correspondingAuthor":true,"prefix":"","firstName":"Frederik","middleName":"H.","lastName":"Igney","suffix":""},{"id":441117049,"identity":"bf6e5645-2225-42a4-9db7-eff5f0ff84b1","order_by":1,"name":"Daniela Reiss","email":"","orcid":"","institution":"Boehringer Ingelheim Pharma GmbH \u0026 Co. KG Discovery Research","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Reiss","suffix":""},{"id":441117051,"identity":"b580567c-24f2-4827-918a-113f566a09a6","order_by":2,"name":"John E. Park","email":"","orcid":"","institution":"Boehringer Ingelheim Pharma GmbH \u0026 Co. KG Discovery Research","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"E.","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2025-03-10 12:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6195356/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6195356/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80710160,"identity":"a7060761-d742-4e0b-9ef5-7dd87eb54f68","added_by":"auto","created_at":"2025-04-16 08:58:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":531954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD40L-stimulated macrophages activate autologous antigen-specific T cells.\u003cbr\u003e\nA\u003c/strong\u003e Influence of peptide loading and CD40L concentration on IFNγ release. Human monocyte-derived macrophages were loaded with MHC-I peptide pool (1 mg/ml) or not, activated with the indicated concentration of CD40L and cocultured with autologous T cells. IFNγ concentration in supernatant was determined after 5 d of coculture. \u003cstrong\u003eB \u003c/strong\u003eInfluence of peptide amount and T cell number on IFNγ release. Macrophages were loaded with the indicated concentrations of CEFX peptide pool, activated or not with CD40L (0.5 mg/ml) and cocultured with the indicated numbers of autologous T cells. IFNγ concentration in supernatant was determined after 5 d of coculture. \u003cstrong\u003eC\u003c/strong\u003eGranzyme B release in supernatants of the experiment in B. Each dot represents an individual donor. Bars represent mean+standard deviation.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6195356/v1/d08fa5d4a413b05ccfc9d217.png"},{"id":80712334,"identity":"75785ed0-a991-4636-b6ca-58fa95501b75","added_by":"auto","created_at":"2025-04-16 09:14:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":767029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of agonists for macrophage stimulation in autologous T cell activation assay. A\u003c/strong\u003e Plate-coated agonist combinations. Macrophages were loaded with MHC-I peptide pool, activated with the indicated plate-bound antibodies and cocultured with autologous T cells. IFNγ concentration in supernatant was determined after 5 d of coculture. CD40L was used in solution. Table shows IFNγ values that were normalized to the IFNγ level of CD40L stimulation (red: higher levels; blue: lower levels). Mean of 2 donors. \u003cstrong\u003eB\u003c/strong\u003e Soluble agonist combinations. As in A, but macrophages were loaded with CEFX peptide pool, and activated with the indicated agonists in solution. Mean of 3-5 donors. \u003cstrong\u003eC\u003c/strong\u003eCombinations of soluble and coated agonists. As in B, but macrophages were activated with the indicated agonists in solution or plate-bound antibodies. Mean of 3 donors.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6195356/v1/a2a1f26fa97c5dc58128e6b4.png"},{"id":80710162,"identity":"0c388c03-2209-44b7-b67c-1a22514eb826","added_by":"auto","created_at":"2025-04-16 08:58:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":719636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConfirmation and characterization of most effective agonist combinations.\u003cbr\u003e\nA\u003c/strong\u003e Confirmation of CD40L + anti-Clec5a combination. Macrophages were loaded with MHC-I peptide pool or not, activated with the indicated combinations of soluble CD40L and plate-bound anti-Clec5a, and cocultured with autologous T cells. IFNγ concentration in supernatant was determined after 5 d of coculture. IFNγ values were normalized to the IFNγ level of CD40L stimulation in presence of MHC-I peptides. \u003cstrong\u003eB\u003c/strong\u003e Confirmation of CD40L and resiquimod combinations. As in A, but macrophages were loaded with CEFX peptide pool, and activated with the indicated agonists in solution or coated to plate. \u003cstrong\u003eC\u003c/strong\u003e Re-activation of CMV- or flu-reactive T cells. As in A, but donors pre-tested for their reactivity to CMV or flu peptide stimulation were used, and macrophages were loaded with CMV or flu peptide, respectively. \u003cstrong\u003eD\u003c/strong\u003eStimulation of M0 vs M2a macrophages. Macrophages were polarized to M0 or M2a, loaded with CEFX peptide pool, activated with the indicated agonist combinations and used for activation of autologous T cells. IFNγ concentrations in supernatant (pg/ml) are shown. Each dot represents an individual donor. Bars represent mean+standard deviation. ULOQ: upper limit of quantification.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6195356/v1/ddd7c77be1d5297e1c04cd84.png"},{"id":80710169,"identity":"0ceaca3c-c0e3-4479-9875-846d2f72ef7d","added_by":"auto","created_at":"2025-04-16 08:58:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2706048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of scFab on tumor cells as model for tumor-specific clustering of agonists.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Schematic representation of tumor cells expressing GPI-anchored scFab agonists and DNA constructs used. \u003cstrong\u003eB\u003c/strong\u003e Co-expression of scFab constructs on HCT116 cells. Exemplary flow cytometry histograms and dot plot for HCT116 that express anti-CD40 and anti-Clec5a (3E12A2). Construct 2 was used for expression of aCD40, and detection was performed by staining of HA tag; aClec5a was expressed using construct 1 and detected via FLAG tag. Filled curve: isotype control staining. Open line: Staining for HA or FLAG tag, respectively. \u003cstrong\u003eC+D\u003c/strong\u003eCD40 agonism of anti-CD40 expressing tumor cell lines. HEK-Blue CD40 reporter cells were incubated with different numbers of HCT116 cells expressing anti-CD40 and/or anti-Clec5a constructs. CD40 agonism was determined via the inducible SEAP reporter activity. CD40L in solution (0.5 mg/ml) was used as control. Wt: wild-type. Mean+SD of triplicates. \u003cstrong\u003eE\u003c/strong\u003eClec5a agonism of anti-CD40 expressing tumor cell lines. PMA-differentiated U937 cells were incubated with different numbers of HCT116 cells expressing anti-CD40 and/or anti-Clec5a constructs. After 24 h CCL3 concentrations in supernatants were determined. Plate-coated anti-Clec5a or isotype control was used as control. Mean+SD of triplicates.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6195356/v1/0b9ad8e6a905425ac31351f6.png"},{"id":80710163,"identity":"d56fb88f-daf2-4efa-b1ab-dc11d87dc9da","added_by":"auto","created_at":"2025-04-16 08:58:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":731090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStimulation of macrophages by tumor-anchored agonists leads to T cell activation. A\u003c/strong\u003e Test of agonists expressed on the cell surface of HCT116 cells for stimulation of macrophages in the autologous T cell activation assay. Macrophages were loaded with CEFX peptide pool and incubated in a triple coculture with autologous T cells and HCT116 cells expressing scFabs to the indicated receptors. IFNγ concentration in supernatant was determined after 3 d of coculture. CD40L in solution in absence of tumor cells was used as control. IFNγ values were normalized to the IFNγ level of CD40L stimulation. \u003cstrong\u003eB\u003c/strong\u003e Test of tumor-anchored anti-CD40 and/or anti-Clec5a. As in A, but using HCT116 cells expressing anti-CD40 and/or anti-Clec5a. IFNγ values were normalized to the IFNγ level of stimulation with HCT116 expressing anti-CD40 and isotype control. \u003cstrong\u003eC\u003c/strong\u003e Granzyme B release in supernatants of the experiment in B. \u003cstrong\u003eD \u003c/strong\u003eSARS-CoV-2 specific activation. Macrophages were loaded with peptides derived from SARS-CoV-2 spike protein and used in a triple coculture with autologous T cells and HCT116 cells expressing scFabs to the indicated receptors. Each dot represents an individual donor. Bars represent mean+standard deviation.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6195356/v1/806a9705d7d49090ed0f2d76.png"},{"id":80710168,"identity":"bd701bb2-5493-437e-bb9e-84f4a669bf8a","added_by":"auto","created_at":"2025-04-16 08:58:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1000810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAgonist combinations can induce inflammatory chemokines and cytokines and repolarize M2a macrophages. A\u003c/strong\u003e Induction of chemokines and cytokines. Macrophages were stimulated either with the indicated agonists or cocultured with agonist-expressing HCT116 tumor cells for 24 h. Table shows concentrations of cytokines and chemokines in the supernatants (pg/ml). Dark blue: lowest level; red: highest levels of the respective cytokine/chemokine; empty: not analysed. Mean of three donors. \u003cstrong\u003eB\u003c/strong\u003e Repolarization of M2a macrophages. M2a-polarized macrophages were stimulated with the indicated agonists. Expression of markers for macrophage polarization were analysed by conventional multicolor flow cytometry. Table shows geometric mean fluorescence intensity (MFI), Red: higher levels; blue: lower levels than control. Mean of three donors.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6195356/v1/a38833835f32d08b46af893e.png"},{"id":80710167,"identity":"097d4df0-a8d0-49dd-b57f-9a0f344767bf","added_by":"auto","created_at":"2025-04-16 08:58:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1267581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT cells that have been activated by macrophages are cytotoxic towards tumor cells. A\u003c/strong\u003e Schematic overview of assay. Only HLA-A2-positive donors that were reactive to CMV or flu peptide stimulation were used. Macrophages were loaded with CMV or flu peptide, respectively, activated with combinations of plate-bound antibodies or soluble agonists, and cocultured with autologous T cells. After 5 d, T cells were separated from these cocultures and incubated with peptide-loaded or peptide-expressing HCT116 tumor cells as target cells. After 48 h, the number of living tumor cells was determined and normalized to a control sample with tumor cells only. Triton was used as control for maximal lysis. \u003cstrong\u003eB\u003c/strong\u003e Summary of killing assay results (N: number of donors). \u003cstrong\u003eC, D\u003c/strong\u003e Results from killing assays. Two independent sets of experiments are shown (C, D). Each dot represents an individual donor. Bars represent mean±standard deviation. No peptide: Macrophages were not loaded with peptide and not stimulated. No stim.: Macrophages were loaded with peptide, but not stimulated.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-6195356/v1/13a30a7bdd634c2a72ac6064.png"},{"id":80711285,"identity":"0886020a-1730-42f9-bb09-3302ae8ed252","added_by":"auto","created_at":"2025-04-16 09:06:57","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":541971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStimulation of macrophages can overcome immunosuppression by TGFb. \u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003eA\u003c/strong\u003e Suppression of IFNg release by TGFb. Macrophages were loaded with CEFX peptide pool and stimulated by adding CD40L or HCT116-CD40 cells or controls. Coculture with autologous T cells was performed in the presence of the indication concentrations of TGFb. IFNg concentration in supernatant was determined after 3 d of coculture. \u003cstrong\u003eB\u003c/strong\u003eOvercoming TGFb-mediated immunosuppression by agonists. As in A, but cocultures were performed in absence or presence of TGFb (50 pg/ml) and macrophages stimulated with the indicated agonists. \u003cstrong\u003eC\u003c/strong\u003eOvercoming immunosuppression by tumor-anchored agonists. As in B, but macrophages were stimulated by addition of agonist-expressing HCT116. TLR7/8 stimulation was achieved by adding resiquimod in solution. Each dot represents an individual donor. Bars represent mean±standard deviation.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-6195356/v1/e5af770311c6c81ae67efad0.png"},{"id":80710161,"identity":"bd33d927-f8c5-4de5-8cf1-83e8a174aaaa","added_by":"auto","created_at":"2025-04-16 08:58:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25392,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6195356/v1/9e325e6949f969f4095f2ddc.docx"}],"financialInterests":"Competing interest reported. The authors are employees of Boehringer Ingelheim.","formattedTitle":"Identification of agonist combinations that stimulate macrophages to induce anti-tumor T cells and overcome immunosuppression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTumor-infiltrating macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) represent the major immune population in most human solid cancers and can reach up to 50% of tumor mass \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. High numbers of these cells are associated with poor prognosis and response to therapy in many tumor types \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, different strategies to therapeutically target this abundant myeloid population are under investigation with the aim to induce immune-mediated destruction of tumors or to restore sensitivity to immune-checkpoint therapy. An early proof of this concept was achieved by the clinical success of the Toll-like receptor (TLR) 4 agonist Bacillus Calmette\u0026thinsp;\u0026minus;\u0026thinsp;Guerin (BCG), which is an approved therapy for bladder cancer \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Similarly, imiquimod is a TLR7 agonist that has been approved for the topical treatment of basal cell carcinoma and genital warts \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. TLRs are pattern recognition receptors that activate innate immune cells, such as macrophages and dendritic cells, and induce inflammatory responses.\u003c/p\u003e \u003cp\u003eColony-stimulating factor 1 receptor (CSF1R) is a key regulator of macrophage survival and differentiation. CSF1R inhibitors have reached late-stage development in a variety of solid tumor types, but several have recently been discontinued due to disappointing efficacy \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The small molecule CSF1R inhibitor pexidartinib (PLX3397) has been approved for the treatment of tenosynovial giant cell tumors, a rare proliferative tumor caused by an overproduction of colony-stimulating factor 1. CD40 is a cell surface receptor that is mainly expressed on macrophages, dendritic cells, B cells and other non-immune cells \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Upon clustering by the ligand CD40L (CD154), antigen-presenting cells secrete proinflammatory cytokines and up-regulate costimulatory molecules such as CD80 and CD86 that are required for costimulation of T cells. Agonistic anti-CD40 antibodies have shown promising preclinical results and initial encouraging clinical data \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, only few myeloid-cell targeting concepts have made it into clinical practice for cancer therapy. Many of these agonists suffer from lack of convincing efficacy or from high systemic toxicity. Toxicity can also restrict the use of agonists to suboptimal doses that result in insufficient immune activation and anti-tumor efficacy.\u003c/p\u003e \u003cp\u003eAn approach to overcome systemic toxicity is to specifically target an agonist to the tumor. Intratumoral administration of an anti-CD40 antibody into superficial lesions was well tolerated at clinically relevant doses and associated with pharmacodynamic responses \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, this route of administration is limited to accessible lesions and a low frequency of injections. To reduce the likelihood of systemic toxicities, bispecific molecules have been developed that target both an activating receptor and a tumor antigen to achieve maximal activity in the presence of the tumor antigen and minimal activity in normal tissues with little antigen expression. A bispecific tumor-targeted anti-CD40 agonistic antibody, which simultaneously bound to CD40 and the tumor antigen carcinoembryonic antigen (CEA), enabled potent in vitro dendritic cell (DC) activation and consecutive T cell cross-priming in a CEA-specific manner \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The bispecific molecule ABBV-428 that targeted CD40 and mesothelin demonstrated enhanced activation of antigen-presenting cells and T cells upon binding to cell-surface mesothelin, and inhibition of tumor cell growth. In a phase I study, ABBV-428 monotherapy displayed a good tolerability profile, but had only minimal clinical activity \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The bispecific fibroblast activation protein (FAP)-targeted CD40 agonist RO7300490 was well tolerated up to the highest dose tested in a phase I study. It achieved a strong and sustained target engagement and immunomodulation in tumor tissue, but no objective responses \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. SBT6050 is a therapeutic comprised of a TLR8 agonist payload conjugated to a HER2-directed monoclonal antibody. It demonstrated single agent efficacy in multiple mouse tumor models without peripheral cytokine production. Preliminary phase I data indicated a manageable safety profile and evidence of myeloid, NK and T cell activation \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. To reduce CD40-related toxicities, also bispecific antibodies have been studied that targeted CD40 activation preferentially to dendritic cells, by coupling the CD40 agonist arm with a CLEC9A-targeting arm. This bispecific reagent demonstrated a superior safety profile compared to the parental anti-CD40 monospecific antibody while triggering potent antitumor activity in mice \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo increase anti-tumor efficacy and maximize the effects of myeloid cell-targeted therapies, combinations of anti-CD40 with other myeloid agonists have been studied in mouse models. Inhibition of CSF1R signaling sensitized TAMs to rapid reprogramming in the presence of a CD40 agonist before their depletion. Combination of anti-CSF1R and anti-CD40 was sufficient to create a proinflammatory tumor milieu that reinvigorated an effective T cell response in transplanted tumors \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In a recent study, combinations of anti-CD40 with various pattern-recognition receptor agonists were tested in a mouse model of pancreatic ductal adenocarcinoma \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Systemic administration of a TLR7/8, STING, NOD2, or Dectin-1 agonist in combination with agonistic anti-CD40 led to high rates of tumor cures and triggered immunological memory. This study demonstrated that cancer immune surveillance in pancreatic tumors that were resistant to checkpoint inhibition could be invoked by coactivation of complementary myeloid signaling pathways.\u003c/p\u003e \u003cp\u003eWe hypothesized that combining coactivation of two receptors with tumor targeting could overcome both challenges of systemic toxicity and low efficacy. Here, we aimed to identify the best combinations of activating receptors on macrophages to convert an immunosuppressive into a proinflammatory tumor microenvironment. We screened a range of agonists on primary human macrophages. Stimulated macrophages were co-cultured with autologous T cells to test their ability to activate antigen-specific T cells. The best combinations were able to overcome TGF-b-mediated immunosuppression in vitro and induced T cells that were cytotoxic towards tumor cells. These investigations will serve as a basis to develop multispecific antibody constructs for tumor-specific activation of macrophages.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eHuman primary cell isolation, macrophage differentiation and polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBuffy coats or leukopaks of healthy blood donors were received from the Institute of Clinical Transfusion Medicine, Ulm, Germany. All experiments were approved by the Ethics Committee of Landes\u0026auml;rztekammer Baden-W\u0026uuml;rttemberg, and informed consent was obtained from all subjects. All methods were carried out in accordance with relevant guidelines and regulations. Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll gradient centrifugation. Monocytes were isolated by positive selection using CD14 MicroBeads (Miltenyi) according to the manufacturer\u0026apos;s protocol. Remaining CD14-negative cells were cryo-preserved for later isolation of autologous T cells. Monocytes were differentiated to macrophages in RPMI-1640 (Gibco, ATCC modifications) with 5 % human serum (Sigma), 1 % Penicillin-Streptomycin (Sigma) and 50 ng/ml M-CSF (BioTechne) in UpCell plates (Thermo Scientific) for 5-7 d at 37 \u0026deg;C and\u0026nbsp;5% CO\u003csub\u003e2\u003c/sub\u003e in an incubator. If not indicated otherwise, these macrophages were used for all assays (M0 macrophages). For polarization to M2a macrophages, human IL-4 and IL-13 (20 ng/ml each; R\u0026amp;D Systems) were added for another 24 h. Autologous T cells were isolated from the CD14-negative fraction of PBMCs using the human Pan T Cell Isolation Kit (Miltenyi).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMacrophage stimulation and autologous T cell activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman monocyte-derived macrophages were removed from UpCell plates by temperature shift and loaded with peptides (1 \u0026micro;g/ml) for 1 h at 37 \u0026deg;C in cell repellent plates (Greiner-bio-one). The following peptide mixes were used (all from JPT Peptide Technologies): CEFX (CEFX Ultra SuperStim Pool), MHC-I (CEFX Ultra SuperStim Pool MHC-I Subset), CMV (Antigen Peptide CMV pp65 - HLA-A*0201 (NLVPMVATV)), Flu (Influenza A MP (58-66) Peptide (GILGFVFTL)), or SARS (PepMix SARS-CoV-2 (Spike Glycoprotein)). Autologous T cells were isolated from the CD14-negative PBMC fraction of the same donor. 2x10\u003csup\u003e4\u003c/sup\u003e macrophages and 10\u003csup\u003e5\u003c/sup\u003e T cells were cocultured in 96-well plates. For stimulation of macrophages, soluble agonists were added to the cocultures (\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e); agonistic antibodies (\u003cstrong\u003eSupplementary Table 2\u003c/strong\u003e) were coated (10\u0026nbsp;mg/ml) to high-binding plates (Costar) before addition of cells. Supernatants of cocultures were harvested after 5 d for analysis of IFNg\u0026nbsp;or granzyme B by ELISA (R\u0026amp;D Systems). For stimulation with tumor-anchored agonists, 2x10\u003csup\u003e3\u003c/sup\u003e scFab-expressing HCT116 cells were added to the coculture, and supernatants were analysed after 3 d. Each condition was performed in triplicates. For normalization, the IFNg concentration of a specific sample was divided by the concentration of the indicated control (usually sample with CD40L stimulation). To investigate the effect of TGFb, the indicated concentrations of TGFb1 (R\u0026amp;D Systems) were added to the T cell:macrophage coculture.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of scFab-expressing HCT116 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFab sequences from antibodies (\u003cstrong\u003eSupplementary Table 3\u003c/strong\u003e) were cloned into pOptiVEC as single chains with a GS linker, followed by a GPI anchor sequence. To enable co-expression, two sets of vectors were generated: one set contained sequences for V5 and FLAG tag in the linker sequence and a hygromycin resistance gene; the other set contained sequences for HA and Myc tag in the linker sequence and a puromycin resistance gene. DNA was synthesized at GeneArt (Thermo Fisher Scientific). HCT116 cells (ATCC) were transfected using Neon Transfection System (Invitrogen) and kept under the respective selection pressure to generate stable cell lines. For co-expression, a vector of the other set was co-transfected into a stable cell line and the second selection pressure added. Expression was analysed by conventional flow cytometry with antibodies against HA (clone 16B12, BV421, Biolegend), and/or Flag tag (clone L5, Alexa-647, Biolegend).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD40 and Clec5a activity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor determining CD40 activity, 5x10\u003csup\u003e4\u003c/sup\u003e HEK-Blue CD40L cells (Invivogen) were seeded in DMEM with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 \u0026micro;g/ml streptomycin. The indicated number of scFab-expressing HCT116 cells were added and incubated for 24 h. CD40L (0.5 mg/ml) was used as positive control. Supernatants were harvested, and QuantiBlue solution (Invivogen) was added. SEAP reporter activity was measured at OD635. To determine Clec5a agonistic activity, U937 cells were differentiated with 10 nM phorbol 12-myristate 13-acetate (PMA; Sigma) for 3 d in 100 mm UpCell plates. 10\u003csup\u003e5\u003c/sup\u003e cells were seeded in 96-well plates (Costar) in medium without PMA, and indicated numbers of HCT116 cells expressing anti-Clec5a constructs were added. After 24 h CCL3 concentrations in supernatants were determined by ELISA (R\u0026amp;D Systems). Plate-coated anti-Clec5a (clone 283834) or isotype control was used as control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMacrophage chemokine/cytokine secretion and repolarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine chemokine and cytokine secretion, macrophages were stimulated either with the plate-bound and/or soluble agonists or cocultured with agonist-expressing HCT116 tumor cells for 24 h. Supernatants were harvested and cytokines and chemokines determined by LegendPlex Human Inflammation Panel 1 and LegendPlex Human Proinflammatory Chemokine Panel (Biolegend). Each condition for each donor was performed in triplicates. To determine repolarization, M2a-polarized macrophages were stimulated with the indicated plate-bound and/or soluble agonists for 48 h. Expression of MHC-II, CD80, CD83, CD163 and CD206 on live cells was analysed by multicolor conventional flow cytometry (all antibodies from BD Biosciences).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT cell cytotoxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePBMCs were tested for HLA-A2 expression and for their reactivity to CMV or Flu peptide stimulation. Only HLA-A2-positive donors that were reactive to CMV or flu peptide stimulation were used. Monocyte-derived macrophages were loaded with the respective peptide, activated with combinations of plate-bound antibodies or soluble agonists, and cocultured with autologous T cells as above. After 5 d, T cells were separated from these cocultures by gently aspirating the T cells in suspension from the adherent macrophages. Replicates were combined to obtain sufficient numbers of T cells as effector cells. As target cells, HCT116 cells stably expressing Renilla luciferase were loaded with the respective peptide. Alternatively, HCT116 cells were used that stably expressed a fusion peptide consisting of the CMV pp65 peptide and the M1 domain of influenza virus. 10\u003csup\u003e5\u003c/sup\u003e T cells were incubated with 10\u003csup\u003e4\u003c/sup\u003e target cells for 48 h (effector:target ratio 10:1). The number of living tumor cells was determined either by Renilla-Glo Luciferase Assay (Promega) or CellTiter-Glo Luminescent Cell Viability Assay (Promega). The signal of T cells in CellTiter-Glo assay was neglectable compared to tumor cell signal at the effector:target ratio used. Values were normalized to a control sample with target cells only. A final concentration of 0.5 % Triton X-100 was added 1 h before readout as control for maximal lysis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCD40L-stimulated macrophages activate autologous antigen-specific T cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe hypothesized that modulation of macrophages in tumors could influence the function of cytotoxic T cells. Therefore, we tested whether human macrophages could directly reactivate antigen-experienced T cells. As model system, we used human monocyte-derived macrophages, and loaded them with a pool of peptides that were derived from common infectious agents and that were known to bind to MHC-I of a broad range of HLA subtypes. Macrophages were cocultured with autologous T cells, and T cell reactivation was quantified by measuring the release of IFNγ\u0026nbsp;(\u003cstrong\u003eFigure 1A\u003c/strong\u003e). In the absence of peptides, IFNγ\u0026nbsp;release was minimal. Peptide-loaded macrophages induced IFNγ\u0026nbsp;release, indicating the reactivation of pre-existing T cells with specificity to the peptide pool of infectious agents. As CD40L is known to stimulate macrophages, we added recombinant CD40L to the cocultures. CD40L stimulation increased IFNγ\u0026nbsp;release, confirming that macrophage modulation could influence T cell reactivation. Donor variability was high, and overall levels of IFNγ\u0026nbsp;were low. Therefore, we switched to a related peptide pool containing MHC-I and MHC-II binding peptides, which resulted in a better assay window (\u003cstrong\u003eFigure 1B\u003c/strong\u003e). Measurement of granzyme B release confirmed the activation of cytotoxic T cells (\u003cstrong\u003eFigure 1C\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStimulation of macrophages by agonist combinations can enhance T cell activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the most effective stimulation of macrophages, we selected a range of receptors from literature. Criteria for selection were expression on macrophages in tumor tissue, an activating function and availability of putative tool agonists (\u003cstrong\u003eSupplementary Table 1\u0026amp;2\u003c/strong\u003e). First, we tested anti-receptor antibodies coated to high-binding plates for cross-linking as single agonists or in combination (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). Peptide-loaded macrophages were cocultured with autologous T cells on these plates. CD40L in solution was used as control. Due to the high donor variability, values were normalized to CD40L stimulation for each donor. Several combinations of antibodies with CD40L induced higher IFNγ\u0026nbsp;release than CD40L alone. Next, we screened a range of putative soluble agonists (\u003cstrong\u003eFigure 2B\u003c/strong\u003e). The TLR7/8 agonist resiquimod was more efficacious than CD40L as single agent, and strongly induced IFNγ\u0026nbsp;when used in combinations. Other effective combinations included CD40L, IL-7, IL-17A and others. As some of the receptors, such as IL-7RA or IL-17RA, are not only expressed on macrophages, but also on T cells, a direct effect of agonists on T cells could not be excluded at this stage. Finally, we also tested combinations of plate-coated antibodies and soluble agonists that had shown some activity in the previous experiments (\u003cstrong\u003eFigure 2C\u003c/strong\u003e). Mainly combinations with anti-TLR4 showed enhanced activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfirmation and additional characterization of most effective agonist combinations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom the screens described above, we selected the most promising agonist combinations for further investigations, mainly based on strength and robustness of response. The majority of hits could be confirmed with additional donors (\u003cstrong\u003eFigure 3A\u0026amp;B\u003c/strong\u003e). With dual stimulation, T cell reactivation remained antigen-dependent, as there was no activation in absence of peptides (\u003cstrong\u003eFigure 3A\u003c/strong\u003e). To further confirm antigen-specific activation, we also analysed the response to a single antigenic peptide. As model antigen, we chose the pp65 antigen from cytomegalovirus (CMV) due to its relatively high prevalence in blood donors, and influenza A matrix protein due to wide-spread influenza vaccination. Donors were pre-tested for HLA-A2 expression and their reactivity to a specific peptide from cytomegalovirus (CMV) or influenza virus (flu). Macrophages from positive donors were loaded with the respective peptide, stimulated with the most promising agonist combinations and used for T cell activation (\u003cstrong\u003eFigure 3C\u003c/strong\u003e). IFNγ responses showed a similar pattern as in previous experiments. Expectedly, the strength of response to these antigens was different as compared to the previously used peptide mix. In tumors, macrophages are more polarized towards an M2 phenotype \u003csup\u003e3\u003c/sup\u003e. Therefore, we compared the ability of M0 and M2a macrophages to activate T cells (\u003cstrong\u003eFigure 3D\u003c/strong\u003e). For the agonists tested, stimulation of M2a macrophages enhanced T cell activation in a similar way as M0 macrophages, indicating that the concept could also work for TAMs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of scFab on tumor cells as model for tumor-specific clustering of agonists.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we investigated whether the concept also worked when agonists were clustered on the cell surface of tumor cells. To circumvent any additional complexity with the characteristics of a specific tumor antigen and respective binders in a multi-specific construct, we expressed scFab fragments directly on the cell surface of the human tumor cell line HCT116 using a glycosylphosphatidylinositol (GPI) anchor. Fab constructs were derived from the sequences of published antibodies (\u003cstrong\u003eSupplementary Table 3\u003c/strong\u003e). To enable co-expression of two different scFab constructs on the same cell, we generated two sets of vectors containing two different selection markers and four different tags (\u003cstrong\u003eFigure 4A\u003c/strong\u003e). Expression was determined by flow cytometry staining of the respective tags (\u003cstrong\u003eFigure 4B\u003c/strong\u003e). Due to the use of standardized constructs, IRES (internal ribosome entry site)-related correlation of Fab expression and selection marker as well as strong selection pressure, expression levels of different scFabs were comparable.\u003c/p\u003e\n\u003cp\u003eTo demonstrate functional activity of tumor-anchored scFabs, we performed a CD40 reporter gene assay. Tumor cells expressing anti-CD40 induced a strong reporter signal in a similar way as the positive control CD40L. Tumor cells that did not express anti-CD40 did not induce any reporter signal (\u003cstrong\u003eFigure 4C\u003c/strong\u003e). Titration of tumor cells expressing anti-CD40 and a second construct confirmed comparable activity of different transfected tumor cells (\u003cstrong\u003eFigure 4D\u003c/strong\u003e). We also tested agonistic activity of two anti-Clec5a scFabs. It has been shown that stimulation of differentiated U937 cells with a monoclonal anti-Clec5a antibody induced CCL3 release \u003csup\u003e24\u003c/sup\u003e. Therefore, we cocultured scFab-expressing HCT116 with differentiated U937 and confirmed that both anti-Clec5a scFabs were agonistic, whereas anti-CD40 alone did not induce CCL3 in this assay (\u003cstrong\u003eFigure 4E\u003c/strong\u003e). We did not directly test the function of other scFabs apart from anti-CD40 or anti-Clec5a, due to the wide range of receptors and pathways involved.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStimulation of macrophages by tumor-anchored scFab leads to T cell activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo mimic tumor-specific clustering of agonists, we stimulated macrophages with HCT116 cell lines that expressed combinations of tumor-anchored anti-receptor scFabs. These macrophages were used to activate autologous antigen-specific T cells in triple cocultures (\u003cstrong\u003eFigure 5A\u0026amp;B\u003c/strong\u003e). Tumor-anchored anti-CD40 induced T cell reactivation as determined by IFNγ\u0026nbsp;release. Similar to the experiments with plate-bound agonists above, several combinations induced higher IFNγ\u0026nbsp;release than CD40-stimulation alone. Mainly combinations with anti-CD40 were effective. For some combinations (CD40+Clec5a), we also measured granzyme B release, which was in line with IFNγ\u0026nbsp;release, confirming the activation of cytotoxic T cells (\u003cstrong\u003eFigure 5C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe also tested reactivation of T cells to an unrelated model antigen, namely the spike glycoprotein from SARS-CoV-2 (\u003cstrong\u003eFigure 5D\u003c/strong\u003e). We hypothesized that due to the pandemic and related vaccinations most donors would be reactive to specific stimulation, so that pre-testing of donors would be unnecessary. However, we observed a very high donor variability. From 6 donors used, we excluded one with no response and one with an extreme response even in absence of additional stimulation. Similar to experiments above, anti-CD40 and anti-TLR4 induced IFNγ release, and several combinations with anti-CD40 resulted in an enhanced response.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAgonist combinations can induce inflammatory chemokines and cytokines and repolarize M2a macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo characterize the direct effect of stimulation on macrophages, we stimulated macrophages in the absence of T cells with the most promising agonists combinations. After stimulation either with plate-bound and/or soluble agonists or by coculture with scFab-expressing tumor cells, the secretion of a panel of inflammatory cytokines and chemokines was analysed (\u003cstrong\u003eFigure 6A\u003c/strong\u003e). CD40 stimulation alone resulted only in modest changes in cytokine/chemokine secretion. In general, strong increases were observed with combinations that were also effective in the T cell activation assays, such as CD40+TLR4, CD40+TLR7/8, CD40+Clec5a, and others.\u003c/p\u003e\n\u003cp\u003eBecause macrophages in tumors are polarized towards an M2 phenotype, we also investigated whether stimulation could repolarize M2a macrophages to a more proinflammatory M1 phenotype. After stimulation with plate-bound and/or soluble agonists, we analysed classical M1/M2 markers by flow cytometry (\u003cstrong\u003eFigure 6B\u003c/strong\u003e). While CD40 stimulation increased MHC-II expression, this effect was not observed with most of the other stimuli or combinations. TLR7/8 stimulation as well as several combinations enhanced the level of costimulatory molecules (CD80, CD83). Changes in the expression of M2 markers (CD163, CD206) were less pronounced.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT cells activated by macrophages are cytotoxic towards tumor cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo demonstrate that appropriate stimulation of macrophages could enable T cell-mediated killing of tumor cells, we analysed the cytotoxicity of activated T cells. Macrophages were loaded with CMV or flu peptide, activated with combinations of plate-bound antibodies or soluble agonists, and cocultured with autologous T cells to induce activation. T cells were separated from these cocultures and their cytotoxicity towards peptide-presenting HCT116 cells was determined (\u003cstrong\u003eFigure 7A\u003c/strong\u003e). Strongest cytotoxicity was observed after stimulation of TLR7/8, TLR4+TLR7/8, and several CD40 combinations (\u003cstrong\u003eFigure 7B\u003c/strong\u003e). Cytotoxicity of activated T cells was dependent on peptide-loading and stimulation of macrophages (\u003cstrong\u003eFigure 7C\u0026amp;D\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStimulation of macrophages can overcome immunosuppression by TGF\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tumor microenvironment, especially in tumors that are refractory to checkpoint inhibitor treatment, is characterized by immunosuppression \u003csup\u003e25\u003c/sup\u003e. One major contributing factor is TGFβ. Therefore, we tested the effect of TGFβ\u0026nbsp;on activation of T cells by CD40-stimulated macrophages (\u003cstrong\u003eFigure 8A\u003c/strong\u003e). IFNγ\u0026nbsp;release was almost completely reduced to baseline by 50 pg/ml TGFβ\u0026nbsp;and completely blocked by higher concentrations. To investigate whether appropriate stimulation of macrophages could overcome TGFβ\u0026nbsp;immunosuppression in vitro, we stimulated macrophages with different agonist combinations and used them for T cell stimulation in the absence or presence of TGFβ\u0026nbsp;(\u003cstrong\u003eFigure 8B\u0026amp;C\u003c/strong\u003e). Although IFNγ release was reduced by TGFβ for all stimulation conditions, IFNγ secretion could be detected even in the presence of TGFβ. With the best agonist combinations, IFNγ levels were reached that were higher than with CD40-stimulation in the absence of TGFβ.\u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003eIn the tumor microenvironment, TAMs represent the largest proportion of immune cells. Therefore, we aimed to identify the best combinations of receptor agonists to activate macrophages in such a way that they promote T cell-mediated killing and immune-mediated destruction of tumor cells. We screened a range of putative agonists and characterized the most effective combinations for their ability to induce cytokine and chemokine release, repolarization, T cell activation, and cytotoxicity. Regarding all assays performed, the combinations that were most effective and fitted best to the concept of tumor-specific macrophage stimulation were: CD40\u0026thinsp;+\u0026thinsp;TLR4, CD40\u0026thinsp;+\u0026thinsp;TLR7/8, CD40\u0026thinsp;+\u0026thinsp;Clec5a, CD40\u0026thinsp;+\u0026thinsp;CSF1R, CD40\u0026thinsp;+\u0026thinsp;CSF2RB and TLR4\u0026thinsp;+\u0026thinsp;TLR7/8. Although combinations with IL-7 or IL-17a showed strong effects in the initial screens, we did not follow them up, because their receptors are not only expressed on macrophages, but also on T cells. Therefore, a direct effect of agonists on T cells could not be excluded, so that other concepts or constructs may be required.\u003c/p\u003e \u003cp\u003eSo far, efficacy of myeloid-targeted therapy in the clinic was rather limited, and treatment responses were potentially determined by patient-specific microenvironmental regulators, as has been shown for anti-CD40 \u003csup\u003e26\u003c/sup\u003e. In our experiments, dual stimulation was more effective than single agonists, and T cell activation was also achieved in donors with low response to CD40L only. Importantly, dual stimulation was also able to overcome immunosuppression by TGFb (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u003cb\u003e\u0026amp;C\u003c/b\u003e). TGFb completely suppressed T cell activation by macrophages that were stimulated via CD40 only. However, stimulation of macrophages with combinations, such as CD40\u0026thinsp;+\u0026thinsp;TLR7/8, CD40\u0026thinsp;+\u0026thinsp;TLR4, or TLR4\u0026thinsp;+\u0026thinsp;TLR7/8, resulted in stronger activation of T cells than with CD40-stimulation in the absence of TGFb. These data suggest that appropriate macrophage stimulation may restore anti-tumor T cell responses and sensitivity to checkpoint inhibitors in tumors that are characterized by an immunosuppressive microenvironment with high macrophage infiltration and high TGFb levels. While TGFb is a major factor, other molecules, such as prostaglandin E2, can also contribute to immunosuppression in the tumor microenvironment \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Therefore, further experiments are needed to demonstrate the value of the approach for patients, e.g. using dissociated patient samples ex vivo.\u003c/p\u003e \u003cp\u003eSystemic stimulation of activating myeloid receptors can lead to severe toxicity \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Therefore, we hypothesized that combining coactivation of two receptors with tumor targeting could result in stronger efficacy and a better safety profile. To mimic tumor-specific clustering of agonists, we expressed scFab fragments directly on the cell surface of a human tumor cell line. Stimulation of macrophages by these tumor-anchored scFabs also led to T cell activation, demonstrating general feasibility of this concept (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Potential drugs could be envisaged as trispecific constructs: In case of two agonistic antibodies, such as anti-CD40 and anti-Clec5a, the construct could be composed of a trispecific antibody with CD40- and Clec5a-binding moieties and a tumor-specific binder, such as anti-FAP. The exact construct geometry and stoichiometry remains to be determined. In case of an agonistic antibody and a soluble small-molecule agonist, such as anti-CD40 and resiquimod, the drug construct may be a bispecific antibody-drug conjugate with a CD40 and a tumor-specific binder and a linked small molecule agonist. Proteinaceous agonists, such as flagellin, offer additional opportunities \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In addition, the characteristics of the tumor-specific antigen need to be considered, such as specificity and density of expression as well as propensity for internalization and clustering.\u003c/p\u003e \u003cp\u003eFor the most effective agonist combinations, we have observed synergistic effects on macrophage stimulation and subsequent T cell activation. For example, anti-Clec5a stimulation alone did not lead to T cell activation, but strongly enhanced the effect of CD40 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). T cell reactivation remained antigen-specific, as there was no activation in the absence of the antigenic peptide. It will be interesting to investigate the underlying signalling pathways and molecular mechanisms. While we have observed upregulation of costimulatory molecules (CD80, CD83) and induction of chemokines and cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), a clear mechanism cannot be derived from these data. Further insight may be gained by transcriptomic analysis of stimulated macrophages and pathway analysis.\u003c/p\u003e \u003cp\u003eThe therapeutic effect of anti-CD40 has mainly been attributed to stimulation of DCs. To increase the therapeutic use of CD40 stimulation, bispecific antibodies have been designed that targeted CD40 activation preferentially to DCs by coupling the CD40 agonist arm with a CLEC9A-targeting arm \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. DCs were also required for the anti-tumor activity of combinations of anti-CD40 with various pattern-recognition receptor agonists in a mouse model of pancreatic ductal adenocarcinoma \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Stimulation of DCs in a tumor by the agonist combinations presented here is likely and may contribute to a therapeutic effect. However, because macrophages are by far more abundant than DCs, the concept of the present paper is to exploit macrophage activation to enhance the anti-tumor immune response. Our experiments demonstrated that T cells could be activated by macrophages in the absence of DCs.\u003c/p\u003e \u003cp\u003eIn one experiment we used peptides from SARS-CoV-2 spike protein as alternative model antigens (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). We hypothesized that due to the pandemic and related vaccinations most donors would be reactive to specific stimulation. Indeed, 5 out of 6 donors responded to stimulation with a large peptide pool derived from spike protein. However, we observed a very high donor variability, potentially caused by the individual patient history of vaccinations and infections. Therefore, like for CMV or flu peptide stimulation, pre-testing of donors may be necessary for use in such experiments.\u003c/p\u003e \u003cp\u003eIn summary, we have identified and characterized agonist combinations that stimulated macrophages to reactivate cytotoxic T cells and could overcome immunosuppression. These investigations will serve as a basis to develop multispecific antibody constructs for tumor-specific activation of macrophages that may represent a promising approach to enhance the anti-tumor immune response and increase survival of patients with macrophage-rich solid tumors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Madlen Hahn, Daniel H\u0026ouml;sch and Janine Schiele for expert technical assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are employees of Boehringer Ingelheim.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFI: Conceptualization, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Visualization, Project administrationDR: Investigation, Conceptualization, Writing \u0026ndash; review \u0026amp; editing JP: Conceptualization, Project administration, Supervision, Writing \u0026ndash; review \u0026amp; editing\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGentles, A. 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(2024).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cancer, immunotherapy, tumor-associated macrophages, T cell activation, tumor microenvironment, CD40","lastPublishedDoi":"10.21203/rs.3.rs-6195356/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6195356/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMacrophages are the major immune cell population in most human solid cancers. Therefore, we hypothesized that appropriate activation of macrophages in tumors could convert an immunosuppressive into a proinflammatory tumor microenvironment and, thus, enable T cell-mediated killing of tumor cells. To identify appropriate activating receptors, we selected putative agonists from literature and used them for stimulation of primary monocyte-derived human macrophages as single agents or in combination. Stimulated macrophages were co-cultured with autologous T cells to test their ability to reactivate antigen-specific T cells. To mimic tumor-specific clustering, we expressed selected agonistic antibodies on the cell surface of tumor cells and used them in coculture assays to determine macrophage stimulation and T cell activation. For the most promising agonists and combinations, we also tested their ability to repolarize M2 to M1 macrophages and determined the induced cytokine and chemokine profile. The most effective agonist combinations were: CD40\u0026thinsp;+\u0026thinsp;TLR4, CD40\u0026thinsp;+\u0026thinsp;TLR7/8, CD40\u0026thinsp;+\u0026thinsp;Clec5a, CD40\u0026thinsp;+\u0026thinsp;CSF1R, CD40\u0026thinsp;+\u0026thinsp;CSF2RB and TLR4\u0026thinsp;+\u0026thinsp;TLR7/8. Macrophages that were stimulated with these combinations activated autologous antigen-specific T cells that were cytotoxic towards tumor cells. These combinations were also able to overcome TGF-beta-mediated immunosuppression in vitro. Tumor-specific activation of macrophages with these agonist combinations may represent a promising approach to enhance the anti-tumor immune response and increase survival of patients with macrophage-rich solid tumors.\u003c/p\u003e","manuscriptTitle":"Identification of agonist combinations that stimulate macrophages to induce anti-tumor T cells and overcome immunosuppression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 08:58:52","doi":"10.21203/rs.3.rs-6195356/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9a210661-ba90-4d4a-a6ff-7be93d9daa8d","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46967364,"name":"Biological sciences/Cancer/Tumour immunology"},{"id":46967365,"name":"Biological sciences/Immunology/Immunotherapy"},{"id":46967366,"name":"Biological sciences/Immunology/Immunotherapy/Immunosuppression"},{"id":46967367,"name":"Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages"}],"tags":[],"updatedAt":"2025-11-18T03:38:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-16 08:58:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6195356","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6195356","identity":"rs-6195356","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}