Multiple Myeloma Bone Marrow Mesenchymal Stromal Cells Inhibit CD8+ T Cell Function in a Process that May Implicate Fibroblast Activation Protein α.

BACKGROUND: Multiple myeloma (MM) is a malignant plasma cell proliferative disorder with limited immunotherapy treatment because of T cell dysfunction. OBJECTIVE: To investigate the immunomodulatory function of bone marrow mesenchymal stromal cells (MM-BMSCs) on CD8+ T cells. METHODS: Proliferation and cytotoxicity were detected by cell counting kit-8 assay. Cell cycle was detected by flow cytometry, and p16 expression was detected by PCR. The expression of fibroblast activation protein α (FAPα) was evaluated by immunohistochemistry. RESULTS: Co-culture of CD8+ T cells with MM-BMSCs decreased the cell survival rate and increased the killing rate (p=0.03, p=0.001, respectively), the percentage of cells in G0/G1 phase and p16 expression (p<0.001). FAPα was mainly in the mesenchymal matrix of the MM microenvironment and elevated in MM derived bone marrow compared to healthy donors (p<0.001). The FAPα inhibitor PT-100, increased survival and the killing rate (p<0.001, p=0.043, respectively), and decreased the percentage of cells in G0/G1 phase and p16 expression (p=0.024, p=0.004, respectively). CONCLUSION: Therefore, MM-BMSCs inhibit the proliferation and cytotoxicity of CD8+ T cells, significantly block the cell cycle and increase p16 expression in co-cultured CD8+ T cells in a cell-cell contact-dependent manner.

Embryonic FAP+ lymphoid tissue organizer cells generate the reticular network of adult lymph nodes.

The induction of adaptive immunity is dependent on the structural organization of LNs, which is in turn governed by the stromal cells that underpin LN architecture. Using a novel fate-mapping mouse model, we trace the developmental origin of mesenchymal LN stromal cells (mLNSCs) to a previously undescribed embryonic fibroblast activation protein-α (FAP)+ progenitor. FAP+ cells of the LN anlagen express lymphotoxin ß receptor (LTßR) and vascular cell adhesion molecule (VCAM), but not intercellular adhesion molecule (ICAM), suggesting they are early mesenchymal lymphoid tissue organizer (mLTo) cells. Clonal labeling shows that FAP+ progenitors locally differentiate into mLNSCs. This process is also coopted in nonlymphoid tissues in response to infection to facilitate the development of tertiary lymphoid structures, thereby mimicking the process of LN ontogeny in response to infection.

A DNA vaccine expressing an optimized secreted FAPα induces enhanced anti-tumor activity by altering the tumor microenvironment in a murine model of breast cancer.

Cancer-associated fibroblasts (CAFs), major components of the tumor microenvironment (TME), promote tumor growth and metastasis and inhibit the anti-tumor immune response. We previously constructed a DNA vaccine expressing human FAPα, which is highly expressed by CAFs, to target these cells in the TME, and observed limited anti-tumor effects in the 4T1 breast cancer model. When the treatment time was delayed until tumor nodes formed, the anti-tumor effect of the vaccine completely disappeared. In this study, to improve the safety and efficacy, we constructed a new FAPα-targeted vaccine containing only the extracellular domain of human FAPα with a tissue plasminogen activator signal sequence for enhanced antigen secretion and immunogenicity. The number of CAFs was more effectively reduced by CD8+ T cells induced by the new vaccine. This resulted in decreases in CCL2 and CXCL12 expression, leading to a significant decrease in the ratio of myeloid-derived suppressor cells in the TME. Moreover, when mice were treated after the establishment of tumors, the vaccine could still delay tumor growth. To facilitate the future application of the vaccine in clinical trials, we further optimized the gene codons and reduced the homology between the vaccine and the original sequence, which may be convenient for evaluating the vaccine distribution in the human body. These results indicated that the new FAPα-targeted vaccine expressing an optimized secreted human FAPα induced enhanced anti-tumor activity by reducing the number of FAPα+ CAFs and enhancing the recruitment of effector T cells in the 4T1 tumor model mice.

Photosensitizer-Encapsulated Ferritins Mediate Photodynamic Therapy against Cancer-Associated Fibroblasts and Improve Tumor Accumulation of Nanoparticles.

Nanoparticles have been widely tested as drug delivery carriers or imaging agents, largely because of their ability to selectively accumulate in tumors through the enhanced permeability and retention (EPR) effect. However, studies show that many tumors afford a less efficient EPR effect and that many nanoparticles are trapped in the perivascular region after extravasation and barely migrate into tumor centers. This is to a large degree attributed to the dense tumor extracellular matrix (ECM), which functions as a physical barrier to prevent efficient nanoparticle extravasation and diffusion. In this study, we report a photodynamic therapy (PDT) approach to enhance tumor uptake of nanoparticles. Briefly, we encapsulate ZnF16Pc, a photosensitizer, into ferritin nanocages, and then conjugate to the surface of the ferritin a single chain viable fragment (scFv) sequence specific to fibroblast activation protein (FAP). FAP is a plasma surface protein widely upregulated in cancer-associated fibroblasts (CAFs), which is a major source of the ECM fiber components. We found that the scFv-conjugated and ZnF16Pc-loaded ferritin nanoparticles (scFv-Z@FRT) can mediate efficient and selective PDT, leading to eradication of CAFs in tumors. When tested in bilateral 4T1 tumor models, we found that the tumor accumulation of serum albumin (BSA), 10 nm quantum dots (QDs), and 50 nm QDs was increased by 2-, 3.5-, and 18-fold after scFv-Z@FRT mediated PDT. Our studies suggest a novel and safe method to enhance the delivery of nanoparticles to tumors.

Aberrant Lck Signal via CD28 Costimulation Augments Antigen-Specific Functionality and Tumor Control by Redirected T Cells with PD-1 Blockade in Humanized Mice.

Purpose: Combination therapy of adoptively transferred redirected T cells and checkpoint inhibitors aims for higher response rates in tumors poorly responsive to immunotherapy like malignant pleural mesothelioma (MPM). Only most recently the issue of an optimally active chimeric antigen receptor (CAR) and the combination with checkpoint inhibitors is starting to be addressed.Experimental Design: Fibroblast activation protein (FAP)-specific CARs with different costimulatory domains, including CD28, Δ-CD28 (lacking lck binding moiety), or 4-1BB were established. CAR-T cells were characterized in vitro and antitumor efficacy was tested in vivo in a humanized mouse model in combination with PD-1 blockade. Finally, the Δ-CD28 CAR was tested clinically in a patient with MPM.Results: All the three CARs demonstrated FAP-specific functionality in vitro Gene expression data indicated a distinct activity profile for the Δ-CD28 CAR, including higher expression of genes involved in cell division, glycolysis, fatty acid oxidation, and oxidative phosphorylation. In vivo, only T cells expressing the Δ-CD28 CAR in combination with PD-1 blockade controlled tumor growth. When injected into the pleural effusion of a patient with MPM, the Δ-CD28 CAR could be detected for up to 21 days and showed functionality.Conclusions: Overall, anti-FAP-Δ-CD28/CD3ζ CAR T cells revealed superior in vitro functionality, better tumor control in combination with PD-1 blockade in humanized mice, and persistence up to 21 days in a patient with MPM. Therefore, further clinical investigation of this optimized CAR is warranted. Clin Cancer Res; 24(16); 3981-93. ©2018 AACR.

Alteration of the Tumor Stroma Using a Consensus DNA Vaccine Targeting Fibroblast Activation Protein (FAP) Synergizes with Antitumor Vaccine Therapy in Mice.

Purpose: Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts and is an interesting target for cancer immune therapy, with prior studies indicating a potential to affect the tumor stroma. Our aim was to extend this earlier work through the development of a novel FAP immunogen with improved capacity to break tolerance for use in combination with tumor antigen vaccines.Experimental Design: We used a synthetic consensus (SynCon) sequence approach to provide MHC class II help to support breaking of tolerance. We evaluated immune responses and antitumor activity of this novel FAP vaccine in preclinical studies, and correlated these findings to patient data.Results: This SynCon FAP DNA vaccine was capable of breaking tolerance and inducing both CD8+ and CD4+ immune responses. In genetically diverse, outbred mice, the SynCon FAP DNA vaccine was superior at breaking tolerance compared with a native mouse FAP immunogen. In several tumor models, the SynCon FAP DNA vaccine synergized with other tumor antigen-specific DNA vaccines to enhance antitumor immunity. Evaluation of the tumor microenvironment showed increased CD8+ T-cell infiltration and a decreased macrophage infiltration driven by FAP immunization. We extended this to patient data from The Cancer Genome Atlas, where we find high FAP expression correlates with high macrophage and low CD8+ T-cell infiltration.Conclusions: These results suggest that immune therapy targeting tumor antigens in combination with a microconsensus FAP vaccine provides two-fisted punch-inducing responses that target both the tumor microenvironment and tumor cells directly. Clin Cancer Res; 24(5); 1190-201. ©2018 AACR.

Fibroblast activation proteins-α suppress tumor immunity by regulating T cells and tumor-associated macrophages.

Fibroblast activation protein-α (FAPα) is a type-II cell-surface-bound integral transmembrane serine protease and selectively overexpressed by tumor-associated stromal fibroblasts (TAFs), which are the main components in the tumor microenvironment, in >90% of malignant epithelial carcinomas. FAPα regulates the immunosuppression of tumor cells in the tumor microenvironment. Regulatory T cells (Tregs) and tumor-associated macrophages (TAMs) are the major immunosuppressive cells in the tumor microenvironment. However, the effect of FAPα on Tregs and TAMs is unknown. The non-enzymatic function of FAPα on Treg and TAM was investigated. In this study, we confirm that FAPα can promote the generation of Tregs and TAMs, which suggests that FAPα plays a immunosuppressive role in the tumor microenvironment and provides evidence for FAP α as a potent immunotherapeutic target for cancer.

FAP positive fibroblasts induce immune checkpoint blockade resistance in colorectal cancer via promoting immunosuppression.

Immune checkpoint blockades that significantly prolonged survival of melanoma patients have been less effective on colorectal cancer (CRC) patients. Growing evidence suggested that fibroblast activation protein-alpha (FAP) on cancer associate fibroblasts (CAFs) has critical roles in regulating antitumor immune response by inducing tumor-promoting inflammation. In this study, we explored the roles of FAP in regulating the tumor immunity and immune checkpoint blockades resistance in CRC experimental systems. We found that CAFs with high FAP expression could induce immune checkpoint blockade resistance in CRC mouse model. Mechanistically, CAFs with high FAP expression promoted immunosuppression in the CRC tumor immune microenvironment by up-regulating CCL2 secretion, recruiting myeloid cells, and decreasing T-cell activity. In human CRC samples, FAP expression was proportional to myeloid cells number, but inversely related to T-cell number. High FAP expression also predicted poor survival of CRC patients. Taken together, our study suggested that high FAP expression in CAFs is one reason leading to immune checkpoint blockades resistance in CRC patients and FAP is an optional target for reversing immune checkpoint blockades resistance.

Frontline Science: Tumor necrosis factor-α stimulation and priming of human neutrophil granule exocytosis.

Neutrophil granule exocytosis plays an important role in innate and adaptive immune responses. The present study examined TNF-α stimulation or priming of exocytosis of the 4 neutrophil granule subsets. TNF-α stimulated exocytosis of secretory vesicles and gelatinase granules and primed specific and azurophilic granule exocytosis to fMLF stimulation. Both stimulation and priming of exocytosis by TNF-α were dependent on p38 MAPK activity. Bioinformatic analysis of 1115 neutrophil proteins identified by mass spectrometry as being phosphorylated by TNF-α exposure found that actin cytoskeleton regulation was a major biologic function. A role for p38 MAPK regulation of the actin cytoskeleton was confirmed experimentally. Thirteen phosphoproteins regulated secretory vesicle quantity, formation, or release, 4 of which-Raf1, myristoylated alanine-rich protein kinase C (PKC) substrate (MARCKS), Abelson murine leukemia interactor 1 (ABI1), and myosin VI-were targets of the p38 MAPK pathway. Pharmacologic inhibition of Raf1 reduced stimulated exocytosis of gelatinase granules and priming of specific granule exocytosis. We conclude that differential regulation of exocytosis by TNF-α involves the actin cytoskeleton and is a necessary component for priming of the 2 major neutrophil antimicrobial defense mechanisms: oxygen radical generation and release of toxic granule contents.

Cyclophosphamide enhances anti-tumor effects of a fibroblast activation protein α-based DNA vaccine in tumor-bearing mice with murine breast carcinoma.

Cyclophosphamide (CY) is a DNA alkylating agent, which is widely used with other chemotherapy drugs in the treatment of various types of cancer. It can be used not only as a chemotherapeutic but also as an immunomodulatory agent to inhibit IL-10 expression and T regulatory cells (Tregs). Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts in the tumor microenvironment. Immunotherapy based on FAPα, as a tumor stromal antigen, typically induces specific immune response targeting the tumor microenvironment. This study evaluated the efficacy of a previously unreported CY combination strategy to enhance the limited anti-tumor effect of a DNA vaccine targeting FAPα. The results suggested CY administration could promote the percentage of splenic CD8+ T cells and decrease the proportion of CD4 + CD25 + Foxp3+ Tregs in spleen. In tumor tissues, levels of immunosuppressive cytokines including IL-10 and CXCL-12 were also reduced. Meanwhile, the CY combination did not impair the FAPα-specific immunity induced by the DNA vaccine and further reduced tumor stromal factors. Most importantly, FAP-vaccinated mice also treated with CY chemotherapy showed a marked suppression of tumor growth (inhibition ratio =80%) and a prolongation of survival time. Thus, the combination of FAPα immunotherapy and chemotherapy with CY offers new insights into improving cancer therapies.

Liposomal Treatment of Experimental Arthritis Can Be Monitored Noninvasively with a Radiolabeled Anti-Fibroblast Activation Protein Antibody.

Rheumatoid arthritis is a chronic autoimmune disorder resulting in synovial inflammation. Fibroblast activation protein (FAP) is overexpressed by fibroblastlike synoviocytes in arthritic joints. Radioimmunoimaging with an anti-FAP antibody might be used to monitor the response to therapy, thus enabling tailored therapy strategies and therapeutic outcomes. The aim of this study was to assess whether a radiolabeled anti-FAP antibody could be used to monitor the efficacy of treatment with long-circulating liposomes (LCL) containing prednisolone phosphate (PLP-LCL) in a mouse model of arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in male DBA/1J mice. Mice were treated with a single injection (10 mg/kg) of PLP-LCL or empty LCL as a control. SPECT and CT images were acquired 24 h after injection of 99mTc-labeled succinimidyl-hydrazinonicotinamide (99mTc-S-HYNIC)-conjugated anti-FAP antibody 28H1 at 2, 5, and 9 d after treatment. The uptake of 99mTc-S-HYNIC-28H1 in all joints was quantified and correlated with macroscopic arthritis scores. RESULTS: Treatment of CIA with PLP-LCL significantly suppressed joint swelling. At just 1 d after treatment, the macroscopic arthritis scores had decreased by 50%. Scores decreased further, to only 10% of the initial scores, at 5 and 9 d after treatment. In contrast, macroscopic arthritis scores had increased up to 600% in untreated mice at 9 d after the injection of empty LCL. 99mTc-S-HYNIC-28H1 uptake ranged from 1.5 percentage injected dose per gram in noninflamed joints to 22.6 percentage injected dose per gram in severely inflamed joints. The uptake of radiolabeled 28H1 in inflamed joints (percentage injected dose) correlated with the arthritis score (Spearman ρ, 0.77; P < 0.0001). Moreover, the uptake of 99mTc-S-HYNIC-28H1 was slightly increased at 9 d after therapy but was not seen macroscopically, indicating that SPECT/CT imaging might be more sensitive than the macroscopic arthritis scoring method. CONCLUSION: SPECT/CT imaging with 99mTc-S-HYNIC-28H1 specifically monitored the response to therapy, and tracer accumulation correlated with the severity of inflammation. In addition, SPECT/CT imaging was potentially more sensitive than the macroscopic arthritis scoring method. This study showed that SPECT/CT with 99mTc-S-HYNIC-28H1 could be used to noninvasively monitor the course of CIA in mice.

Protein Nanocage Mediated Fibroblast-Activation Protein Targeted Photoimmunotherapy To Enhance Cytotoxic T Cell Infiltration and Tumor Control.

Carcinoma-associated fibroblasts (CAFs) are found in many types of cancer and play an important role in tumor growth and metastasis. Fibroblast-activation protein (FAP), which is overexpressed on the surface of CAFs, has been proposed as a universal tumor targeting antigen. However, recent studies show that FAP is also expressed on multipotent bone marrow stem cells. A systematic anti-FAP therapy may lead to severe side effects and even death. Hence, there is an urgent need of a therapy that can selectively kill CAFs without causing systemic toxicity. Herein we report a nanoparticle-based photoimmunotherapy (nano-PIT) approach that addresses the need. Specifically, we exploit ferritin, a compact nanoparticle protein cage, as a photosensitizer carrier, and we conjugate to the surface of ferritin a FAP-specific single chain variable fragment (scFv). With photoirradiation, the enabled nano-PIT efficiently eliminates CAFs in tumors but causes little damage to healthy tissues due to the localized nature of the treatment. Interestingly, while not directly killing cancer cells, the nano-PIT caused efficient tumor suppression in tumor-bearing immunocompetent mice. Further investigations found that the nano-PIT led to suppressed C-X-C motif chemokine ligand 12 (CXCL12) secretion and extracellular matrix (ECM) deposition, both of which are regulated by CAFs in untreated tumors and mediate T cell exclusion that prevents physical contact between T cells and cancer cells. By selective killing of CAFs, the nano-PIT reversed the effect, leading to significantly enhanced T cell infiltration, followed by efficient tumor suppression. Our study suggests a new and safe CAF-targeted therapy and a novel strategy to modulate tumor microenvironment (TME) for enhanced immunity against cancer.

Improvement of anti-tumor immunity of fibroblast activation protein α based vaccines by combination with cyclophosphamide in a murine model of breast cancer.

Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts (CAFs), which are the main type of cells in the tumor microenvironment. CAFs exert immunosuppressive activity, which can weaken the effects of cancer immunotherapy and mainly account for poor outcomes with therapeutic vaccines. To better target and destroy CAFs, a FAPα vaccine using a modified vaccinia ankara (MVA) vector was constructed and used with a DNA vaccine reported in our previous work for heterologous prime-boost immunizations in mice. This strategy to generate anti-tumor immunity partly reduced 4T1 tumor growth through producing FAPα-specific cytotoxic T lymphocyte responses in a preventive model, but the effect required improvement. Combining the FAPα-based cancer vaccines (CpVR-FAP/MVA-FAP) with cyclophosphamide (CY), which can be used not only as a chemotherapeutic but also an immunomodulatory agent to promote a shift from immunosuppression to immunopotentiation, resulted in markedly enhanced tumor growth inhibition compared with the CpVR-FAP/MVA-FAP group. This strategy achieved synergistic effects in a therapeutic model by improving the tumor inhibition rate by 2.5-fold (90.2%), significantly enhancing cellular immunity and prolonging the survival of 4T1 tumor-bearing mice by 35% compared with the PBS group. Furthermore, CAFs, stromal factors and immunosuppressive factors such as IL-10 and Tregs were also markedly decreased by the CY combination. These results indicated that FAPα-targeted MVA boosting in combination with CY is an effective approach to improving specific anti-tumor immune responses through overcoming immunosuppression. This study may offer important advances in research on clinical cancer immunotherapies by modulating immunosuppressive factors.

Enhancement of fibroblast activation protein α-based vaccines and adenovirus boost immunity by cyclophosphamide through inhibiting IL-10 expression in 4T1 tumor bearing mice.

Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts (CAFs) of more than 90% of malignant epithelia carcinomas. CAFs are the main type of cells in the tumor microenvironment which offer nutrition and protection to the tumor and regulate immunosuppression. To eliminate CAFs, a vaccine targeting FAPα may be used with a heterologous prime-boost strategy to enhance the FAPα-specific cellular immunity. Here, a FAP vaccine using a recombinant adenovirus (rAd) vector was constructed as well as a DNA vaccine reported in our previous work. Although the DNA prime-rAd boost strategy enhanced FAPα-specific immune responses, improvement of anti-tumor immunity effects was not observed. Examination of immunosuppressive factors revealed that high expression of the IL-10 cytokine was considered the main cause of the failure of the prime-boost strategy. However, heterologous vaccination in combination with a low-dose of cyclophosphamide (CY), which was reported to reduce IL-10 production and promote a shift from immunosuppression to immunopotentiation, resulted in enhanced effects in terms of numbers of effector T cells and tumor growth inhibition rates, compared to the CY alone or DNA alone group. Tumor growth was inhibited markedly when the prime-boost strategy was combined with CY in both the prophylactic and therapeutic settings and the survival time of 4T1 tumor bearing mice was also prolonged significantly. With the reduction of IL-10, enhancement of the anti-tumor effect by the prime-boost strategy was observed. These results suggest that FAPα-targeted rAd boosting in combination with CY is an attractive approach to overcoming immunosuppression in cancer vaccines.

RG7386, a Novel Tetravalent FAP-DR5 Antibody, Effectively Triggers FAP-Dependent, Avidity-Driven DR5 Hyperclustering and Tumor Cell Apoptosis.

Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946-57. ©2016 AACR.

The application of the fibroblast activation protein α-targeted immunotherapy strategy.

Cancer immunotherapy has primarily been focused on attacking tumor cells. However, given the close interaction between tumor cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME), CAF-targeted strategies could also contribute to an integrated cancer immunotherapy. Fibroblast activation protein α (FAP α) is not detectible in normal tissues, but is overexpressed by CAFs and is the predominant component of the stroma in most types of cancer. FAP α has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a post-proline bond. When all FAP α-expressing cells (stromal and cancerous) are destroyed, tumors rapidly die. Furthermore, a FAP α antibody, FAP α vaccine, and modified vaccine all inhibit tumor growth and prolong survival in mouse models, suggesting FAP α is an adaptive tumor-associated antigen. This review highlights the role of FAP α in tumor development, explores the relationship between FAP α and immune suppression in the TME, and discusses FAP α as a potential immunotherapeutic target.

Anti-tumor effects of DNA vaccine targeting human fibroblast activation protein α by producing specific immune responses and altering tumor microenvironment in the 4T1 murine breast cancer model.

Fibroblast activation protein α (FAPα) is a tumor stromal antigen overexpressed by cancer-associated fibroblasts (CAFs). CAFs are genetically more stable compared with the tumor cells and immunosuppressive components of the tumor microenvironment, rendering them excellent targets for cancer immunotherapy. DNA vaccines are widely applied due to their safety. To specifically destroy CAFs, we constructed and examined the immunogenicity and anti-tumor immune mechanism of a DNA vaccine expressing human FAPα. This vaccine successfully reduced 4T1 tumor growth through producing FAPα-specific cytotoxic T lymphocyte responses which could kill CAFs, and the decrease in FAPα-expressing CAFs resulted in markedly attenuated expression of collagen I and other stromal factors that benefit the tumor progression. Based on these results, a DNA vaccine targeting human FAPα may be an attractive and effective cancer immunotherapy strategy.

Immunization of stromal cell targeting fibroblast activation protein providing immunotherapy to breast cancer mouse model.

Unlike heterogeneous tumor cells, cancer-associated fibroblasts (CAF) are genetically more stable which serve as a reliable target for tumor immunotherapy. Fibroblast activation protein (FAP) which is restrictively expressed in tumor cells and CAF in vivo and plays a prominent role in tumor initiation, progression, and metastasis can function as a tumor rejection antigen. In the current study, we have constructed artificial FAP(+) stromal cells which mimicked the FAP(+) CAF in vivo. We immunized a breast cancer mouse model with FAP(+) stromal cells to perform immunotherapy against FAP(+) cells in the tumor microenvironment. By forced expression of FAP, we have obtained FAP(+) stromal cells whose phenotype was CD11b(+)/CD34(+)/Sca-1(+)/FSP-1(+)/MHC class I(+). Interestingly, proliferation capacity of the fibroblasts was significantly enhanced by FAP. In the breast cancer-bearing mouse model, vaccination with FAP(+) stromal cells has significantly inhibited the growth of allograft tumor and reduced lung metastasis indeed. Depletion of T cell assays has suggested that both CD4(+) and CD8(+) T cells were involved in the tumor cytotoxic immune response. Furthermore, tumor tissue from FAP-immunized mice revealed that targeting FAP(+) CAF has induced apoptosis and decreased collagen type I and CD31 expression in the tumor microenvironment. These results implicated that immunization with FAP(+) stromal cells led to the disruption of the tumor microenvironment. Our study may provide a novel strategy for immunotherapy of a broad range of cancer.

Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage.

Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.

Unravelling the immunological roles of dipeptidyl peptidase 4 (DPP4) activity and/or structure homologue (DASH) proteins.

Dipeptidyl peptidase (DPP) 4 (CD26, DPP4) is a multi-functional protein involved in T cell activation by co-stimulation via its association with adenosine deaminase (ADA), caveolin-1, CARMA-1, CD45, mannose-6-phosphate/insulin growth factor-II receptor (M6P/IGFII-R) and C-X-C motif receptor 4 (CXC-R4). The proline-specific dipeptidyl peptidase also modulates the bioactivity of several chemokines. However, a number of enzymes displaying either DPP4-like activities or representing structural homologues have been discovered in the past two decades and are referred to as DPP4 activity and/or structure homologue (DASH) proteins. Apart from DPP4, DASH proteins include fibroblast activation protein alpha (FAP), DPP8, DPP9, DPP4-like protein 1 (DPL1, DPP6, DPPX L, DPPX S), DPP4-like protein 2 (DPL2, DPP10) from the DPP4-gene family S9b and structurally unrelated enzyme DPP2, displaying DPP4-like activity. In contrast, DPP6 and DPP10 lack enzymatic DPP4-like activity. These DASH proteins play important roles in the immune system involving quiescence (DPP2), proliferation (DPP8/DPP9), antigen-presenting (DPP9), co-stimulation (DPP4), T cell activation (DPP4), signal transduction (DPP4, DPP8 and DPP9), differentiation (DPP4, DPP8) and tissue remodelling (DPP4, FAP). Thus, they are involved in many pathophysiological processes and have therefore been proposed for potential biomarkers or even drug targets in various cancers (DPP4 and FAP) and inflammatory diseases (DPP4, DPP8/DPP9). However, they also pose the challenge of drug selectivity concerning other DASH members for better efficacy and/or avoidance of unwanted side effects. Therefore, this review unravels the complex roles of DASH proteins in immunology.