The AAPS Journal ( # 2017) DOI: 10.1208/s12248-017-0152-y

Review Article Theme: Precision Medicine: Implications for the Pharmaceutical Sciences Guest Editors: Marilyn N. Martinez and Adel Karara

Nanotechnology as a Delivery Tool for Precision Cancer Therapies Bhawna Sharma,1 Rachael M. Crist,1 and Pavan P. Adiseshaiah1,2

Received 14 August 2017; accepted 19 September 2017 Abstract.

Genomic analyses from patients with cancer have improved the understanding of the genetic elements that drive the disease, provided new targets for treating this relentless disease, and offered criteria for stratifying patient populations that will benefit most from treatments. In the last decade, several new targeted therapies have been approved by the FDA based on these omics findings, leading to significantly improved survival and quality of life for select patient populations. However, many of these precision medicines, e.g., nucleic acid-based therapies and antibodies, suffer from poor plasma stability, suboptimal pharmacokinetic properties, and immunological toxicities that prohibit their clinical translation. Nanotechnology is being explored as a delivery platform that can enable the successful delivery of these precision medicine treatments without these limitations. These precision nanomedicines are able to protect the cargo from degradation or premature/burst release prior to accumulation at the tumor site and improve the selectivity to cancer cells by incorporating ligands that can target receptors overexpressed on the cancer cell surface. Here, we review the development of several precision nanomedicines based on genomic analysis of clinical samples, actively targeted nanoparticle delivery systems in the clinic, and the pathophysiological barriers of the tumor microenvironment. Successful translation of these precision nanomedicine initiatives will require an effective collaboration between basic and clinical investigators to match the right patient with the right therapies and to deliver them at therapeutic concentrations which will improve overall treatment responses.

KEY WORDS: cancer therapeutics; genomics; nanomedicine; oncology; precision medicine.

INTRODUCTION Precision, or personalized, medicine involves genetic and other testing that is used to inform treatment strategies specific to an individual patient, in an effort to improve overall recovery and survival. Successful translation of precision medicine depends on clinicians being able to identify a set of genes/markers that can best inform the choice of treatment regimens, allowing them to devise strategies that can be tailored to enhance overall outcomes. Completion of the human genome sequencing project has provided new insights into relevant biomarkers and has pushed precision medicine to the forefront of medical research. The field of oncology, in particular, has benefited immensely from identification of several driver genes (oncogenes and tumor suppressor genes) and requirements for activation of signaling pathways (e.g.,

1

Nanotechnology Characterization Laboratory, Cancer Technology Program, Leidos Biomedical Research, Inc., National Laboratory for Cancer Research, Frederick, 21702, USA. 2 To whom correspondence should be addressed. [email protected])

Research Frederick Maryland (e-mail:

EGFR, Wnt-β-catenin, PI3K-AKT) through sequence analysis of clinical samples. The stratification of patients with cancer can be mapped to identify genetic aberrations (e.g., mutations in oncogenes or tumor suppressor genes) that could benefit from targeted treatment in a select population afflicted with the disease. Several targeted therapies have received FDA approval based on genomic information obtained from patients with cancer. The successful translation of these targeted therapies has improved the progression-free and overall survival of patients when used as a single agent or in combination with other cytotoxic agents. For example, erlotinib, which targets EGFR, is used for treating lung cancer (in combination with carboplatin and paclitaxel) and pancreatic cancer (in combination with gemcitabine) (1,2). A list of additional FDAapproved targeted therapies for cancer is summarized in Table I (3). In addition, there are a number of approved antibody-drug conjugates that take advantage of these previously approved cell surface receptor targets. For example, Kadcyla, which targets Her2, is approved to treat patients with breast cancer in combination with paclitaxel or docetaxel (4). In addition, genomic analysis has helped in identifying patient populations that could benefit from these targeted 1550-7416/17/0000-0001/0 # 2017 American Association of Pharmaceutical Scientists

Sharma et al. Table I. FDA-Approved Therapies Targeting Genetic Aberrations

Indication

Gene

Treatment

Bone cancer Brain cancer/glioma Breast cancer

RANKL VEGF HER2

Denosumab Bevacizumab Trastuzumab, pertuzumab, ado-trastuzumab emtansine, lapatinib Palbociclib, ribocictib Everolimus Bevacizumab Venclexta (venetoclax) Nivolumab Imatinib Bevacizumab Cetuximab, panitumumab Nivolumab Rydapt Ibrutinib Bosutinib Alectinib Cetuximab, erlotinib, gefitinib, lapatinib Crizotinib Pembrolizumab Bevacizumab Vemurafenib, dabrafenib Ipilimumab Nivolumab, pembrolizumab Niraparib, rucaparib, olaparib Bevacizumab Everolimus Erlotinib

Cervical cancer Chronic lymphocytic leukemia (CLL) Chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) Colorectal cancer Head and neck Leukemia, MDS

Lung cancer

Melanoma

Ovarian cancer Pancreatic cancer

CDK4/6 inhibitor mTOR VEGF BCL-2 PD-1 Bcr-Abl tyrosine kinase VEGF EGFR PD-1 FLT3 Btk Bcr-Abl kinase ALK EGFR ROS1 PD-1 VEGF BRAF V600E mutation CTLA-4 PD-1 PARP VEGF mTOR EGFR

https://www.centerwatch.com/drug-information/fda-approved-drugs/; https://www.cancer.gov/about-cancer/treatment/clinical-trials/nci-supported/nci-match

therapies. For example, patients with pancreatic ductal adenocarcinoma harboring wild-type KRAS have an improved median overall survival of 9.2 months when treated with gemcitabine and erlotinib. Patients with mutant KRAS demonstrate a median overall survival of only 5.2 months, further demonstrating that treatment can be optimized through genomic analyses that identify patients who will benefit from a particular therapy (5). In another clinical study, patients with colorectal cancer harboring mutations in KRAS and BRAF conferred resistance to EGFR-targeted therapies (e.g., cetuximab and panitumumab), providing evidence that selecting patients with wild-type KRAS/BRAF provides the most favorable treatment response (6,7). Several diagnostic kits (e.g., Cologuard for the detection of KRAS mutation in patients with colorectal cancer; OvaDx, a panel of genes for the detection of ovarian cancer; and BRAF V600 mutation RT-PCR test for patients with melanoma) have been developed for the detection of genetic mutations from patient samples using minimally invasive techniques. This type of genomic analysis can ascertain which patient population could benefit most from a treatment, or conversely would experience negative effects from a therapy due to gene mutations. The results from these simple tests are used to inform a patient’s treatment regimen to significantly improve overall outcomes.

Precision medicine takes a holistic approach, utilizing all the available tools (i.e., all the -omics) to understand which targeted therapies could be used to improve the overall and progression-free survival for which class of patients with cancer. The increased application of molecular profiling approaches (e.g., genomics, transcriptome, proteomics, and metabolomics) on tumor biopsy samples—detecting mutations in oncogene(s) and/or tumor suppressor gene(s), identifying increased/decreased production of key gene transcripts or proteins, and monitoring changes in metabolite profiles—has improved the prognosis for many patients (8). It requires cross-disciplinary expertise in genomic sequencing and analysis, bioinformatics, as well as expertise to identify the relevance of these genetic variations to clinical treatments (9). The data gathered from these Bomics^ platforms can also provide vital information about additional targets that may be clinically viable but have not yet been tested. These datasets can be used not only to inform the development of novel chemical entities but also to formulate novel delivery platforms (e.g., nanotechnology) to improve drug pharmacokinetics and accumulation at the tumor site. Appropriately tailored nanotechnology platforms can overcome the challenges associated with delivery of many active pharmaceutical ingredients (APIs) used in precision therapies, e.g., small interfering RNA (siRNA), microRNA (miRNA), DNA oligos, and plasmids. Nanotechnology

Precision Nanomedicine for Cancer platforms often decrease the toxicity and increase the selective delivery of their cargo to the diseased sites, thus improving therapeutic response. Several cancer driver genes have been successfully identified, and the delivery of these precision medicines is being explored using nanotechnology (e.g., siKRAS G12D LODER). The translation of these precision nanomedicine therapies will require a thorough understanding of nanoparticles’ interaction with immune cells, biodistribution, and clearance routes to avoid off target toxicities. In addition, understanding the complexity of cancer (initiation, progression, tumor microenvironment or pathophysiological barriers, and drug resistance) is critical to developing effective treatment strategies. A nanoparticle’s design characteristics (e.g., size, surface chemistry, drug release) can be optimized for the therapeutic being delivered, as well as the type and stage of cancer to overcome the pathophysiological barriers of drug transport. Herein, we describe several precision nanomedicines in clinical development, as well as describe approaches to optimizing precision treatment strategies. Precision Medicine: Unraveling the Cancer Genome The genetic (e.g., mutations) and cellular (e.g., fibroblast, immune cells, endothelial cells) heterogeneity that exists within and between primary and metastatic sites, as well as among different patient populations, can make it difficult to identify and validate target signals (10,11). A recent whole-genome sequencing of four treatment-naïve patients with pancreatic cancer alluded to the presence of the same mutations within the driver genes of every metastatic lesion. This is encouraging, as it can be harnessed to provide improved efficacy for this patient population using targeted agents (12). In addition, the study found that most of the tumor heterogeneity constituted mutations in the passenger genes, which have limited or unknown functional roles in tumor development and potential therapeutic response. Genomic data analysis has resulted in the identification of several driver genes that are now used as a guide for selecting a treatment strategy (13) (Table I). For example, the chromosomal translocation of BCR-ABL in chronic myelogenous leukemia (CML) has led to the development of imatinib, a kinase inhibitor, which has improved the survival of patients with CML to over 95% (14). Similarly, identification of mutations in BRAF (V600E) and EGFR (exon 19 deletion or exon 21 substitution; L858R) in melanoma and lung cancers, respectively, has led to the development of targeted therapies (e.g., vemurafenib to target BRAF mutation; erlotinib and gefitinib to target EGFR mutation) that have improved patient overall survival (1,15). Unfortunately, the presence of a gene mutation does not guarantee that the targeted treatment strategy will result in a positive therapy response. For example, approximately 8% of patients with colorectal cancer are predisposed with the presence of a BRAF V600E mutation and are associated with a poor prognosis (16). In a pilot phase II trial, patients with metastatic BRAF-mutated colorectal cancer were treated with the single-agent vemurafenib, but did not show clinical activity. Their resistance to vemurafenib was partly attributed to the presence of additional KRAS and NRAS mutations in

low frequency (17). A couple of plausible mechanisms of resistance have been proposed since the surprising poor response of vemurafenib was observed in patients with colorectal cancers (CRCs). First, vemurafenib is a substrate of the drug efflux pump, P-glycoprotein, which has enhanced expression in CRC in comparison to melanoma, and could have resulted in the poor therapeutic response (18). Second, preclinical studies have indicated that a single-agent BRAF inhibition in CRC may not be sufficient in inhibiting the MAPK pathway, but was enough in melanoma, confirming the limited vemurafenib activity observed in the clinic for CRC, perhaps due to the feedback EGFR reactivation (19,20). Recently, basket clinical trials are being employed to more widely identify patient populations that could benefit from targeted treatments based on key genetic aberrations (21). Whereas traditional clinical trials focus on select cancer indications (e.g., approval of vemurafenib for BRAF mutation; olaparib for BRCA1/2 alterations), basket clinical trials focus on select genetic mutations regardless of the cancer type (13). Indeed, the FDA recently approved Keytruda (pembrolizumab), an anti-PD-1 inhibitor for unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) solid cancers regardless of the tumor location (e.g., colorectal, breast, pancreatic, small cell lung cancer, biliary, esophageal, endometrial, or prostate cancers). In another example, larotrectinib treatment of adult and pediatric patients with cancer harboring tropomyosin receptor kinase (TRK) fusion protein (NAVIGATE and SCOUT clinical trials) led to a 76% objective response rate, which was durable and consistent in patients with TRK fusion protein. This positive response rate led to the recent FDA designation of breakthrough therapy for larotrectinib (LOXO-101) (22). This designation, along with the recent approval of Keytruda—an example of a personalized immunotherapy—further emphasizes the role of genomics in treating this lethal disease (23,24). Despite the encouraging basket clinical results leading to the approval of novel therapies for several solid cancers, it is prudent to remain cautious as still other unknown genetic elements/signaling pathways may override the therapeutic response and enhance tumor progression (e.g., vemurafenib in colon cancer) (20). Two independent reports from clinical trials with checkpoint inhibitors provided clues to the presence of additional underlying genetic aberrations that can lead to the hyperprogression and clinical deterioration in patients with cancer (25). In one study, loss-of-function mutations in JAK1/2 in patients with melanoma and colorectal cancer that received Keytruda resulted in resistance to PD-1 therapy (26). The proposed mechanism is that the loss of JAK1/2 function leads to insensitivity to interferon gamma signaling and provides a selective advantage for cancer cell proliferation, resulting in the resistance to PD-1 blockade therapy (26). In another study, a subset of patients treated with checkpoint inhibitors showed accelerated tumor growth as monitored by time-to-treatment failure (25). Genomic analysis from these negative responders confirmed the amplification of MDM2/MDM4 or EGFR aberrations that correlated with enhanced tumor growth and hyperprogression. These examples highlight the importance of careful genomic analysis as a standard practice prior to devising a treatment strategy.

Sharma et al. Table II. Clinical Trials of Nanoformulations for the Delivery of Personalized Medicines

Nano-intervention Small molecule Aurora B kinase inhibitor in polymeric nanoparticle, AZD2811 Nanoparticle albumin bound rapamycin, targeting mTOR mutations, ABI-009

siRNA/miRNA/DNAi/plasmid Liposomal siRNA for BCL2

Liposomal formulation of PNT2258

Target

Indication

Clinical trial identifier

Aurora B kinase

Advanced solid tumors

Phase I; NCT02579226

mTOR

Several advanced cancers (perivascular epithelioid cell tumors, bladder cancer, CNS neoplasm)

Phase Phase Phase Phase Phase

II; NCT02646319 I; NCT00635284 (28) II; NCT02494570 I/II; NCT02009332 I; NCT02975882

BCL2

Malignant solid tumors

BCL2

Relapsed/refractory non-Hodgkin lymphoma Glioblastoma, recurring glioblastoma

Phase Phase Phase Phase Phase

I; NCT01191775 (29) II; NCT01733238 II; NCT02226965 II; NCT0237803 II; NCT01733238 (30)

siRNA targeting BCL2L12 conjugated BCL2L12 to gold nanoparticles, NU-0129 Rexin-G, matrix-targeted nanoparticle Cyclin G1 with dominant negative cyclin G1 construct

Several advanced cancers (breast cancer, Phase I/II; NCT00505271 osteosarcoma, sarcoma, pancreatic Phase II; NCT00572130 (31) cancer) Phase I/II; NCT00505713 (31) Phase I/II; NCT00504998 (32) Phase I; NCT00121745 Multiple myeloma, B cell Phase I/II; NCT01435720 (33) lymphoma and plasma cell leukemia

SNS01-T, siRNA targeting elF5A and plasmid expressing apoptotic mutant elF5A with polyethylenimine Liposomal siRNA targeting EPHA2 (34) Liposomal FUS1 gene delivery

elF5A

EPHA2 FUS1

Advanced cancer Lung cancer

LiPlaCis, liposomal Grb-2 anti-sense oligonucleotide

Grb-2

Leukemia

PEGylated lipopolymer formulation for IL-12 gene delivery, EGEN-001 Liposomal microRNA, miR-34, miR-RX34

IL-12

Ovarian cancer

miR-RX34

Phase I; NCT03020017

Solid tumors or hematological malignancies K R A S Pancreatic cancer

Polymeric siRNA for mutant KRAS, siG12D LODER Liposomal siRNA targeting MYC, DCR-MYC

Mutant G12D MYC

Liposomal RNAi therapeutic targeting PKN3, Atu027 Liposomal siRNA targeting PLK1, TKM-080301

PKN3 PLK1

Advanced solid tumors (multiple myeloma and lymphoma, hepatocellular carcinoma) Advanced solid tumors (pancreatic cancer) Liver cancer

Liposomal siRNA targeting VEGF-A and KSP, ALN-VSP02

VEGF-A and KSP

Solid tumors

Phase Phase Phase Phase Phase Phase Phase

I; NCT01591356 I; NCT00059605 (35) I/II; NCT01455389 II; NCT02781883 I; NCT01159028 I/II; NCT02923986 II; NCT01118052

Phase Phase Phase Phase Phase Phase

I; NCT01829971 (36) I/II; NCT02862145 I; NCT01188785 (37) II; NCT01676259 I; NCT02110563 Ib/II NCT02314052

Phase I/II; NCT01808638 (38) Phase I; NCT00938574 (39) Phase I/II; NCT01262235 (40) Phase I; NCT01437007 Phase II; NCT02191878 Phase I; NCT01158079 (41) Phase I; NCT00882180 (42)

https://clinicaltrials.gov/ct2/results?term=nanoparticle%2C+cancer%2C+target&pg=1

Nanomedicine Delivery of Targeted Therapies for Precision Oncology Omics-Driven Cancer Nanomedicine Omics analysis of patients with cancer has provided researchers and pharmaceutical companies valuable information (cancer and stage-specific datasets) that is being used to identify targets for novel precision medicines (e.g., siRNA,

miRNA, DNA, CRISPR-CAS9). Unfortunately, systemic delivery of these nucleic acid-based therapies often results in rapid degradation by serum nucleases, with a blood half-life of 5– 10 min, thus providing no therapeutic advantage (27). Delivery of these agents using nanoplatforms, however, can circumvent this and other immunotoxicological issues, dramatically improving therapeutic efficacy. In fact, many of these nanoformulated nucleic acid-based therapies are now in early phase clinical trials for a variety of cancer indications (Table II).

Precision Nanomedicine for Cancer Despite the delivery challenges, siRNA and other nucleic acid-based treatments can provide several therapeutic advantages. These treatments possess the ability to target any gene, reducing off-target toxicity and enhancing potency. To overcome the delivery challenges associated with nucleic acid-based therapies, various nanoparticle platforms have been used (43,44). These nanoparticle platforms shield the nucleic acids from serum nucleases, prolonging the plasma half-life. Nanoplatforms can also be appropriately tailored with the addition of polyethylene glycol (PEG) or other similar polymer moieties on the nanoparticle surface to reduce immune cell recognition and accumulation in the mononuclear phagocytic systems (e.g., liver, spleen) (45). Additionally, immunotoxicities (e.g., cytokine overexpression, hypersensitivity, etc.) can often be mitigated by chemical modification of the nucleotide bases in addition to modifying the nanoparticle platform (46). Systemic delivery is the preferred route of administration for nanoformulations containing nucleic acids, as the nanoparticles can efficiently accumulate at both the primary and metastatic cancer sites (46). Many of the degradants can then be rapidly cleared by the kidneys and liver (44). Select strategies being explored to deliver nucleic acid-based therapies are described below. An siRNA therapy in clinical trials is ALN-VSP, a 100 nm lipid-based nanoformulation encapsulating a 1:1 ratio of two different chemically modified siRNAs that target vascular growth factor A (VEGFA) and kinesin spindle protein (KSP). The plasma pharmacokinetic analysis from phase I clinical trials showed a similar maximum concentration (Cmax) and area under the curve (AUC) for KSP and VEGF siRNA following a single systemic injection of ALNVSP (NCT01158079) (42,46). The treatment normalizes the tumor vasculature as evaluated by VEGF mRNA levels and correlated with a decrease in tumor blood flow as monitored by DCE-MRI. The decrease in KSP mRNA levels, which impacts the mitotic cell cycle, was also observed via extrahepatic tumor biopsy (42). The pharmacodynamic effect observed in biopsy samples from patients confirmed the successful delivery of both siRNAs, suggesting stability of the nanoparticles during systemic circulation. Although intravenous infusion of the ALN-VSP nanoparticles often resulted in cytokine activation (e.g., IL-10, IL-1Ra, IL-6, TNF, and G-CSF) and dose-limiting toxicities such as thrombocytopenia (low platelet counts) and hypokalemia (low potassium level), at least one patient benefited from the experimental treatment. A complete treatment response was observed in a patient with endometrial cancer with metastases in hepatic and abdominal lymph node, an especially encouraging result given the disease had progressed following standard chemotherapy. One of the first targeted siRNA-containing nanoparticles in clinical trials was CALAA-01 (NCT00689065). It is a 75 nm cationic cyclodextrin-based PEGylated polymer with a human transferrin protein ligand to target cancer cells (46). The cargo of CALAA-01 is an siRNA that targets the M2 subunit of ribonucleotide reductase (RRM2), which is overexpressed in several cancers and is involved in nucleic acid metabolism (46). Clinical biopsy samples confirmed dose-dependent CALAA-01 accumulation in tumors and not in adjacent normal tissues, and accumulation coincided

with the knockdown of RRM2 at the transcript and protein levels (46,47). Even though CALAA-01 demonstrated encouraging results in a phase I trial, 21% of the patients stopped treatment due to toxicity, stemming from the transferrin-targeted ligand and drug instability (48). The dose-limiting toxicities of CALAA-01 were hypersensitivity reaction, fever, lymphopenia, and diarrhea (46). In addition, CALAA-01 treatment induced activation of cytokines (e.g., IL-6, IL-10, TNF, IFNγ). Due to these severe adverse events and dose-limiting toxicities, further clinical development of CALAA-01 was not pursued. DNA oligonucleotides are also being used as precision treatments and have been delivered using liposomal formulations. For example, PNT2258 utilizes a 130 nm liposome to encapsulate a 24-mer DNA oligonucleotide (DNA interference; PNT100) that targets the 5′-untranscribed regulatory region of an anti-apoptotic protein, BCL2 (49). In a phase II clinical trial (Wolverine study; NCT02226965) conducted in patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL) and in patients with Richter’s transformation (Brighton study), interim findings showed only modest efficacy (8%) as a single agent (49). However, in four patients with DLBCL, two patients showed a complete response, one patient showed a partial response, and one patient showed signs of disease stabilization, indicating that a subset of patients with DLBCL might be responsive to PNT2258 treatment. Several adverse events related to PNT2258 infusion were reported, such as back/flank pain, nausea, vomiting, diarrhea, elevated aspartate aminotransferase (AST), and thrombocytopenia that eventually led to its discontinued development (29). Lipid-based nanoparticles are also being used to deliver plasmid DNA samples, such as SGT-53 and EGEN-001. SGT53 is a cationic charged anti-transferrin-targeted lipoplex containing a plasmid DNA encoding for human wild-type p53 gene (50). In phase Ia clinical trials, p53 transgene was detected by PCR in malignant metastatic biopsy specimens from three patients, indicating successful delivery of the plasmid containing p53 using a targeted immunoliposome formulation (NCT00470613) (51). EGEN-001 is an immunotherapeutic agent consisting of IL-12 plasmid encapsulated in polyethyleneglycol-polyethyleneamine-cholesterol nanoparticles, which enables uptake by ovarian cancer cells (52). An initial phase I clinical trial in patients with platinum-resistant recurrent ovarian cancer demonstrated reasonable safety as a single agent (NCT01118052) (52). Both formulations experienced several adverse events, such as fatigue, fever, chills, leukopenia, etc., but continue evaluation in phase II clinical trials (Table II). Nanotechnology strategies are also being used in the delivery of the newly discovered gene editing clustered regularly interspaced short palindromic repeat (CRISPR/ CAS9) system, which has the potential to provide exciting new avenues for treating genetic diseases including cancer (53). In a recent example, DNA nanoparticles with a partial sequence homology to the single guide RNA (sgRNA) demonstrated targeting to GFP in a U2OS-GFP tumor model (54). The DNA nanoparticles were coated with a cationic polymer polyethylenimine to promote endosomal escape following cellular uptake to enhance the delivery of CAS9/

Sharma et al. sgRNA to the nucleus. The intratumoral delivery of the construct resulted in a modest 25% decrease in GFP fluorescence at the site of injection (54). To take advantage of precision, gene modification systems would require systemic delivery of the construct to correct the mutated gene at both primary and metastatic tumor sites to ensure a complete response. The nanotech delivery of CRISPR/CAS9 systems is still at an early stage of development. Although nanomedicines for nucleic acid delivery have shown, at best, only modest improvements in overall survival and patient care to date, there are many factors that likely contribute to their limited success. Most of the patients enrolled in these trials have been previously treated with standard-of-care therapies. Responses could be limited due to the development of clonal populations/heterogeneity of cancer cells with drug resistance and/or more aggressive phenotypes (46). Patients were not stratified based on underlying changes in genomics, nor were they assessed for drug resistance, which would unduly impact the efficacy of these nanoparticle treatments. The majority of clinical trials to deliver nucleic acid therapies have been halted because of hematological and immunological toxicities. Most of these first-generation nanoparticles have resulted in induction of cytokines and other dose-limiting toxicities contributed mainly by the nucleic acid. Now however, there is a significant body of knowledge regarding strategies to overcome these toxicities. Second-generation nanoparticles are using chemical modification of the nucleic acids combined with appropriately tailored nanoparticle physicochemical properties to circumvent these toxicities (45,55). The eventual clinical translation of these formulations stands to dramatically improve patient outcomes. Tumor-Targeted Nanomedicines An improved understanding of tumor biology has led researchers to exploit potential targets (e.g., dysregulated oncogenes, activation of signaling pathways, and cancer cell surface proteins) for selective delivery of therapeutic agents using novel nanoformulations (13). Nanotechnology has taken advantage of the existing knowledge of targeted therapies (Table I) by incorporating targeting ligands (e.g., aptamers, small molecules, antibody fragments) that bind to overexpressed receptors on cancer cells to increase tumor specificity and reduce off-target drug toxicities (56). A number of ligand-targeted nanoparticles are in clinical trials for cancer therapy. These are summarized in Table III. Based on the tumor biology of prostate cancer, plasma PSA levels are considered a surrogate marker for early detection of the disease (63). Utilizing this overexpression, a PSMA-targeted polymeric nanoparticle encapsulating docetaxel (BIND-014) was tested in phase I and II studies with the intention of selective targeting to prostate tumor cells and neovasculature that overexpress the PSMA receptor (60). In a phase I clinical trial (NCT01300533) and preclinical toxicokinetic studies in mouse, rat, and monkey, BIND-014 was retained in the vascular compartment which correlated to improved efficacy in multiple cancer types (e.g., cervical cancer and cholangiocarcinoma), suggestive of altered pharmacokinetics of the particles (60,64). Enhanced anti-tumor activity observed with BIND-014 for cancer indications that

did not respond to Taxotere suggest increased nanoparticle uptake by the cancer cells/neovasculature due to the presence of the PSMA targeting ligand on the nanoparticles and/or increased nanoparticle accumulation via the enhanced permeation and retention (EPR) effect (60). In a subsequent phase II clinical trial (NCT01812746), BIND-014 demonstrated anti-tumor activity in three patients with chemotherapy-naïve metastatic castration-resistant prostate cancer, an increase in median OS (13.4 months), and a 50% reduction in PSA levels in 30% of PSA evaluable patients (65). In another example, sub-10 nm cRGD targeted silica nanoparticles (C-dots) were labeled with dual 124I PET and Cy5 optical imaging agents for rapid, non-invasive detection of cancer lesions and to enable demarcation of tumor margins for surgical resection (66). The C-dot particles were stable and well tolerated in first-in-human studies in patients with metastatic melanoma, with selective uptake and accumulation of the labeled nanoparticles at the tumor site. Systemic administration of 124I-cRGDY-PEG-C-dots did not result in any appreciable accumulation in off-target or mononuclear phagocytic organs—liver, lung, spleen, or bone marrow. A low level (< 0.001%/g) of radiotracer was observed in the gastrointestinal tracts, which is in line with low level expression of αvβ3 integrin in the intestine and tissues of vascular origin (67). These ultrasmall nanoparticles enabled rapid renal clearance, and the particles did not show any toxic or adverse events, suggesting utility in early cancer diagnosis (66). Another actively targeted nanoparticle undergoing clinical evaluation is MM-302, a HER2-targeted, PEGylated liposomal doxorubicin designed to treat patients with breast cancer with HER2 receptor expression. In preclinical and early phase I clinical trials, MM-302 alone or in combination with trastuzumab (which binds to a different HER2 epitope) demonstrated an improved efficacy and safety profile in a HER2-positive advanced/metastatic breast cancer patient population (59). Based on positive findings from these initial clinical studies, a phase II clinical trial (HERMIONE; NCT02213744) was initiated in anthracycline-naïve patients with locally advanced or metastatic breast cancer positive for HER2 expression. Unfortunately, patients treated with MM302 in combination with trastuzumab showed no clear benefit in terms of median progression-free survival over the comparator treatment (chemotherapy of physician’s choice in combination with trastuzumab). Notably, no serious adverse events, such as cardiac toxicity, were observed in either the monotherapy or combination treatment arms. The few examples noted above provide encouraging insight that actively targeted nanoparticles do promote selectivity to the tumor in the clinical setting. To further improve the delivery of ligand-targeted nanoparticles to the tumor, it is important to understand the disease heterogeneity and differences in receptor expression within the primary and between primary and metastatic sites. Evaluating receptor expression on cancer cells both spatially and temporally due to heterogeneity will allow for improved receptor targeting based upon different stages of disease development and different cancer types. An approach that targets multiple surface receptors in different cell populations within the tumor can also increase the therapeutic potential of these

Precision Nanomedicine for Cancer Table III. Clinical Trials of Actively Targeted Nanoparticles for the Delivery of Precision Medicines

Targeted NP

Targeting ligand Therapeutic agent Indication

Clinical trial identifier

EGFR targeting EDV-DOX nanocells Anti-EGFR antibody-coated immunoliposomal doxorubicin TargomiRs, anti-EGFR antibody targeted EDV nanocells with miR-16 EGFR-targeting EDVmitoxantrone nanocells, ErbituxEDVsMIT MM-302, HER2 targeted antibody liposomal doxorubicin conjugate BIND-014, PSMA targeted docetaxel nanoparticle

EGFR

Doxorubicin

Recurrent gliobastoma multiforme

Phase I; NCT02766699

EGFR

Doxorubicin

Solid tumors

Phase I; NCT01702129 (57)

EGFR

miR-16

Lung cancer, malignant mesothelioma

Phase I; NCT02369198 (58)

EGFR

Mitoxantrone

Pediatric solid and CNS tumors

Phase I; NCT02687386

HER2

Doxorubicin

Breast cancer

Phase II/III; NCT02213744 (59)

PSMA

Docetaxel

Advanced and metastatic solid tumors (prostate cancer, NSCLC, urothelial carcinoma, cholangiocarcinoma, cervical cancer, squamous cell carcinoma of head and neck) Solid tumors (glioblastoma, pancreatic cancer, recurrent tumors)

SGT-53, anti-transferrin Transferrin antibody bound liposomal p53 plasmid delivery

p53 plasmid

SGT-94, anti-transferrin Transferrin antibody bound liposome RB94 gene delivery CALAA-01, transferrin targeted Transferrin polymer based RRM2 siRNA nanoparticles

RB94 gene

Solid tumors

Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase

RRM2 siRNA

Solid tumors

Phase I; NCT00689065 (48)

targeted nanomedicines. Additionally, employing ligandtargeted radiopharmaceuticals may help in identifying tumor mass at primary and metastatic sites and in selecting the patient population which might respond to ligand-targeted nanotherapeutics due to the differences in receptor expression. Indeed, this novel approach is being explored for the folate-targeted radioimaging agent etarfolatide and has enabled the identification of patients that are folate receptor positive for lung, kidney, brain, or ovarian cancers (68). Nanomedicines Tailored to the Tumor Microenvironment Thus far, precision medicines have been described as specific entities (i.e., nucleic acid-based therapies, antibodies, or small molecules) that are used to target either oncogenes or signaling pathways involved in disease progression. And, nanotechnology is being exploited as the ideal platform for delivery of these personalized therapies, as described in the previous sections. These genes and signaling pathways are identified by genomic, proteomic, metabolomic, and other screenings of a patient or population of patients. However, another often overlooked approach to achieving precision treatments involves personalized screening or characterization of the tumor microenvironment. The tumor microenvironment will vary based on the cancer type, previous treatments, patient genetics, or other factors (e.g., physiological or pathological barriers). A thorough understanding of the tumor microenvironment can allow for a more precise selection of appropriate therapies, such as vascular

II; NCT01812746 I; NCT01300533 (60) II; NCT01792479 II; NCT02283320 II; NCT02479178 I; NCT00470613 (61) I; NCT02354547 II; NCT02340117 II; NCT02340156 I; NCT01517464 (62)

normalization agents, etc. Furthermore, nanoparticle platforms can be appropriately tailored to deliver cytotoxics and/ or precision medicines with greater efficiency to a more defined tumor microenvironment, thus providing an enhanced therapeutic benefit. Successful delivery of nanomedicines depends on improved pharmacokinetic profiles and overcoming the pathophysiological barriers of the tumor microenvironment, which may have both interpatient and intrapatient heterogeneity (69–72). For precision medicine to work, nanomaterials that are utilized for drug delivery should be tailored to accumulate preferentially in the tumor interstitium in comparison to the normal tissue. There are several factors that should be considered for a successful translation of a nano-oncology drug, for example, normalizing the tumor vasculature and stroma for increased nanoparticle accumulation at the tumor site and optimizing the nanoparticle size and other physicochemical characteristics for optimal tumor-targeted delivery. The tumor microenvironment is highly heterogeneous, composed of cancer cells as well as both cellular (e.g., fibroblast, endothelial cells, immune cells) and acellular stromal matrix (e.g., collagen, glycosaminoglycan, hyaluronan) (73). The tumor heterogeneity within a patient’s primary tumor and between primary and metastatic sites can significantly impact nanomedicine delivery and treatment outcomes (74,75). Effective translation of a nanomedicine requires fine tuning of the nanoparticle characteristics to enable homogeneous extravasation in the tumor, overcoming several transport barriers (71,76). Nanoparticle accumulation

Sharma et al. at the tumor site varies significantly from one cancer indication to another depending on particle size and other physicochemical parameters (69). For example, the distribution of an indium-labeled liposome (roughly the same size as Doxil) demonstrated significantly different levels of accumulation in patients with breast cancer (5 ± 3% injected dose/kg) in comparison to patients with head and neck cancers (33 ± 16% injected dose/kg) (77). The effectiveness of 100 nm PEGylated liposomal doxorubicin (Doxil), as monitored by overall survival, tumor response rates, and reduced cardiotoxicity, was evident in patients afflicted with Kaposi scarcoma and patients with ovarian cancer that were cisplatin responsive (78,79). On the contrary, patients with metastatic breast cancer and multiple myeloma demonstrated reduced cardiotoxicity, but did not show significantly improved overall survival (80). Several reviews highlight the importance of nanoparticle physicochemical properties to biodistribution and tumor accumulation (70,81,82). The size of a patient’s tumor can impact an efficient and homogeneous delivery of nanomedicine into the tumor (81,83). Larger tumors tend to be more necrotic with decreased vessel density and high interstitial fluid pressure in the center compared with the tumor periphery, which can severely reduce nanomedicine delivery (84). Several strategies have been employed to overcome this pathophysiological barrier by improving the vessel perfusion in the tumor (e.g., anti-angiogenic agents) or relieving the solid stress (e.g., losartan, PEG-hyaluronidase, saridegib/vismodegib) to increase the extravasation of nanomedicine at the tumor site (71,85). Anti-angiogenic therapies targeting the proangiogenic factors (e.g., VEGF, bFGF, PDGF) when used at an appropriate dose can lead to remodeling of the tumor vasculature, improve vessel perfusion, and increase drug delivery to the tumor, which is especially beneficial for hyperpermeable tumors (e.g., glioma) (85). Clinically, treatment with anti-angiogenic therapies in patients with rectal cancer and glioblastoma has demonstrated normalization of tumor vessels with a decrease in vessel size and permeability/ leakiness and increased drug delivery (86,87). The cessation of anti-angiogenic therapy has been shown to cause these improved tumor vessels to revert back to the abnormal vessel structure, evident in a glioblastoma clinical trial, suggesting that anti-angiogenic therapy should be continued to improve the delivery of precision medicine at the tumor site (87,88). Several preclinical studies have shown that anti-angiogenic therapies improve the delivery of small nanoparticles (approx. 10–20 nm) in comparison to larger nanoparticles (approx. 100 nm) (89,90). To gain a clinical benefit from antiangiogenic agents in the patient population, the dose and schedule of an anti-angiogenic treatment should be modulated depending on the type of cancer by employing either a non-invasive imaging modality or biomarker analysis (e.g., VEGF level) to improve overall survival. Another aspect of tumor biology that should be considered to improve the delivery of precision medicine in addition to tumor vasculature is the modulation of the tumor stroma. The desmoplastic stroma in the tumors (e.g., pancreatic, breast, melanoma cancers) can form a pathobiological barrier that can impede the accumulation of large size nanoparticles,

but may allow the extravasation of small size nanoparticles (91). Several agents (e.g., losartan, PEGylated hyaluronidase, inhibitors of Hedgehog signaling pathways) have been employed to relieve the solid stress in fibrotic tumors to decompress the vessels thereby enhancing nanomedicine accumulation (71,91,92). In a small randomized clinical trial using the Hedgehog signaling inhibitor vismodegib, an improved median overall survival in patients was seen in patients with pancreatic cancer when combined with gemcitabine and nab-paclitaxel, further suggesting that normalizing the tumor stroma and reducing the solid stress can increase the delivery of precision nanomedicines (93). In an orthotopic model of pancreatic cancer, animals that were injected with losartan (an FDA-approved anti-hypertensive drug that is also known to have anti-fibrotic activity) demonstrated an increase in the accumulation of PEGylated liposomal doxorubicin (Doxil) in comparison to animals that received Doxil alone (91). The increase in the transport of larger size nanoparticles (e.g., 100 nm Doxil) was due to the reduced solid stress/vessel compression by the tumor stroma, which led to improved vessel permeability (91). These few examples further emphasize the need to optimize both the physicochemical properties of the nanoparticle and the tumor microenvironment for a successful translation of precision nanomedicine in the clinic. CONCLUSIONS Precision medicine for cancer has advanced substantially in the last decade due to improvements in high-throughput genome sequencing and profiling of transcripts and proteins. This has led to the discovery of many new targets in the fight against cancer, including highly potent and selective inhibitors, siRNA gene knockdown therapies, and antibodytargeted receptors. As stand-alone treatments, however, many of these therapies suffer from poor pharmacokinetics (e.g., short half-life), dose-limiting toxicities (e.g., hypersensitivity, lymphopenia, thrombocytopenia, hypokalemia, increase in AST, cytokine release), and adverse reactions in patients (e.g., fever, chills, infusion-related back pain, nausea, diarrhea). Many of these limitations, though, can be overcome by selecting an appropriately tailored nanoformulation for the delivery of these precision medicines (45,55). The identification of receptor overexpression on cancer cells has led to the development of several actively targeted nanoformulations to improve selectivity to the cancer cells and reduce off-target toxicities, and these are now being tested in clinical trials for various cancer indications. The translation of these novel therapeutics to the clinic has been possible because of the alignment of datasets obtained from a multitude of cancer patients with disease management. Enhanced collaborations among different stakeholders (basic and clinical translational labs, nanoformulation scientists, and oncologists) also help to promote the bench-to-bedside translation of these novel personalized medicines. Successful translation of precision nanomedicines depends upon evaluation of several criteria, in addition to the underlying genetic aberrations—thorough understanding and characterization of tumor anatomy, stage of the disease, receptor expression, prior chemotherapy regimens, and tumor microenvironment, all of which can substantially

Precision Nanomedicine for Cancer impact novel nanotherapy responses. The homogeneous distribution of precision nanomedicines to the tumor can be achieved by tailoring nanoparticle physicochemical characteristics and normalizing the tumor microenvironment (vessels and/or stroma), which together can enhance therapeutic efficacy and improve progression-free survival (89). Finally, development of a workflow to understand the genetic drivers for this highly heterogeneous disease will enable the delivery of a well-characterized precision nanomedicine in a patient population that could immensely benefit in the clinic. Funding Information This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.

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