Exhausted CD8+ T (TEX) cell responses are maintained by precursors of exhausted T (TPEX) cells that possess high self-renewal and developmental potential. TPEX cells also drive the proliferative burst of effector T cells upon therapeutic immune checkpoint blockade (ICB). However, the spatial context and signals that regulate their differentiation and function are not well defined. Here we identify developmental and functional compartmentalization of TPEX and TEX cells across secondary lymphoid organs during chronic infection. We show that stem-like CD62L+ TPEX and effector-like CX3CR1+ TEX cells constitute a distinct developmental lineage that is promoted by the lymph node (LN) microenvironment and dependent on the transcription factor KLF2. LNs act as a niche in which migratory dendritic cells provide antigen and costimulatory signals to maintain the proliferative fitness of CD62L+ TPEX cells and generation of CX3CR1+ TEX cells. Moreover, LNs exclusively drive the proliferative burst and systemic dissemination of CX3CR1+ TEX cells during ICB. Thus, our findings identify a unique role for LNs in the maintenance of T cell differentiation and function during systemic chronic infection and ICB therapy. Here the authors show a function for lymph nodes in the maintenance of effector T cell differentiation and function during chronic infection and checkpoint blockade, identifying a spatial component in the regulation of exhausted T cell fitness.

Vaccination can prevent infections and modulate immune-related diseases. Although conventional vaccines primarily stimulate CD4+ T cells and antibody production, effective antitumor immunity requires activation of CD8+ T cells. Photochemical internalization (PCI) is a promising technology that can facilitate cytosolic antigen delivery, promoting CD8+ T-cell responses. In this study, we studied early immune and cutaneous reactions after PCI-based vaccination with the photosensitizer disulphonated tetraphenyl chlorine. Mice were vaccinated intradermally with ovalbumin antigen and disulphonated tetraphenyl chlorine, followed by light treatment. We assessed cutaneous inflammatory reactions through histology, immunohistochemistry, fluorescence microscopy, and real-time PCR. Systemic inflammatory and immune reactions were analyzed in blood, lymph nodes, and spleen by flow cytometry, clinical chemistry, and ELISA. Disulphonated tetraphenyl chlorine was retained in cutaneous structures and accumulated in draining lymph nodes, and light activation triggered dose- and time-dependent cutaneous inflammatory reactions, including infiltration of innate myeloid CD11b+ and GR1+ macrophages, cross-presenting dendritic cells, and neutrophils, alongside enhanced local and systemic production of proinflammatory cytokines. High disulphonated tetraphenyl chlorine doses triggered severe cutaneous and systemic reactions, but PCI-treated skin showed a high degree of plasticity and healing. These mechanistic insights into local and systemic effects of PCI-based vaccination may contribute to translating PCI into clinical practice and wider application.

Abstract Metastatic colorectal cancer remains a leading cause of cancer-related deaths, with a 5-year survival rate of only 15%. T cell–engaging bispecific antibodies (TCBs) represent a class of biopharmaceuticals that redirect cytotoxic T cells toward tumor cells, thereby turning immunologically “cold” tumors into “hot” ones. The carcinoembryonic antigen (CEA) is an attractive tumor-associated antigen that is overexpressed in more than 98% of patients with colorectal cancer. In this study, we report the comparison of four different TCB formats employing the antibodies F4 (targeting human CEA) and 2C11 (targeting mouse CD3ε). These formats include both antibody fragment–based and IgG-based constructs, with either one or two binding specificities of the respective antibodies. The 2 + 1 arrangement, using an anti-CEA single-chain diabody fused to an anti-CD3 single-chain variable fragment, emerged as the most potent design, showing tumor killing at subnanomolar concentrations across three different CEA+ cell lines. The in vitro activity was three times greater in C57BL/6 mouse colon adenocarcinoma cells (MC38) expressing high levels of CEA compared with those expressing low levels, highlighting the impact of CEA density in this assay. The optimal TCB candidate was tested in two different immunocompetent mouse models of colorectal cancer and showed tumor growth retardation. Ex vivo analysis of tumor infiltrates showed an increase in CD4+ and CD8+ T cells upon TCB treatment. This study suggests that bivalent tumor targeting, monovalent T-cell targeting, and a short spatial separation are promising characteristics for CEA-targeting TCBs.

Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy -associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune -infiltrated from immune -depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and nonsmall cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture.

While cellular metabolism was proposed to be a driving factor of the activation and differentiation of B cells and the function of the resulting antibody-secreting cells (ASCs), the study of correlations between cellular metabolism and functionalities has been difficult due to the absence of technologies enabling the parallel measurement. Herein, we performed single-cell transcriptomics and introduced a direct concurrent functional and metabolic flux quantitation of individual murine B cells. Our transcriptomic data identified lactate metabolism as dynamic in ASCs, but antibody secretion did not correlate with lactate secretion rates (LSRs). Instead, our study of all splenic B cells during an immune response linked increased lactate metabolism with acidic intracellular pH and the upregulation of apoptosis. T cell-dependent responses increased LSRs, and added TLR4 agonists affected the magnitude and boosted LSR high B cells in vivo , while resulting in only a few immunoglobulin-G secreting cells (IgG-SCs). Therefore, our observations indicated that LSR high cells were not differentiating into IgG-SCs, and were rather removed due to apoptosis.

Secondary lymphedema is a common sequel of oncologic surgery and presents a global health burden still lacking pharmacological treatment. The infiltration of the lymphedematous extremities with CD4+T cells influences lymphedema onset and emerges as a promising therapy target. Here, we show that the modulation of CD4+FOXP3+CD25+regulatory T (Treg) cells upon anti-CTLA4 treatment protects against lymphedema development in patients with melanoma and in a mouse lymphedema model. A retrospective evaluation of a melanoma patient registry reveals that anti-CTLA4 reduces lymphedema risk; in parallel, anti-CTLA4 reduces edema and improves lymphatic function in a mouse-tail lymphedema model. This protective effect of anti-CTLA4 correlates with a systemic expansion of Tregs, both in the animal model and in patients with melanoma. Our data thus show that anti-CTLA4 with its lymphedema-protective and anti-tumor properties is a promising candidate for more diverse application in the clinics.

Atypical chemokine receptor 3 (ACKR3) is a scavenger of the chemokines CXCL11 and CXCL12 and of several opioid peptides. Additional evidence indicates that ACKR3 binds two other non-chemokine ligands, namely the peptide hormone adrenomedullin (AM) and derivatives of the proadrenomedullin N-terminal 20 peptide (PAMP). AM exhibits multiple functions in the cardiovascular system and is essential for embryonic lymphangiogenesis in mice. Interestingly, AM-overexpressing and ACKR3-deficient mouse embryos both display lymphatic hyperplasia. Moreover, in vitro evidence suggested that lymphatic endothelial cells (LECs), which express ACKR3, scavenge AM and thereby reduce AM-induced lymphangiogenic responses. Together, these observations have led to the conclusion that ACKR3-mediated AM scavenging by LECs serves to prevent overshooting AM-induced lymphangiogenesis and lymphatic hyperplasia. Here, we further investigated AM scavenging by ACKR3 in HEK293 cells and in human primary dermal LECs obtained from three different sources in vitro. LECs efficiently bound and scavenged fluorescent CXCL12 or a CXCL11/12 chimeric chemokine in an ACKR3-dependent manner. Conversely, addition of AM induced LEC proliferation but AM internalization was found to be independent of ACKR3. Similarly, ectopic expression of ACKR3 in HEK293 cells did not result in AM internalization, but the latter was avidly induced upon co-transfecting HEK293 cells with the canonical AM receptors, consisting of calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP)2 or RAMP3. Together, these findings indicate that ACKR3-dependent scavenging of AM by human LECs does not occur at ligand concentrations sufficient to trigger AM-induced responses mediated by canonical AM receptors.

Antibody-based therapeutics represent an important class of biopharmaceuticals in cancer immunotherapy. CD3 bispecific T-cell engagers activate cytotoxic T-cells and have shown remarkable clinical outcomes against several hematological malignancies. The absence of a costimulatory signal through CD28 typically leads to insufficient T-cell activation and early exhaustion. The combination of CD3 and CD28 targeting products offers an attractive strategy to boost T-cell activity. However, the development of CD28-targeting therapies ceased after TeGenero's Phase 1 trial in 2006 evaluating a superagonistic anti-CD28 antibody (TGN1412) resulted in severe life-threatening side effects. Here, we describe the generation of a novel fully human anti-CD28 antibody termed "E1P2" using phage display technology. E1P2 bound to human and mouse CD28 as shown by flow cytometry on primary human and mouse T-cells. Epitope mapping revealed a conformational binding epitope for E1P2 close to the apex of CD28, similar to its natural ligand and unlike the lateral epitope of TGN1412. E1P2, in contrast to TGN1412, showed no signs of in vitro superagonistic properties on human peripheral blood mononuclear cells (PBMCs) using different healthy donors. Importantly, an in vivo safety study in humanized NSG mice using E1P2, in direct comparison and contrast to TGN1412, did not cause cytokine release syndrome. In an in vitro activity assay using human PBMCs, the combination of E1P2 with CD3 bispecific antibodies enhanced tumor cell killing and T-cell proliferation. Collectively, these data demonstrate the therapeutic potential of E1P2 to improve the activity of T-cell receptor/CD3 activating constructs in targeted immunotherapeutic approaches against cancer or infectious diseases.

Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule that supports T cell activation, leukocyte migration, and (lymph)angiogenesis and has been shown to contribute to the pathology of various immune-mediated disorders, including asthma and corneal graft rejection. In contrast to monoclonal antibodies (mAbs) targeting ALCAM’s T cell expressed binding partner CD6, no ALCAM-targeting mAbs have thus far entered clinical development. This is likely linked with the broad expression of ALCAM on many different cell types, which increases the risk of eliciting unwanted treatment-induced side effects upon systemic mAb application. Targeting ALCAM in surface-exposed tissues, such as the lungs or the cornea, by a topical application could circumvent this issue. Here, we report the development of various stability- and affinity-improved anti-ALCAM mAb fragments with cross-species reactivity towards mouse, rat, monkey, and human ALCAM. Fragments generated in either mono- or bivalent formats potently blocked ALCAM–CD6 interactions in a competition ELISA, but only bivalent fragments efficiently inhibited ALCAM–ALCAM interactions in a leukocyte transmigration assay. The different fragments displayed a clear size-dependence in their ability to penetrate the human corneal epithelium. Furthermore, intranasal delivery of anti-ALCAM fragments reduced leukocyte infiltration in a mouse model of asthma, confirming ALCAM as a target for topical application in the lungs.

Tumor-associated lymph vessels and lymph node involvement are critical staging criteria in several cancers. In skin squamous cell carcinoma, lymph vessels play a role in cancer development and metastatic spread. However, their relationship with the cancer stem cell niche at early tumor stages remains unclear. To address this gap, we studied the lymph vessel localization at the cancer stem cell niche and observed an association from benign skin lesions to malignant stages of skin squamous cell carcinoma. By co-culturing lymphatic endothelial cells with cancer cell lines representing the initiation and promotion stages, and conducting RNA profiling, we observed a reciprocal induction of cell adhesion, immunity regulation, and vessel remodeling genes, suggesting dynamic interactions between lymphatic and cancer cells. Additionally, imaging analyses of the cultured cells revealed the establishment of heterotypic contacts between cancer cells and lymph endothelial cells, potentially contributing to the observed distribution and maintenance at the cancer stem cell niche, inducing downstream cellular responses. Our data provide evidence for an association of lymph vessels from the early stages of skin squamous cell carcinoma development, opening new avenues for better comprehending their involvement in cancer progression.

BACKGROUND:In this study, we describe the generation of a fully human monoclonal antibody (named '7NP2') targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types of solid neoplasms.METHODS:7NP2 was isolated from a synthetic antibody phage display library and was improved by one round of mutagenesis-based affinity maturation. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin (IL)-12 was generated and the anticancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. Furthermore, the safety of the fully human product (named IL12-7NP2) was evaluated in Cynomolgus monkeys.RESULTS:Biodistribution analysis in tumor-bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent antitumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates.CONCLUSIONS:The results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2.

To ensure proper immune function, most leukocytes constantly move within tissues or between them using the blood and lymphatic vessels as transport routes. While afferent lymphatic vessels transfer leukocytes from peripheral tissues to draining lymph nodes (dLNs), efferent lymphatics return lymphocytes from LNs back into the blood vascular circulation. Over the last decades, great progress has been made in our understanding of leukocyte migration into and within the lymphatic compartment, leading to the approval of new drugs targeting this process. In this review, we first introduce the anatomy of the lymphatic vasculature and the main cell types migrating through lymphatics. We primarily focus on dendritic cells (DCs) and T cells, the most prominent lymph-borne cell types, and discuss the functional significance as well as the main molecules and steps involved in their migration. Additionally, we provide an overview of the different techniques used to study lymphatic trafficking.

Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as Mycobacterium tuberculosis. The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose, Mycobacterium bovis BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated in vitro for analysis of their activation of T cells in vitro or in vivo after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1β in vivo. Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of M. bovis BCG.

Neutrophils are the first nonresident effector immune cells that migrate to a site of infection or inflammation; however, improper control of neutrophil responses can cause considerable tissue damage. Here, we found that neutrophil responses in inflamed or infected skin were regulated by CCR7-dependent migration and phagocytosis of neutrophils in draining lymph nodes (dLNs). In mouse models of Toll-like receptor–induced skin inflammation and cutaneous Staphylococcus aureus infection, neutrophils migrated from the skin to the dLNs via lymphatic vessels in a CCR7-mediated manner. In the dLNs, these neutrophils were phagocytosed by lymph node–resident type 1 and type 2 conventional dendritic cells. CCR7 up-regulation on neutrophils was a conserved mechanism across different tissues and was induced by a broad range of microbial stimuli. In the context of cutaneous immune responses, disruption of CCR7 interactions by selective CCR7 deficiency of neutrophils resulted in increased antistaphylococcal immunity and aggravated skin inflammation. Thus, neutrophil homing to and clearance in skin-dLNs affects cutaneous immunity versus pathology.

Tumour-draining lymph nodes (LNs) undergo massive remodelling including expansion of the lymphatic sinuses, a process that has been linked to lymphatic metastasis by creation of a pre-metastatic niche. However, the signals leading to these changes have not been completely understood. Here, we found that extracellular vesicles (EVs) derived from melanoma cells are rapidly transported by lymphatic vessels to draining LNs, where they selectively interact with lymphatic endothelial cells (LECs) as well as medullary sinus macrophages. Interestingly, uptake of melanoma EVs by LN-resident LECs was partly dependent on lymphatic VCAM-1 expression, and induced transcriptional changes as well as proliferation of those cells. Furthermore, melanoma EVs shuttled tumour antigens to LN LECs for cross-presentation on MHC-I, resulting in apoptosis induction in antigen-specific CD8(+) T cells. In conclusion, our data identify EV-mediated melanoma-LN LEC communication as a new pathway involved in tumour progression and tumour immune inhibition, suggesting that EV uptake or effector mechanisms in LECs might represent a new target for melanoma therapy.

Particulate antigen‐delivery systems are instrumental for therapeutic vaccination, aiming at improving the safety and efficacy of treatments by targeting specialized antigen‐presenting cells (APCs). However, the induction of potent adaptive immune responses, especially cellular immunity, remains a major challenge. Herein, a novel nanoparticulate antigen‐delivery system based on amphiphilic cyclodextrins (CDs) is developed as a platform for therapeutic cancer vaccination. Supramolecular nanosized CD structures are formed in aqueous media and loaded with peptide antigens. The nanoparticle's adjuvant capacity is tested in cell experiments with murine bone marrow‐derived dendritic cells (BMDCs) or macrophages and T cells. Peptide‐loaded nanoparticles cause upregulation of costimulatory molecules on BMDCs and facilitate activation and proliferation of antigen‐specific T‐lymphocytes in vitro. Correct processing for major histocompatibility complex (MHC) class‐I antigen presentation is demonstrated using a capped version of the ovalbumin‐derived peptide SIINFEKL (c‐SFL). After immunization of mice with peptide‐loaded CD nanoparticles, the frequencies of antigen‐specific and cytokine‐producing CD8 + T cells are increased. This work sheds light on the immune‐stimulating properties of amphiphilic CD nanoparticles and reveals their considerable potential as carriers for cancer vaccines.

Background T cell activation is initiated through engaging T cell receptor (TCR)/CD3 complex upon recognizing antigenic peptides presented by major histocompatibility complex (pMHC).1,2 However, TCR/CD3 complex alone is not sufficient for full T cell activation. In fact, the absence of an additional signal induces T cell exhaustion and thus impaired activation of T cells.3 CD28 is a crucial co-stimulatory receptor that enhances T cell proliferation, survival and production of key cytokines such as IL-2, IFN- γ, and TNF- α.4–7 Despite the potential key role of CD28 in cancer immunotherapy, there have been safety concerns following the TeGenero disaster in 2006.8 Here we describe the generation of a fully human anti-CD28 antibody named "VE19ZH". VE19ZH features a unique combination of desirable properties in comparison to other currently available mAbs such as TGN1412. Methods We isolated a new fully human antibody (VE19ZH) against human CD28 by phage display technology. Binding was validated by flow cytometry on primary human T cells. The co-stimulatory effect in combination with anti-CD3 (OKT3) was assessed in vitro by proliferation of human PBMCs and cytokine release. To rule out the undesirable super-agonistic effect observed by TGN1412, VE19ZH was tested for its capability to activate human PBMCs in the absence of TCR/CD3 signaling. Cross-reactivity against CTLA-4 and mouse CD28 were evaluated by ELISA and flow cytometry. Finally, the binding epitope of VE19ZH was revealed using PepSpot™ technology. Results VE19ZH is a fully human antibody that binds selectively to both human and murine CD28. VE19ZH IgG4 exhibited a strong and significant co-stimulatory effect on human PBMCs when combined with anti-human CD3 (OKT3). Unlike TGN1412, VE19ZH did not activate T cells without TCR/CD3 signaling. In addition, VE19ZH was shown to bind to CTLA-4. VE19ZH binds to an epitope similar to the natural ligand (CD80/CD86) as revealed by PepSpot™ technology. In vitro killing activity was validated using BiTE formats and showed synergism with low concentrations of CD3 bispecifics. Conclusions VE19ZH is a promising module for cancer immunotherapy with unique properties: (i) Fully human mAb for minimal immunogenicity (ii) Potent co-stimulator for full T cell activation (iii) Conventional agonist of CD28 and not super-agonistic like TGN1412 (iv) cross reacts with mouse CD28 for better assessment in immunocompetent mouse models (v) Binds to human CTLA-4 for potential checkpoint inhibition. The potential of VE19ZH to boost T cell response via CD28 activation and CTLA-4 blockade is currently being investigated in vitro and in vivo. References Davis MM, Bjorkman PJ. T-cell antigen receptor genes and T-cell recognition. Nature 1988;334(6181):395–402. La Gruta NL, et al, Understanding the drivers of MHC restriction of T cell receptors. Nat Rev Immunol 2018;18(7):467–478. Mueller DL, Jenkins MK, Schwartz RH. Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu Rev Immunol, 1989;7:445–80. Jenkins MK, et al. CD28 delivers a costimulatory signal involved in antigen-specific IL-2 production by human T cells. J Immunol 1991;147(8):2461–6. June CH, et al. T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression. Mol Cell Biol 1987;7(12):4472–81. Martin PJ, et al. A 44 kilodalton cell surface homodimer regulates interleukin 2 production by activated human T lymphocytes. J Immunol 1986;136(9):3282 Weiss A, Manger B, Imboden J. Synergy between the T3/antigen receptor complex and Tp44 in the activation of human T cells. J Immunol 1986;137(3):819–25. Suntharalingam G, et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med 2006;355(10):1018–28.

Background Fibroblast Activation Protein (FAP) has been described as the "next billion-dollar nuclear theranostics target"1, since more than 28 different tumor types have successfully been imaged in patients with radiolabeled FAP ligands. 2-3 FAP can be found in the tumor microenvironment (TME) of most malignant solid tumors, while being absent in most healthy tissues. Thus, it is an attractive target for both imaging and therapeutic applications. Monoclonal antibodies targeting TME antigens have been considered for the delivery of bioactive payloads, such as proinflammatory cytokines. Antibody-cytokine fusions (also called immunocytokines) may exploit the tumor-homing properties of the antibody moiety, in order to concentrate the cytokine payload at the site of disease and enhance the therapeutic index.4 Interleukin-12 (IL12) have been extensively studied in oncology. IL12 strongly promotes NK cells, CD4+ and CD8+ T cells to produce interferon-gamma (IFN-g), one of the most relevant mediators of anti-cancer immunity.5 Methods In this work, we describe the generation of a novel anti-FAP antibody, called 7NP2. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin-12 was generated and the anti-cancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. To prepare for future clinical trials, a fusion protein consisting of human IL12 linked to the 7NP2 antibody was further investigated in a toxicology study in Cynomolgus monkeys. Results Biodistribution analysis in tumor bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent anti-tumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates. Conclusions The results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2. References Calais J. FAP: The next billion dollar nuclear theranostics target?. Journal of Nuclear Medicine 2020;61(2). doi:10.2967/jnumed.119.241232. Kratochwil C, et al, 68Ga-FAPI PET/CT: Tracer uptake in 28 different kinds of cancer. Journal of Nuclear Medicine 2019;60(6). doi: 10.2967/jnumed.119.227967. Backhaus P, et al.Translational imaging of the fibroblast activation protein (FAP) using the new ligand [68Ga]Ga-OncoFAP-DOTAGA. European Journal of Nuclear Medicine and Molecular Imaging 2022;49(6). doi:10.1007/s00259-021-05653-0. Neri D. Antibody–Cytokine Fusions: Versatile Products for the Modulation of Anticancer Immunity. Cancer Immunology Research 2019. doi: 10.1158/2326-6066.CIR-18-0622. Puca E, et al. The antibody-based delivery of interleukin-12 to solid tumors boosts NK and CD8+ T cell activity and synergizes with immune checkpoint inhibitors. International Journal of Cancer 2019. doi:10.1002/ijc.32603. Ethics Approval Mouse experiments were performed under a project license (license number 04/2018) granted by the Veterinäramt des Kantons Zürich, Switzerland, in compliance with the Swiss Animal Protection Act (TSchG) and the Swiss Animal Protection Ordinance (TSchV). Procedures on Cynomolgus monkeys (including housing, health monitoring, restrain, dosing, etc) and ethical revision were performed according to the current Italian legislation (Legislative Decree March 4th, 2014 n. 26) enforcing the 2010/63/EU Directive on the protection of animals used for biomedical research.

T cell migration via afferent lymphatics to draining lymph nodes (dLNs) depends on expression of CCR7 in T cells and CCL21 in the lymphatic vasculature. Once T cells have entered lymphatic capillaries, they slowly migrate into contracting collecting vessels. Here, lymph flow picks up, inducing T cell detachment and rapid transport to the dLNs. We find that the atypical chemokine receptor 4 (ACKR4), which binds and internalizes CCL19 and CCL21, is induced by lymph flow in endothelial cells lining lymphatic collectors, enabling them to scavenge these chemokines. In the absence of ACKR4, migration of T cells to dLNs in TPA-induced inflammation is significantly reduced. While entry into capillaries is not impaired, T cells accumulate in the ACKR4-deficient dermal collecting vessel segments. Overall, our findings identify an ACKR4-mediated mechanism by which lymphatic collectors facilitate the detachment of lymph-borne T cells in inflammation and their transition from crawling to free-flow toward the dLNs.