Antitumor synergism between PAK4 silencing and immunogenic phototherapy of engineered extracellular vesicles.

Immunotherapy has revolutionized the landscape of cancer treatment. However, single immunotherapy only works well in a small subset of patients. Combined immunotherapy with antitumor synergism holds considerable potential to boost the therapeutic outcome. Nevertheless, the synergistic, additive or antagonistic antitumor effects of combined immunotherapies have been rarely explored. Herein, we established a novel combined cancer treatment modality by synergizing p21-activated kinase 4 (PAK4) silencing with immunogenic phototherapy in engineered extracellular vesicles (EVs) that were fabricated by coating M1 macrophage-derived EVs on the surface of the nano-complex cores assembled with siRNA against PAK4 and a photoactivatable polyethyleneimine. The engineered EVs induced potent PAK4 silencing and robust immunogenic phototherapy, thus contributing to effective antitumor effects in vitro and in vivo. Moreover, the antitumor synergism of the combined treatment was quantitatively determined by the CompuSyn method. The combination index (CI) and isobologram results confirmed that there was an antitumor synergism for the combined treatment. Furthermore, the dose reduction index (DRI) showed favorable dose reduction, revealing lower toxicity and higher biocompatibility of the engineered EVs. Collectively, the study presents a synergistically potentiated cancer treatment modality by combining PAK4 silencing with immunogenic phototherapy in engineered EVs, which is promising for boosting the therapeutic outcome of cancer immunotherapy.

Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities.

PURPOSE: The epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors. EGFR-specific monoclonal antibodies (mAbs), such as cetuximab and panitumumab, have been approved for the treatment of colorectal and head and neck cancer. To increase tissue penetration, we constructed single-chain fragment variable (scFv) antibodies derived from these mAbs and evaluated their potential for targeted cancer therapy. The resulting scFv-based EGFR-specific immunotoxins (ITs) combine target specificity of the full-size mAb with the cell-killing activity of a toxic effector domain, a truncated version of Pseudomonas exotoxin A (ETA'). METHODS: The ITs and corresponding imaging probes were tested in vitro against four solid tumor entities (rhabdomyosarcoma, breast, prostate and pancreatic cancer). Specific binding and internalization of the ITs scFv2112-ETA' (from cetuximab) and scFv1711-ETA' (from panitumumab) were demonstrated by flow cytometry and for the scFv-SNAP-tag imaging probes by live cell imaging. Cytotoxic potential of the ITs was analyzed in cell viability and apoptosis assays. Binding of the ITs was proofed ex vivo on rhabdomyosarcoma, prostate and breast cancer formalin-fixed paraffin-embedded biopsies. RESULTS: Both novel ITs showed significant pro-apoptotic and anti-proliferative effects toward the target cells, achieving IC50 values of 4 pM (high EGFR expression) to 460 pM (moderate EGFR expression). Additionally, rapid internalization and specific in vitro and ex vivo binding on patient tissue were confirmed. CONCLUSIONS: These data demonstrate the potent therapeutic activity of two novel EGFR-specific ETA'-based ITs. Both molecules are promising candidates for further development toward clinical use in the treatment of various solid tumors to supplement the existing therapeutic regimes.

Recombinant anti-EGFR immunotoxin 425(scFv)-ETA' demonstrates anti-tumor activity against disseminated human pancreatic cancer in nude mice.

Pancreatic carcinoma is the fifth leading cause of cancer-related deaths in North America and Europe. Major reasons for the high mortality rate include the inability to detect pancreatic cancer at an early stage, extensive local invasion, and early formation of lymphatic and hematogenous metastases. Consequently, novel and effective therapies need to be developed urgently in order to improve the outcome of patients. Since overexpression of the epidermal growth factor receptor (EGFR) in pancreatic tumors correlates with advanced clinical staging, increased tumor size and reduced patient survival, this receptor represents an appropriate target for immunotherapy. We recently generated the recombinant immunotoxin 425(scFv)-ETA' by genetically fusing the anti-EGFR single chain variable fragment 425(scFv) to a truncated version of Pseudomonas aeroginosa exotoxin A (ETA'). The 425(scFv)-ETA' fusion protein was functionally expressed in the periplasmic space of Escherichia coli and was purified using a combination of metal-ion affinity and anion exchange chromatography. The protein showed specific binding to and toxicity against the EGFR-positive, metastatic pancreatic carcinoma cell line L3.6pl, but not to control cell systems. We report the anti-tumor activity of this recombinant immunotoxin in a disseminated human pancreatic cancer nude mouse model. After intravenous (i.v.) injection of L3.6pl cells into immunodeficient nude mice, both single (20 microg on day 1 after challenge) and repeated (10 microg on days 1, 2, 3 and 4 after tumor cell injection) i.v. administration of 425(scFv)-ETA' resulted in a significant reduction in the average number of lung metastases from 56.25 per animal in the control groups to 0.875 per animal (single injection) and 0.286 per animal (repeated injection), respectively, in the experimental groups. In summary, this is the first report showing an in vivo anti-tumor effect caused by the recombinant immunotoxin 425(scFv)-ETA' against disseminated growing metastatic human pancreatic carcinoma cells. Our data suggest that EGFR-specific antibody toxins could be suitable for further clinical investigation in the development of therapies for pancreatic carcinoma.