Optimization strategies for expression of a novel bifunctional anti-PD-L1/TGFBR2-ECD fusion protein.

The novel anti-PD-L1/TGFBR2-ECD fusion protein (BR102) comprises an anti-PD-L1 antibody (HS636) which is fused at the C terminus of the heavy chain to a TGF-ß1 receptor Ⅱ ectodomain (TGFBR2-ECD), and which can sequester the PD-1/PD-L1 pathway and TGF-ß bioactivity in the immunosuppressive tumor microenvironment. In the expression of TGFBR2-ECD wild-type fused protein (BR102-WT), a 50 kDa clipped species was confirmed to be induced by proteolytic cleavage at a "QKS" site located in the N-terminus of the ectodomain, which resulted in the formation of IgG-like clipping. The matrix metalloproteinase-9 was determined to be associated with BR102-WT digestion. In addition, it was observed that the N-glycosylation modifications of the fusion protein were tightly involved in regulating proteolytic activity and the levels of cleavage could be significantly suppressed by MMP-inhibitors. To avoid proteolytic degradation, eliminating protease-sensitive amino acid motifs and introducing potential glycosylation were performed. Three sensitive motifs were mutated, and the levels of clipping were strongly restrained. The mutant candidates exhibited similar binding affinities to hPD-L1 and hTGF-ß1 as well as highly purified BR102-WT2. Furthermore, the mutants displayed more significant proteolytic resistance than that of BR102-WT2 in the lysate incubation reaction and the plasma stability test. Moreover, the bifunctional candidate Mu3 showed an additive antitumor effect in MC38/hPD-L1 bearing models as compared to that of with anti-PD-L1 antibody alone. In conclusion, in this study, the protease-sensitive features of BR102-WT were well characterized and efficient optimization was performed. The candidate BR102-Mutants exhibited advanced druggability in drug stability and displayed desirable antitumor activity.

Targeting the αv integrin/TGF-ß axis improves natural killer cell function against glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-ß activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-ß signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-ß axis as a potentially useful therapeutic target in GBM.

Structure-based discovery and redesign of TGF-ß1 Elbow epitope recognition by its type-II receptor in hypertrophic scarring biotherapy.

Transforming growth factor-ß1 (TGF-ß1) signaling pathway has been implicated in the fibroblast activation of hypertrophic scarring (HS). Previously, we proposed a new biotherapeutic strategy to combat HS by disrupting the intermolecular interaction of TGF-ß1 with its cognate type-II receptor (TßR-II). Here, we further demonstrate that the binding site of TGF-ß1 to TßR-II is not overlapped with the conformational wrist epitope and linear knuckle epitope that are traditionally recognized as the functional binding sites of bone morphogenetic protein-2 (BMP-2) to its type-II receptor (BMPR-II), which can thus be regarded as a new functional site we called elbow epitope. Structural, energetic, and dynamic investigations reveal that the elbow epitope consists of two sequentially discontinuous, spatially vicinal segments Loop30-34 and Turn90-95 ; they cannot work effectively to independently interact with TßR-II. Rational redesign of the epitope is performed using an integrated in silio-in vitro method based on crystal and modeled structure data. In the procedure, the two epitope segments are split from the interface of TGF-ß1-TßR-II complex and then connected with each other in a head-to-tail manner by adding a flexible poly-(Gly)n linker between them, thus resulting in a series of combined peptides. We found that the peptide affinity reaches maximum at n = 2, which shares a consistent binding mode with the elbow epitope at native complex interface. The linker of either too long (n > 2) or too short (n < 2) cannot properly place the gap space between the two segments, thus impairing the binding compatibility of designed peptides with TßR-II active site.

Cancer immunotherapy via targeted TGF-ß signalling blockade in TH cells.

Cancer arises from malignant cells that exist in dynamic multilevel interactions with the host tissue. Cancer therapies aiming to directly kill cancer cells, including oncogene-targeted therapy and immune-checkpoint therapy that revives tumour-reactive cytotoxic T lymphocytes, are effective in some patients1,2, but acquired resistance frequently develops3,4. An alternative therapeutic strategy aims to rectify the host tissue pathology, including abnormalities in the vasculature that foster cancer progression5,6; however, neutralization of proangiogenic factors such as vascular endothelial growth factor A (VEGFA) has had limited clinical benefits7,8. Here, following the finding that transforming growth factor-ß (TGF-ß) suppresses T helper 2 (TH2)-cell-mediated cancer immunity9, we show that blocking TGF-ß signalling in CD4+ T cells remodels the tumour microenvironment and restrains cancer progression. In a mouse model of breast cancer resistant to immune-checkpoint or anti-VEGF therapies10,11, inducible genetic deletion of the TGF-ß receptor II (TGFBR2) in CD4+ T cells suppressed tumour growth. For pharmacological blockade, we engineered a bispecific receptor decoy by attaching the TGF-ß-neutralizing TGFBR2 extracellular domain to ibalizumab, a non-immunosuppressive CD4 antibody12,13, and named it CD4 TGF-ß Trap (4T-Trap). Compared with a non-targeted TGF-ß-Trap, 4T-Trap selectively inhibited TH cell TGF-ß signalling in tumour-draining lymph nodes, causing reorganization of tumour vasculature and cancer cell death, a process dependent on the TH2 cytokine interleukin-4 (IL-4). Notably, the 4T-Trap-induced tumour tissue hypoxia led to increased VEGFA expression. VEGF inhibition enhanced the starvation-triggered cancer cell death and amplified the antitumour effect of 4T-Trap. Thus, targeted TGF-ß signalling blockade in helper T cells elicits an effective tissue-level cancer defence response that can provide a basis for therapies directed towards the cancer environment.

Profibrotic Activation of Human Macrophages in Systemic Sclerosis.

OBJECTIVE: Genome-wide gene expression studies implicate macrophages as mediators of fibrosis in systemic sclerosis (SSc), but little is known about how these cells contribute to fibrotic activation in SSc. We undertook this study to characterize the activation profile of SSc monocyte-derived macrophages and assessed their interaction with SSc fibroblasts. METHODS: Plasma and peripheral blood mononuclear cells (PBMCs) were obtained from whole blood from SSc patients (n = 24) and age- and sex-matched healthy controls (n = 12). Monocytes were cultured with autologous or allogeneic plasma to differentiate cells into macrophages. For reciprocal activation studies, macrophages were cocultured with fibroblasts using Transwell plates. RESULTS: The gene expression signature associated with blood-derived human SSc macrophages was enriched in SSc skin in an independent cohort and correlated with skin fibrosis. SSc macrophages expressed surface markers associated with activation and released CCL2, interleukin-6, and transforming growth factor ß under basal conditions (n = 8) (P < 0.05). Differentiation of healthy donor monocytes in plasma from SSc patients conferred the immunophenotype of SSc macrophages (n = 13) (P < 0.05). Transwell experiments demonstrated that coculture of SSc macrophages with SSc fibroblasts induced fibroblast activation (n = 3) (P < 0.05). CONCLUSION: These data demonstrate that the activation profile of SSc macrophages is profibrotic. SSc macrophages are activated under basal conditions and release mediators and express surface markers associated with both alternative and inflammatory macrophage activation. These findings also suggest that activation of SSc macrophages arises from soluble factors in local microenvironments. These studies implicate macrophages as likely drivers of fibrosis in SSc and suggest that therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.

TGF-ß induces ST2 and programs ILC2 development.

The molecular pathways underlying the development of innate lymphoid cells (ILCs) are mostly unknown. Here we show that TGF-ß signaling programs the development of ILC2s from their progenitors. Specifically, the deficiency of TGF-ß receptor II in bone marrow progenitors results in inefficient development of ILC2s, but not ILC1s or ILC3s. Mechanistically, TGF-ß signaling is required for the generation and maintenance of ILC2 progenitors (ILC2p). In addition, TGF-ß upregulates the expression of the IL-33 receptor gene Il1rl1 (encoding IL-1 receptor-like 1, also known as ST2) in ILC2p and common helper-like innate lymphoid progenitors (CHILP), at least partially through the MEK-dependent pathway. These findings identify a function of TGF-ß in the development of ILC2s from their progenitors.

The Sweet Kiss Breaching Immunological Self-Tolerance.

Metabolic alterations leading to overactivation of nutrient-energy-sensing pathways have been linked to altered immunological self-tolerance. Now, Zhang and colleagues (Immunity, 2019) have identified a key role for high glucose consumption in exacerbating autoimmunity in mice via induction of T helper (Th)17 cells. This reveals a novel mechanism underlying effects of diet during autoimmunity development with major translational implications.

Engineering the TGFß Receptor to Enhance the Therapeutic Potential of Natural Killer Cells as an Immunotherapy for Neuroblastoma.

PURPOSE: The ability of natural killer (NK) cells to lyse allogeneic targets, without the need for explicit matching or priming, makes them an attractive platform for cell-based immunotherapy. Umbilical cord blood is a practical source for generating banks of such third-party NK cells for "off-the-shelf" cell therapy applications. NK cells are highly cytolytic, and their potent antitumor effects can be rapidly triggered by a lack of HLA expression on interacting target cells, as is the case for a majority of solid tumors, including neuroblastoma. Neuroblastoma is a leading cause of pediatric cancer-related deaths and an ideal candidate for NK-cell therapy. However, the antitumor efficacy of NK cells is limited by immunosuppressive cytokines in the tumor microenvironment, such as TGFß, which impair NK cell function and survival. EXPERIMENTAL DESIGN: To overcome this, we genetically modified NK cells to express variant TGFß receptors, which couple a mutant TGFß dominant-negative receptor to NK-specific activating domains. We hypothesized that with these engineered receptors, inhibitory TGFß signals are effectively converted to activating signals. RESULTS: Modified NK cells exhibited higher cytotoxic activity against neuroblastoma in a TGFß-rich environment in vitro and superior progression-free survival in vivo, as compared with their unmodified controls. CONCLUSIONS: Our results support the development of "off-the-shelf" gene-modified NK cells, that overcome TGFß-mediated immune evasion, in patients with neuroblastoma and other TGFß-secreting malignancies.

Gut microbiota translocation promotes autoimmune cholangitis.

Gut microbiota and bacterial translocation have been implicated as significant contributors to mucosal immune responses and tolerance; alteration of microbial molecules, termed pathogen-associated molecular patterns (PAMP) and bacterial translocation are associated with immune pathology. However, the mechanisms by which dysregulated gut microbiota promotes autoimmunity is unclear. We have taken advantage of a well-characterized murine model of primary biliary cholangitis, dnTGFßRII mice, and an additional unique construct, toll-like receptor 2 (TLR2)-deficient dnTGFßRII mice coined dnTGFßRIITLR2-/- mice to investigate the influences of gut microbiota on autoimmune cholangitis. Firstly, we report that dnTGFßRII mice manifest altered composition of gut microbiota and that alteration of this gut microbiota by administration of antibiotics significantly alleviates T-cell-mediated infiltration and bile duct damage. Second, toll-like receptor 2 (TLR2)-deficient dnTGFßRII mice demonstrate significant exacerbation of autoimmune cholangitis when their epithelial barrier integrity was disrupted. Further, TLR2-deficiency mediates downregulated expression of tight junction-associated protein ZO-1 leading to increased gut permeability and bacterial translocation from gut to liver; use of antibiotics reduces microbiota translocation to liver and also decreases biliary pathology. In conclusion, our data demonstrates the important role of gut microbiota and bacterial translocation in the pathogenesis of murine autoimmune cholangitis.

SMAD4 promotes TGF-ß-independent NK cell homeostasis and maturation and antitumor immunity.

SMAD4 is the only common SMAD in TGF-ß signaling that usually impedes immune cell activation in the tumor microenvironment. However, we demonstrated here that selective deletion of Smad4 in NK cells actually led to dramatically reduced tumor cell rejection and augmented tumor cell metastases, reduced murine CMV clearance, as well as impeded NK cell homeostasis and maturation. This was associated with a downregulation of granzyme B (Gzmb), Kit, and Prdm1 in Smad4-deficient NK cells. We further unveiled the mechanism by which SMAD4 promotes Gzmb expression. Gzmb was identified as a direct target of a transcriptional complex formed by SMAD4 and JUNB. A JUNB binding site distinct from that for SMAD4 in the proximal Gzmb promoter was required for transcriptional activation by the SMAD4-JUNB complex. In a Tgfbr2 and Smad4 NK cell-specific double-conditional KO model, SMAD4-mediated events were found to be independent of canonical TGF-ß signaling. Our study identifies and mechanistically characterizes unusual functions and pathways for SMAD4 in governing innate immune responses to cancer and viral infection, as well as NK cell development.

miR-130a and miR-145 reprogram Gr-1+CD11b+ myeloid cells and inhibit tumor metastasis through improved host immunity.

Tumor-derived soluble factors promote the production of Gr-1+CD11b+ immature myeloid cells, and TGFß signaling is critical in their immune suppressive function. Here, we report that miR-130a and miR-145 directly target TGFß receptor II (TßRII) and are down-regulated in these myeloid cells, leading to increased TßRII. Ectopic expression of miR-130a and miR-145 in the myeloid cells decreased tumor metastasis. This is mediated through a downregulation of type 2 cytokines in myeloid cells and an increase in IFNγ-producing cytotoxic CD8 T lymphocytes. miR-130a- and miR-145-targeted molecular networks including TGFß and IGF1R pathways were correlated with higher tumor stages in cancer patients. Lastly, miR-130a and miR-145 mimics, as well as IGF1R inhibitor NT157 improved anti-tumor immunity and inhibited metastasis in preclinical mouse models. These results demonstrated that miR-130a and miR-145 can reprogram tumor-associated myeloid cells by altering the cytokine milieu and metastatic microenvironment, thus enhancing host antitumor immunity.

Tumor-Specific T-Cells Engineered to Overcome Tumor Immune Evasion Induce Clinical Responses in Patients With Relapsed Hodgkin Lymphoma.

Purpose Transforming growth factor-ß (TGF-ß) production in the tumor microenvironment is a potent and ubiquitous tumor immune evasion mechanism that inhibits the expansion and function of tumor-directed responses; therefore, we conducted a clinical study to discover the effects of the forced expression of a dominant-negative TGF-ß receptor type 2 (DNRII) on the safety, survival, and activity of infused tumor-directed T cells. Materials and Methods In a dose escalation study, eight patients with Epstein Barr virus-positive Hodgkin lymphoma received two to 12 doses of between 2 × 107 and 1.5 × 108 cells/m2 of DNRII-expressing T cells with specificity for the Epstein Barr virus-derived tumor antigens, latent membrane protein (LMP)-1 and LMP-2 (DNRII-LSTs). Lymphodepleting chemotherapy was not used before infusion. Results DNRII-LSTs were resistant to otherwise inhibitory concentrations of TGF-ß in vitro and retained their tumor antigen-specific activity. After infusion, the signal from transgenic T cells in peripheral blood increased up to 100-fold as measured by quantitative polymerase chain reaction for the transgene, with a corresponding increase in the frequency of functional LMP-specific T cells. Expansion was not associated with any acute or long-term toxicity. DNRII-LSTs persisted for up to ≥ 4 years. Four of the seven evaluable patients with active disease achieved clinical responses that were complete and ongoing in two patients at > 4 years, including in one patient who achieved only a partial response to unmodified tumor-directed T cells. Conclusion TGF-ß-resistant tumor-specific T cells safely expand and persist in patients with Hodgkin lymphoma without lymphodepleting chemotherapy before infusion. DNRII-LSTs can induce complete responses even in patients with resistant disease. Expression of DNRII may be useful for the many other tumors that exploit this potent immune evasion mechanism.