Immunotherapies for NSCLC: Are We Cutting the Gordian Helix?

Chemotherapy is currently the standard-of-care for non-oncogene-driven advanced non-small cell lung cancer (NSCLC). Due to improvements in chemotherapeutic choices and supportive care, patients currently typically undergo multiple lines of chemotherapy as their disease progresses. Although treatments have improved over recent years, limited benefits are seen, especially in patients receiving later-line chemotherapy, as response rates can be low, response duration short and survival poor. Molecular-targeted therapies have provided improvement in outcomes. However, these treatments only offer a clear benefit in subsets of tumors harbouring the appropriate genomic alteration (mutation, amplification, translocation). Recent advances in immunotherapy have highlighted the potential of immuno-oncology-based treatments for NSCLC, offering the potential to provide durable responses and outcomes regardless of histology or mutation status. Blocking inhibitory pathways such as the cytotoxic lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) checkpoint pathways with monoclonal antibodies has generated antitumor immune responses that are transforming cancer therapeutics. PD-1 and programmed cell death ligand-1(PD-L1) antibodies have shown durable responses in NSCLC, with a favourable safety profile and manageable side-effects. The activity of immune checkpoint inhibitors is currently been assessed in treatment-naïve patients with PD-L1-positive advanced NSCLC. Combinatorial approaches with other immune checkpoint inhibitors, chemotherapy, or targeted-agents are being explored in ongoing clinical trials, and may improve outcome in NSCLC. The emerging data not only offer the hope of a better cancer therapy but also provide evidence that changes our understanding on how the host immune system interacts with human cancer. It is therefore conceivable that agents blocking the CTLA-4/PD-1/PD-L1 axis will provide valuable additions to the growing armamentarium of targeted-agents.

Brain Metastases in NSCLC - are TKIs Changing the Treatment Strategy?

Non-small-cell lung cancer (NSCLC) ranks as a leading cause of cancer-related death globally. Brain metastases are a frequent complication of NSCLC, with 25-40% of patients developing brain metastases during the course of the disease, often within the first 2 years after diagnosis of the primary tumor. Improvements in neurological symptoms and performance status have been reported with whole-brain radiation therapy (WBRT) in combination with steroid therapy in NSCLC patients. In addition, a survival benefit has been reported for patients with a single brain metastasis treated with stereotactic radiosurgery, while the clinical outcome is improved with surgery followed by WBRT versus WBRT alone. However, due to their poor performance status, many patients with brain metastases are not eligible for surgery or radiosurgery. Furthermore, the role of systemic chemotherapy for the treatment of brain metastases is controversial due to the impenetrable nature of the blood brain barrier (BBB), with reported response rates to chemotherapy ranging from 15-30% (overall survival [OS] 6-8 months). Response rates of brain metastases to EGFR tyrosine kinase inhibitor (TKI) treatment (e.g. gefitinib, erlotinib, afatinib) in patients with NSCLC harboring EGFR mutations reach 60-80%, with a complete response rate as high as 40%. Median OS is 15-20 months, and progression-free survival in the brain reaches 6.6-11.7 months, demonstrating an improved clinical outcome. Metastatic involvement of the CNS appears to be a relatively common complication in patients with ALK-positive NSCLC and the CNS represents a dominant site of progression in ALK-positive patients treated with the ALK TKI crizotinib. In addition, CNS progression on crizotinib contributes substantially to the high levels of morbidity and mortality observed among patients with ALK-rearrangements, a finding that is consistent with low CNS penetration of the drug. Second-generation ALK inhibitors (ceritinib, alectinib) are well-tolerated and demonstrate excellent intracranial activity. The various reports of dramatic and prolonged responses in brain metastases patients treated with EGFR and ALK TKIs suggest that these agents may be a valid treatment option for patients with asymptomatic brain metastases from NSCLC, especially for those with EGFR-activating mutations or harboring ALK rearrangement. However, larger phase III studies are required to fully define the activity of these agents and their place in the therapeutic armamentarium of brain metastases.

Ofatumumab in combination with ICE or DHAP chemotherapy in relapsed or refractory intermediate grade B-cell lymphoma.

Standard treatment of transplant-eligible patients with relapsed diffuse large B-cell lymphoma (DLBCL) consists of rituximab and platinum-based chemotherapy, either ifosfamide, carboplatin, and etoposide (ICE) or dexamethasone, cytarabine, and cisplatin (DHAP), with autologous transplant consolidation for those with chemosensitive disease. Nonetheless, outcomes are suboptimal for patients failing rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). We performed a multi-center phase II trial investigating the safety and efficacy of ofatumumab, a monoclonal antibody against CD20, combined with ICE or DHAP second-line therapy in patients with relapsed or refractory DLBCL, grade 3b follicular lymphoma, or transformed follicular lymphoma. Sixty-one patients were treated with either ofatumumab-ICE (35) or ofatumumab-DHAP (26). The overall response rate (ORR) was 61%, and the complete response (CR) rate was 37%. In patients with 2 or 3 adverse risk factors according to the second-line, age-adjusted, international prognostic index, the ORR was 59% and CR 31%, and in patients with early-relapsing or primary refractory disease, the ORR was 55% and CR 30%. Toxicity was largely hematologic, and stem cell mobilization was successful in 43 of 45 patients. Substitution of ofatumumab for rituximab in standard second-line regimens following failure of R-CHOP is a promising approach. This trial was registered at www.clinicaltrials.gov as NCT00823719.

Combretastatin dinitrogen-substituted stilbene analogues as tubulin-binding and vascular-disrupting agents.

Several stilbenoid compounds having structural similarity to the combretastatin group of natural products and characterized by the incorporation of two nitrogen-bearing groups (amine, nitro, serinamide) have been prepared by chemical synthesis and evaluated in terms of biochemical and biological activity. The 2',3'-diamino B-ring analogue 17 demonstrated remarkable cytotoxicity against selected human cancer cell lines in vitro (average GI 50 = 13.9 nM) and also showed good activity in regard to inhibition of tubulin assembly (IC 50 = 2.8 microM). In addition, a single dose (10 mg/kg) of compound 17 caused a 40% tumor-selective blood flow shutdown in tumor-bearing SCID mice at 24 h, thus suggesting the potential value of this compound and its corresponding salt formulations as new vascular-disrupting agents.

Design, synthesis, and biological evaluation of combretastatin nitrogen-containing derivatives as inhibitors of tubulin assembly and vascular disrupting agents.

A series of analogs with nitro or serinamide substituents at the C-2'-, C-5'-, or C-6'-position of the combretastatin A-4 (CA4) B-ring was synthesized and evaluated for cytotoxic effects against heart endothelioma cells, blood flow reduction to tumors in SCID mice, and as inhibitors of tubulin polymerization. The synthesis of these analogs typically featured a Wittig reaction between a suitably functionalized arylaldehyde and an arylphosphonium salt followed by separation of the resultant E- and Z-isomers. Several of these nitrogen-modified CA4 derivatives (both amino and nitro) demonstrate significant inhibition of tubulin assembly as well as cytotoxicity and in vivo blood flow reduction. 2'-Aminostilbenoid 7 and 2'-amino-3'-hydroxystilbenoid 29 proved to be the most active in this series. Both compounds, 7 and 29, have the potential for further pro-drug modification and development as vascular disrupting agents for treatment of solid tumor cancers and certain ophthalmological diseases.

Pharmacokinetics and splenic accumulation of N-acetylamino-3-chloro-N-(2-diethylamino-ethyl) benzamide after a single administration to rats.

The purpose of this study was to measure the pharmacokinetics and tissue accumulation of N-acetylamino-3-chloro-N-(2-diethylamino-ethyl) benzamide (NACPA) after oral or intravenous administration at a single dose of 25 mg/kg to female W/Fu rats. The serum pharmacokinetics of NACPA were characterized by rapid absorption, distribution and elimination. However, in comparison with its parent compound, 4-amino-3-chloro-N-(2-diethylamino-ethyl) benzamide (3-CPA), NACPA displayed a higher Cmax (mean+/-SD, 201+/-21 vs 33.6+/-0.5 nmol/ml, p < 0.05), and a longer elimination half-life (50+/-0.8 vs 36.6+/-1.1 min, p < 0.05) following intravenous administration. Bioavailability of NACPA was significantly greater than that of 3-CPA (50% compared with 14%, p < 0.05). The tissue accumulation of NACPA was generally higher than that of 3-CPA. NACPA was deposited at higher concentrations in the spleen than in the kidney and liver. Cellular pharmacokinetics indicated that NACPA accumulated more readily in lymphocyte related cells than in liver related cells. Furthermore, incubation of human peripheral lymphocytes with NACPA resulted in inhibition of lymphocyte proliferation, INF-gamma production and chemotaxis. All these results suggest that NACPA may be a good candidate drug for oral administration for immune modulation.

Neural progenitor cell lines inhibit rat tumor growth in vivo.

Current therapies for gliomas often fail to address their infiltrative nature. Conventional treatments leave behind small clusters of neoplastic cells, resulting in eventual tumor recurrence. In the present study, we have evaluated the antitumor activity of neural progenitor cells against gliomas when stereotactically injected into nucleus Caudatus of Fisher rats. We show that the rat neural progenitor cell lines HiB5 and ST14A, from embryonic hippocampus and striatum primordium, respectively, are able to prolong animal survival and, in 25% of the cases, completely inhibit the outgrowth of N29 glioma compared with control animals. Delayed tumor outgrowth was also seen when HiB5 cells were inoculated at the site of tumor growth 1 week after tumor inoculation or when a mixture of tumor cells and HiB5 cells were injected s.c. into Fisher rats. HiB5 cells were additionally coinoculated together with two alternative rat gliomas, N32 and N25. N32 was growth inhibited, but rats inoculated with N25 cells did not show a prolonged survival. To evaluate the possibility of the involvement of the immune system in the tumor outgrowth inhibition, we show that HiB5 cells do not evoke an immune response when injected into Fisher rats. Furthermore, the rat neural progenitor cells produce all transforming growth factor beta isotypes, which could explain the observed immunosuppressive nature of these cells. Hence, some neural progenitor cells have the ability to inhibit tumor outgrowth when implanted into rats. These results indicate the usefulness of neural stem cells as therapeutically effective cells for the treatment of intracranial tumors.

Combretastatin family member OXI4503 induces tumor vascular collapse through the induction of endothelial apoptosis.

The mechanism of tumor cell killing by OXI4503 was investigated by studying vascular functional and morphological changes post drug administration. SCID mice bearing MHEC5-T hemangioendothelioma were given a single dose of OXI4503 at 100 mg/kg. Tumor blood flow, measured by microsphere fluorescence, was reduced by 50% at 1 hr, and reached a maximum level 6-24 hr post drug treatment. Tumor vascular permeability, measured by Evan's blue and hemoglobin, increased significantly from 3 hr and peaked at 18 hr. The elevated tumor vessel permeability was accompanied by an increase in vascular endothelial growth factor (VEGF) from 1 hr post drug treatment. Immunohistochemical staining for CD31 and laminin showed that tumor blood vessels were affected as early as 3 hr but more prominent from 6 hr. From 12 hr, the vessel structure was completely destroyed. Histopathological and double immunohistochemical staining showed morphological change and induction of apoptosis in endothelial cells at 1-3 hr, followed by tumor cell necrosis from 6-72 hr. There were no statistically significant changes of Evan's blue and hemoglobin contents in liver tissue over the time course. These results suggest that OXI4503 selectively targets tumor blood vessels, and induces blood flow shutdown while it enhances tumor blood vessel permeability. The early induction of endothelial cell apoptosis leads to functional changes of tumor blood vessels and finally to the collapse of tumor vasculature, resulting in massive tumor cell necrosis. The time course of the tumor vascular response observed with OXI4503 treatment supports this drug for development as a stand alone therapy, and also lends support for the use of the drug in combination with other cancer therapies.

Insulin induces SOCS-6 expression and its binding to the p85 monomer of phosphoinositide 3-kinase, resulting in improvement in glucose metabolism.

The suppressors of cytokine signaling (SOCS) family is thought to act largely as a negative regulator of signaling by cytokines and some growth factors. Surprisingly, the SOCS-6 transgenics had no significant defects in the cytokine signaling and hematopoietic system but displayed significant improvements in glucose metabolism. Insulin stimulation of Akt/protein kinase B was also potentiated. Biochemical analysis showed that, after insulin stimulation, SOCS-6 interacted with the monomeric p85 subunit of class-Ia phosphoinositide (PI) 3-kinase but not with p85/p110 dimers. Furthermore, SOCS-6 expression is transiently increased by serum and insulin in normal fibroblasts. However, both the mRNA and protein of SOCS-6 were rapidly degraded after induction by insulin. The degradation of the SOCS-6 protein was partially inhibited by a proteasome inhibitor, suggesting a proteasome-mediated degradation mechanism. In contrast, SOCS-6-associated p85 was not degraded and could be recruited to the newly synthesized SOCS-6 molecules in the presence of insulin, suggesting that SOCS-6 expression and its interaction with p85, but not the degradation, is regulated by insulin. The phenotype of SOCS-6 transgenic mice bears a striking resemblance to p85 knock-out mouse models in which glucose metabolism stimulated by insulin is significantly improved despite reduced activation of PI 3-kinase. This suggests that monomeric p85 might play a physiologically important role in attenuating signaling through PI 3-kinase-dependent pathways in unstimulated cells. Therefore, our results indicate that SOCS-6 may provide a dynamically regulated mechanism by which insulin can transiently overcome the negative effects that p85 monomers have on signaling via PI 3-kinase-dependent signaling pathways.

LEPREL1, a novel ER and Golgi resident member of the Leprecan family.

We have identified a novel protein, Leprecan-like 1 (LEPREL1), with profound similarity to the Leprecan family of proteoglycans. The genomic organization of the Leprecan gene family was found to be highly conserved. Expression analysis shows that LEPREL1 is expressed in most tissues as a 3.4 kb transcript encoding an 80 kDa protein. A LEPREL1 specific antibody stains many cell types including adipocytes and neuroendocrine cells of the gastrointestinal epithelium. Muscle tissue contains a specific 6.5 kb transcript and a 200 kDa protein. The 3.4 kb LEPREL1 transcript encodes a 708 amino acid protein containing a signal sequence, four tetratricopeptide repeats (TPRs), a leucine zipper, a P-loop, a prolyl 4-hydroxylase alpha domain (P4Halpha), and a C-terminal KDEL ER-retention motif. LEPREL1 is localized to the ER and Golgi network and over-expressing it affects normal protein disulfide isomerase staining patterns in the ER.

Identification and characterization of a human smad3 splicing variant lacking part of the linker region.

Smad3 is one of the signal transducers that are activated in response to transforming growth factor-beta (TGF-beta). We have identified and characterized a splicing variant of smad3. The splicing variant (smad3-Delta3) lacks exon 3 resulting in a truncated linker region. We could detect mRNA expression of smad3-Delta3 in all investigated human tissues. Real-time PCR analyses demonstrated that the fraction of smad3-Delta3 mRNA compared to normal smad3 varies between tissues. The amount of spliced mRNA was estimated to represent 0.5-5% of the normal smad3 mRNA. When smad3-Delta3 is overexpressed in a fibrosarcoma cell line, the Smad3-Delta3 is translocated to the nucleus upon TGF-beta stimulation and binds the Smad responsive element. Using a CAGA luciferase reporter system, we demonstrate that Smad3-Delta3 has transcriptional activity and we conclude that Smad3-Delta3 possesses functional transactivating properties.

Mesenchymal progenitor cell-mediated inhibition of tumor growth in vivo and in vitro in gelatin matrix.

A preformed gelatin matrix containing adherent rat colon carcinoma cells was transplanted subcutaneously into rats to analyze the outgrowth of the tumor and the inflammatory response. The gelatin matrix simplifies the precise localization of the tumor cells early after implantation and allows the gelatin piece with a growing tumor to be dissected for analysis in vitro, after various times in vivo. The immortalized mesenchymal progenitor cell line MPC1cE was cocultured with rat colon carcinoma cells in vivo in gelatin matrix. The mesenchymal progenitor cells inhibited the outgrowth of the rat colon carcinoma and a complete inhibition was seen if the number of mesenchymal progenitor cells were at least equal to the number of tumor cells. The mixture of tumor cells and mesenchymal progenitor cells induced more infiltration of monocytes and granulocytes than tumor cells or mesenchymal progenitor cells alone. Infiltration of T cells and CD31+ endothelial cells correlated to the presence of tumor cells and not to mesenchymal progenitor cells. These findings suggest that tumor cell culture in vivo in a gelatin matrix is effective for early localization of tumor cells in vivo and that mesenchymal progenitor cells effectively inhibit the growth of the tumor cells in vivo.

Oxi4503, a novel vascular targeting agent: effects on blood flow and antitumor activity in comparison to combretastatin A-4 phosphate.

Oxi4503, which is the diphosphate prodrug of combretastatin A1, is a novel vascular targeting agent from the combretastatin family. Another member of this family, Combretastatin A-4 phosphate (CA4P), is a well-characterized vascular targeting agent already being evaluated in clinical trials. The potential for tumor vascular targeting by Oxi4503 was assessed in a mouse system. This approach aims to shut down the established tumor vasculature, leading to the development of extensive tumor cell necrosis. The vascular effects of Oxi4503 were assessed in the s.c. implanted MDA-MB-231 adenocarcinoma and the MHEC5-T hemangio-endothelioma in SCID mice and in a range of normal tissues. Blood flow was measured by i.v. injection of fluorescence beads, while quantitative fluorescence microscopy was used to measure the spatial heterogeneity of blood flow in tumor sections. Oxi4503 induced the shutdown of tumor blood vessels in a dose-dependent pattern with an ED50 at 3 mg/kg in contrast to 43 mg/kg of CA4P. Quantitative fluorescence microscopy showed that Oxi4503 increased the spatial heterogeneity in tumor blood flow. Oxi4503 affected peripheral tumor regions less than central regions, although this was not as pronounced as seen with CA4P, where only central regions were affected. The vascular shutdown induced by administration of Oxi4503 at a dose of 6 mg/kg resulted in extensive cell loss 24 hours following treatment, which translated into a significant effect on tumor growth. Tumor growth was completely repressed at doses above 12.5 mg/kg of Oxi4503, while doses above 25 mg/kg showed tumor regression and even complete regression in some animals. These results are promising for the use of Oxi4503 as a tumor vascular targeting agent. Moreover the potent antitumor effect when administered as a single agent suggests a different activity profile than CA4P.

Synthesis, in vitro, and in vivo evaluation of phosphate ester derivatives of combretastatin A-4.

Combretastatin A-4 disodiumphosphate (CA4P), a prodrug formulation of the natural product combretastatin A-4 (CA4), is currently in clinical investigation for the treatment of cancer. In vivo, CA4P is rapidly enzymatically converted to CA4, a potent inhibitor of tubulin polymerization (IC(50)=1-2 microM), and rapidly causes bloodflow shutdown in tumor tissues. A variety of alkyl and aryl di- and triesters of CA4P have been synthesized and evaluated as potential CA4 prodrugs and/or stable CA4P analogues.