Bifunctional immune checkpoint-targeted antibody-ligand traps that simultaneously disable TGFß enhance the efficacy of cancer immunotherapy.

A majority of cancers fail to respond to immunotherapy with antibodies targeting immune checkpoints, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) or programmed death-1 (PD-1)/PD-1 ligand (PD-L1). Cancers frequently express transforming growth factor-ß (TGFß), which drives immune dysfunction in the tumor microenvironment by inducing regulatory T cells (Tregs) and inhibiting CD8+ and TH1 cells. To address this therapeutic challenge, we invent bifunctional antibody-ligand traps (Y-traps) comprising an antibody targeting CTLA-4 or PD-L1 fused to a TGFß receptor II ectodomain sequence that simultaneously disables autocrine/paracrine TGFß in the target cell microenvironment (a-CTLA4-TGFßRIIecd and a-PDL1-TGFßRIIecd). a-CTLA4-TGFßRIIecd is more effective in reducing tumor-infiltrating Tregs and inhibiting tumor progression compared with CTLA-4 antibody (Ipilimumab). Likewise, a-PDL1-TGFßRIIecd exhibits superior antitumor efficacy compared with PD-L1 antibodies (Atezolizumab or Avelumab). Our data demonstrate that Y-traps counteract TGFß-mediated differentiation of Tregs and immune tolerance, thereby providing a potentially more effective immunotherapeutic strategy against cancers that are resistant to current immune checkpoint inhibitors.

Milk derived colloid as a novel drug delivery carrier for breast cancer.

Triple negative breast cancer has an extremely poor prognosis when chemotherapy is no longer effective. To overcome drug resistance, novel drug delivery systems based on nanoparticles have had remarkable success. We produced a novel nanoparticle component 'MDC' from milk-derived colloid. In order to evaluate the anti-cancer effect of MDC, we conducted in vitro and in vivo experiments on cancer cell lines and a primary tumor derived breast xenograft. Doxorubicin (Dox) conjugated to MDC (MDC-Dox) showed higher cancer cell growth inhibition than MDC alone especially in cell lines with high EGFR expression. In a mouse melanoma model, MDC-Dox significantly suppressed tumor growth when compared with free Dox. Moreover, in a primary tumor derived breast xenograft, one of the mice treated with MDC-Dox showed partial regression, while mice treated with free Dox failed to show any suppression of tumor growth. We have shown that a novel nanoparticle compound made of simple milk-derived colloid has the capability for drug conjugation, and serves as a tumor-specific carrier of anti-cancer drugs. Further research on its safety and ability to carry various anti-cancer drugs into multiple drug-resistant primary breast models is warranted.

Inhibition of TGF-ß enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy.

EGF receptor (EGFR)-targeted monoclonal antibodies (mAb), such as cetuximab, execute their antitumor effect in vivo via blockade of receptor-ligand interactions and engagement of Fcγ receptors on immune effector cells that trigger antibody-dependent cell-mediated cytotoxicity (ADCC). We show that tumors counteract the in vivo antitumor activity of anti-EGFR mAbs by increasing tumor cell-autonomous expression of TGF-ß. We show that TGF-ß suppresses the expression of key molecular effectors of immune cell-mediated cytotoxicity, including Apo2L/TRAIL, CD95L/FasL, granzyme B, and IFN-γ. In addition to exerting an extrinsic inhibition of the cytotoxic function of immune effectors, TGF-ß-mediated activation of AKT provides an intrinsic EGFR-independent survival signal that protects tumor cells from immune cell-mediated apoptosis. Treatment of mice-bearing xenografts of human head and neck squamous cell carcinoma with cetuximab resulted in emergence of resistant tumor cells that expressed relatively higher levels of TGF-ß compared with untreated tumor-bearing mice. Although treatment with cetuximab alone forced the natural selection of TGF-ß-overexpressing tumor cells in nonregressing tumors, combinatorial treatment with cetuximab and a TGF-ß-blocking antibody prevented the emergence of such resistant tumor cells and induced complete tumor regression. Therefore, elevated levels of TGF-ß in the tumor microenvironment enable tumor cells to evade ADCC and resist the antitumor activity of cetuximab in vivo. Our results show that TGF-ß is a key molecular determinant of the de novo and acquired resistance of cancers to EGFR-targeted mAbs, and provide a rationale for combinatorial targeting of TGF-ß to improve anti-EGFR-specific antibody therapy of EGFR-expressing cancers.

Adenylate kinase 3 sensitizes cells to cigarette smoke condensate vapor induced cisplatin resistance.

BACKGROUND: The major established etiologic risk factor for bladder cancer is cigarette smoking and one of the major antineoplastic agents used for the treatment of advanced bladder cancer is cisplatin. A number of reports have suggested that cancer patients who smoke while receiving treatment have lower rates of response and decreased efficacy of cancer therapies. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the effect of cigarette smoke condensate (CSC) vapor on cisplatin toxicity in urothelial cell lines SV-HUC-1 and SCaBER cells. We showed that chronic exposure to CSC vapor induced cisplatin resistance in both cell lines. In addition, we found that the expression of mitochondrial-resident protein adenylate kinase-3 (AK3) is decreased by CSC vapor. We further observed that chronic CSC vapor-exposed cells displayed decreased cellular sensitivity to cisplatin, decreased mitochondrial membrane potential (ΔΨm) and increased basal cellular ROS levels compared to unexposed cells. Re-expression of AK3 in CSC vapor-exposed cells restored cellular sensitivity to cisplatin. Finally, CSC vapor increased the growth of the tumors and also curtail the response of tumor cells to cisplatin chemotherapy in vivo. CONCLUSIONS/SIGNIFICANCE: The current study provides evidence that chronic CSC vapor exposure affects AK3 expression and renders the cells resistant to cisplatin.

Resistance of cancers to immunologic cytotoxicity and adoptive immunotherapy via X-linked inhibitor of apoptosis protein expression and coexisting defects in mitochondrial death signaling.

The ability of cancers to evade immune surveillance and resist immunotherapy raises a fundamental question of how tumor cells survive in the presence of a competent immune system. Studies to address this question have primarily focused on mechanisms by which tumor cells avoid recognition by or induce tolerance in the immune system. However, little is known about whether cancer cells also acquire an intrinsic ability to resist killing by immune effectors. We find that cancer cells enhance their ability to withstand an attack by cytotoxic immune effector cells via acquisition of specific genetic alterations that interfere with the shared mitochondrial death signaling pathway entrained by granzyme B, IFN-gamma, and Apo2 ligand/tumor necrosis factor-related apoptosis inducing ligand (Apo2L/TRAIL), three key mediators of immunologic cell-mediated cytotoxicity. We show that the coexistence of specific mitochondrial signaling defects (either deletion of Bax, overexpression of Bcl-x(L), or deletion of Smac) with expression of X-linked inhibitor of apoptosis protein decreases the sensitivity of cancer cells to IFN-gamma/Apo2L/TRAIL- or granzyme B-induced apoptosis, lymphocyte-mediated cytotoxicity in vitro, and adoptive cellular immunotherapy in vivo. Conversely, negating X-linked inhibitor of apoptosis protein expression or function in tumor cells with defective mitochondrial signaling enables direct activation of caspase-3/-7 by granzyme B or Apo2L/TRAIL, and restores their susceptibility to immunologic cytotoxicity. These findings identify an important mechanism by which cancers evade elimination by immune effector cells and suggest that cancer immunotherapy might be improved by concurrent strategies to alleviate or circumvent the intrinsic mitochondrial death signaling defects that help cancer cells resist immunologic cytotoxicity.

Elimination of hepatic metastases of colon cancer cells via p53-independent cross-talk between irinotecan and Apo2 ligand/TRAIL.

The majority of colorectal cancers have lost/inactivated the p53 tumor suppressor gene. Using isogenic human colon cancer cells that differ only in their p53 status, we demonstrate that loss of p53 renders tumor cells relatively resistant to the topoisomerase I inhibitor, irinotecan. Whereas irinotecan-induced up-regulation of the proapoptotic proteins PUMA and Noxa requires p53, we find that irinotecan inhibits Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 and 5 (STAT3/5) signaling in both p53-proficient and p53-deficient tumor cells. We show that irinotecan inhibits JAK2-STAT3/5-dependent expression of survival proteins (Bcl-x(L) and XIAP) and cooperates with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) to facilitate p53-independent apoptosis of colon cancer cells. Whereas xenografts of p53-deficient colon cancer cells are relatively resistant to irinotecan compared with their p53-proficient counterparts, combined treatment with irinotecan and Apo2L/TRAIL eliminates hepatic metastases of both p53-proficient and p53-deficient cancer cells in vivo and significantly improves the survival of animals relative to treatment with either agent alone. Although the synergy between chemotherapy and Apo2L/TRAIL has been ascribed to p53, our data demonstrate that irinotecan enhances Apo2L/TRAIL-induced apoptosis of tumor cells via a distinct p53-independent mechanism involving inhibition of JAK2-STAT3/5 signaling. These findings identify a novel p53-independent channel of cross-talk between topoisomerase I inhibitors and Apo2L/TRAIL and suggest that the addition of Apo2L/TRAIL can improve the therapeutic index of irinotecan against both p53-proficient and p53-deficient colorectal cancers, including those that have metastasized to the liver.