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Supplementary Figure 1. Systemic neutrophilia in tumor bearing mice. Supplementary Figure 2. Increased metastasis does not result from increased intravasation or post-extravasation events. Supplementary Figure 3. NK-mediated clearance of B16-F10-GFP+ cells. Supplementary Figure 4. Ly6G+ neutrophils are more abundant in spleens of 4T1 bearing mice. Supplementary Figure 5. Splenocyte conditioned medium activates endothelial cells and induces MMP-9 secretion.

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Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies

Despite the significant therapeutic advances provided by immune-checkpoint blockade and chimeric antigen receptor T cell treatments, many malignancies remain unresponsive to immunotherapy. Bispecific antibodies targeting tumor antigens and activating T cell receptor signaling have shown some clinical efficacy; however, providing co-stimulatory signals may improve T cell responses against tumors. Here, we developed a trispecific antibody that interacts with CD38, CD3 and CD28 to enhance both T cell activation and tumor targeting. The engagement of both CD3 and CD28 affords efficient T cell stimulation, whereas the anti-CD38 domain directs T cells to myeloma cells, as well as to certain lymphomas and leukemias. In vivo administration of this antibody suppressed myeloma growth in a humanized mouse model and also stimulated memory/effector T cell proliferation and reduced regulatory T cells in non-human primates at well-tolerated doses. Collectively, trispecific antibodies represent a promising platform for cancer immunotherapy. Wu et al. develop trispecific antibodies that recognize CD38, CD3 and CD28 and induce T cell activation and co-signaling. They show in mice and non-human primates that by engaging multiple targets, these antibodies induce enhanced tumor-cell killing.

Autophagy is the cellular process by which cytoplasmic contents are degraded and recycled through a lysosomal pathway. In cancer, autophagy can contribute to both tumor promotion and tumor suppression, and emerging evidence supports a role in resistance to immunotherapy. We investigated whether inhibition of autophagy with a small molecule inhibitor of the class III phosphoinositide 3-kinase Vps34 could stimulate anti-tumor immune responses and potentiate the efficacy of immune checkpoint blockade (ICB). Treatment with the Vps34 inhibitor (Vps34i) inhibited autophagy and was weakly cytotoxic to B16F10, MC38, EMT6 and CT26 mouse syngeneic tumor cell lines. In certain cancers, autophagy has been linked to lysosomal degradation of MHC I proteins, thereby downregulating expression and contributing to immune evasion. Notably, we observed minimal upregulation of MHC I surface expression on a subset of mouse and human cell lines. We further explored whether autophagy inhibition would result in release of any soluble immune stimulatory factors from mouse tumor cell lines. Inhibition of Vps34 markedly increased the secretion of several proinflammatory chemokines, including CCL5 and CXCL10. Increased chemokine secretion was also observed in a panel of human cancer cell lines, including NCI-H2009 lung cancer and PC-3 prostate cancer, confirming that the response is observed in human cancer cell lines derived from different tissue origins. As the chemokines CCL5 and CXCL10 are critical to the development of robust anti-tumor immunity, we next tested whether Vps34i treatment could potentiate the anti-tumor efficacy of ICB. We explored the activity of Vps34i as a single agent and in combination with anti-PD-1 antibody in three different syngeneic tumor models, including EMT6, CT26 and MC38. No single agent activity was observed in these models, however, combination of anti-PD-1 and Vps34i significantly inhibited the growth of CT26 tumors compared to control. Anti-tumor efficacy of combination treatment was not significantly different compared to single agents, and no complete tumor regressions were observed. No significant increases in CCL5 and CXCL10 were observed in the serum of combination treated mice, potentially explaining the lack of robust efficacy. Our results suggest that inhibition of autophagy promotes the secretion of proinflammatory cytokines from mouse and human cancer cells in vitro. As the autophagy pathway exhibits cross talk with other major cell signaling pathways, the tumor contexture may be a critical determinant of the role of autophagy in anti-tumor immune responses. Future experiments will explore which tumor contexts are sensitive to combination of autophagy inhibition and ICB. Citation Format: Marie Bernardo, Yu-an Zhang, Martin Graf, Jane Cheng, Fangxian Sun, Virna Cortez-Retamozo, Sukhvinder Sidhu, Eladio Marquez, Donald Jackson, Jack Pollard, Timothy R. Wagenaar, Donald Shaffer. Inhibition of the autophagy protein Vps34 induces tumor cell secretion of proinflammatory chemokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5590.

BackgroundSAR444245 is a non-alpha IL-2 Synthorin TM molecule designed with a site-specific non-natural amino acid serving as a bioconjugation site for a single PEG. The non-natural amino acid is positioned to enable the PEG bioconjugation to obscure block binding to the IL-2 alpha receptor, while retaining near-native affinity with the intermediate affinity βγ IL-2 receptor. The non-alpha features of SAR444245 minimize activation of immune suppressive regulatory CD4+ T cells, while retaining activity on CD8+ T cells and NK cells expressing the IL-2 βγ receptors. NK cells exert anti-tumor activity through antibody dependent cellular cytotoxicity (ADCC) of IgG antibodies as well as antibody independent mechanisms.MethodsHere, we utilized a panel of human primary PBMC based immunoassays and transcriptomic analysis to evaluate whether SAR444245 may improve ADCC function of IgG1 anti-tumor target antibodies.ResultsWe characterized the ability of SAR444245 to enhance the cytolytic function of NK cells towards the prototypic NK target cell K562 as well as to modulate NK cell ADCC in combination with EGFR or CD20-targeting antibodies. In vitro assays demonstrated that SAR444245 can activate NK cells, promote NK cell proliferation and improve cytotoxicity of NK cells against K562 cells and across a panel of human EGFR and CD20 positive cell lines. In PBMC based ADCC assays with 1ug/ml of antibody, SAR444245 improved ADCC function maximally by 9-fold for an anti-EGFR antibody and at 5-fold for an anti-CD20 antibody. SAR444245 exhibited dose-dependent enhancement of NK cell ADCC function. Notably, this activity was observed in cell lines expressing varying levels of EGFR and CD20. SAR444245 treatment was associated with dose dependent increases in NK cell degranulation and IFN-γ production. Transcriptomic profiling revealed that SAR444245 had broad effects on NK cell biology leading to changes in inhibitory and activating receptors.ConclusionsIn summary, these results indicate that SAR444245 can enhance the cytolytic activity of NK cells and enhance the ADCC effect of tumor-directed antibodies by activating NK cells.

Senescence is an important p53-controlled tumor suppressor program that not only opposes the proliferation of cancer cells but also promotes their immune-mediated clearance in certain contexts. In hepatocellular cancer, p53 induction promotes an innate immune cell-mediated clearance of senescent cells wherein natural killer (NK) cells seem to play the primary sentinel role. Whether NK cells also surveil cancer cells in other tumor types when p53 is activated to promote a senescence response is unknown. To identify the role that NK and other innate immune cell types have on the surveillance and destruction of lung adenocarcinoma cells, we developed an orthotopic transplantation model where p53 gene function could be restored to induce senescence after successful engraftment of tumor cells in the mouse lung. Contrary to precedent, we found that NK cells actually limited the efficient clearance of tumor cells from the mouse lung after p53 restoration. Instead, activation of p53 induced the infiltration of monocytes, neutrophils, and interstitial macrophages. Loss of NK cells further promoted expansion of these inflammatory cell types and tumor clearance after p53 restoration. These observations suggest that NK cell responses to p53 activation in lung adenocarcinoma is distinct from those found in other tumor types and that diverse innate immune cell populations may play context-dependent roles during tumor immune surveillance. Further, our data provide an impetus to understand the broader mechanisms that regulate cancer cell destruction by multiple cell types of the innate immune system and distinct cancer contexts.

The success of early cancer immunotherapies has led to the development of novel therapeutic approaches including multi-specific antibodies engaging innate and adaptive effector cells. To evaluate the antitumor potency of these multi-specific cell engagers, we have established a protocol to select human PBMCs from donors of a commercial frozen source by their potency to produce inflammatory cytokines and the capacity to engraft into NOD-Scid IL2Rgammanull (NSG)mice. The selected human PBMCs were used to reconstitute NSG mice, or as donors of effector cells that upon in vitro expansion were adoptively transferred to NSG mice for humanization. We have employed these humanization strategies to evaluate T cell engagers, which are typically multi-specific antibodies directed against the T cell and one or multiple tumor-associated antigen (TAA), whose therapeutic strategy is to 1) engage T cells, 2) activate the T cells and 3) engage tumor cells, and thus induce tumor cell killing. We will present representative data from several humanization approaches using liquid and solid orthotopic tumor models to assess the effectiveness of multi-specific T cell engagers in mediating antitumor responses. Thus, pre-clinical evaluations rely on the development of optimized tumor bearing humanized models that mirror key properties of a human setting, to assess the therapeutic potential of anti-tumor multi-specific biologics. Citation Format: Liqing Cheng, Jane Chen, Qunyan Yu, Natalia Malkova, Virna F. Cortez-Retamozo, Fangxian Sun, Sukhvinder Sidhu. Donor selection strategies to optimize humanized mouse models for the evaluation of T cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2712.

Current HER2-targeted therapies have markedly improved the outcome of cancer patients with HER2 overexpressing tumors. However, these patients may eventually relapse or develop treatment resistance. In addition, HER2-low patients that are not eligible for treatment constitute a significant portion of the breast cancer patients. To address these unmet needs, we have developed a novel HER2-targeting T cell engager, SAR443216. This is a trispecific antibody with binding sites for HER2, CD3 and CD28, and containing a mutated IgG4-Fc which lacks effector functions. CD28 binding contributes to T cell activation, including activation of IL-2 and NFκB pathways, as well as induction of anti-apoptotic protein, Bcl-xL. In the presence of HER2-positive cancer cells, SAR443216 is able to activate primary human CD4 and CD8 T cells, resulting in T cell proliferation and secretion of cytokines and granzyme B. Moreover, it has potent in vitro T cell-dependent cellular cytotoxicity (TDCC) against a panel of HER2-expressing cancer cell lines, including those that are HER2-low. The potency of in vitro TDCC is largely correlated with HER2 surface expression in the target cells. Finally, in a HER2-low breast cancer xenograft model, SAR443216 also exhibited significant anti-tumor activity in immuno-deficient NSG mice reconstituted with primary human T cells. Thus, SAR443216 represents a promising new drug for cancer patients with HER2-expressing tumors, including those who are currently ineligible for stand-of-care therapy. Citation Format: Wenwen Sha, Sri Vadde, Zhili Song, Edward Seung, Zhen Xing, Liqing Chen, Virna Cortez-Retamozo, Sukhvinder Sidhu, Dinesh Bangari, Lan Wu, Ronnie Wei, Zhi-yong Yang, Gary Nabel, Vasiliki Pelekanou, Michele Sanicola-Nadel, Serena Masciari, Dmitri Wiederschain, Lily Pao. SAR443216, a novel trispecific T cell engager with potent T cell-dependent cytotoxicity for HER2-low tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1825.

A bona fide portrayal of tumor growth Bone has a well-established role in advanced cancer. It provides a supportive microenvironment for the growth of metastatic cells that escape the primary tumor, which ultimately leads to loss of bone mass. Engblom et al. show that bone may also contribute to early-stage tumorigenesis through a mechanism that leads to an increase in bone mass (see the Perspective by Zhang and Lyden). In mouse models of lung adenocarcinoma, primary tumor cells remotely activated bone-resident cells called osteoblasts, which have a bone-building function. The activated osteoblasts in turn triggered production of a certain type of neutrophil that infiltrates the primary tumor and promotes its growth. Patients with early-stage lung cancer were also found to have an increase in bone density, consistent with the findings in mice. Science , this issue p. eaal5081 ; see also p. 1127

Summary Macrophages frequently infiltrate tumors and can enhance cancer growth, yet the origins of the macrophage response are not well understood. Here we address molecular mechanisms of macrophage production in a conditional mouse model of lung adenocarcinoma. We report that overproduction of the peptide hormone Angiotensin II (AngII) in tumor-bearing mice amplifies self-renewing hematopoietic stem cells (HSCs) and macrophage progenitors. The process occurred in the spleen but not the bone marrow, and was independent of hemodynamic changes. The effects of AngII required direct hormone ligation on HSCs, depended on S1P 1 signaling, and allowed the extramedullary tissue to supply new tumor-associated macrophages throughout cancer progression. Conversely, blocking AngII production prevented cancer-induced HSC and macrophage progenitor amplification and thus restrained the macrophage response at its source. These findings indicate that AngII acts upstream of a potent macrophage amplification program and that tumors can remotely exploit the hormone's pathway to stimulate cancer-promoting immunity.

Immune cells promote the initial metastatic dissemination of carcinoma cells from primary tumors. In contrast to their well-studied functions in the initial stages of metastasis, the specific roles of immunocytes in facilitating progression through the critical later steps of the invasion–metastasis cascade remain poorly understood. Here, we define novel functions of neutrophils in promoting intraluminal survival and extravasation at sites of metastatic dissemination. We show that CD11b+/Ly6G+neutrophils enhance metastasis formation via two distinct mechanisms. First, neutrophils inhibit natural killer cell function, which leads to a significant increase in the intraluminal survival time of tumor cells. Thereafter, neutrophils operate to facilitate extravasation of tumor cells through the secretion of IL1β and matrix metalloproteinases. These results identify neutrophils as key regulators of intraluminal survival and extravasation through their cross-talk with host cells and disseminating carcinoma cells. SIGNIFICANCE: This study provides important insights into the systemic contributions of neutrophils to cancer metastasis by identifying how neutrophils facilitate intermediate steps of the invasion–metastasis cascade. We demonstrate that neutrophils suppress natural killer cell activity and increase extravasation of tumor cells., Human Frontier Science Program (Strasbourg, France) (fellowship LT00728/2008-L), Charles King Trust Foundation, Massachusetts Institute of Technology. Ludwig Center for Cancer Research, Cancer Research Institute (New York, N.Y.) (Irvington Fellowship), National Institutes of Health (U.S.) (grant P01 CA080111), National Institutes of Health (U.S.) (grant CA163109)

Tissue macrophages and dendritic cells derive from hematopoietic stem cells, which exist in the bone marrow and generate intermediate precursor populations with increasingly restricted lineage potentials. There exists several precursors committed to the macrophage and dendritic cell lineages; these cells exhibit distinct tropism and function and respond differentially in pathophysiologic conditions. In this review, we consider experimental contexts in which macrophage and dendritic cell responses in tissue are not only dictated by the local environment, but also by the quantity and quality of newly recruited lineage precursor cells. Consequently, we discuss whether therapeutic control of macrophage and dendritic cell responses in tissue may be achieved through manipulation of their lineage precursors.

One of the most intriguing aspects of male reproductive physiology is the ability to generate spermatogenic cells – which are ‘foreign’ to the host – without triggering immune activation. After leaving the testis, spermatozoa enter the epididymis where they mature and are stored. In this study, we report a previously unrecognized dense network of dendritic cells (DCs) located at the base of the epididymal epithelium. This network was detected in transgenic mice expressing CD11c-EYFP and CX3CR1-GFP reporters. Epididymal DCs (eDCs) establish intimate interactions with the epithelium and project long dendrites between epithelial cells toward the lumen. We show that isolated eDCs express numerous leukocyte markers described previously in other organs that are in contact with the external environment, and present and cross-present ovalbumin to T cellsin vitro. eDCs are, therefore, strategically positioned to regulate the complex interplay between immune tolerance and activation, a balance that is fundamental to male fertility.

Repulsive guidance molecule (RGM) family members RGMa, RGMb/Dragon, and RGMc/hemojuvelin were found recently to act as bone morphogenetic protein (BMP) coreceptors that enhance BMP signaling activity. Although our previous studies have shown that hemojuvelin regulates hepcidin expression and iron metabolism through the BMP pathway, the role of the BMP signaling mediated by Dragon remains largely unknown. We have shown previously that Dragon is expressed in neural cells, germ cells, and renal epithelial cells. In this study, we demonstrate that Dragon is highly expressed in macrophages. Studies with RAW264.7 and J774 macrophage cell lines reveal that Dragon negatively regulates IL-6 expression in a BMP ligand-dependent manner via the p38 MAPK and Erk1/2 pathways but not the Smad1/5/8 pathway. We also generated Dragon knockout mice and found that IL-6 is upregulated in macrophages and dendritic cells derived from whole lung tissue of these mice compared with that in respective cells derived from wild-type littermates. These results indicate that Dragon is an important negative regulator of IL-6 expression in immune cells and that Dragon-deficient mice may be a useful model for studying immune and inflammatory disorders.

Monitoring Monocyte Reservoirs Monocytes are cells of the immune system that are recruited to sites of tissue injury and inflammation where they help to resolve the infection and are important for tissue repair. The bone marrow and blood are believed to be the primary reservoirs from which monocytes are mobilized after injury. Swirski et al. (p. 612 ; see the Perspective by Jia and Pamer ) now demonstrate that the spleen also serves as a critical reservoir of monocytes that are recruited during ischemic myocardial injury. Monocytes in the spleen are very similar in phenotype to blood-derived monocytes and are mobilized to the injured heart, where they represent a large fraction of the total monocytes that are recruited. The chemoattractant, angiotensin II, is required for optimal monocyte mobilization from the spleen and emigration into injured tissue.

Tumor-associated macrophages (TAMs) invade the tumor stroma in many cancers, yet their role is incompletely understood. To visualize and better understand these critical cells in tumor progression, we screened a portfolio of rationally selected, injectable agents to image endogenous TAMs ubiquitously in three different cancer models (colon carcinoma, lung adenocarcinoma, and soft tissue sarcoma). AMTA680, a functionally derivatized magneto-fluorescent nanoparticle, labeled a subset of myeloid cells with an “M2” macrophage phenotype, whereas other neighboring cells, including tumor cells and a variety of other leukocytes, remained unlabeled. We further show that AMTA680-labeled endogenous TAMs are not altered and can be tracked noninvasively at different resolutions and using various imaging modalities, e.g., fluorescence molecular tomography, magnetic resonance imaging, and multiphoton and confocal intravital microscopy. Quantitative assessment of TAM distribution and activity in vivo identified that these cells cluster in delimited foci within tumors, show relatively low motility, and extend cytoplasmic protrusions for prolonged physical interactions with neighboring tumor cells. Noninvasive imaging can also be used to monitor TAM-depleting regimen quantitatively. Thus, AMTA680 or related cell-targeting agents represent appropriate injectable vehicles for in vivo analysis of the tumor microenvironment.

Eosinophils are multifunctional leukocytes that degrade and remodel tissue extracellular matrix through production of proteolytic enzymes, release of proinflammatory factors to initiate and propagate inflammatory responses, and direct activation of mucus secretion and smooth muscle cell constriction. Thus, eosinophils are central effector cells during allergic airway inflammation and an important clinical therapeutic target. Here we describe the use of an injectable MMP-targeted optical sensor that specifically and quantitatively resolves eosinophil activity in the lungs of mice with experimental allergic airway inflammation. Through the use of real-time molecular imaging methods, we report the visualization of eosinophil responses in vivo and at different scales. Eosinophil responses were seen at single-cell resolution in conducting airways using near-infrared fluorescence fiberoptic bronchoscopy, in lung parenchyma using intravital microscopy, and in the whole body using fluorescence-mediated molecular tomography. Using these real-time imaging methods, we confirmed the immunosuppressive effects of the glucocorticoid drug dexamethasone in the mouse model of allergic airway inflammation and identified a viridin-derived prodrug that potently inhibited the accumulation and enzyme activity of eosinophils in the lungs. The combination of sensitive enzyme-targeted sensors with noninvasive molecular imaging approaches permitted evaluation of airway inflammation severity and was used as a model to rapidly screen for new drug effects. Both fluorescence-mediated tomography and fiberoptic bronchoscopy techniques have the potential to be translated into the clinic.

Introduction: The development of specific radiolabeled probes towards molecular markers in vivo has gained interest as targeted imaging allows for a more accurate detection of diseases. We investigate the feasibility of targeted imaging of cancer antigens using the variable domain of single chain camelid antibodies (Nanobodies®) labeled with 99mTechnetium. Nanobodies against carcinoembryonic antigen (CEA) were used as a model. Methods: His6-CEA1 Nanobodies were generated and labeled with 99mTc at their His-tag using Tc(I)-tricarbonyl (Isolink, Mallinckrodt, B.V., Petten, The Netherlands). The normal biodistribution was assessed in healthy athymic mice by ex vivo analysis at 1 and 3 h. In vivo targeting was evaluated in the same mouse model bearing the CEA-positive LS174T tumour or a CEA-negative A431 (human skin carcinoma) control tumour. Pinhole SPECT imaging was performed at 3 hours after intravenous injection of 90 MBq 99mTc-His6-CEA1 using a dual-headed gamma camera equipped with pinhole collimators. Results: Radiolabeling efficiency was > 95%. General biodistribution showed intense renal uptake and marked liver accumulation. Using pinhole-SPECT, the average uptake of 99mTc- His6-CEA1 in LS174T (CEA positive) was significantly higher compared to the A431 (CEA negative) control tumour: respectively 3.2 ± 0.6 %IA/cm3 and 1.1 ± 0.2 %IA/cm3 (p < 0.05). Conclusion: This study presents effective labeling of Nanobodies with 99mTc using Tc(I)-carbonyl chemistry and shows their potential as a new type of specific probes for imaging antigen expression.

Checkpoint blockade immunotherapies can be extraordinarily effective, but might benefit only the minority of patients whose tumors are pre-infiltrated by T cells. Here, using lung adenocarcinoma mouse models, including genetic models, we show that autochthonous tumors that lacked T cell infiltration and resisted current treatment options could be successfully sensitized to host antitumor T cell immunity when appropriately selected immunogenic drugs (e.g., oxaliplatin combined with cyclophosphamide for treatment against tumors expressing oncogenic Kras and lacking Trp53) were used. The antitumor response was triggered by direct drug actions on tumor cells, relied on innate immune sensing through toll-like receptor 4 signaling, and ultimately depended on CD8(+) T cell antitumor immunity. Furthermore, instigating tumor infiltration by T cells sensitized tumors to checkpoint inhibition and controlled cancer durably. These findings indicate that the proportion of cancers responding to checkpoint therapy can be feasibly and substantially expanded by combining checkpoint blockade with immunogenic drugs.

Nanobodies are the smallest fragments of naturally occurring single-domain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies are strictly monomeric, very stable, and highly soluble entities. We identified a nanobody with subnanomolar affinity for the human tumor-associated carcinoembryonic antigen. This nanobody was conjugated to Enterobacter cloacae β-lactamase, and its site-selective anticancer prodrug activation capacity was evaluated. The conjugate was readily purified in high yields without aggregation or loss of functionality of the constituents. In vitro experiments showed that the nanobody–enzyme conjugate effectively activated the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug 7-(4-carboxybutanamido) cephalosporin mustard at the surface of carcinoembryonic antigen-expressing LS174T cancer cells. In vivo studies demonstrated that the conjugate had an excellent biodistribution profile and induced regressions and cures of established tumor xenografts. The easy generation and manufacturing yield of nanobody-based conjugates together with their potent antitumor activity make nanobodies promising vehicles for new generation cancer therapeutics.

Antigen variation is a successful defense system adopted by several infectious agents to evade the host immune response. The principle of this defense strategy in the African trypanosome paradigm involves a dense packing of variant surface glycoproteins (VSG) exposing only highly variable and immuno-dominant epitopes to the immune system, whereas conserved epitopes become inaccessible for large molecules. Reducing the size of binders that target the conserved, less-immunogenic, cryptic VSG epitopes forms an obvious solution to combat these parasites. This goal was achieved by introducing dromedary Heavy-chain antibodies. We found that only these unique antibodies recognize epitopes common to multiple VSG classes. After phage display of their antigen-binding repertoire, we isolated a single domain antibody fragment with high specificity for the conserved Asn-linked carbohydrate of VSG. In sharp contrast to labeled concanavalin-A that stains only the flagellar pocket where carbohydrates are accessible because of less dense VSG packing, the single domain binder stains the entire surface of viable parasites, irrespective of the VSG type expressed. This corroborates the idea that small antibody fragments, but not larger lectins or conventional antibody fragments, are able to penetrate the dense VSG coat to target their epitope. The diagnostic potential of this fluorescently labeled binder was proven by the direct, selective, and sensitive detection of parasites in blood smears. The employment of this binder as a molecular recognition unit in immuno-toxins designed for trypanosomosis therapy becomes feasible as well. This was illustrated by the specific trypanolysis induced by an antibody::β-lactamase fusion activating a prodrug.

Uncontrolled inflammation is one of the leading causes of kidney failure. Pro-inflammatory responses can occur in the absence of infection, a process called sterile inflammation. Here we show that the purinergic receptor P2Y14 (GPR105) is specifically and highly expressed in collecting duct intercalated cells (ICs) and mediates sterile inflammation in the kidney. P2Y14 is activated by UDP-glucose, a damage-associated molecular pattern molecule (DAMP) released by injured cells. We found that UDP-glucose increases pro-inflammatory chemokine expression in ICs as well as MDCK-C11 cells, and UDP-glucose activates the MEK1/2-ERK1/2 pathway in MDCK-C11 cells. These effects were prevented following inhibition of P2Y14 with the small molecule PPTN. Tail vein injection of mice with UDP-glucose induced the recruitment of neutrophils to the renal medulla. This study identifies ICs as novel sensors, mediators and effectors of inflammation in the kidney via P2Y14.

The epithelium that lines the epididymal duct establishes the optimal milieu in which spermatozoa mature, acquire motility, and are stored. This finely tuned environment also protects antigenic sperm against pathogens and autoimmunity, which are potential causes of transient or permanent infertility. The epididymal epithelium is pseudostratified and contains basal cells (BCs) that are located beneath other epithelial cells. Previous studies showed that in the mouse epididymis, BCs possess macrophage-like characteristics. However, we previously identified a dense population of cells belonging to the mononuclear phagocyte (MP) system (comprised of macrophages and dendritic cells) in the basal compartment of the mouse epididymis and showed that a subset of MPs express the macrophage marker F4/80. In the present study, we evaluate the distribution of BCs and MPs in the epididymis of transgenic CD11c-EYFP mice, in which EYFP is expressed exclusively in MPs, using antibodies against the BC marker keratin 5 (KRT5) and the macrophage marker F4/80. Immunofluorescence labeling for laminin, a basement membrane marker, showed that BCs and most MPs are located in the basal region of the epithelium. Confocal microscopy showed that in the initial segment, both BCs and MPs project intraepithelial extensions and establish a very intricate network. Flow cytometry experiments demonstrated that epididymal MPs and BCs are phenotypically distinct. BCs do not express F4/80, and MPs do not express KRT5. Therefore, despite their proximity and some morphological similarities with peritubular macrophages and dendritic cells, BCs do not belong to the MP system.

Tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) can control cancer growth and exist in almost all solid neoplasms. The cells are known to descend from immature monocytic and granulocytic cells, respectively, which are produced in the bone marrow. However, the spleen is also a recently identified reservoir of monocytes, which can play a significant role in the inflammatory response that follows acute injury. Here, we evaluated the role of the splenic reservoir in a genetic mouse model of lung adenocarcinoma driven by activation of oncogenic Kras and inactivation of p53. We found that high numbers of TAM and TAN precursors physically relocated from the spleen to the tumor stroma, and that recruitment of tumor-promoting spleen-derived TAMs required signaling of the chemokine receptor CCR2. Also, removal of the spleen, either before or after tumor initiation, reduced TAM and TAN responses significantly and delayed tumor growth. The mechanism by which the spleen was able to maintain its reservoir capacity throughout tumor progression involved, in part, local accumulation in the splenic red pulp of typically rare extramedullary hematopoietic stem and progenitor cells, notably granulocyte and macrophage progenitors, which produced CD11b + Ly-6C hi monocytic and CD11b + Ly-6G hi granulocytic cells locally. Splenic granulocyte and macrophage progenitors and their descendants were likewise identified in clinical specimens. The present study sheds light on the origins of TAMs and TANs, and positions the spleen as an important extramedullary site, which can continuously supply growing tumors with these cells.

Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes-but not the noninflammatory subset-depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.

Rationale: Monocytes recruited to ischemic myocardium originate from a reservoir in the spleen, and the release from their splenic niche relies on angiotensin (Ang) II signaling. Objective: Because monocytes are centrally involved in tissue repair after ischemia, we hypothesized that early angiotensin-converting enzyme (ACE) inhibitor therapy impacts healing after myocardial infarction partly via effects on monocyte traffic. Methods and Results: In a mouse model of permanent coronary ligation, enalapril arrested the release of monocytes from the splenic reservoir and consequently reduced their recruitment into the healing infarct by 45%, as quantified by flow cytometry of digested infarcts. Time-lapse intravital microscopy revealed that enalapril reduces monocyte motility in the spleen. In vitro migration assays and Western blotting showed that this was caused by reduced signaling through the Ang II type 1 receptor. We then studied the long-term consequences of blocked splenic monocyte release in atherosclerotic apolipoprotein (apo)E −/− mice, in which infarct healing is impaired because of excessive inflammation in the cardiac wound. Enalapril improved histologic healing biomarkers and reduced inflammation in infarcts measured by FMT-CT (fluorescence molecular tomography in conjunction with x-ray computed tomography) of proteolytic activity. ACE inhibition improved MRI-derived ejection fraction by 14% on day 21, despite initially comparable infarct size. In apoE −/− mice, ischemia/reperfusion injury resulted in larger infarct size and enhanced monocyte recruitment and was reversible by enalapril treatment. Splenectomy reproduced antiinflammatory effects of enalapril. Conclusion: This study suggests that benefits of early ACE inhibition after myocardial infarction can partially be attributed to its potent antiinflammatory impact on the splenic monocyte reservoir.

Objective: To test a novel self-activating viridin (SAV) prodrug that slowly releases wortmannin, a potent phosphoinositide 3-kinase inhibitor, in a model of antibody-mediated inflammatory arthritis. Methods: The SAV prodrug was administered to K/BxN mice or to C57BL/6 (B6) mice that had been injected with K/BxN serum. Ankle thickness was measured, and histologic changes were scored after a 10-day disease course (serum-transfer arthritis). Protease activity was measured by a near-infrared imaging approach using a cleavable cathepsin–selective probe. Further near-infrared imaging techniques were used to analyze early changes in vascular permeability after serum injection, as well as neutrophil–endothelial cell interactions. Neutrophil functions were assessed using an oxidative burst assay as well as a degranulation assay. Results: SAV prevented ankle swelling in mice with serum-transfer arthritis in a dose-dependent manner. It also markedly reduced the extent of other features of arthritis, such as protease activity and histology scores for inflammation and joint erosion. Moreover, SAV was an effective therapeutic agent. The underlying mechanisms for the antiinflammatory activity were manifold. Endothelial permeability after serum injection was reduced, as was firm neutrophil attachment to endothelial cells. Endothelial cell activation by tumor necrosis factor α was impeded by SAV, as measured by the expression of vascular cell adhesion molecule. Crucial neutrophil functions, such as generation of reactive oxygen species and degranulation of protease-laden vesicles, were decreased by SAV administration. Conclusion: A novel SAV prodrug proved strongly antiinflammatory in a murine model of antibody-induced inflammatory arthritis. Its activity could be attributed, at least in part, to the inhibition of neutrophil and endothelial cell functions., National Institutes of Health (U.S.) (Grant P01-AI-054904), National Institutes of Health (U.S.) (Grant R01-EB-001872), National Institutes of Health (U.S.) (Grant R01-AR-046580), National Institutes of Health (U.S.) (Grant R24-CA-92782), National Institutes of Health (U.S.) (Grant P50-CA-86355)

Over the last years there has been a growing interest in the use of antibodies and antibody fragments as therapeutic entities to treat cancer. Identification of the smallest antibody fragment still capable of binding to antigen has progressed from full antibody molecules to Fab and recombinant single chain Fv fragments. A further reduction to single-domain binding proteins based upon immuno-globulin VH and VH-like domains is being pursued since they offer exciting prospects in the development of novel immunotherapeutics. Unfortunately, the poor solubility and reduced antigen affinity requires additional engineering. By serendipity, it was discovered that this engineering is already performed in nature. Part of the humoral immune response of camels and llamas is based largely on heavy-chain antibodies where the light chain is totally absent. These unique antibody isotypes interact with the antigen by virtue of only one single variable domain, referred to as VHH. Here we describe the properties of camel VHH that offer added value over conventional antibody fragments as treatment modality for tumour patients.

Antigen variation is a successful defense system adopted by several infectious agents to evade the host immune response. The principle of this defense strategy in the African trypanosome paradigm involves a dense packing of variant surface glycoproteins (VSG) exposing only highly variable and immuno-dominant epitopes to the immune system, whereas conserved epitopes become inaccessible for large molecules. Reducing the size of binders that target the conserved, less-immunogenic, cryptic VSG epitopes forms an obvious solution to combat these parasites. This goal was achieved by introducing dromedary Heavy-chain antibodies. We found that only these unique antibodies recognize epitopes common to multiple VSG classes. After phage display of their antigen-binding repertoire, we isolated a single domain antibody fragment with high specificity for the conserved Asn-linked carbohydrate of VSG. In sharp contrast to labeled concanavalin-A that stains only the flagellar pocket where carbohydrates are accessible because of less dense VSG packing, the single domain binder stains the entire surface of viable parasites, irrespective of the VSG type expressed. This corroborates the idea that small antibody fragments, but not larger lectins or conventional antibody fragments, are able to penetrate the dense VSG coat to target their epitope. The diagnostic potential of this fluorescently labeled binder was proven by the direct, selective, and sensitive detection of parasites in blood smears. The employment of this binder as a molecular recognition unit in immuno-toxins designed for trypanosomosis therapy becomes feasible as well. This was illustrated by the specific trypanolysis induced by an antibody::beta-lactamase fusion activating a prodrug.

Meeting abstracts B7-H4 is a member of the B7 family of co-regulatory receptors. It is believed to negatively regulate T cell function and has been associated with poor prognosis in renal cell and ovarian cancers. We performed an unbiased analysis of TCGA gene expression data and identified triple

The variable domain of functional heavy chain antibodies (VHH) devoid of light chains, found in camels, constitute the smallest intact antigen-binding domain fragment. Two camel single-domain fragments, cAb-Lys2 and cAb-Lys3, recognizing an overlapping epitope of lysozyme with a dissociation constant of 2 nM and 65 nM, respectively, and a bivalent cAb-Lys3 were investigated for their ability to target transgenic tumors expressing lysozyme on their membrane. Biodistribution studies revealed that these non-immunogenic monomeric and bivalent camel single-domain antigen binders specifically target lysozyme-expressing tumors and metastatic lesions. The excess of antibody is rapidly eliminated from the blood circulation and no cAb retention was observed in normal organs. The tumor to organ cAb-ratios at 2 and 8 hr were in the (2.1-10.8):1 and (6.2-23.7):1 range, respectively. The degree and specificity of tumor retention is independent of the affinity of the recombinant camel single-domain fragments for their antigen and from their univalent monomeric (15 kDa) or bivalent format (33 kDa). This study demonstrates the successful and specific in vivo targeting of tumors by camel single-domain fragments. It may open perspectives for their future use as tumor-targeting vehicle, due to their small size, soluble behaviour and because they are non-immunogenic and interact with epitopes that are less antigenic for conventional antibodies.

Optical projection tomography is a new ex vivo imaging technique that allows imaging of whole organs in three dimensions at high spatial resolutions. In this Letter we demonstrate its capability to tomographically visualize molecular activity in whole organs of mice. In particular, eosinophil activity in asthmatic lungs is resolved using a Born-normalized fluorescence optical projection tomography and employing a near-IR molecular probe. The possibility to achieve molecularly sensitive imaging contrast in optical projection tomography by means of targeted and activatable imaging reporter agents adds a new range of capabilities for investigating molecular signatures of pathophysiological processes and a wide variety of diseases and their development.

The development of specific radiolabeled probes towards molecular markers in vivo has gained interest as targeted imaging allows for a more accurate detection of diseases. We investigate the feasibility of targeted imaging of cancer antigens using the variable domain of single chain camelid antibodies (Nanobodies) labeled with 99mTechnetium. Nanobodies against carcinoembryonic antigen (CEA) were used as a model. Methods. His6-CEA1 Nanobodies were generated and labeled with 99mTc at their His-tag using Tc(I)-tricarbonyl (Isolink, Mallinckrodt, B.V., Petten, The Netherlands). The normal biodistribution was assessed in healthy athymic mice by ex vivo analysis at 1 and 3 h. In vivo targeting was evaluated in the same mouse model bearing the CEA-positive LS1 74T tumour or a CEA-negative A431 (human skin carcinoma) control tumour. Pinhole SPCT imaging was performed at 3 hours after intravenous injection of 90 MBq 99mTc-His6-CEA1 using a dual-headed gamma camera equipped with pinhole collimators Results. Radiolabeling efficiency was >95%. General biodistribution showed intense renal uptake and marked liver accumulation. Using pinhole-SPCT, the average uptake of 99mTc-His6-CEA1 in LS174T (cea positive) was significantly higher compared to the A431 (CEA negative) control tumour: respectively 3.2 + 0.6%IA/cm3 and 1.1+0.2IA/cm3 (p Conclusion. This study presents effective labelilng of Nanobodies with 99Tc using Tc(I)-carbonyl chemistry and shows their potential as a new type of specific probes for imaging antigen expression.